WO2024073453A2

WO2024073453A2 - Tissue processing cartridge and instrument

Info

Publication number: WO2024073453A2
Application number: PCT/US2023/075173
Authority: WO
Inventors: Stevan Jovanovich; John BASHKIN; David Eberhart
Original assignee: S2 Genomics Inc
Current assignee: S2 Genomics Inc
Priority date: 2022-09-26
Filing date: 2023-09-26
Publication date: 2024-04-04
Anticipated expiration: 2025-03-26
Also published as: WO2024073453A3

Abstract

Systems, methods, and apparatus are described to collect and prepare cells, nuclei, subcellular components, and biomolecules from specimens including tissues. Some of the systems provided herein comprise an instrument and cartridge. The instrument can comprise at least one cartridge interface configured to engage at least one cartridge. In some cases, the cartridge comprises a functional unit (e.g., a processing chamber comprising a grinder assembly) and at least one cartridge bay or vessel bay, wherein the cartridge bay or vessel bay comprises attachment features permitting at least one functional unit to engage with the cartridge. The functional unit or units can include at least one of a filtration unit, a magnetic processing unit, a dissociation chamber, a processing chamber, an output unit, a flowcell unit, a tangential flow filtration unit, and a waste unit.

Description

TISSUE PROCESSING CARTRIDGE AND INSTRUMENT

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/377,137, filed on September 26, 2022, which is incorporated herein by reference in its entirety.

FIELD OF INVENTION

[0002] This disclosure relates to the field of sample preparation from biological materials. More specifically, the disclosure relates to the processing of solid tissues into single cells or single nuclei with optional magnetic bead purification into purified cell-types that may be diluted to a specific titer and counter for bioanalysis. The disclosure also relates to the production of bulk NGS libraries from tissues and the labeling of surface epitopes of cells.

BACKGROUND

[0003] Traditional sequencing approaches generally involve sequencing nucleic acids collected en masse from large quantities of cells, yielding a global view of the average genotype or gene expression of the sample. In contrast, single-cell sequencing can provide a detailed snapshot of individual cells within such a sample at a DNA or RNA level. In some cases, singlecell sequencing is accomplished by tagging the nucleic acids in an individual cell with a molecular tag that identifies their cell of origin.

[0004] Preparation of high-quality single-cell or single-nuclei suspensions from solid tissues continues to be a major experimental bottleneck and customer pain point. Protocols can be timeconsuming and difficult to execute reproducibly. Different tissues and cell types have differing dissociation requirements to produce single-cell suspensions. Manual processing can lead to inconsistent yields, viabilities, and cell-type representation, which can be daunting experimental challenges. There is a need in the art for improved systems for processing tissues and cells.

SUMMARY

[0005] This disclosure provides systems, instruments, cartridges, and related methods for processing tissues. Generally, the compositions and systems provided herein can be used to process tissues into cell suspensions, subcellular organelle suspensions, or nuclei suspensions. In some cases, this disclosure provides modular cartridges that comprise bays with attachment features that can be used to engage with one or more functional units provided herein. Exemplary functional units include, but are not limited to, at least one processing chamber comprising an internal surface functionalized with grinding features; at least one filtration unit comprising a filter; at least one magnetic processing unit comprising magnetic particles; at least one output unit comprising a separable output tube; at least one flowcell unit; at least one tangential flow filtration unit; at least one waste unit; or a combination thereof.

[0006] In some embodiments, this disclosure provides a Sample Processing System that can be used for tissue processing. A Tissue Processing System embodiment can be implemented as a flexible, extensible system that can process solid or liquid tissue and other samples into single cells, nuclei, organelles, and biomolecules with mechanical and enzymatic or chemical processes. In some cases, the system produces single nuclei, subcellular components or organelles, biomolecules (e.g., DNA; RNA; proteins; carbohydrates; lipids); metabolites; and other biological components, including natural products for bioanalysis. In some embodiments, the Tissue Processing System performs affinity or other purifications to enrich or deplete cell types, organelles such as nuclei, mitochondria, ribosomes, or other organelles, or extracellular fluids or remove debris. In some embodiments the Tissue Processing System can perform bulk Next Generation Sequencing (NGS) library preparation. In some embodiments, the Tissue Processing System processes tissue into single-nuclei (or sincle cell or single subcellular organelle) libraries for sequencing including Sanger, NGS, single nuclei NGS, and other nucleic acid sequencing technolgies, or protoeomics, or other analytical methods.

[0007] In some embodiments the Sample Processing System can be integrated with downstream bioanalysis to create a sample-to-answer system. In some embodiments, a Tissue Processing System processing embodiment is integrated with a nucleic acid bioanalysis system to sequence nucleic acids from FFPE preserved tissues. Integrated is used to mean, in some cases, that the workflows directly interface with, or in other contexts that the physical system directly interfaces with, or is incorporated into a system, instrument, or device, or the output of one system is compatible with the input of the next system. In some embodiments, the Tissue Processing System is integrated with a nucleic acid sequencer to produce a sample-to-answer system. In other embodiments the Tissue Processing System is integrated with an optical module to produce a sample-to-answer system for cell surface markers, enyzmes, metabolites, cell health, or other cellular components.

[0008] The Sample Processing System can have multiple subsystems and modules that perform processing or analysis. In some embodiments, one or more cartridges performs one or more steps in the processing workflow. In some embodiments the cartridges have multiple processing sites such as processing chambers that can process more than one sample. In some embodiments a cap couples mechanical disruption on the cartridge from a Physical Dissociation Subsystem. In some embodiments reagents from an Enzymatic and Chemical Dissociation Subsystem are delivered to the cartridge by a Fluidic Subystem to regions that are used as Processing Chambers and Post-Processing Chambers to disrupt or dissociate specimen and process the cells, subcellular components, and biomolecules for bioanalysis. In some embodiments, the Fluidic Subsystem uses syringe pumps, piezopumps, on-cartrige pumps and valves, vacuum (negative or positive pressure), pressure, pneumatics, or other components capable of moving fluids.

[0009] The addition of fluids can be controlled by a Fluidic Subsystem with the complete system controlled by software in a Control Subsystem which can include the user interface through a device comprised of monitor, embedded display, touch screen; or through audio commands through the system or an accessory devices such as a cell phone or microphone. In some instances the Control Subsytem can include interfaces to laboratory information management systems, other instruments, databases, analysis software, email, text, and other applications. In some instances, the Control Subsystem can include control software and scripts that contol the operation and in some embodiemtns the scripts can be revised, created, or edited by the operator.

[0010] In some embodiments, the amount of dissociation is monitored at intervals during the dissociation and in some instances the yield is determined during or after processing using a Measurement Subsystem. The degree of dissociation can be determined inside the main dissociation compartment and/or in a separate compartment or channel, and/or in the external instrument. For example, the Measurement Subsystem can be an optical imaging device to image cells or nuclei or tissue using brightfiled, phase contrast, flourescence, chemiluminescence, nearfield, or other optical readouts, or an electrical measurement, such as impedance measurement of the change in conductivity when a cell passes through a sensor, or other types of measurements. [0011] In some embodiments, cell or organelle or other imaging or labeling solutions, such as cell type specific antibodies, stains, or other reagents, can be added to the tissue or single cells or nuclei before, during, or after processing. The imaging can capture cells, subcellular structures, cell health assays of apoptosis, necrosis, or cytoxicity, or histological or other data. In some embodiments the images can be analyzed to direct the operation and workflow of the Sample Processing System through decisions trees, hash tables, machine learning, or artificial intelligence. In other embodiments the imaging or labeling solutions can contain DNA or other barcodes.

[0012] In some embodiments, single cells or nuclei in suspension or on surfaces are further processed using a tangential flow module to purify cell types, nuclei, nucleic acids, or other biomolecules, change buffers, or remove debris.

[0013] In some embodiments, single cells or nuclei in suspension or on surfaces are further processed using magnetic bead or particle technologies using a Magnetic Processing module to purify or deplete cell types, nuclei, nucleic acids, or other biomolecules, or change buffers or perform one or more reactions in series. In some embodiments, the Magnetic Processing module can use magnetic and paramagnetic particles or beads or surfaces or other sizes and shapes, to separate single ccells, or cell types, or nuclei , or other biocomponents comprised of subcellular components and biomolecules such as macromolecules and nucleic acids, comprised of DNA and RNA; proteins; carbohydrates; lipids; biomolecules with multiple types of macromolecules; metabolites; and other biological components, including natural products for bioanalysis. In some embodiments the beads have a surface chemistry that facilitates the purification of the biologicals in conjunction with the chemical conditions. In other embodiments the beads have affinity molecules comprised of antibodies, aptamers, biomolecules, etc. that specifically purify certain biologicals such as cell types, nucleic acids, nuclei, or other components of tissue or samples.

[0014] The term singulated cells is used to mean single cells in suspension or on a surface or in a well including a microwell or nanowell such that they can be processed as single cells. Similarly, singulated nuclei and singulated organelles refer to single nuclei or organelles in suspension or on a surface or in a well including a microwell or nanowell such that they can be processed as single entities.

[0015] In some embodiments, the specimen is added to a cartridge which performs both physical and enzymatic dissociation of the tissue. In some embodiments the Tissue Processing System performs tituration and other physical dissociation modalities as a step or steps in the process of singulating cells The physical dissociation modalities include passing the specimen through screens, filters, orifices, grinding, blending, sonication, smearing, pestles, bead beating, and other methods known to one skilled in the art to physically disrupt tissue to help produce single cells or nuclei or nucleic acids or other biomolecules.

[0016] In some embodiments, the Sample Processing System is a Tissue Processing System embodiment. In some embodiments, the Tissue Processing System described can input formalin- fixed, paraffin embedded (FFPE) or optimal cutting temparature (OCT) samples, or other primary or secondary samples, and output single nuclei ready for single nuclei analysis or for additional processing, .g, to library preparation, or many other applications. In some embodiments, there is a cartridge that inputs FFPE or OCT tissue and/or other specimens and outputs a single nuclei suspension. In some embodiments, there is a device that holds the input FFPE or OCT tissue to retain the tissue during some of the processing steps.

[0017] In some embodiments, the Sample Processing System, such as a Tissue Processing System embodiment, uses enzymes to assist in the process of singulating cells or nuclei including enzymes to preserve nucleic acids and prevent clumping. The enzymes are comprised of but not limited to collagenases (e.g, collagenases type I, II, in, IV, and others), elastase, trypsin, papain, tyrpLE, hyaluronidase, chymotrypsin, Accutase™, neutral protease, pronase, liberase, clostripain, caseinase, neutral protease (Dispase®), DNAse, protease XIV, RNase inhibitors, or other enzymes, biochemicals, or chemicals such as Triton X-100, Nonidet P40, detergents, surfactants, etc. In other embodiments, different reagents or mixtures of reagents are applied sequentially to dissociate deparaffinized, rehydrated FFPE specimens into single-cell or single nuclei suspensions. In other embodiments, reagents containing detergents or surfactants are applied to dissociate deparaffinized, rehydrated FFPE specimens into single nuclei suspensions. [0018] In some embodiments the Tissue Processing System produces suspensions of known titers. In some embodiments the Tissue Processing System monitors the amount of singulation of a sample and adjusts the treatment time and concentration of enzymes, chemicals, mechanical disruption, or other dissociation agents by monitoring of the dissociation, for example by the production of single cells or nuclei. The monitoring can be in real time, in intervals, or endpoints or any combinations thereof.

[0019] The Tissue Processing System can in some embodiments select from sets of reagents to deparaffinize, rehydrate, reverse crosslinks, and dissociate tissue and in some embodiments can adjust the production of single nuclei or cells by monitoring by the system, in some instances in real time, at intervals, or as an endpoint the titer, quality, or other attributes of the single nuclei suspensions.

[0020] The Tissue Processing System has advantages over existing technology and can produce single cells, single nuclei, or biomolecules from tissue in an automated and standardized instrument that can in some embodiments process the specimens into bulk or sincle cell NGS libraries or other preparations. The Tissue Processing System can enable users, e.g., researchers, clinicians, forensic scientists, and many disciplines to perform identical processing on biosamples, reducing user variability, and throughput constraints of manual processing.

[0021] Embodiments of the Tissue Processing System can prepare single nuclei suspensions or single cells or nucleic acids for analysis by methods comprised of bulk and single cell/nuclei DNA sequencing, RNA sequencing, DNA microarrays, mass spectrometry, Raman spectroscopy, electrophysiology, flow cytometry, mass cytometry, and many other analytical methods well known to one skilled in the art including multidimensional analysis (e.g., LC/MS, CE/MS, etc.) and multi-‘omics (e.g., genomic and proteomic analysis, genomic and cell surface analysis, etc.). [0022] The Tissue Processing System embodiment described is compatible with commercially available downstream library preparation and analysis by NGS sequencers. The term NGS is used to connote either NGS or nanopore or , single molecule sequencing or other sequencing methods or sample preparation methods as appropriate without limitation. As contemplated herein, next generation sequencing refers to high-throughput sequencing, such as massively parallel sequencing (e.g., simultaneously (or in rapid succession) sequencing any of at least 1,000, 100,000, 1 million, 10 million, 100 million, or 1 billion polynucleotide molecules). Sequencing methods may include, but are not limited to: high-throughput sequencing, pyrosequencing, sequencing-by-synthesis, single-molecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing-by-ligation, sequencing-by-hybridization, RNA-Seq (Illumina), Digital Gene Expression (Helicos), next generation sequencing, Single Molecule Sequencing by Synthesis (SMSS) (Helicos), massively-parallel sequencing, Clonal Single Molecule Array (Solexa), shotgun sequencing, Maxam-Gilbert or Sanger sequencing, primer walking, sequencing using PacBio, SOLiD, Ion Torrent, Genius (GenapSys) or nanopore (e.g, Oxford Nanopore, Roche) platforms and any other sequencing methods known in the art.

[0023] In another aspect provided herein is an apparatus, composition of matter, or article of manufacture, and any improvements, enhancements, and modifications thereto, as described in part or in full herein and as shown in any applicable Figures, including one or more features in one or more embodiment.

[0024] In another aspect provided herein is an apparatus, composition of matter, or article of manufacture, and any improvements, enhancements, and modifications thereto, as described in part of in full herein and as shown in any applicable Figures, including each and every feature. [0025] In another aspect provided herein is a method or process of operation or production, and any improvements, enhancements, and modifications thereto, as described in part or in full herein and as shown in any applicable Figures, including one or more feature in one or more embodiment.

[0026] In another aspect provided herein is a method or process of operation or production, and any improvements, enhancements, and modifications thereto, as described in part or in full herein and as shown in any applicable Figures, including each and every feature.

[0027] In another aspect provided herein is a product, composition of matter, or article of manufacture, and any improvements, enhancements, and modifications thereto, produced or resulting from any processes described in full or in part herein and as shown in any applicable Figures.

[0028] In some embodiments the single-cell or nuclei suspension is prepared for a bioanalysis module for downstream analysis including but not limited to sequencing, next generation sequencing, proteomic, genomic, gene expression, gene mapping, carbohydrate characterization and profiling, lipid characterization and profiling, flow cytometry, imaging, DNA or RNA microarray analysis, metabolic profiling, functional, or mass spectrometry, or combinations thereof. [0029] In another aspect provided herein is a data analysis system that correlates, analyzes, and visualizes the analytical information of a sample component such as its degree of single cell or nuclei dissociation, with the processing step and measures the change over time, and/or amount of enyzmatic activity, and/or physical and /or chemical or enzymatic disruptions of the original biological specimen.

[0030] In another aspect provided herein is a data analysis system that correlates, analyzes, and visualizes the analytical information of a sample component such as its degree of single cell or nuclei dissociation, with the processing step and measures the change over time, and/or amount of enyzmatic/chemical activity, and/or physical disruptions of the original biological specimen and adjusts the processing parameters from the analytical information.

[0031] In another aspect provided herein is a data analysis system that correlates, analyzes, and visualizes the analytical information of a sample component such as its level of apoptosis or necrosis, presence and amount of surface markers, metabolites, enzyme activity, or other parameters. In some embodiments, the measurements are made over time and the processing step and/or amount of enyzmatic/chemical activity, and/or physical disruptions of the original biological specimen and other processing parameters are adjusted from the analytical information.

[0032] The Tissue Processing System is a novel platform that automates and standardizes the processing FFPE tissues into single nuclei or single cell suspensions. This will have broad impacts. Process standardization will be critical for comparison of data from lab to lab or research to researcher. The Human Cell Atlas project intends to freely share the multi-national results in an open database. However, with no standardization of the complete process, direct comparisons will greatly suffer from widely varying impacts of the first processing step of producing singlecells or nuclei from tissue. Additionally, when single-cell or nuclei sequencing becomes clinically relevant, the standardization and de-skilling of the production of single-cells or nuclei from FFPE tissues will be required to be performed by an automated instrument such as the Tissue Processing System.

[0033] In another aspect, provided herein is a system comprising: (a) an instrument comprising: (i) one or more cartridge interfaces configured to engage a cartridge; (ii) a fluidics module comprising: (1) one or more containers containing one or more liquids and/or gasses and/or solids that may be dissolved to form liquids; (2) one or more fluid lines connecting the containers with fluid ports in the cartridge interface; and (3) one or more pumps configured to move liquids and/or gasses into and/or out of the fluid port(s); (iii) a mechanical module comprising an actuator; (iv) optionally, a magnetic processing module comprising a source of magnetic force, wherein the magnetic force is positioned to form a magnetic field in the processing chamber; (v) optionally, a measurement module; (vi) optionally, a control module comprising a processor and memory, wherein the memory comprises code that, when executed by the processor, operates the system; and (b) one or more cartridges, each engaged with one of the cartridge interfaces, wherein each cartridge comprises: (i) a sample inlet port; (ii) one or more cartridge ports communicating with the fluid ports in the cartridge interface; (iii) a processing chamber communicating with the sample inlet port and with at least one cartridge port, and comprising a tissue disruptor configured for mechanical disruption of tissue, wherein the tissue disruptor engages with and is actuated by the actuator when the cartridge is engaged with the cartridge interface; (iv) an optional strain chamber communicating with the processing chamber configured to separate cells and/or nuclei from disrupted tissue; (v) an optional post-processing chamber communicating with the strain chamber, optionally communicating with one or more cartridge ports and configured to perform one or more processing steps on separated cells and/or nuclei when required; (vi) optionally, one or more waste chambers fluidically connected with the processing chamber; and one or more tangential flow filtration modules connected with the processing and/or post-processing chambers. In some embodiments the tissue disruptor comprises a grinder, a pestle or a variable orifice. In another embodiment the system further comprises a barcode reader. In another embodiment the system comprises a measurement module (vii) that performs optical imaging to measure titer, clumping, and/or viability of cells or nuclei or properties of biomolecules. In another embodiment the system comprises a measurement module (viii) and a control system (ix), wherein the measurement module measures, and one or more time points, characteristics of a sample in the processing chamber, and control system comprises code that determines a state of the sample, e.g., viability or degree of single cell or nuclei dissociation or degree of deparaffinization or rehydration, etc., and optionally adjusts processing parameters. In another embodiment the system further comprises (c) a device to hold one or more FFPE tissues during the cartridge processing. In another embodiment the system further comprises (d) an analysis module, wherein an input port of the analysis module is in fluid communication with the processing chamber. In another embodiment the analysis module performs an analysis selected from one or more of: DNA sequencing, next generation DNA sequencing, proteomic analysis, genomic analysis, gene expression analysis, gene mapping, carbohydrate characterization and profiling, lipid characterization and profiling, flow cytometry, imaging, DNA or RNA microarray analysis, metabolic profiling, functional analysis, and mass spectrometry. In another embodiment the cartridge interface comprises a means of positioning the cartridge in the instrument that engages the fluidic module and the mechanical module and optionally is temperature controlled. In another embodiment the cartridge is disposable. [0034] In another aspect provided herein is a method comprising: (a) providing a FFPE tissue sample to a processing chamber; (b) automatically performing deparaffinization, rehydration, mechanical and enzymatic/ chemi cal disruption of the tissue in the processing chamber to produce disrupted tissue comprising released nuclei and/or cells and debris; (c) automatically moving the disrupted tissue into an optional strain chamber comprising a strainer and/or filter and separating the released nuclei and/or cells from the debris therein; and (d) automatically moving the released cells and/or nuclei into a post-processing chamber. In another embodiment (e) further comprises performing at least one processing step on the released cells and/or nuclei in the processing chamber. In another embodiment processing comprises one or more automatically performed processes selected from: (I) deparaffinizing FFPE tissue; (II) rehydrating deparaffinized FFPE tissue; (III) enyzmatic or chemical or physical treatement for antigen presentation or other pretreatment; (IV) isolating cell or nuclei suspensions; (V) isolating protein; (VI) converting RNA into cDNA; (VII) preparing one or more libraries of adapter tagged nucleic acids; (VIII) performing PCR; (IX) isolating individual cells or individual nuclei in nanodrops or nanoboluses; and (X) outputting released cells and/or nuclei into output vessels such as 8 well strip tubes, microtiter plates, Eppendorf tubes, a chamber in the cartridge, or other vessels capable of receiving the cell suspensions, libraries, or other output. In another embodiment the method further comprises: (e) automatically capturing the released cells and/or nuclei in the postprocessing chamber or other chamber by binding to magnetically attractable particles comprising moieties having affinity for the cells and/or nuclei and applying a magnetic force to the processing chamber to immobilize the captured cells and/or nuclei. In another embodiment the method further comprises: (f) automatically monitoring cell and/or nuclei titer in the processing chamber and, when the titer reaches a desired level, exchanging a dissociation solution used to dissociate the tissue for a buffer.

[0035] In another aspect provided herein is a cartridge comprising: (i) a sample inlet port; (ii) one or more cartridge ports configured to communicate with fluid ports in a cartridge interface; (iii) a processing chamber communicating with the sample inlet port and with at least one cartridge port, and comprising a tissue disruptor configured for mechanical disruption of tissue, wherein the tissue disruptor engages with and is actuated by the actuator when the cartridge is engaged with the cartridge interface; (iv) a post-processing chamber containing one or more strainers, optionally communicating with one or more cartridge ports and configured to perform one or more processing steps on separated cells; and (v) optionally, one or more waste chambers fluidically connected with the post-processing chamber. In another embodiment the cartridge further comprises a cap that opens and closes the sample inlet port. In another embodiment the cap comprises a tissue disruptor element that moves about rotationally and back and forth along an axis. In another embodiment the cartridge further comprises a holder that retains the FFPE tissue when required duing processing. In another embodiment the cartridge further comprises a top piece and a bottom piece connected by collapsible element which allow the top piece and/or the bottom piece to move relative to the holder. In another embodiment the holder comprises one or more a mesh screens or fdters. In another embodiment the holder comprises two surfaces each with a mesh screen or filter. In another embodiment the holder comprises two surfaces each with a mesh screen or filter or porous material that are joined by magnetic forces, or connected through a hinge or connected by snap-together features. In another embodiment the cartridge further comprises a grinding element for grinding tissue in the processing chamber. In another embodiment the cartridge further comprises a pestle element for disrupting the tissue in the processing chamber. In another embodiment the cartridge further comprises a barcode comprising information about the cartridge and/or its use. In another embodiment the cartridge further comprises a plunger configured to move slideably within the processing chamber.

[0036] In some embodiments, provided herein is a modular cartridge and a system that engages the modular cartridge. The modular cartridge comprises a frame, which can be made of a single piece of material. The frame comprises at least one functional unit attached thereto and formed therein, at least in part. A functional unit performs a function, and can include, without limitation, at least one processing chamber comprising an internal surface functionalized with grinding features; a filtration unit comprising a filter; a magnetic processing unit optionally comprising magnetic particles; an output unit optionally comprising a separable output tube; a flowcell unit; a tangential flow filtration unit; or a waste unit. The frame further comprises a plurality of bays comprising attachment features. The attachment features are configured to accept or engage the functional units. In some embodiments, all the attachment features are configured the same, and each of the different functional units alo is configured to be positioned in any of the bays. In other embodiments, a plurality of the attachment features are different, and functional units can only engage the attachment feature they also are configured for. In some embodiments, an attachment feature at one or more positions is dedicated to accept a particular kind of functional unit. For example, an attachment feature and a magnetic separation functional unit can each be configured to only mate with each other. The functional units will be fluidically connected through fluidic conduits, which may be positioned in the cartridge, or in the system, wherein they engage the functional units when cartridge is engaged with a cartridge interface. In this way, a variety of cartridge configurations is contemplated. Typically, the assembled cartridge will comprise a processing chamber comprising grinding feaures for grinding tissue. Typically, the assembled cartridge also will comprise an output chamber. Other functional units will depend on the purpose to which the cartridge is to be put. For example, a filter chamber may typically be included to filter tissue debris. A magnetic processing unit may be included to capture and wash certain types of cells or subcellular organelles. A waste chamber may included if the ground tissue is to be fractionated. A tangential flow filtration unit may be inlcuded to purify cells or subcellular organelles. A flowcell may be inlcuded to examiner the contents of sample after release of cells and/or subcellular organelles. These functional units can be positioned in any order in the vessel bays and fluidically connected in any arrangement. Accordingly, the modular cartridge of this disclosure is quite versatile.

INCORPORATION BY REFERENCE

[0037] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0039] Figure 1 shows an exemplary modular cartridge.

[0040] Figure 2 shows a high level depiction of a modular cartridge and the functional units of a single bay of an instrument to operate the modular cartridge to dissociate tissues into many potential biological products.

[0041] Figure 3 shows shows a high level depiction of a modular cartridge and the functional units of a single bay of an instrument with an optical detector to operate the modular cartridge to dissociate tissues into many potential biological products including titered purified single cells and nuclei.

[0042] Figure 4 shows the overall workflow of processing a solid tissue sample in a cartridge using the Singulator 100 or 200 systems into single cell or single nuclei suspensions with downstream analysis for single cell genomics, multi-omics, or single cell biology applications.

[0043] Figure 5 depicts an exemplary modular cartridge and a 8-cartridge instrument, not to scale.

[0044] Figure 6 shows an exemplary modular cartridge configured with five modular functional units with pinch valves and the interactions with cannulas and valve actuators from the front (6 A) and back views (6B). [0045] Figure 7 shows an exemplary modular cartridge configured with three modular functional units with pinch valves and the interactions with cannulas and valve actuators from the front (7 A) and back views (7B) as well as a view of the modular frame with a molded in Dissociation Chamber (an embodiment of a Processing Chamber) and Output Chamber (7C).

[0046] Figure 8 shows a close-up of a modular Dissociation Chamber with a rotor with grinding features from the front and back.

[0047] Figure 9 shows exemplary rotors that (A) have a center post on the rotor, (B) have a center post on the stator, (C) have side gap setting features on the side of the rotor, and (D) have side setting swirls on the side of the rotor.

[0048] Figure 10 shows designs of rotor and stator teeth patterns.

[0049] Figure 11 shows an exemplary modular Filtration Chamber holding three filters with two tubing connections and a port to connect to a cannula.

[0050] Figure 12 shows an exemplary modular Processing Chamber with two dip tubes and a port to connect to a cannula that can also be used for as a Magnetic Processing Chamber.

[0051] Figure 13 shows an exemplary pinch valve.

[0052] Figure 14 shows an exemplary modular Output Chamber with an engaged centrifuge tube.

[0053] Figure 15 shows an exemplary modular Waste Chamber with an input port and a port to connect to a cannula.

[0054] Figure 16 shows an exemplary Tangential Flow module without (16A) and with (16B) an optical interrogation region.

[0055] Figure 17 shows an exemplary non-modular cartridge on the left and a detail of the cap showing the teeth on the bottom of the rotor on the right.

[0056] Figure 18 shows some of the tissues that have been dissociated into single cells or nuclei.

[0057] Figure 19 shows an exemplary Singulator 100 with enclosure and other parts removed to illustrate the major components of the instrument.

[0058] Figure 20 shows the viability of single cell suspensions produced on a Singulator 100 system from fresh mouse solid tissues.

[0059] Figure 21 shows the nuclei titer per mg of tissue from exemplar tissues produced on a Singulator 100 system from fresh mouse solid tissues. Note that the spleen is for a two-fold dilution of the sample.

[0060] Figure 22 shows scRNA-Seq analysis of human lung showing cells isolated from a normal lung biopsy from a human patient with squamous cell carcinoma.

[0061] Figure 23 shows a conceptual design of a flow cell integrated on the modular cartridge. [0062] Figure 24 shows an illustrative workflow to produce bulk DNA sequencing libraries from tissues on a modular cartridge by end-polishing, A-tailing, and ligation with size selection of the library.

[0063] Figure 25 shows shows an illustrative workflow to produce bulk DNA sequencing libraries from tissues on a modular cartridge by tagmentation.

[0064] Figure 26 shows an illustrative workflow to produce bulk RNA sequencing libraries from tissues on a modular cartridge.

[0065] Figure 27 shows a conceptual modular cartridge configured for processing FFPE into single cells or nuclei.

[0066] Figure 28 shows an exemplary tissue ring for holding FFPE curls during processing.

[0067] Figure 29 shows an overview of single cell and nuclei sequencing applications from solid tissues using single cell and other metrics.

[0068] Figure 30 shows scRNA-Seq analysis of human lung showing cells isolated from a normal lung biopsy from a human patient with squamous cell carcinoma.

[0069] Figure 31 shows snRNA-Seq of human liver clustered and annotated for 18 cell types.

[0070] Figure 32 shows an exemplary computer system.

[0071] Figure 33A shows an examplarly modular cartridge in section view with three cartridge modules connected by a macroscale fluidic device.

[0072] Figure 33B shows an examplarly fluidic device.

DETAILED DESCRIPTION OF THE INVENTION

[0073] It will be readily apparent to one of ordinary skill in the art that the embodiments and implementations are not necessarily inclusive or exclusive of each other and may be combined in any manner that is non-conflicting and otherwise possible, whether they be presented in association with a same, or a different, embodiment or implementation. The description of one embodiment or implementation is not intended to be limiting with respect to other embodiments and/or implementations. Also, any one or more function, step, operation, or technique described elsewhere in this specification may, in alternative implementations, be combined with any one or more function, step, operation, or technique described in the summary. Thus, the embodiment and implementations described herein are illustrative rather than limiting.

[0074] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having” or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of features is not necessarily limited only to those features but may include other features not expressly listed or inherent to such process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive-or and not to an exclusive-or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present). Both plural and singular means may be included.

[0075] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a centrifuge tube” includes a plurality of centrifuge tubes, including mixtures thereof.

[0076] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[0077] As used herein, the term “about” a number refers to that number plus or minus 10% of that number. For example, "about 10", would include values from 9 to 11, unless otherwise indicated by the context in which the term is used. The term “about” a range refers to that range minus 10% of its lowest value and plus 10% ofits greatest value. Similarly, when the term "about" is used before a non-numerical term that is a stand-in for a numerical value (e g , horizontal, perpendicular, aligned), the term "about" refers to the value of the non-numerical term (e.g., 90 degrees, 1800 degrees) plus or minus 10% of that value.

[0078] Whenever the term "at least," "greater than, " "greater than or equal to, " "no more than, " "less than," or "less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term term applies to each of the numerical values in that series of numerical values, unless otherwise specified. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0079] Whenever the term "no more than," "less than," or "less than or equal to" precedes the first numerical value in a series of two or more numerical values, the term "no more than," "less than," or "less than or equal to" applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0080] Specimen: The term “specimen,” as used herein, refers to an in vitro cell, cell culture, virus, bacterial cell, fungal cell, plant cell, bodily sample, FFPE sample, or tissue sample that contains genetic material. In certain embodiments, the genetic material of the specimen comprises RNA. In other embodiments, the genetic material of the specimen is DNA, or both RNA and DNA. In certain embodiments the genetic material is modified. In certain embodiments, a tissue specimen includes a cell isolated from a subject. A subject includes any organism from which a specimen can be isolated. Non-limiting examples of organisms include prokaryotes, eukaryotes, or archaebacteria, including bacteria, fungi, animals, plants, or protists. The animal, for example, can be a mammal or a non-mammal. The mammal can be, for example, a rabbit, dog, pig, cow, horse, human, or a rodent such as a mouse or rat. In particular aspects, the tissue specimen is a human tissue sample. The tissue specimen can be liquid, for example, a blood sample, red blood cells, white blood cells, platelets, plasma, serum. The specimen, in other non-limiting embodiments, can be saliva, a cheek, throat, or nasal swab, a fine needle aspirate, a tissue print, cerebral spinal fluid, mucus, lymph, feces, urine, skin, spinal fluid, peritoneal fluid, lymphatic fluid, aqueous or vitreous humor, synovial fluid, tears, semen, seminal fluid, vaginal fluids, pulmonary effusion, serosal fluid, organs, bronchio-alveolar lavage, tumors, frozen cells, or constituents or components of in vitro cell cultures. In other aspects, the tissue specimen is a solid tissue sample or a frozen tissue sample or a biopsy sample such as a fine needle aspirate or a core biopsy or a resection or other clinical or veternary specimen. In other aspects, the tissue specimen is an optimal cutting temperature compound (OCT) or FFPE or other preserved sample such as a biopsy sample such as a fine needle aspirate or a core biopsy or a resection or other clinical or veterinary specimen. In still further aspects, the specimen comprises a virus, bacteria, or fungus. The specimen can be an ex vivo tissue or sample or a specimen obtained by laser capture microdissection. The specimen can be a fixed specimen, including as set forth by U.S. Published Patent Application No. 2003/0170617 filed Jan. 28, 2003.

[0081] The term cartridge can mean many different embodiments. At times cartridge is used to represent a physically integrated cartridge 200 and at other times specifically for a modular cartridge 205. In turn a modular cartridge 205 which has one or more functional modules can have many embodiments, designs, and forms, such as a linear layout or circular or rectangle on vertical. The connections between the modules of the modular cartridge 205 can be of many types, capillary, microfluidic, tubing, channels, etc. and can be as varied as individual tubes connecting modules, to microfluidic circuits with 0, 1, 5, 10 or more valves. The modules of the modular cartridge 205 can have many functionalities, e.g., dissociation, filtration, tangential flow filtration, preforming reactions, magnetic processing, waste, detection, sample preparation, column preparation, optical detection, mass spec detection, fluorescent detection, raman detection, analysis including Al, and output. In some embodiments, the modular cartridge 205 can perform one or more steps of a workflow. In some embodiments, the modular cartridge 205 can perform all the steps in a workflow from preparation of a raw biological sample to a readout of one or more biological or chemical properties of the sample.

[0082] In some embodiments, the modular cartridge 205 can prepare single cells that can be analyzed further for biomolecules including one or more polynucleotides or polypeptides or other macromolecules. In some embodiments, the polynucleotides can include a single-stranded or double-stranded polynucleotide. In some embodiments, the polypeptide can include an enzyme, antigen, hormone or antibody. In some embodiments, the one or more biomolecules can include RNA, mRNA, cDNA, DNA, genomic DNA, microRNA, long noncoding RNA, ribosomal RNA, transfer RNA, chloroplast DNA, mitochondrial DNA, or other nucleic acids including modified nucleic acids and complexes of nucleic acids with proteins or other macromolecules.

Overview

[0083] This disclosure provides, in some embodiments, an advanced single or multi-sample (e.g., 1-, 2-, 4-, 8-, 9-, 10-, 11-, 12-, or 15- sample) cartridge-based Sample Processing System 50 which can be a Tissue Processing System 80 that can process biopsy-sized or other sized samples, including solid tissue specimens 130, to produce single cell and nuclei suspensions and optionally to purify cells, or cell types or nuclei with paramagnetic bead processing, or tangential flow filtration, or purify nucleic acids, or prepare NGS bulk libraries, or perform other cell or molecular biology or chemistry sample preparation or assays.

[0084] Next-generation sequencing (NGS), mass spectrometry, fluorescent activated cell sorting (FACS), and other modem high-throughput analysis systems have revolutionized life and medical sciences. The progression of information has been from the gross level of organism, to tissue, and now to single cell and nuclei analysis. Single cell analysis of genomic, proteomic including protein expression, carbohydrate, lipid, and metabolism of individual cells is providing fundamental scientific knowledge and revolutionizing research and clinical capabilities.

[0085] Figure 1 shows an exemplary modular cartridge 205 that can be operated by a Sample Processing System 50 to prepare many types samples including filtered single cell suspensions 1102 and filtered nuclei suspensions 1103 from specimens 101 including tissue specimens 120 and solid tissue specimens 130. The modular cartridge 205 configuration shown in Figure 1 can also perform magnetic bead 685 processing to create purified single cell suspensions 1100 or nuclei suspensions 1101 with antibody -coated magnetic beads 686 or create bulk NGS libraries 1205 from DNA 1073 or RNA 1074 or purify subcellular components 1060 such as nuclei 1050, macromolecules 1071, or biomolecules 1070.

[0086] Figure 2 shows a high-level depiction of the upper instrument cartridge interface 1501 and lower instrument cartridge interface 1502 of a single bay module of a Sample Processing System 50 to operate a modular cartridge 205. The upper instrument cartridge interface 1501 system shows an example with actuators for a z-axis stepper motor 2110, a rotary motor 2120, pinch valve 491 actuators, syringe pump 2130 with a vacuum accumulator 1520 and valves to connect the vacuum to different Camillas 1416 and chamber, fluidic connections which can be to spring-loaded cannulas 1416, or reagents which may be in a reagent container 1426 which may be a cassette. The reagents can include magnetic bead purification reagents, which can be in a temperature-controlled reagent storage chamber 1419, including paramagnetic beads 685 with different surface chemistries with different properties including hydrophilic, hydrophobic, affinity capture paramagnetic beads 685 which can functionalized with antibodies 686, or concanavalin A 687, or oligonucleotides 680, or polyT paramagnetic beads 688, or oligo-capture paramagnetic beads 689, or aptamer capture paramagnetic beads 690, or lectin capture paramagnetic beads 691, or any other affinity capture paramagnetic beads 692; buffers; dissociation reagents comprising collagenases (e.g., collagenases type I, II, III, IV, and others), elastase, trypsin, papain, tyrpLE, hyaluronidase, chymotrypsin, Accutase™, neutral protease, pronase, liberase, clostripain, caseinase, neutral protease (Dispase®), DNAse, protease XIV, or other enzymes; antibodies which can be unlabeled or have labels including fluorescent or other labels; RNase inhibitor; DNase; reagents for library preparation comprising fragmentase, reverse transcriptase, klenow fragment, NEBNext® End Repair Module, T4 DNA polymerase, DNA ligase, RNA ligase, NEBNext® dA-Tailing Module, RNA polymerase, Tn5, transposases; other enzymes comprising DNA modifying enzymes, DNA gyrase, DNA topoisomerase, transcription factors, fusion of Proteins A and/or Gto Micrococcal Nuclease, mung bean nuclease, other nuclease and DNA or RNA modifying enzymes; histone binding proteins and other non-enzymatic proteins; other biomolecules including carbohydrates and lipids; spike in controls; chemicals comprising buffers, ethanol, isopropanol, other alcohols, PEG, salts, xylene, xylene replacements, bisulfite, water, Triton X-100, Nonidet P40, detergents, and surfactant.

[0087] The instrument lower cartridge interface 1502 in Figure 2 shows temperature control of the Dissociation Chamber 441 (e.g., 4°C or 37°C), a moveable magnet in a temperature controlled interface to a Magnetic Processing Chamber 905 (e.g., 4°C or 37°C), and a 4°C controlled Output Chamber with a centrifuge tube 855. A vacuum accumulator 1520 and a thermal processor which can be a Peltier 1530 or circulating liquid system 1540, or circulating air system 1550, or resistive heat 1560 or many other temperature control devices (e.g., thermal/mag subassembly (instrument), heating/cooling subassembly (instrument)) are shown the instrument lower cartridge interface 1502.

[0088] Figure 3 shows a Sample Processing System 50 configured with an optical module 2600 that can produce purified titered single cell suspensions 1310 or purified titered single nuclei suspensions 1320 and many other outputs. In some embodiments, one or more Sample Processing System 50 instruments operate one or more modular cartridges 205 simultaneously. In some embodiments, one or more Sample Processing System 50 instruments operate one or more modular cartridges 205 sequentially. Sequential operation of the modular cartridges 205 generally involves initiating the process of a first cartridge 205 followed by initiating the process of a second cartridge 205. In some cases, sequential operation involves waiting for the first cartridge 205 to complete its process before initiating the process of the second modular cartridge 205. In some cases, sequential operation involves initiating the second cartridge 205 before the process of the first cartridge 205 is completed, e.g., while the first cartridge 205 is still operating. [0089] The optical module 2600 can have many different emobodiments. In some embodiments, the optical module 2600 is a multi -wavelength fluorescent imager that ‘stares’ at an optical detection area 2720 of a flow cell 2620. The flow cell 2620 can be resuable or replaceable and can be incorporated into the instrument or, in some embodiments, be incorporated into the modular cartridge 205 as a separate cartridge module or integrated with another cartridge module such as a tangential flow filtration module 2700. The optical module 2600 can use many different detectors for imaging comprised of CCD, CMOS, e.g., Hanamatsu digital CMOS detector, C13949-50U, avalanche photodiodes, multipixel photon counters, silicon photomultipliers, and other detectors. In one embodiment, an OEM high-resolution scientific CMOS board level camera (e.g., Hamamatsu Cl 1440-52U30 with appropriate optics) can be for multi-color fluorescence imaging with LED or laser through a lens onto a flow cell. The emitted fluorescence will pass through the optical system, including bandpass and LED/laser blocking filters, before being focused on the CMOS array of the board camera. The optical module 2600 can be mounted on a mechanical auto-focusing stage to adjust the focus in steps of 16 mm to allow 2.5x imaging through the depth of the flowcell 2620.

[0090] The devices, systems, and methods provided herein also provide improved (and higher) sample throughput, optimized protocols for many different tissues and species, simplified automated workflows with seamless integration with downstream single-cell sequencing library preparation 1200 or integrated production of bulk sequencing libraries 1205, and the ability to process FFPE specimens 150. The devices, systems and methods provided herein can be used in a variety of applications including but not limited to detection of cellular heterogeneity, cellular organization, cell-cell interactions, brain structure and function, tumor progression and resistance, immuno-oncology , and development, among other areas of biology.

[0091] In some instances, solid tissue specimens 120, including fresh, frozen, OCT, or FFPE preserved specimens 150, are added to the cartridge 205 (e.g., a single-use cartridge) by the operator who inserts a cartridge 205 into an instrument bay and selects the protocol for that sample. In some embodiments, additional cartridges 205 can be added any time there is an available bay. In some cases, the methods provided herein use (or the systems or devices provided herein include) one or more Singulator systems (e.g., Singulator™ 100 and 200 systems) (Figure 4) automate the processing of solid tissues into single cells or nuclei suspensions. In some cases, a single sample is processed in a disposable injection-molded cartridge 200 (e.g., using the Singulator™ 100); in some cases, two samples are processed in two cartridges 200 simultaneously (e.g., using the Singulator 200). In some cases, filtered singulated cell suspensions 1102 or filtered nuclei suspensions 1103 are produced from a wide range of tissues from organisms ranging from human to rodent to insect to plants.

[0092] In some cases, this disclosure provides an advanced next-generation automated, programmable, Tissue Processing System 80 to dissociate and process solid tissue specimens 120. The Tissue Processing Systems 80 may be scalable to process 1 or more solid tissues simultaneously into single cell or nuclei suspensions in disposable cartridges 200 which can be modular cartridges 205, with improved dissociation processes, real-time optimization, and optional paramagnetic bead processing to integrate workflows for cell enrichment/depletion and purification or molecular biology including single cell or bulk DNA and RNA sequencing as well as many other applications.

[0093] The devices and system described herein, in some cases, is an 8-cartridge Singulator instrument 2080 to operate a modular cartridge 205 (Figure 5). This disclosure may comprise producing single cell and nuclei suspensions for scRNA-Seq, snRNA-Seq, ATAC-Seq, CITE- Seq, TILs or other cell-type purification, or produce bulk RNA or DNA library preparation, or label and detect cell surface markers, or many other applications.

[0094] The devices and systems provided herein may be the first fully automated, extensible, cartridge-based system for tissue dissociation and filtration, for processing of samples from as little as 1 mg, in parallel, with the flexibility to isolate cells or nuclei, and to post-process cells, nuclei, or nuclei acids using integrated magnetic bead handling and/or tangential flow filtration or other modules. The dissociation process and downstream workflows can be optimized for multiple sequencing applications. In some embodiments, the integration of tissue dissociation with additional downstream processes (e.g., RBC lysis and magnetic bead purification of cell types) permits new automated workflows, experimental efficiency, user convenience, and consistency — and improves automation, integration and simplification of workflows. Process

[0095] In some embodiments, the overall process may comprise one or more of the following processes in any combination or order:

[0096] Dissociation. In some embodiments, upon insertion, the modular cartridge 205 docks with the instrument’s cartridge interface 1500. Enzymes to dissociate the tissue 410 or nuclei isolation reagents 412 may be added to the Dissociation Chamber 441 through cannula 1416 and Dissociation Chamber reagent addition port 470; small volume reagents, e.g., RNase inhibitor, etc., may be added through the cannula or manually added to the Dissociation Chamber 441. The mechanical disruption pressure of disruptors, including rotors 353 and improved self-centering disruptors 420, may be adjusted in real-time by force sensors 2115 or by IR camera 261, or video camera 2613, or other detectors. The bottom of the Dissociation Chamber 441 can be optically clear and be monitored by an IR camera 2612 for temperature verification and control, and imaged by a video camera 2613 for display to the operator or for analysis such as degree of dissociation. The dissociation process may be optimized in real-time from the images or by the force sensor 2115. In some embodiments, the Dissociation Chamber is a Processing Chamber. In some embodiments, the Dissociation Chamber is a pre-processing chamber.

[0097] In some embodiments, the Dissociation Chamber 441 is designed to process tissue, nuclei, cells, or specimen in volume > 1 mL, > 5 m , or more. In some embodiments, the Dissociation Chamber 441 is designed to process tissue, nuclei, cells, or specimen in volume < 1 mL, < 0.5 mL, < 0.1 ml, or less. In some embodiments, the Dissociation Chamber 441 is designed to process tissue, nuclei, cells, or specimen in volume more than 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL or more. In some embodiments the Dissociation Chamber 441 is designed to process tissue, nuclei, cells, or specimen in volume less than 1 mL, 0.9 mL, 0.8 mL, 0.7 mL, 0.6 mL, 0.5 mL, 0.4 mL, 0.3 mL, 0.2 mL, 0.1 mL, 0.05 mL, 0.01 mL, 0.005 mL, 0.001 mL, or less.

Filtration. In some embodiments, following dissociation, the single cell or nuclei suspensions are moved through a Filtration Chamber 450. The chamber may have one to three or more filters 341 to remove clumps and filter to the appropriate size range. The Filtration Chamber 450 may either output suspensions directly into Output Chamber 850 with a centrifuge tube 855 or into an Output Chamber 850 without a centrifuge tube 855 on the cartridge 205, or deliver the sample for magnetic processing, or tangential flow filtration, or deliver it for further processing, e.g., red blood cell lysis, antibody labeling, etc., or other processes. Solutions may be added to wash the filters 341 as needed, or to added to the sample, or vacuum can be applied by filtration cannula 2928 connecting to a pump or other reagent delivery system, or vacuum including a vacuum accumulator 1520. In some embodiments, the Filtration Chamber 450 is designed to process tissue, nuclei, cells, or specimen in volume > 1 mL, > 5 mL, or more. In some embodiments, the Filtration Chamber 450 is designed to process tissue, nuclei, cells, or specimen in volume < 1 mL, < 0.5 mL, < 0.1 ml, or less. In some embodiments, the Filtration Chamber 450 is designed to process tissue, nuclei, cells, or specimen in volume more than 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL or more. In some embodiments the Filtration Chamber 450 is designed to process tissue, nuclei, cells, or specimen in volume less than 1 mL, 0.9 mL, 0.8 mL, 0.7 mL, 0.6 mL, 0.5 mL, 0.4 mL, 0.3 mL, 0.2 mL, 0.1 mL, 0.05 mL, 0.01 mL, 0.005 mL, 0.001 mL, or less.

[0098] Magnetic Processing. If desired, the single cell or nuclei suspensions can be further processed in a Processing Chamber 460. For example, RBC lysis reagents and stop reagent can be added to the Processing Chamber 460, mixed, and incubated. The Processing Chamber 460 can also be used as a Magnetic Processing Chamber 905 (MPC) with paramagnetic bead 685 addition. A Magnetic Processing Chamber 905 can be a macrofluidic device or microfluidic, i.e., less than 1 mL volumes. In some embodiment, two or more Processing Chambers 460 or Magnetic Processing Chambers 905 can be used; for example a first Magnetic Processing Chamber 905 that receives one to ten mL and captures cells or subcellular organelles or nucleic acid on paramagnetic beads 685 and a second Magnetic Processing Chamber 905 that is designed to perform reactions or purifications in volume less than one mL or less than 0.5 mL or less than 0.1 mL, or smaller volumes. In some embodiments, the first Magnetic Processing Chamber is designed to reduce volume from 5 ml to 200 ul, 100 ul, 50 ul or less. In some embodiments, the second Magnetic Processing Chamber is designed to perform reactions with reagents in volume less than 0.5 mL, less than 0.1 mL, or smaller volumes. In some embodiments, the Magnetic Processing Chamber 905 is designed to process tissue, nuclei, cells, or specimen in volume > 1 mL, > 5 mL, or more. In some embodiments, the Magnetic Processing Chamber 905 is designed to process tissue, nuclei, cells, or specimen in volume < 1 mL, < 0.5 mL, < 0.1 ml, or less. In some embodiments, the Magnetic Processing Chamber 905 is designed to process tissue, nuclei, cells, or specimen in volume more than 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL or more. In some embodiments the Magnetic Processing Chamber 905 is designed to process tissue, nuclei, cells, or specimen in volume less than 1 mL, 0.9 mL, 0.8 mL, 0.7 mL, 0.6 mL, 0.5 mL, 0.4 mL, 0.3 mL, 0.2 mL, 0.1 mL, 0.05 mL, 0.01 mL, 0.005 mL, 0.001 mL, or less.

[0099] Antibody-functionalized paramagnetic beads 686 can be resuspended either by mixing using fluidics as described below, or by use of a miniaturized ‘vortex’ agitation using a vibrating or rotating agitator. In some embodiments, the magnetic beads 685 are added to the single cell or nuclei or other biological suspensions, mixed, and targets captured within modular cartridge 205 with the moveable on-instrument magnet 910. After washing to enrich or deplete single cell types, or remove debris such as myelin, the cells can be eluted or moved into a buffer of choice. The Processing Chamber 460 can be used to perform reactions that do not require paramagnetic beads 685.

[0100] Tangential Flow Filtration. If desired, the single cell or nuclei suspensions may be processed in a Tangential Flow Filtration module 2700 in the modular cartridge 205 to remove debris that is smaller than the single cells or nuclei of interest. In the Tangential Flow Filtration module 2700, preferably fdtered single cell or nuclei are input to the module and passed into a region with preferably two tangential flow filters 2710, e.g., 1, 2 , 5, 10, 20 mm filters, with upper buffer region 2707 and lower buffer region 2708 that have buffer or media or other liquids circulated by syringe pumps 2130 to process the sample in tangential flow region 2706. The circulation of buffer can acts as a concentration gradient to withdraw small particles of debris that can pass through the filters and are removed from the sample, or to concentrate the sample, or to change the buffer or media. In some embodiments, tangential flow filtration can be used before optical readout and dilution. In some embodiments, the Tangential Flow Filtration module 2700 is designed to process tissue, nuclei, cells, or specimen in volume > 1 mL, > 5 mL, or more. In some embodiments, the Tangential Flow Filtration module 2700 is designed to process tissue, nuclei, cells, or specimen in volume < 1 mL, < 0.5 mL, < 0.1 ml, or less. In some embodiments, the Tangential Flow Filtration module 2700 is designed to process tissue, nuclei, cells, or specimen in volume more than 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL or more. In some embodiments the Tangential Flow Filtration module 2700 is designed to process tissue, nuclei, cells, or specimen in volume less than 1 mL, 0.9 mL, 0 8 mL, 0.7 mL, 0.6 mL, 0.5 mL, 0.4 mL, 0.3 mL, 0.2 mL, 0.1 mL, 0.05 mL, 0.01 mL, 0.005 mL, 0.001 mL, or less.

Modular Cartridge

[0101] In some embodiments, tissue processing occurs in an integrated cartridge 200, particularly a disposable cartridge. In some embodiments, solid tissues are processed into single cell and nuclei suspensions in single-sample, injection-molded integrated plastic cartridges (Figure 4). The cartridges can have two or more primary chambers. In some embodiments, after the sample is added, the Sample Processing System 50 can add liquids at any time to the Dissociation Chamber 441 or Output Chamber 850; liquids can be retained on the cartridge during processing until the resulting single cell or nuclei suspension is pipetted out. Generally, nothing from the cartridge enters the instrument, preventing sample-to- sample carryover or contamination. [0102] This disclosure provides an exemplary modular cartridge 205 design; one example of a linear modular cartridge 205 is shown in Figure 6A and Figure 6B. In a modular design, functional modules or functional units or vessels can be molded into a modular frame 2905, or designed as ‘stand-alone’ modules to be irreversibly ‘clicked’ into a planar or other modular frame 2905, or designed to be placed into a receptacle or vessel bay in modular frame 2905 that holds the functional module in position. In some embodiments, the functional modules are attached by clicking or snapping into the modular frame 2905 whereby one or more flexure tabs on the functional module fit into one of more receiver spaces or slots in the modular frame 2905 to retain the functional module. Figure 6B shows two snap-in connectors 2906 on Waste Chamber 430 attaching it to the modular frame 2905. In other embodiments, the functional modules twist into the modular frame 2905, for example, a 90° twist with one or more tabs engaging one or more stops that can have features to also fix the functional module’s vertical position. In other embodiments, the functional modules screw into the modular frame 2905 to establish both a fixed rotational and vertical position; the functional modules can have either male or female thread features. In other embodiments, the functional modules are press fit into the modular frame 2905 which has a complementary shape or the functional modules are adhesively attached or glued to the modular frame 2905. In some embodiments, the functional modules are fit into the modular frame 2905 and a clip is inserted to affix the functional modules. In some embodiments, the functional modules are fit into the modular frame 2905 and top device attached to the frame by screws, glue, adhesive, snaps or other mechanisms to affix the functional modules in place.

[0103] Modular units can be attached to attachment features by any mechamisn, including, for example, mechanical attachment, magnetic attraction, and adhesion. Some attachment methods include, for example, threaded coupling, push-pull connectors, twist locks, clamps, slide-and- lock mechanisms, and quarter-turn fasteners. In a press-fit or friction-fit, the attachment feature typically includes a recess or a hole, typically with a tapered configuration. The stand-alone unit can have a configuration that has a complementary shape. Complementary shapes can be, for example, circular, conical, or cylindrical. Alternatively, the aperture can comprise ridges. In another configuration, the attachment feature and the stand-alone unit have complementary threads, such as a lightbulb and socket configuration. The stand-alone unit can be screwed into the attachment feature. In another configuration, the stand-alone unit can have a snap-in feature. When pressed into the attachment feature, the snap-in feature clears a lip of the attachment feature and snaps outward, creating an attachment. In another configuration, the locking mechanism can by a bayonet mount. Alternatively, the attachment feature and the stand-alone unit can be attached through an adhesive such as a glue, or a tape. [0104] In some embodiments, the modular units are connected by tubing 2158, which can be used to create pinch valves 491. In other implementations the modular units can be connected by a fluidic device 2907 which can be a microfluidic device or microchip device. Figure 33A shows an example of a modular cartridge 205 in section view with three cartridge modules, Dissociation Chamber 441, Filtration Chamber 450, and Output Chamber 850 with a centrifuge tube 855, connected by a macroscale fluidic device 2907. Figure 33B shows the fluidic device 2907 that serves to route fluids from Filtration Chamber 450 to Output Chamber 850 with two twist valves 2908 that can direct flows. In this example, fluidic device 2907 has macrofluidic channels that are formed between two layers of material and has a dip tube 2917 that can access samples in the bottom of Filtration Chamber 450. As shown in Figure 33B, when vacuum is applied to cannula seat 2916 by a cannula, as shown by the white arrow, dip tube 2917 will pull material from the bottom of Filtration Chamber 450 through the dip tube 2917 and through the fluidic device 2907 and open twist valve 2908 into Output Chamber 850 as shown by the dark arrows. In this example, fluidic device 2907 is formed from an upper fluidic device layer 2911 and a lower fluidic device layer 2912 that can be assembled by methods comprising adhesion, compression of a compliant gasket plane comprising a separate elastomer fdm or a adhesive- backed film or an injection molded elastomer overmolded onto the fluidic article, or ultrasonic welding and twist valves 2908 are then inserted. Twist valves 2908 can be controlled by actuators on the upper cartridge interface 1501 and can have channels on the bottom surface, such as straight or T patterns to connect fluidic channels as desired. While Figure 33A and Figure 33B show a macrofluidic fluidic layer 2907, the channels can also be microfluidic scale as desired.

[0105] In some embodiments, the functional modules on modular cartridge 205 can all be macrofluidic with volumes of greater than one milliliter. In other embodiments, one of more of the functional modules can be microfluidic with volumes of microliters to less than one microliter. In some embodiments a microfluidic chip can be a functional module. For example, Figure 23 shows a flow cell 2620 which may have channels of 100 micrometers wide leading to an optical area 2720 which may have a total volume of 10 microliters, or 100 microliters and therefore be a microfluidic device. Both macrofluidic and microfluidic devices can be connected together as described herein and used as appropriate. For example, tissue sample dissociation can be performed in the macroscale and, after filtration, a first macroscale Magnetic Processing Chamber 905 could reduce the volume to 100 microliters for further processing in a microscale Magnetic Processing Chamber 905 to perform reactions such as labeling reactions or DNA library preparation with costly reagents which preclude use of milliliter volumes.

[0106] In some embodiments the modular units can be connected by capillaries. Valve actuators 2116 on the instrument cartridge interface 1500 can open or close the pinch valves 491 by pressing tubing 2158 on valve seats 492. In some embodiments, the Dissociation Chamber 441 can be irreversibly ‘clicked’ into a planar or other frame 2905. In some embodiments, the Dissociation Chamber 441 can be engaged with a planar or other frame 2905. In some embodiments, the Dissociation Chamber 441 can also be molded into the modular frame 2905 or be placed into modular frame 2905. The functional modular units can be enclosed in a cartridge enclosure 2910 for ease of handling. In some embodiments the modular frame 2905 can be part of cartridge enclosure 2910. In some embodiments, cannulas 1416 from the instrument cartridge interface 1500 connect to cannula seats 2916 on cartridge ports 2915 to provide reagents, pressure, or vacuum to modular cartridge 205. To improve the sealing of the cannulas 1416, in some embodiments, the cartridge cannula seats 2916 can be overmolded with a pliant material such as a rubber. As well known to ones skilled in the art, the valve actuators 2116 can be implemented using devices comprising stepper motors 2117, pnuematically-driven actuators 2118, rotary motors 2120 with a cam 2157, or many other embodiments.

[0107] The example in Figure 6 shows an exemplary modular cartridge 205 with five modular units: Dissociation Chamber 441, Filtration Chamber 450, Processing Chamber 460 shown as a Magnetic Processing Chamber 905, Output Chamber 850 with a centrifuge tube 855, and a Waste Chamber 430.

[0108] The modular approach may simplify molding fabrication of complex cartridges since many of the component modules can be individually fabricated which means that constraints in molding multiple functions into a single cartridge, as in cartridge 200 as illustrated in Figures 4 and Figure 17 are relaxed; for example, different side pulls can be on each functional module of the modular cartridge 205 from many different directions, sometimes impossible with an integrated injection molded cartridge 200. The functional modules of modular cartridge 205 can include injection molded parts, or 3D printed parts, or machined parts or many other fabricated of the modules. The modular appoach allows over-molding on individual components to create seals, and/or decouples the optimization and production of the different functional elements. Each cartridge module can be independently developed and optimized. The modular cartridge 205 allows a common cartridge framework and modular frame 2905 to be configured to perform multiple workflows to meet the multiple needs of single cell biology. The examples shown here are of linear modular cartridges 205 that minimize the width of the cartridge which can be advantageous for some instrument designs to minimize the width of the instrument. In some embodiments, the modular cartridges are positioned in a linear sequence. In some embodiments, the modular cartriges are positioned in many different configurations, e.g., square, round, rectangular, are possible in a horizontal plane, or a cartridge may be vertical or any combination. [0109] Figure 7A and B show the front and back respectives of an exemplary embodiment of a modular cartridge 205 with three functional modules, a Dissociation Chamber 441 and a Output Chamber 850 molded into modular frame 2905 as shown in Figure 7C, and the Filtration Chamber 450 clicked into the modular frame. Tubing 2159 connects the chambers.

[0110] Mechanical disruption in the Dissociation Chamber 441 (Figure 8) may, for example, use a spring-loaded cap 210 containing rotor 353 with grinding features 220 with cap coupler 211 which is used by the instrument to rotate the rotor 353, or move it up or down; the spring (not shown) can pulls the rotor 353. The sample is loaded by removing cap 210 and placing the sample in the Dissociation Chamber 441 before replacing cap 210. Reagents, e.g. enzyme cocktails 405 to digest extracellular matrix for cellular production or nuclei isolation solution 412 for nuclei production or chaotrophs 414, e.g., guanidine HC1 or sodium isothiocynate, for nucleic acid 1072 production, or other reagents, can be added through cannula 1416 connecting to reagent addition port 470 for enzymatic or chemical processing of the tissue specimen 120.

[oni] In some embodiments, the modular cartridge 205 can use the rotor for biopsy and other small samples described in detail in International Application No. PCT/US2017/063811 International Application No. PCT/US2019/035097 and International Application No. PCT/US2023/014338, with a center ‘tooth’ and rings of 500 mm ‘teeth’ (in the cap), with complementary rings of 500 mm ‘teeth’ on the bottom of the Dissociation Chamber 441 to mechanically disrupt tissues.

[0112] Currently, the ‘side gap’ 221 between the rotor and inside Dissociation Chamber wall is not mechanically fixed and can vary as the rotor is spun or lowered. This could lead to the mechanical destruction of some fragile cell types and contribute to run-to-run variability [0113] The cartrige module described herein can be used to process tissue into single-cell suspensions or nuclei and are, in some cases, single-use. In some embodiments, cartridge inputs specimen (e.g., fresh tissue) or FFPE tissue specimen or OCT tissue specimen and output singulated cells or nuclei. In some embodiments, the Tissue Processing System combines the mechanical disruption of specimen on cartridge, adds reagents such as chemicals, detergents, enzymatic or chemical dissolution solutions and other fluids according to the protocols, and controls sample movement, pressures, and temperature. In some embodiments, the Tissue Processing System can move or rotate mechanical tissue disruptor elements comprised of without limitation a syringe plunger, pestle, Dounce pestle, or grinder, using a z axis stepper with a rotary motor coupled through the cap. In some embodiments, the term plunger is at times used to refer to combination of shaft/piston and rotor with optional disruption features (e.g., teeth) 355 with spring in sheath. [0114] In some embodiments, the mechanical tissue disruptor elements have features on the bottom of the rotor or grinder that can mechanically disrupt tissue at the bottom or floor of processing Chamber which in some embodiments may have complementary features to aid in the disruption of the tissue. In some embodiments, the mechanical tissue disruptor elements does not have features on the bottom of the rotor or grinder but can be flat and mechanically disrupt tissue against a flat surface at the bottom or floor of processing Chamber. Disruption also occurs in the ‘side gap’ between the rotor and the side wall of processing Chamber in some embodiments.

[0115] In some embodiments, disposable cartridge process multiple types of preserved FFPE or OCT tissues with mechanical disruption and enzymatic or chemical dissociation that can be adjusted according to the tissue type and condition of the FFPE tissue, such as age, or chemical process. In some embodiments, the cartridge can be designed to process tissue as quickly and as gently as possible, not expose the operator to the tissue being processed, and be manufacturable at low cost. Multiple mechanical methods may be needed to accommodate the wide range of tissues and their individual requirements: designs are shown that can be readily adapted to multiple different mechanical disruption methods comprising variable orifice, grinding with rotating plungers, pestles, and straining and filtering using a plunger as well as other mechanical methods without limitation.

[0116] In some caes, cartridges can be designed for 3D printing, injection molding in plastics with single or double pulls and low labor assembly, or layered assembly of fluidic and other layers, combinations of methods, and other methods well known to one skilled in the art. In some embodiments, fluids can be delivered to cartridge by pumps such as a syringe pump or by vacuum or can be preloaded onto cartridge or many combinations. In some embodiments, flexible tubing can connect chambers and creates simple pinch valves to direct flow. In other embodiments, channels are created in the cartridge and valves can be incorporated such as pneumatic valves, or other valves.

[0117] In some embodiments, the cartridge includes a processing chamber comprising a stator comprising teeth arranged in an annular array. In some embodiments, the processing chamber further comprises a first processing port from which a cell, nuclei organelle suspension can be removed from the processing chamber. In some embodiments, the cartridge further includes a post-processing chamber and a vacuum chamber. The vacuum chamber comprises a vacuum port.

[0118] In some embodiments, the cartridge further include a grinder assembly. In some embodiments, the grinder assembly includes a plunger comprising a piston and a rotor positioned at an end of the piston. In some embodiments, the rotor comprises on a bottom

-Tl- surface, grinding elements, e.g., teeth, including a central tooth and an annular array of three rings of teeth. In some embodiments, the teeth can have a blunt or sharp shape. In some embodiments they may take the shape of a trapazoid in cross section. In some embodiments, the outermost ring of teeth is positioned at the edge of the rotor. In some embodiments, the grinding assembly further includes a sleeve or sheath around the piston. In some embodiments, the grinding assembly further includes a cap to position the plunger in the processing chamber. In some embodiments, the cap further comprises a slot or other mechanism configured to engage a key of an actuator to actuate the grinder. In some embodiments, the grinder assembly includes a spring which biases the rotor toward the cap so that positive pressure must be asserted on the plunger by the actuator to press the rotor against the stator. In some embodiments, the annular rings of teeth in the rotor and the stator are positioned complementary to one another so that when the grinder is pressed against the stator the rings of the stator mesh with the rings of the rotor (e.g., are staggered against). That is, in an exemplary embodiment, teeth in the stator do not touch teeth in the rotor. This configuration facilitates rotation of the rotor against the stator so that teeth from one part do not collide with teeth from another part. The number of rings of teeth in each of the rotor and the stator can be determined by a skilled artisan. Factors influencing the determination include the total surface area of the stator and the face of the rotor, as well as the size of the teeth. In certain embodiments the number of rings of teeth in the stator and/or the rotor can be any of none, one, two, three, four, five, or six. In one embodiment teeth can have a trapezoidal cross-section. The processing chamber can have a cylindrical shape. The stator can have a radius between, for example, 5 mm and 25 mm, e.g., about 12 mm. The processing chamber can have a volume less than 1 ml, or between about 1 mL and 50 mL, for example, between about 10 mL and about 30 mL, e g., about 15 mL. The rotor and the sidewalls of the processing chamber can be configured so that when the plunger is inserted into the processing chamber there is a gap between the sidewall of the processing chamber and an edge of the rotor. The size of the gap can be optimized to allow passage of whole cells, nuclei or organelles between the sidewall and the rotor. In certain embodiments, the teeth can have a height of about 500 microns and a width of about 1 mm to 2 mm.

[0119] Spin rates for the dissociation can be 10-200 rpm. Total revolutions of the grinding element can be 5-500. In an exemplary protocol, the spin rate is about 45 rpm (slow) or about 150 rm (fast), with about 4 seconds of revolution, about 1-2 second pause, then about another 4 seconds, then repeat (about 16 seconds total rotation time) at each vertical displacement step of the stepper motor, sequentially going lower towards the bottom of the cartridge, about 9 vertical displacements in all, and at the bottom-most step, there are about 3 repetitions of the rotation periods rather than 2.

[0120] In some embodiments, rotors are provided that maintain uniform gaps on the sides and/or bottom. Figure 9 shows four designs. Designs with a center post 223 to eliminate wobble and maintain a fixed ‘side gap’ 221 (i.e., the gap between the side of rotor 353 and the side of the Dissociation Chamber 441) and a fixed ‘bottom gap’ 222 (i.e., the gap between the bottom of the rotor 353 and the bottom of the Dissociation Chamber 441) are shown with a center post 223 on rotor 353 and a center hole 224 on the stator 354 (Panel A) or a center post 223 on the stator 354 and a center hole 224 on the bottom of the rotor 353 ( Panel B). The insert in panels A and B shows a section of the rotor 353 and stator 354. In two other designs, the side gap 221 is set by gap setting bumps 228 (Panel C) or gap setting swirls 229 (Panel D) to minimize cell or nuclei damage from possible side gap variability as rotor 353 is spun; in these two designs the bottom gap 222 may be controlled by a force sensor 2115 or combined with the bottom gap 222 setting features from Figure 9A or 9B or other embodiments.

[0121] In addition, the grinding features 220 (tooth) shapes and patterns of the rotor 353 and stator 354 (Figure 10) can be optimized for different tissues and sample sizes. For example, designs with narrower or smaller teeth can be used to optimize yields from large samples (50-200 mg) or for yields from biopsy-sized samples, or by sequencing for cell representation, or for ambient mRNA release.

[0122] A Filtration Chamber 450 (Figure 11) may connect to the Dissociation Chamber 441 via tubing 2929 to filtration center input port 2920. The dissociated tissue may be pulled by vacuum through tubing 2920 into the Filtration Chamber 450 and through one, two, three or more filters 341, e. , 145, 40, and 20 mm for nuclei. In some embodiments, the filters are designed to filter different sizes of debris, nuclei, cells, or specimen. In some embodiments, the filters are designed to filter the same sizes of debris, nuclei, cells, or specimen. For example, the size of debris, nuclei, cells, or specimen is larger than 5 mm, 10 mm, 20 mm, 30 mm, 40 mm, 50 mm, 60 mm, 70 mm, 80 mm, 90 mm, 100 mm, 110 mm, 120 mm, 130 mm, 140 mm, 150 mm, 160 mm, 170 mm, 180 mm, 190 mm, or 200 mm. In some instances, the size of debri, nuclei, cells, or specimen is smaller than 5 mm, 10 mm, 20 mm, 30 mm, 40 mm, 50 mm, 60 mm, 70 mm, 80 mm, 90 mm, 100 mm, 110 mm, 120 mm, 130 mm, 140 mm, 150 mm, 160 mm, 170 mm, 180 mm, 190 mm, or 200 mm. In some embodiments, the filter has a pore size of more than 5 mm, 10 mm, 20 mm, 30 mm, 40 mm, 50 mm, 60 mm, 70 mm, 80 mm, 90 mm, 100 mm, 110 mm, 120 mm, 130 mm, 140 mm, 150 mm, 160 mm, 170 mm, 180 mm, 190 mm, or 200 mm. For example, the filter has a pore size of less than 5 mm, 10 mm, 20 mm, 30 mm, 40 mm, 50 mm, 60 mm, 70 mm, 80 mm, 90 mm, 100 mm, 110 mm, 120 mm, 130 mm, 140 mm, 150 mm, 160 mm, 170 mm, 180 mm, 190 mm, or 200 mm.

[0123] In some embodiments, one or more of the filters is ultrasonically welded to the bottom of top cap 2925. The top cap 2925 of Filtration Chamber 450 may have a fluidic connection via port 2921 with cannula seat 2916 for a cannula (not shown) to deliver wash reagents to the filters 341 or to pull vacuum or apply pressure when mixing materials from downstream modules, e.g., Processing Chamber 460 which can be used as Magnetic Processing Chamber 905. Filtered single cell suspensions 1102 or filtered nuclei suspensions 1103 or filtered nucleic acids 1072 may be moved through filtration output tubing 2124 (not shown) connected to the filtration bottom output port 2923 either directly to an output centrifuge tube 855 in modular cartridge 205, or to the Magnetic Processing Chamber 905 or an Output Chamber 850 or other chambers or tubes as needed. Filter surface areas may be optimized for <5, 10, 15, 20, 25, 30, 35, 40, 45, 50 mg or larger samples. Filter surface areas may be optimized for <1, <0.1, <0.01 mg or smaller samples. Filter surface areas may be optimized for <20 mg samples. Tissues with varied characteristics, e.g., soft or fibrous, may be dissociated and different sets of filters used to match tissue characteristics.

[0124] A Processing Chamber 460 , in some cases, which can be a Magnetic Processing Chamber (MPC) 905 (Figure 12) can accept sample input from the Filtration Chamber 450 and output concentrated, purified single-cell suspensions 1100 or other materials after performing labeling reactions or molecular biology reactions such as bulk sequencing library construction. In some embodiments, the MPC top cap 2925 has three ports, one can be a reagent input port 2932 to connect with MPC cannula 2938 (not shown) to deliver beads, vacuum, or wash or other fluids from the upper cartridge interface 1501, and two ports with fluidic dip tubes that extend to the bottom of the chamber. In some embodiments, the fluid dip tubes are notched at the lower end to allow the dip tubes to be in contact with the floor of the Magnetic Processing Chamber 905. A MPC sample input port 2933 connects filtration output tubing 2924 from Filtration Chamber 450 to MPC sample input dip tube 2934 and can be used to move the sample from Filtration Chamber 450 into the Magnetic Processing Chamber 905.

[0125] The MPC outlet port 2935 with MPC sample output dip tube 2936 can provide air or other gases for bubble mixing or vacuum to move the sample to MPC output tubing 2937 connected to MPC output port 2935 or to move washes to an on-cartridge Waste Chamber 430. [0126] In some embodiments, the workflow can begin by pulling the filtered sample from the Filtration Chamber 450 into the Processing Chamber 460 used as Magnetic Processing Chamber 905 by vacuum on MPC output tubing 2937 from a downstream module or by using vacuum from MPC cannula 2938 connected to cannula seat 2916 on MPC reagent input port 2932 while closing MPC pinch valve 2962 with actuator 2963 (Figure 6B). Paramagnetic beads 685 with antibodies for cell-type pulldowns (e.g., Dynabeads, FlowComp, ThermoFisher) may be added by MPC cannula 2938 and the sample mixed.

[0127] Multiple mixing modes can be employed. In some embodiments, the solution is first pulled into Filtration Chamber 450 through the filtration output tubing 2924 by applying vacuum from upstream on the Filtration Chamber 450 through filtration cannula 2938 with filtration pinch valve 2918 closed. The solution is then pulled back into Magnetic Processing Chamber 905 by applying vacuum on cannula 2938 with pinch valve 2962 closed. The process can be repeated as many times as necessary to fully mix the sample. In another embodiment, magnetic beads 685 can be captured and released onto opposite sides of the chamber or can be moved circularly to create a vortex by moving magnet(s) 910. Another embodiment is bubble mixing where air or other gases are bubbled through dip tubes 2934 and 2936 to create mixing.

[0128] A movable neodymium or other magnet 910 may be used to capture the cells, nuclei, or other materials bound to magnetic beads 685 to the bottom or wall of the MPC 905. The liquid can then be pulled to an on-cartridge Waste Chamber 430 or to off-cartridge waste by vacuum through MPC sample output dip tube 2936. Wash fluid can be added via MPC cannula 2938, magnet 910 moved away from the Magnetic Processing Chamber 905 to release the magentic beads 685 with bound cells or nuclei to allow mixing, and the sample purified by successive rounds of capture and washing. With some magnetic beads 685, cells may be released from the beads by addition of the appropriate buffers or for beads with cleavable linkers, cells or other biological materials can be released by cleavage of the linkers. The balance between particle size, time for separation, and required magnetic force can be optimized for a range of paramagnetic particles from <0.1 to >10 mm.

[0129] In some cases, while the device can be implemented a Magnetic Processing Chamber 905 that it has utility even if magnetic processing is not in the workflow. For example, to mix two solutions or to perform a red blood cell lysis or other reaction when used as a Processing Chamber 460 without paramagnetic beads 685.

[0130] Simple pinch valves 491 (Figure 13) can open and close fluidic circuits to direct flows; for example, to direct samples and liquids, sending washes to waste for paramagnetic bead processing, or moving samples for further on-board sample preparation or output. The flexible tubing 493 connecting cartridge chambers may be held in a tubing support 494 to enable mechanical actuators 2116 on the instrument cartridge interface 1500 to clamp down on tubing 493 to push the tubing 493 against valve seats 492 to close the pinch valve 491 or the actuators 2116 can release the flexible tubing 493 to open these simple, reliable, normally open pinch valves 491. The pinch valve can comprise a fluidic conduit in contact with a valve seat, and a valve actuator, e.g., comprising a wedge, that, upon actuation, presses the conduit against the valve seat, thereby closing the valve.

[0131] The Output Chamber 850 (Figure 14) can have many embodiments. The output chamber can comprise a holder adapted to engage an attachment feature, and confiured to hold a removable vessel. In certain embedments, a centrifuge tube 855 screwed into a top cap 2940 which has an Output Chamber input port 2941 connecting via tubing (not shown) to the appropriate upstream chamber, e.g., Filtration Chamber 450 or Magnetic Processing Chamber 905, and an Output Chamber output port 2942 which connects to Output Chamber cannula 2945 on the upper cartridge interface 1501. In some embodiments, the output chamber top cap 2942 connects to centrifuge tube 855 which may screw together, or snap together, or click in, or engaged with, or be attached by glue or other methods well know to one skilled in the art. In some embodiments, centrifuge tube 855 is reversibly engagble with the output chamber top cap 2942.

[0132] An optional on-cartridge Waste Chamber 430 (Figure 15) can hold waste from paramagnetic bead processing or applications such as FFPE deparaffinization. The Waste Chamber 430 may be molded into modular frame 2905 or snapped into modular frame 2905. The waste chamber top cap 2940 may have two or more connections: a Waste Chamber input port 2951 connecting to the upstream module, e.g. , Magnetic Processing Chamber 905 or Dissociation Chamber 441, and a Waste Chamber output port 2952 connecting to a waste chamber cannula 2955 (not shown) or to vacuum from the instrument. The waste chamber top cap 2940 of waste chamber 430 connects to Waste Chamber body 2945 which may screw together, or snap together, or click in, or engaged with, or be attached by glue or other methods well know to one skilled in the art. In some embodiments, the Waste Chamber 430 is designed to process tissue, nuclei, cells, or specimen in volume > 1 mL, > 5 mL, or more. In some embodiments, the Waste Chamber 430 is designed to process tissue, nuclei, cells, or specimen in volume < 1 mL, < 0.5 mL, < 0.1 ml, or less. In some embodiments, the Waste Chamber 430 is designed to process tissue, nuclei, cells, or specimen in volume more than 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL or more. In some embodiments the Waste Chamber 430 is designed to process tissue, nuclei, cells, or specimen in volume less than 1 mL, 0.9 mL, 0.8 mL, 0.7 mL, 0.6 mL, 0.5 mL, 0.4 mL, 0.3 mL, 0.2 mL, 0.1 mL, 0.05 mL, 0.01 mL, 0.005 mL, 0.001 mL, or less.

[0133] An optional tangential flow module 2700 (Figure 16A and B) can be used to remove small debris from the sample, concentrate the sample, or change buffers. The tangential flow module 2700 (Figure 16A) is connected by input port 2703 to the appropriate upstream chamber, e.g., the Filtration Chamber 450 or Magnetic Processing Chamber 905, by tubing or other fluidic connectors. Dissociated single cell and nuclei suspensions can be moved into the tangential flow module 2700 to the tangential flow region 2706 by vacuum or pressure. The appropriate buffer is circulated through the upper buffer region 2707 and the lower buffer region 2708 which is separated by the tangential flow region 2706 by tangential flow filters 2710. The dissociated single cell and nuclei suspensions can be concentrated and the buffer exchanged. In some embodiments, multiple tangential flow regions 2706 can be implemented with different tangential flow filters 2710 or different buffers. In some embodiments, an optical interrogation region 2720 (Figure 16B) can be incorporated into the tangential flow module 2700. In some embodiments, optical interrogation region 2720 is a flowcell 2620. In some embodiments, the tangential flow module 2700 is designed to process tissue, nuclei, cells, or specimen in volume > 1 mL, > 5 mL, or more. In some embodiments, the tangential flow module 2700 is designed to process tissue, nuclei, cells, or specimen in volume < 1 mL, < 0.5 mL, < 0.1 ml, or less. In some embodiments, the tangential flow module 2700 is designed to process tissue, nuclei, cells, or specimen in volume more than 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL or more. In some embodiments the tangential flow module 2700 is designed to process tissue, nuclei, cells, or specimen in volume less than 1 mL, 0.9 mL, 0.8 mL, 0.7 mL, 0.6 mL, 0.5 mL, 0.4 mL, 0.3 mL, 0.2 mL, 0.1 mL, 0.05 mL, 0.01 mL, 0.005 mL, 0.001 mL, or less.

[0134] In an embodiment shown in Figures 6A and 6B, a port in a dissociation chamber is fluidically connected via a fluidic channel to a port in a filtration channel. A port in the filtration chamber is fluidically connected via a fluidic channel to a port in a magnetic processing chamber. A port in the magnetic processing chamber fluidically connected via a fluidic channel to a port in an output chamber. A port in the output chamber is fluidically connected via a fluidic channel to a port in a waste chamber. When engaged with an instrument, ports in each of the filtration chamber, magnetic processing chamber, output chamber and waste chamber are fluidically connected via cannulae to a source of positive or negative pressure. Typically, this will vaccuum. [0135] To move liquid between chambers, suction can be applied via a cannula to draw liquid from a fluidically adjacent container. Where vacuum would pull from chambers in series, or from chambers on opposite sides of the subject chamber, a valve, such as a pinch valve, can be closed next to the unwanted chamber, preventing liquid from being pulled from that chamber.

Methods

[0136] This disclosure also provides, in some embodiments, a method for purifying cells or subcellular organelles from a tissue comprising: (I) providing a system of any of the preceding claims, wherein the system comprises a cartridge engaged therewith, wherein the cartridge comprises a tissue sample and dissociation reagent in the processing chamber; (II) using the disruptor actuator to operate the grinder assembly to grind the tissue, thereby releasing cells and/or subcellular organelles; and (III) using the pressure source to move the released cells and/or subcellular organelles from the processing chamber directly or indirectly to an output receptacle of the output unit.

[0137] In some embodiments, the method further comprises, before moving the released cells and/or subcellular organelles into the output receptacle, performing at least one of the following operations:- using the pressure source to move the released cells and/or subcellular organelles from the processing chamber into the filtration unit to separate debris from the cells and/or subcellular organelles; - using the pressure source to move the released cells and/or subcellular organelles from either the processing chamber or the filtration unit into a magnetic processing unit, capturing cells and/or subcellular organelles in the magnetic processing unit by using the magnetic force to attract cells and/or subcellular organelles that are associated with magnetic or paramagnetic particles, and, optionally, moving unattracted material of the magnetic processing chamber, and optionally washing the attracted cells and/or subcellular organelles using the pressure source to move liquid into the magnetic processing chamber; - using the pressure source to move at least some of the released cells and/or subcellular organelles into a flowcell and detecting living and/or dead cells and/or subcellular organelles using the measurement module; - using the pressure source to move the cells and/or subcellular organelles into a tangential flow device to remove contaminants; or - using the pressure source to move waste from a functional unit into the waste unit.

[0138] In some embodiments, the tangential flow device comprises two tangential flow filters connecting with upper and lower buffer regions functioning as a concentration gradient.

[0139] In some embodiments, grinding the tissue comprises executing a computer script indicating one or more of: a number of times the grinder assembly is moved in a Z direction, a pressure with which the grinder assembly presses the tissue in the Z direction, a number of rotations of the grinder assembly in the clockwise or counterclockwise directions.

[0140] In some embodiments, the tissue is fresh tissue or preserved tissue (e.g., FFPE tissue). [0141] In some embodiments, the method further comprises isolating and sequencing nucleic acids from the purified cells and/or subcellular organelles.

[0142] Also provided herein is a a method for preparing titered, purified single cells and/or subcellular organelles performed in a system described herein, comprising: (a) withdrawing cells and/or subcellular organelles from the processing chamber or a functional unit of the cartridge; (b) staining the cells and/or subcellular organelles in a known volume; (c) moving the stained cells and/or subcellular organelles into the measurement module; (d) using the measurement module, determining titer and/or viability of the stained cells and/or subcellular organelles; (e) if the determined titer is greater than a desired titer, then, adding liquid to the processing chamber or functional unit from which the cells and/or subcellular organelles were withdrawn, and adding liquid to achieve the desired titer; or, if the determined titer is less than a desired titer, then, concentrating the cells and/or subcellular organelles in the cartridge, and, adding liquid to the cells and/or subcellular organelles to achieve the desired titer; and (f) optionally, moving the cells and/or subcellular organelles at the desired titer into an output receptacle of the output unit.

[0143] In some embodiments, cells and/or subcellular organelles are concentrated by immobilizing the cells and/or subcellular organelles in the magnetic processing unit by capture on magnetic or paramagnetic particles and immobilization using the magnetic force, and removing liquid from the magnetic processing unit to achieve the desired titer, optionally adding liquid as necessary.

[0144] In some embodiments, immobilizing comprises capturing cells and/or subcellular organelles on magnetic or paramagnetic particles derivatized with antibodies specific for the cells and/or subcellular organelles; and, immobilizing the particles by apply magnetic force to the magnetic processing unit; separating waste from the immobilized particles by withdrawing the waste from the magnetic processing unit; optionally, releasing the captured cells and/or subcellular organelles from the antibodies (e g., by adjusting pH); and resuspended the cells and/or subcellular organelles in a liquid.

[0145] In some embodiments, using a measurement module comprises moving the cells and/or subcellular organelles into a flowcell and interrogating the flowcell with the optical detector.

[0146] This disclosure also provides, in some embodiments, a method for detecting surface proteins on cells or subcellular organelles from a tissue comprising: (I) providing a system described herein, wherein the system comprises a cartridge engaged therewith, wherein the cartridge comprises a tissue sample and dissociation reagent in the processing chamber; (II) using the disruptor actuator to operate the grinder assembly to grind the tissue, thereby releasing cells and/or subcellular organelles; (III) using the pressure source to move the released cells and/or subcellular organelles from the processing chamber directly or indirectly to a magnetic processing unit; (IV) using the magnetic separation assembly, immobilizing the cells and/or subcellular organelles in the magnetic processing unit; (V) labeling one or more proteins on the surface of the cells and/or subcellular organelles; (VI) using the pressure source to move the labeled cells and/or subcellular organelles into a flowcell in optical communication with the optical detector of the measurement module; and (VII) using the optical detector to detect the labeled proteins.

[0147] This disclosure also provides, in some embodiments, a method for determining health of cells or subcellular organelles from a tissue comprising: (I) providing a system described herein, wherein the system comprises a cartridge engaged therewith, wherein the cartridge comprises a tissue sample and dissociation reagent in the processing chamber; (II) using the disruptor actuator to operate the grinder assembly to grind the tissue, thereby releasing cells and/or subcellular organelles; (III) using the pressure source to move the released cells and/or subcellular organelles from the processing chamber directly or indirectly to a magnetic processing unit; (IV) using the magnetic separation assembly, immobilizing the cells and/or subcellular organelles in the magnetic processing unit; (V) labeling the cells and/or subcellular organelles with a marker that differentiates healthy cells and/or organelles from necrotic, late stage apoptosis, and/or early necrotic cells and/or subcellular organelles; (VI) using the pressure source to move the labeled cells and/or subcellular organelles into a flowcell in optical communication with the optical detector of the measurement module; and (VII) using the optical detector to detect labeled and/or unlabeled cells and/or subcellular organelles.

[0148] This disclosure also provides, in some embodiments, a method for purifying DNA or RNA from cells and tissue, comprising: (I) providing a system described herein, wherein the system comprises a cartridge engaged therewith, wherein the cartridge comprises a tissue sample and dissociation reagent in the processing chamber; (II) releasing DNA or RNA from the cells by using the disruptor actuator to operate the grinder assembly to grind the tissue, wherein the processing chamber comprises one or more reagents to disrupt cells and/or subcellular organelles thereby releasing DNA or RNA; (III) using the pressure source to move the released cells and/or subcellular organelles from the processing chamber directly or indirectly to a magnetic processing unit; (IV) using the magnetic separation assembly, immobilizing the released to DNA or RNA using particles that capture nucleic acids (e.g., SPRI beads), removing unbound material and, optionally, washing the particles; (V) optionally, moving the washed particles or released DNA or RNA into the output receptacle of the output unit.

[0149] In some embodiments, the method further comprieses: (VI) in the magnetic processing unit, performing one or more of: reverse transcribing the RNA to produce cDNA, polishing the DNA or RNA, and repairing DNA or cDNA and ligating DNA or cDNA to DNA sequencing adapters, or tagmentation, or any other molecular biology enzymatic reaction on nucleic acid.

[0150] In some embodiments, the method further comprises performing PCR on DNA in the magnetic processing chamber or other chamber.

[0151] In some embodiments, the method further comprises performing a sizing cut on a library.

[0152] This disclosure provides, in some embodiments, a method for purifying cells or subcellular organelles from a tissue comprising: (a) providing a system of any of claims 108-114, wherein the system comprises a cartridge engaged therewith, wherein the cartridge comprises a tissue sample or cell sample within the at least one processing chamber or dissociation chamber; (b) using a disruptor actuator to operate a grinder assembly to grind the tissue within the processing chamber, thereby releasing cells and/or subcellular organelles; and (c) using a pressure source to move the released cells and/or subcellular organelles from the processing chamber directly or indirectly to an output receptacle of the output unit.

[0153] In some embodiments, the method further comprises, before moving the released cells and/or subcellular organelles into the output receptacle, performing at least one of the following operations: (a) using the pressure source to move the released cells and/or subcellular organelles from the processing chamber into the fdtration unit to separate debris from the cells and/or subcellular organelles; (b) using the pressure source to move the released cells and/or subcellular organelles from either the processing chamber or the filtration unit into a magnetic processing unit, capturing cells and/or subcellular organelles in the magnetic processing unit by using the magnetic force to attract cells and/or subcellular organelles that are associated with magnetic or paramagnetic particles, and, optionally, moving on attracted material of the magnetic processing chamber, and optionally washing the attracted cells and/or subcellular organelles using the pressure source to move liquid into the magnetic processing chamber; (c) using the pressure source to move at least some of the released cells and/or subcellular organelles into a flowcell and detecting living and/or dead cells and/or subcellular organelles using the measurement module; (d) using the pressure source to move the cells and/or subcellular organelles into a tangential flow device to remove contaminants; or (e) using the pressure source to move waste from a functional unit into the waste unit.

[0154] In some embodiments, the method further comprises using a first magnetic processing chamber to reduce a volume of the sample and a second magnetic processing chamber to perform reactions.

[0155] In some embodiments, the method furtehr comprises using the first magnetic processing chamber to reduce a volume of a sample to 0.001, 0.005, 0.01, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, or 0.5 of its initial volume.

[0156] In some embodiments, the cartridge comprises a tissue fragment and wherein the cartridge comprises a filtration unit with a pore size of 100-500 microns, e.g., 100-400 microns, 200-500 microns, 100-300 microns.

[0157] In some embodiments, the method produces a single cell suspension.

[0158] In some embodiments, the method produces a single cell suspension and the cartridge comprises a first filter with pores that are 50 to 500 microns in diameter, e.g., 50-200 microns, 100-500 microns, 200-500 microns, 150 -400 microns and/or a second filter with pores 20-200 microns in diameter, e g., 20-100 microns, 40-200 microns, 100-200 microns or 20-150 microns in diameter. [0159] In some embodiments, the method produces a single sub-cellular organelle suspension (e.g., a single nuclei suspension).

[0160] In some embodiments, the method produces a single sub-cellular organelle suspension (e.g., a single nuclei suspension) and the filtration unit comprises a first filter with pores 50 to 500 microns in diameter, e.g., 50-200 microns, 100-500 microns, 200-500 microns, 150 -400 microns in diameter; and/or a second filter comprising pores that are 5-80 microns in diameter, e.g., 5-50 microns, 20 - 80 microns, 10-80 microns.

[0161] In some embodiments, the method produces a single cardiomyocyte suspension.

[0162] In some embodiments, the method produces a single cardiomyocyte suspension and the filtration unit comprises a first filter with pores 50 to 500 microns in diameter, e.g., 50-200 microns, 100-500 microns, 200-500 microns, 150 -400 microns in diameter; and/or a second filter comprising pores that are 200-300 microns in diameter, 150-400 microns in diameter, or 200-600 microns in diameter.

[0163] This disclosure provides, in some embodiments, a method of preparing a cartridge for use in an instrument that produces single cell suspensions, single nuclei suspensions or single subcellular organelle suspensions, the method comprising: (a) providing a cartridge described herein; and (a) attaching one or more of the plurality of the stand-alone units to the cartridge via the attachment features.

[0164] Also provided herein, in some embodiments, is a method of preparing a cartridge for use in an instrument that produces single cell suspensions, single nuclei suspensions or single subcellular organelle suspensions, the method comprising: (a) providing a first and second cartridge described herein; (a) attaching a first set of the stand-alone units to the attachment features in the vessel bays of the first cartridge; (a) attaching a second set of the stand-alone units to the attachment features in the vessel bays of the second cartridge, wherein the first set and the second set of stand-alone units comprise different combinations of stand-alone units. In some embodiments, the first and second cartridges each comprise a common region situated a common location on the first and second cartridge that interfaces with an integrated feature of the instrument. In some embodiments, the integrated feature is a thermal controller, a magnetic field, an optical detector, a measurement device, fluidics subassembly, actuators, cannulae, tubing, pressure source, positive or negative pressure, reagent source, wash buffer source, fluorescent detector, regulatable magnetic field; fluid dispenser, or fluid aspirator.

Systems

[0165] This disclosure provides, in some embodiments, is a system comprising an instrument engaged with the cartridge described herein. In some embodiments, the system is engaged with the cartridge via tubing or cannulae. In some embodiments, the cartridge comprises fluidic ports in communication with fluid ports situated in the instrument. In some embodiments, the instrument comprises a fluidics subassembly comprising: (a) at least one pressure source of positive and/or negative pressure, wherein the at least one pressure source optionally comprises separate vacuum source and pump (e.g., syringe pump); (b) at least one reagent container for containing liquids, fluidically connected to the at least one pressure source, optionally contained in a reagent cassette; or (c) at least one cannula communicating with the at least one pressure source and, directly or indirectly through one or more fluidic lines, with at least one port of a functional unit, or of the processing chamber, when the at least one cartridge is engaged with the at least one cartridge interface.

[0166] In some embodiments, the instrument comprises an actuator subassembly comprising: (a) a disruptor actuator configured to engage the grinder assembly; or (b) at least one valve actuator (e.g., a pinch valve actuator) configured to actuate one or more valves to control fluid flow in fluidic conduits in the at least one cartridge.

[0167] In some embodiments, the instrument comprises magnetic separation assembly(s) comprising a source of magnetic force that is reversibly engageable with a functional unit engaged with a cartridge when the cartridge is engaged with the cartridge interface.

[0168] In some embodiments, the system comprises a thermal assembly configured to regulate temperature of functional units engaged with the plurality of vessel bays or frame.

Kit

[0169] This disclosure provides, in some embodiments, a kit comprising the cartridge described herein and a plurality of stand-alone vessels. In some embodiments, the plurality of stand-alone vessels comprises: (a) at least one processing chamber comprising an internal surface functionalized with grinding features; (b) at least one filtration unit comprising a filter or strainer; (c) at least one magnetic processing unit comprising magnetic particles; (d) an output unit comprising a separable output tube; (e) a flowcell unit; (f) a tangential flow filtration unit; or (g) a combination thereof.

Instrument

[0170] One or more instruments may operate one or more modular cartridges 205 simultaneously. Each single-use cartridge 205 may use a single instrument bay. In some embodiments, a single-use cartridge 205 can process two or more samples. The instrument bay may deliver reagents 411 using a pump which can be a syringe pump 2130, pull vacuum on ports, use z-axis 2110 and rotary motors 2120 with force sensors 2115 for mechanical disruption, control and monitor temperature with a thermistor, thermocouple, or IR camera 2612, capture video of the dissociation process with a visible camera 2613, operate pinch valves 491, move a magnet 910 with an actuator 935, enumerate cells or nuclei or debris or detect fluorescent on a optics module 2600, or perform sample-to-answer assays.

[0171] In some embodiments of the system, the cartridge-instrument interface 1500 is varied. Figure 2 shows one embodiment of the key elements for the instrument’s cartridge interface 1500. To maintain a narrow footprint, fluidic connections and mechanical interactions (tissue disruption, valves) can be accessed from the top (or a first side or surface of the instrument), and thermal control, optical readout, and magnetics can be accessed from the bottom (or second side or surface of the instrument).

[0172] In one design embodiment, the modular cartridge 205 is inserted into a receiving dock of the lower cartridge interface 1502 by the user. The receiving dock may use one of many temperature regulating subsystem 1475 embodiments to set the temperature of the Dissociation Chamber 441, or other chambers, or output tubes 855 such as using circulating liquids from constant temperature reservoirs (e.g., 4 and 37°C), or Peltier heating or cooling, or resistive heating, or other methods well known to one skilled in the art. The receiving dock may have optical 2613 and/or IR 2612 cameras beneath or on the side of the Dissociation Chamber 441 (not shown) to visualize dissociation and monitor temperature respectively. Magnets 910 may be moved by magnetic actuators 935 beneath or on the side of the Magnetic Processing Chamber 905. Other embodiments, as illustrated in Figures 3, 4, and 5, have the elements of the cartridge interface 1500 in different configurations.

[0173] After insertion of cartridge 205, the upper cartridge interface 1501 may move down and engage spring-loaded, cannulas 1416, e.g., 2 (+/- 1) mm ID, stainless steel cannulas 1416, against over-molded flexible cannula seats 2916, e.g., rubber or other compliant materials, on the fluidic/vacuum ports 2915 of the cartridge 205 to create robust fluidic and vacuum seals. The mechanical disruptor assembly on the upper cartridge interface 1501 engages the disruptor rotor 353 in the cartridge cap 210; the assembly may have a rotary motor 2120 on a stage with its z- position independently controlled by a stepper 2119 to operate the spring-loaded rotor 353 in the cap 210, with a force sensor 2115 to measure and in some embodiments adjust pressure on the tissue in real-time. The upper cartridge interface 1501 may have actuators 2116 to open and close cartridge pinch valves 491.

[0174] Single cell sequencing can depend on the quality of the single cells or nuclei used. In some embodiments, this disclosure provides fully automated, higher throughput systems, that process solid tissues into high quality single cells or nuclei, for optimized scRNA-Seq, snRNA- Seq, ATAC-Seq, CITE-Seq and other single cell or single nuclei workflows. In some embodiments, the systems have the capability to magnetically enrich or deplete cell types and dilute cell suspensions in the proper buffer, ready for the next processing steps. [0175] High-throughput single-cell sequencing and genomics can be accomplished using nanodroplets as highly parallel reactors or using combinatorial indexing to process eukaryotic mRNA from single cells into cDNAs with the incorporation of cell and molecular barcodes. In some embodiments, provided herein, single-cell genomics yields information useful for transcriptome sequencing (scRNA-Seq and snRNA-Seq), single-cell DNA sequencing (DNA- Seq), chromatin accessibility (ATAC-Seq) assays, cell surface analysis (CITE-Seq), multiome, and many other applications.

[0176] The output of the Tissue Processing System 80 can also be used for cell biology, such as FACS sorting, growth of organoids, cell health assays, e.g., apoptosis and necrosis, single cell proteomics, single cell metabolomics, and many other applications (Figure 4).

Software

[0177] The system operation is controlled by a Control System 700 wtih control software 725 which controls electronics 710 through computer 720 to operate instrument and interact with the user through graphic user interface 740 and a touchscreen interface 730 on a tablet 750 or through a mouse and keyboard and screen or other devices including virtual reality devices. LabScript™ is the S2 software development platform used both for R&D and commercial products. LabScript is designed to accelerate instrument development and commercialization. Existing software modules can be expanded to accommodate eight or more sample bays and operate all associated devices.

[0178] The software modules are: LabScript Host, a rapid development system to develop, maintain, and run scripts, with utilities that allow scripts to interact with users and other software. The system defines a standardized scripting interface and provides all tools needed to quickly create and test scripts. LabScript Scripts, coded in any Net language, compiled to standardized DLLs. Scripts have access to LabScript libraries and can launch other scripts. The host provides full support for script development, execution, testing, and coordination. Once the scripting logic is developed, the scripting host layer is replaced by an executable, dramatically shortening the normal development cycle. LabScript Library includes a variety of components as pre-compiled DLL’s and allow intercommunication with hardware components (pumps, valves, sensors, controllers), and with coordination functions— scheduling, image and statistical analysis, database access, etc. The Singulator Graphical User Interface 740 (GUI) guides the user through protocols selection, modification, and execution with optional instructional videos of the operation. The Singulator 200 GUI 740, which operates two cartridge bays, can be expanded to operate four, eight, 16 or more instrument bays, with operators able to ‘zoom into’ any instrument bay for protocol selection or modification, or real-time video and log displays. The Coordinator layer orchestrates resource use, preventing conflicts while running multiple samples in parallel and enabling random access of the instrument bays. Lab Script MetaData of sample and run parameters are collected in logs and available for export to LIMS or for bioinformatic analysis.

Singulator systems

[0179] Singulator systems can, in some embodiments, comprise a Singulator instrument (Figure 4), protocols, valve-less cartridges (Figure 17), and reagent formulations to standardize processing solid tissues into doubly-filtered single-cells or nuclei suspensions.

[0180] In some embodiments, the cartridge 200 is the heart of the system: all or most processing can occur and is contained within the cartridge 200. The inj ection-molded commercial cartridge 200 (Figure 17) has a Dissociation Chamber 441, a Vacuum Trap 468, and a Output Chamber 850, two embedded filters 341, and/or a cap 210 with a spring-loaded rotor disruptor 353. In some cases, cartridge 200 can be particularly designed to process cells and/or nuclei; in some cases, cartridges 200 process cells and/or nuclei for samples less than 20 mg, less than 30 mg, less than 50 mg, or less than 100 mg. In some embodiments, the injection-molded commercial cartridge 200 can be a modular cartridge 205 with many of the elements reconfigured and new functional modules added. In some embodiments the modular cartridge elements are connected by tubing 493 or by an integrated fluidic device such as a microchip 2705. In some embodiments, the modular cartridge elements are connected by capillaries.

[0181] In some embodiments, the instrument (Figure 4) operates cartridge 200 and can add reagents to different chambers at programmed times, mechanically mix the sample, control the mechanical disruption, and move dissociated cells or nuclei through filters 341 into the Output Chamber 850 on the cartridge where additional reagents can be added.

[0182] In some embodiments, the operator first removes cap 210 and then places a solid tissue specimen 120 (<1 to >300 mg) in the appropriate cartridge 200, e.g., for cells or for nuclei, replaces cap 210, and inserts the cartridge 200 into the instrument 80. For cells the operator also loads single-use reagents 411 onto the instrument’s Single-Shot Mechanism 1240. In some embodiments, after the operator selects or modifies a protocol, the system automatically processes the sample into dual-filtered single cell cells suspensions 1102 in 20-60 min depending on tissue type or into dual-filtered nuclei suspensions 1103 in ~5.5 min. The Singulator 100 has been validated for a wide range of tissues (Figure 18).

Singulator 100 and 200 Operation. Referring to Figure 19, when a cartridge 200 is inserted into the instrument 80, it docks with three spring-loaded cannulas 1416 that connect to the instrument’s fluidic subsystem, which has a syringe pump 2130 and six-way valves 2140. The system delivers the appropriate enzymatic or chemical reagents (S2’s or user-provided) from a ‘single shot’ mechanism 1240 for enzymes (for cells) or from a temperature-controlled reagent module 1419 (for nuclei) to the cartridge’s Dissociation Chamber 441. The cartridge 200 is temperature-controlled at ~6°C or 37°C by Peltier thermoelectric device 1440. When the cartridge 200 is inserted into the instrument 80, the Dissociation Chamber 441 is aligned with the instrument’s rotary motor 2120. The spring-loaded rotor 353 in cap 210 is engaged by the instrument to raise, lower, and rotate the disruption rotor 353 for selectable modes of mixing and mechanical tissue disaggregation. An auto-mince routine eliminates manual mincing for many tissue types, increasing reproducibility and convenience. After processing, the user removes the cartridge, pierces a foil seal 465, and pipettes out filtered single-cell suspension 1102 or filtered nuclei suspension 1103 for further processing.

For single cells, the tissue specimen 120 is typically incubated at 37°C in a tissue-specific enzyme formulation (e.g., collagenase, elastase, protease, DNase, etc.) with incubation and mixing for 10-60 or 20-60 min followed by one or more mechanical disruption cycles. The mechanical tissue disruption results from slow rotation of the rotor 353 in cap 210, which has 500 mm rounded ‘teeth’ 355 on the bottom (Figure 17 right) and the complementary teeth 355 on the bottom stator 354 of Dissociation Chamber 441, and from dissociation by displacement of the dissociated sample through a ‘side gap’ 221 (e.g., 250 mm ) between the side of the rotor 353 and the Dissociation Chamber 441 wall. The descent of the rotor 353 as it dissociates a tissue specimen 120 can be controlled by real-time force sensor 2115 feedback. Different disaggregation protocols can be preprogrammed for many standard tissues and can be customized for additional tissues. After dissociation, the suspension is pulled by vacuum on the trap port 467 through 145 and 70 mm filters 341 into Output Chamber 850, and the Dissociation Chamber 441 is washed twice with buffer to facilitate complete recovery of the dissociated cells.

The Singulator 100 processes solid tissues into single cell suspensions from a broad range of organisms, including human, rodents, insects, and worms, typically for single cell sequencing applications (Figure 18). Figure 20 shows cell viabilities range from 76-95% for fresh mouse tissues with good reproducibility. Cell yields are tissue-dependent and range from -5,000- >1,000,000 cells/mg tissue for mouse tissues. Similar results are found with human tissues.

For nuclei, the tissue specimen 120 is mechanically disrupted in a nuclei isolation solution 412, e.g., Nuclei Isolation Reagent (#100-063-396, S2 Genomics), containing a proprietary formulation with 0.1% NP-40, at ~6°C using rotor 353 with a 150 mm side gap 221. About 90% of tissues can be processed with a single mechanical disruption cycle using a single protocol; additional protocols include a second disruption cycle for fibrous and other difficult tissues. Disrupted tissue is pulled by vacuum through 145 mm and 40 mm filters 341 into the Output Chamber 850. The Dissociation Chamber 441 is washed with an osmo-protecting nuclei storage solution 413, e.g., Nuclei Storage Reagent (#100-063-623, S2 Genomics) without detergent. Figure 21 shows the yields of nuclei per mg of input mouse tissue. Computer Systems

[0183] Models provided herein can be executed by programmable digital computer.

[0184] Figure 31 shows an exemplary computer system. The computer system 9901 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 9905, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 9901 also includes memory or memory location 9910 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 9915 (e.g., hard disk), communication interface 9920 (e. ., network adapter) for communicating with one or more other systems, and peripheral devices 9925, such as cache, other memory, data storage and/or electronic display adapters. The computer readable memory 9910, storage unit 9915, interface 9920 and peripheral devices 9925 are in communication with the CPU 9905 through a communication bus (solid lines), such as a motherboard. The storage unit 9915 can be a data storage unit (or data repository) for storing data. The computer system 9901 can be operatively coupled to a computer network (“network”) 9930 with the aid of the communication interface 9920. The network 9930 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 9930 in some cases is a telecommunication and/or data network. The network 9930 can include one or more computer servers, which can enable distributed computing, such as cloud computing.

[0185] The CPU 9905 can execute a sequence of machine-readable instructions, which can be embodied in a program or software (code). The instructions may be stored in a memory location, such as the computer readable memory 9910. The instructions can be directed to the CPU 9905, which can subsequently program or otherwise configure the CPU 9905 to implement methods of the present disclosure.

[0186] The storage unit 9915 can store files, such as drivers, libraries, and saved programs. The storage unit 9915 can store user data, e g., user preferences, log files, video or other images, and user programs. The computer system 9901 in some cases can include one or more additional data storage units that are external to the computer system 9901, such as located on a remote server that is in communication with the computer system 9901 through an intranet or the Internet.

[0187] The computer system 9901 can communicate with one or more remote computer systems through the network 9930.

[0188] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 9901, such as, for example, on the computer readable memory 9910 or electronic storage unit 9915. The machine executable or machine-readable code can be provided in the form of software. During use, the code can be executed by the processor 9905. In some cases, the code can be retrieved from the storage unit 9915 and stored on the memory 9910 for ready access by the processor 9905. In some situations, the electronic storage unit 9915 can be precluded, and machine-executable instructions are stored on memory 9910. The code can be used to communicate and issue instructions to electronic devices, e.g., circuit boards 9940, modules, or subsystems, on the instrument ,for example, the rotary DC motor relay board or the heater relay board driving pettier to accomplish tasks such as rotating a motor or controlling the temperature of the cartridge.

[0189] The computer system 9901 can communicate with one or more remote computer systems through the network 9930.

[0190] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 9901, such as, for example, on the computer readable memory 9910 or electronic storage unit 9915. The machine executable or machine-readable code can be provided in the form of software. During use, the code can be executed by the processor 9905. In some cases, the code can be retrieved from the storage unit 9915 and stored on the memory 9910 for ready access by the processor 9905. In some situations, the electronic storage unit 9915 can be precluded, and machine-executable instructions are stored on memory 9910. The code can be used to communicate and issue instructions to electronic devices, e.g., circuit boards 9940, modules, or subsystems, on the instrument ,for example, the rotary DC motor relay board or the heater relay board driving peltier to accomplish tasks such as rotating a motor or controlling the temperature of the cartridge.

[0191] Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks.

[0192] The computer system 9901 can include or be in communication with an electronic display 9935 that comprises a user interface (UI) 9940 for providing, for example, input parameters for methods described herein. Examples of UIs include, without limitation, a graphical user interface (GUI) and web-based user interface. EXEMPLARY EMBODIMENTS

[0193] 1. A system comprising:

(a) an instrument comprising:

(i) at least one cartridge interface configured to engage at least one cartridge, wherein the at least one cartridge comprises a processing chamber comprising a grinder assembly and at least one cartridge bay having at least one functional unit engaged therewith, wherein the functional unit or units include at least one of a filtration unit, a magnetic processing unit, an output unit, a flowcell unit, a tangential flow filtration unit, and a waste unit;

(ii) an actuator subassembly comprising:

(1) a disruptor actuator configured to engage the grinder assembly; and

(2) at least one valve actuator (e g., a pinch valve actuator) configured to actuate one or more valves to control fluid flow in fluidic conduits in the at least one cartridge;

(iii) a fluidics subassembly comprising:

(1) at least one pressure source of positive and/or negative pressure, wherein the at least one pressure source optionally comprises separate vacuum source and pump (e.g., syringe pump);

(2) at least one reagent container for containing liquids, fluidically connected to the at least one pressure source, optionally contained in a reagent cassette;

(3) at least one cannula communicating with the at least one pressure source and, directly or indirectly through one or more fluidic lines, with at least one port of a functional unit, or of the processing chamber, when the at least one cartridge is engaged with the at least one cartridge interface;

(vi) optionally, one or more magnetic separation assembly(s) comprising a source of magnetic force that is reversibly engageable with a functional unit engaged with a cartridge when the cartridge is engaged with the cartridge interface;

(vii) optionally, athermal assembly configured to regulate temperature of functional units engaged with cartridge bays;

(viii) optionally, a measurement module comprising an optical detector and, optionally, a flowcell fluidically connected to the pressure source;

(ix) optionally, a control subsystem comprising a digital computer comprising a processor and memory, wherein the memory comprises code that, when executed by the processor, instructs the system to perform one or more operations; and

(b) optionally, at least one cartridge engaged with the at least one cartridge interface. [0194] 2. The system of embodiment 1, further comprising at least one motor configured to operate the disruptor actuator, the valve actuator, and the optional magnetic assembly.

[0195] 3. The system of any of the preceding embodiments, further comprising a flowcell fluidically connected with the at least one pressure source and at least one fluid line fluidically connected with the processing chamber and/or at least one functional unit in the cartridge, wherein the flowcell is configured for optical interrogation by the optical detector.

[0196] 4. The system of any of the preceding embodiments, further comprising a waste chamber fluidically connected with the at least one pressure source.

[0197] 5. The system of any of the preceding embodiments, wherein the disruptor actuator comprises a linear driver (e.g., a stepper motor or a pneumatic driver) that drives the actuator in an up-down (Z axis) direction, and a rotary motor that rotates the actuator around a Z axis.

[0198] 6. The system of any of the preceding embodiments, wherein the valve actuator is configured to close or open a valve by releasably pressing a pinching element against a flexible tube in the cartridge, which flexible tube fluidically connects (A) the processing chamber with a functional unit or (B) functional units with each other.

[0199] 7. The system of any of the preceding embodiments, wherein the at least one pressure source comprises a vacuum source communicating with a vacuum accumulator, which vacuum accumulator is fluidically connected to one or more cannulae through one or more vacuum valves. [0200] 8. The system of any of the preceding embodiments, wherein the at least one pressure source comprises a pump fluidically connected through at least one valve to at least one of the reagent containers and at least one cannula, wherein the pump is configured to draw liquid from the reagent container and push liquid to the cannula.

[0201] 9 The system of any of the preceding embodiments, wherein the at least one reagent container containing one or more of a reagent for dissociating tissue, paramagnetic beads, and wash reagents.

[0202] 10. The system of any of the preceding embodiments, wherein the at least one cannula is configured to move from a disengaged position to an engaged position in which the cannula meets with a port in a functional unit or the processing chamber.

[0203] 11. The system of any of the preceding embodiments, wherein the source of magnetic force is a magnet or electromagnet.

[0204] 12. The system of any of the preceding embodiments, wherein reversibly engaging the source of magnetic force comprises moving the source toward or away from the functional unit. [0205] 13. The system of any of the preceding embodiments, wherein the thermal assembly comprises a Peltier device. [0206] 14. The system of any of the preceding embodiments, wherein the thermal assembly comprises temperature regulatory elements configured to reversibly engage the processing chamber or at least one functional unit, e.g., by moving the elements toward or away from the processing chamber or functional unit.

[0207] 15. A method for purifying cells or subcellular organelles from a tissue comprising:

(I) providing a system of any of the preceding embodiments, wherein the system comprises a cartridge engaged therewith, wherein the cartridge comprises a tissue sample and dissociation reagent in the processing chamber;

(II) using the disruptor actuator to operate the grinder assembly to grind the tissue, thereby releasing cells and/or subcellular organelles; and

(III) using the pressure source to move the released cells and/or subcellular organelles from the processing chamber directly or indirectly to an output receptacle of the output unit.

[0208] 16. The method of embodiment 15, further comprising, before moving the released cells and/or subcellular organelles into the output receptacle, performing at least one of the following operations:

- using the pressure source to move the released cells and/or subcellular organelles from the processing chamber into the filtration unit to separate debris from the cells and/or subcellular organelles;

- using the pressure source to move the released cells and/or subcellular organelles from either the processing chamber or the filtration unit into a magnetic processing unit, capturing cells and/or subcellular organelles in the magnetic processing unit by using the magnetic force to attract cells and/or subcellular organelles that are associated with magnetic or paramagnetic particles, and, optionally, moving unattracted material of the magnetic processing chamber, and optionally washing the attracted cells and/or subcellular organelles using the pressure source to move liquid into the magnetic processing chamber;

- using the pressure source to move at least some of the released cells and/or subcellular organelles into a flowcell and detecting living and/or dead cells and/or subcellular organelles using the measurement module;

- using the pressure source to move the cells and/or subcellular organelles into a tangential flow device to remove contaminants; or

- using the pressure source to move waste from a functional unit into the waste unit. [0209] 17. The method of embodiment 15-16, wherein the tangential flow device comprises two tangential flow filters connecting with upper and lower buffer regions functioning as a concentration gradient.

[0210] 18. The method of any of embodiments 15-17, wherein grinding the tissue comprises executing a computer script indicating one or more of: a number of times the grinder assembly is moved in a Z direction, a pressure with which the grinder assembly presses the tissue in the Z direction, a number of rotations of the grinder assembly in the clockwise or counterclockwise directions.

[0211] 19. The method of any of embodiments 15-18, wherein the tissue is fresh tissue or preserved tissue (e.g., FFPE tissue).

[0212] 20. The method of any of embodiments 15-19, further comprising isolating and sequencing nucleic acids from the purified cells and/or subcellular organelles.

[0213] 21. A method for preparing titered, purified single cells and/or subcellular organelles performed in a system of any of embodiment 1-14, comprising:

(a) withdrawing cells and/or subcellular organelles from the processing chamber or a functional unit of the cartridge;

(b) staining the cells and/or subcellular organelles in a known volume;

(c) moving the stained cells and/or subcellular organelles into the measurement module;

(d) using the measurement module, determining titer and/or viability of the stained cells and/or subcellular organelles,

(e) if the determined titer is greater than a desired titer, then, adding liquid to the processing chamber or functional unit from which the cells and/or subcellular organelles were withdrawn, and adding liquid to achieve the desired titer; or, if the determined titer is less than a desired titer, then, concentrating the cells and/or subcellular organelles in the cartridge, and, adding liquid to the cells and/or subcellular organelles to achieve the desired titer; and

(f) optionally, moving the cells and/or subcellular organelles at the desired titer into an output receptacle of the output unit.

[0214] 22. The method of embodiment 21, wherein cells and/or subcellular organelles are concentrated by immobilizing the cells and/or subcellular organelles in the magnetic processing unit by capture on magnetic or paramagnetic particles and immobilization using the magnetic force, and removing liquid from the magnetic processing unit to achieve the desired titer, optionally adding liquid as necessary.

[0215] 23. The method of embodiment 21-22, wherein immobilizing comprises capturing cells and/or subcellular organelles on magnetic or paramagnetic particles derivatized with antibodies specific for the cells and/or subcellular organelles; and, immobilizing the particles by apply magnetic force to the magnetic processing unit; separating waste from the immobilized particles by withdrawing the waste from the magnetic processing unit; optionally, releasing the captured cells and/or subcellular organelles from the antibodies (e.g., by adjusting pH); and resuspended the cells and/or subcellular organelles in a liquid.

[0216] 24. The method of any of embodiments 21-23, wherein using a measurement module comprises moving the cells and/or subcellular organelles into a flowcell and interrogating the flowcell with the optical detector.

[0217] 25. A method for detecting surface proteins on cells or subcellular organelles from a tissue comprising:

(I) providing a system of any of embodiments 1-14, wherein the system comprises a cartridge engaged therewith, wherein the cartridge comprises a tissue sample and dissociation reagent in the processing chamber;

(II) using the disruptor actuator to operate the grinder assembly to grind the tissue, thereby releasing cells and/or subcellular organelles;

(III) using the pressure source to move the released cells and/or subcellular organelles from the processing chamber directly or indirectly to a magnetic processing unit;

(IV) using the magnetic separation assembly, immobilizing the cells and/or subcellular organelles in the magnetic processing unit;

(V) labeling one or more proteins on the surface of the cells and/or subcellular organelles;

(VI) using the pressure source to move the labeled cells and/or subcellular organelles into a flowcell in optical communication with the optical detector of the measurement module; and

(VII) using the optical detector to detect the labeled proteins.

[0218] 26. A method for determining health of cells or subcellular organelles from a tissue comprising:

(IV) using the magnetic separation assembly, immobilizing the cells and/or subcellular organelles in the magnetic processing unit; (V) labeling the cells and/or subcellular organelles with a marker that differentiates healthy cells and/or organelles from necrotic, late stage apoptosis, and/or early necrotic cells and/or subcellular organelles;

(VII) using the optical detector to detect labeled and/or unlabeled cells and/or subcellular organelles.

[0219] 27. A method for purifying DNA or RNA from cells and tissue, comprising:

(II) releasing DNA or RNA from the cells by using the disruptor actuator to operate the grinder assembly to grind the tissue, wherein the processing chamber comprises one or more reagents to disrupt cells and/or subcellular organelles thereby releasing DNA or RNA;

(IV) using the magnetic separation assembly, immobilizing the released to DNA or RNA using particles that capture nucleic acids (e g., SPRI beads), removing unbound material and, optionally, washing the particles;

(V) optionally, moving the washed particles or released DNA or RNA into the output receptacle of the output unit.

[0220] 28. The method of embodiment 27, further comprising:

(VI) in the magnetic processing unit, performing one or more of: reverse transcribing the RNA to produce cDNA, polishing the DNA or RNA, and repairing DNA or cDNA and ligating DNA or cDNA to DNA sequencing adapters, or tagmentation, or any other molecular biology enzymatic reaction on nucleic acid.

[0221] 29. The method of embodiment 27 or 28, further comprising performing PCR on DNA in the magnetic processing chamber or other chamber.

[0222] 30. The method of embodiment 28, further comprising performing a sizing cut on a library.

[0223] 31. A cartridge for dissociating tissue, comprising:

(a) a processing chamber comprising a stator, a side wall, a top orifice, and a first processing chamber port positioned in the side wall; and (b) a grinder assembly comprising a plunger comprising a rotor having a side, the grinder assembly slidably positioned in the processing chamber through the top orifice; wherein: (i) the rotor comprises a side that comprises a plurality of gap setting features on the side, wherein, when the rotor is positioned in the processing chamber, the gap setting features maintain a gap between the side of the rotor and the side wall of the processing chamber. [0224] 32. The cartridge of embodiment 31, wherein the rotor has a thickness between about one millimeters and about fifty millimeters.

[0225] 33. The cartridge of embodiment 31-32, wherein the gap setting features are configured as bumps, swirls or stripes.

[0226] 34. The cartridge of any of embodiments 31-33, wherein the gap setting features are positioned at a top of the side of the rotor.

[0227] 35. The cartridge of any of embodiments 31-34, wherein the gap setting features are configured as stripes oriented obliquely with respect to the side of the rotor.

[0228] 36. The cartridge of any of embodiments 31-35, wherein the rotor comprises between three and thirty-six gap setting features.

[0229] 37. The cartridge of any of embodiments 31-36, wherein each gap setting feature is separated from an adjacent gap setting feature by between 10 degrees and 120 degrees, e.g., between 30 degrees and 60 degrees.

[0230] 38. The cartridge of any of embodiments 31-37, wherein comprising a gap between the rotor and the side wall of about 1 micron and 1,000 microns.

[0231] 39. A cartridge for dissociating tissue, comprising:

(a) a processing chamber comprising a stator, a side wall, a top orifice, and a first processing chamber port positioned in the side wall; and

(b) a grinder assembly comprising a plunger comprising a rotor, the grinder assembly slidably positioned in the processing chamber through the top orifice; wherein:

(c) the stator comprises a plurality of teeth arranged in a spaced-apart array of rings; and

(d) the rotor comprises a plurality of teeth arranged in a spaced-apart array of rings, wherein one ring of teeth is positioned at or substantially at a circumference of the rotor;

(e) either the rotor comprises a center post and the stator comprises a center hole, , or the stator comprises a center post and the rotor comprises a center hole; and

(f) wherein, when the rotor contacts the stator:

(1) the center post mates with the center hole, and

(2) the rings in the stator and the rings in the rotor are positioned such that rings of teeth in the stator mesh with the one or more central teeth and rings of teeth in the rotor. [0232] 40. The cartridge of embodiment 39, wherein the center post has a height of between about 0.5 mm and about 3 mm.

[0233] 41. The cartridge of embodiment 39 or 40, wherein the center post and hole set a gap between a grinding feature of the stator and a grinding feature of the rotor of between about 0.005 mm and about 0.5 mm.

[0234] 42. The cartridge of any of embodiments 39-41, wherein a plurality of the teeth have a trapezoidal cross-section.

[0235] 43. The cartridge of any of embodiments 39-42, wherein the stator and the rotor each comprise three rings of teeth.

[0236] 44. The cartridge of any of embodiments 39-43, wherein the rotor comprises an inner ring comprising six teeth, a middle ring comprising six teeth and an outer ring comprising 11 teeth.

[0237] 45. The cartridge of any of embodiments 39-44, wherein the rotor comprises teeth at a density of about 1 tooth per 0.0025 mm² to about 1 tooth per 0.10 mm², e g., about 1 tooth per 0.05 mm²

[0238] 46. The cartridge of any of embodiments 39-45, wherein the stator comprises an inner ring comprising four teeth, a middle ring comprising six teeth and an outer ring comprising 10 teeth.

[0239] 47. The cartridge of any of embodiments 39-46, wherein the stator comprises teeth at a density of about 1 tooth per 0.002 mm² to about 1 tooth per 0.08 mm², e.g., about 1 tooth per 0.04 mm²

[0240] 48. The cartridge of any of embodiments 39-47, wherein one or more teeth have a height of about 500 microns and a width of about 1 mm to 2 mm.

[0241] 49. The cartridge of any of embodiments 39-48, wherein the processing chamber has a volume between 0.05 mL and 100 mL, e.g., between 2 mL and 5 mL, between 10 mL and 50 mL, or between 10 mL and 20 mL.

[0242] 50. The cartridge of any of embodiments 39-49, wherein the processing chamber has a cross-sectional area of between about 78 mm² (e.g., radius of about 5 mm) and about 1256 mm² (e.g., radius of about 20 mm), e.g., about 452 mm² (e.g., radius of about 12 mm).

[0243] 51. The cartridge of any of embodiments 39-50, comprising a tissue sample no greater than 20 mg, no greater than 10 mg, no greater than 5 mg, no greater than 2 mg, or no greater than 1 mg.

[0244] 52. The cartridge of any of embodiments 39-51, comprising a gap between the rotor and the sidewall of about 1 micron and 500 microns. [0245] 53. The cartridge of any of embodiments 39-52, wherein the first processing chamber port is positioned above a top of the rotor when the rotor is fully depressed.

[0246] 54. The cartridge of any of embodiments 39-52, wherein the grinder assembly further comprises a cap attached to the plunger and configured to cover the orifice and position the grinder assembly in the processing chamber.

[0247] 55. The cartridge of any of embodiments 39-54, wherein the plunger is spring-biased toward the cap.

[0248] 56. The cartridge of any of embodiments 39-55, wherein the cap comprises a key slot to engage an actuator.

[0249] 57. The cartridge of any of embodiments 39-56, wherein the rotor of the plunger is biased toward the cap (e.g., spring biased).

[0250] 58. The cartridge of any of embodiments 39-57, wherein the rotor has sufficient clearance from the processing chamber walls to allow liquid, cells and nuclei to pass around the rotor during depression, and the first processing port is positioned above the rotor when fully depressed.

[0251] 59. A cartridge adapted to interface with an instrument wherein a. the cartridge comprises a frame comprising (i) at least one functional unit and (ii) a plurality of vessel bays; b. the at least one functional unit comprises:

(i) at least one processing chamber or dissociation chamber comprising an internal surface functionalized with grinding features;

(ii) at least one filtration unit comprising a filter or strainer;

(iii) at least one magnetic processing unit comprising paramagnetic or magnetic particles;

(iv) an output unit comprising a separable output tube;

(v) a flowcell unit;

(vi) a tangential flow filtration unit; or

(vii) a combination thereof; and c. the vessel bays comprise attachment features that are structured to engage with a plurality of stand-alone units, wherein the plurality of stand-alone units are structured to attach to the frame.

[0252] 60. The cartridge of embodiment 59, wherein the vessel bay is not engaged with or attached to a stand-alone unit.

[0253] 61. The cartridge of embodiment 59 or 60, wherein the frame, the at least one functional unit and the plurality of vessel bays are molded together as a single unit. [0254] 62. The cartridge of any of embodiments 59-61, wherein the frame, the at least one functional unit and the plurality of vessel bays are injected-molded as a single unit.

[0255] 63. The cartridge of any of embodiments 59-62, wherein the frame, the at least one functional unit and the plurality of vessel bays comprise a polymeric material.

[0256] 64. The cartridge of any of embodiments 59-63, wherein the at least one functional unit is an independent unit that is attached to and locked into, clipped into, or snapped into the frame via the attachment features.

[0257] 65. The cartridge of any of embodiments 59-64, wherein the at least one functional unit is an independent unit that is attached to and locked into, clipped into, or snapped into the frame via the attachment features and is removable.

[0258] 66. The cartridge of any of embodiments 59-65, wherein the at least one functional unit is an independent unit that is attached to and locked into, clipped into, clicked into or snapped into the frame via the attachment features and is not manually removable without a tool or is irreversibly attached to the frame.

[0259] 67. The cartridge of any of embodiments 59-66, wherein the at least one functional unit is an independent unit that is removably attached to, removably snapped into, or removably twisted into the attachment feature in the frame.

[0260] 68. The cartridge of any of embodiments 59-67, wherein the attachment features comprise: a. a press-fit comprising a recess or hole with a shape ( optionally conical or cylindrical), that can receive a complementary-shaped projection (optionally, conical or cylindrical, where applicable) that is situated on a stand-alone unit; b. a press-fit comprising a recess or aperture functionalized with ridges or threads that can receive a projection situated on a stand-alone unit that comprises complementary ridges, grooves, or threads, where applicable; c. a press-fit comprising a projection with a shape that is complementary to a recess or hole present in a stand-alone unit, wherein the shape is optionally conical or cylindrical; d. a press-fit comprising a projection with functionalized with ridges or threads that are complementary to ridges, grooves, or threads present within a hole or recess within a stand-alone unit; e. a click-in function that can click into a click-in feature present on a stand-alone unit; f. a click-in function that can click into a snap-in feature present on a stand-alone unit; g. a twist function that can twist into or receive a twist feature on a stand-alone unit, wherein the twist function or feature comprises complementary ridges, grooves or threads; h. an adhesive, epoxy adhesive or glue; or i. a flange.

[0261] 69. The cartridge of any of embodiments 59-68, wherein a plurality of stand-alone units are attached to the vessel bays via the attachment features and the plurality of stand-alone units comprise: a. at least one processing chamber comprising an internal surface functionalized with grinding features; b. at least one filtration unit comprising a filter; c. at least one magnetic processing unit comprising magnetic particles; d. an output unit comprising a separable output tube; e. a flowcell unit; f. a tangential flow filtration unit; g. a waste unit; or h. a combination thereof.

[0262] 70. The cartridge of any of embodiments 59-69, wherein the at least one functional unit comprises: at least one processing chamber comprising an internal surface functionalized with grinding features.

[0263] 71. The cartridge of any of embodiments 59-70, wherein the at least one functional unit comprises at least one processing chamber comprising an internal surface functionalized with grinding features and wherein the plurality of stand-alone units comprises an output unit comprising a separable output tube, wherein the stand-alone unit is attached to a vessel bay.

[0264] 72. The cartridge of any of embodiments 59-71, wherein the grinding features comprise a plurality of teeth or ridges.

[0265] 73. The cartridge of any of any of embodiments 59-72, the cartridge further comprising apertures designed to engage with tubing or cannulae.

[0266] 74. The cartridge of any of embodiments 59-73, wherein the cartridge comprises tubing or cannulae.

[0267] 75. The cartridge of any of embodiments 59-74, wherein the cartridge further comprises a tissue disruptor within the at least on processing chamber.

[0268] 76. The cartridge of any of embodiments 59-75, wherein at least one of the at least one functional unit is fluidically connected to at least one of the stand-alone units.

[0269] 77. The cartridge of any of embodiments 59-76, wherein the least one processing chamber comprises a stator and a rotor, with, optionally, each comprising teeth. [0270] 78. The cartridge of any of embodiments 59-77, wherein the rotor comprises teeth at a density of about 1 tooth per 0.0025 mm² to about 1 tooth per 0.10 mm², e g., about 1 tooth per 0.05 mm²

[0271] 79. The cartridge of any of embodiments 59-78, wherein the stator comprises teeth at a density of about 1 tooth per 0.002 mm² to about 1 tooth per 0.08 mm², e.g., about 1 tooth per 0.04 mm²

[0272] 80. The cartridge of any of embodiments 59-79, wherein one or more teeth have a height of about 500 microns and a width of about 1 mm to 2 mm.

[0273] 81. The cartridge of any of embodiments 59-80, wherein at least one of the functional units or stand-alone units has a volume between 1 ul, 20 ul, 50 ul, 100 ul, 500 ul, 1 mL, 2 mL, 5 m , 5 mL and 100 mL, e.g., between 10 mL and 50 mL, e.g. between 10 mL and 20 mL.

[0274] 82. The cartridge of any of embodiments 59-81, wherein the processing chamber has a volume between 1 ul, 20 ul, 50 ul, 100 ul, 500 ul, 1 mL, 2 mL, 5 mL, 5 mL and 100 mL, e.g., between 10 mL and 50 mL, e.g. between 10 mL and 20 mL.

[0275] 83. The cartridge of any of embodiments 59-82, wherein: a. at least one of the functional units or stand-alone units comprises a flow cell that, optionally, has a volume between 1 ul and 1 mL, e.g., 1 ul- 0.5 ml; 10 ul-.l ml; 1 ul -.2 mL; 1 ul - 100 ul; b. at least one of the functional units or stand-alone units comprises a microfluidic device, microfluidic chip, laminar or microcapillary with a volume 1 ul - 1 mL, e.g., 1 ul-.l mL, 10 ul-.l mL, 50 ul-.2 mL, 10 ul- 50 ul, .1 mL- 1 mL, or 5 ul - 500 ul; c. the cartridge comprises a microfluidic device attached to the cartridge that facilities transfer of fluids from or within the functional units or stand-alone units; d. the cartridge comprises a microfluidic device or chip; e. the cartridge comprises a flow cell with channels that are 10 - 1000 uM in width, length or depth, e.g., 10-100, 50-500, 50-1000 uM; f. the cartridge comprises a magnetic processing chamber that is situated within the flowcell; g. the cartridge comprises a magnetic processing chamber comprising a detection unit; h. the cartridge comprises a polymer frame or polypropenol frame and, optionally, a functional unit that is glass, a window, optically transparent, quartz or translucent; or i. the cartridge comprises a functional unit that is glass, a window, optically transparent, quartz or translucent, and, optionally, the frame is a polymer frame.

[0276] 84. The cartridge of any of embodiments 59-83, wherein the processing chamber, at least one functional unit, or at least one of the stand-alone units, has a cross-sectional area of between about 12.6 mm² (e.g., radius about 2 mm), 78 mm² (e.g., radius of about 5 mm) and about 1256 mm² (e.g., radius of about 20 mm), e.g., about 452 mm² (e.g., radius of about 12 mm). [0277] 85. The cartridge of any of embodiments 59-84, wherein the output tube comprises a single-cell, single-nuclei, or single-sub-cellular organelle suspension.

[0278] 86. The cartridge of any of embodiments 59-85, wherein the filtration unit comprises a filter or strainer having pores no greater than about 40 microns (e.g., no greater than about 30 microns, no greater than about 20 microns), and an optional second strainer having pores 40 - 200 microns, less than 40 microns, greater than 5 microns, or 100-500 microns.

[0279] 87. The cartridge of any of embodiments 59-86, wherein the filtration unit comprises two or more filters or strainers.

[0280] 88. The cartridge of any of embodiments 59-87, wherein the two or more filters or strainers comprise different pore sizes.

[0281] 89. The cartridge of any of embodiments 59-88, wherein a pore size of one filter is

100 to 500 microns and a pore size of a different filter is less than 50 microns, or between 2 microns and 40 microns.

[0282] 90. The cartridge of any of embodiments 59-89, wherein the two or more filters or strainers comprise a first filter with first pores and a second filter with second pores, wherein the first pores comprise pores with a larger diameter than at least one of the second pores.

[0283] 91. The cartridge of any of embodiments 59-90, wherein the two or more filters or strainers further comprise a third filter with third pores that comprise pores with a diameter smaller than a diameter of at least one of the second pores.

[0284] 92. The cartridge of any of embodiments 59-91, wherein the first filter is situated upstream of the second filter, which is, optionally upstream of the third filter if present.

[0285] 93. The cartridge of any of embodiments 59-92, wherein the filtration unit comprises two or more filters in a stacked configuration, vertical stacked configuration, or horizontal stacked configuration.

[0286] 94. The cartridge of any of embodiments 59-93, wherein the filtration unit comprises a first filter with pores that are 50 to 500 microns in diameter, e g., 50-200 microns, 100-500 microns, 200-500 microns, 150 -400 microns.

[0287] 95. The cartridge of any of embodiments 59-94, wherein the filtration unit comprises a first filter with pores that are 50 to 500 microns in diameter, e.g., 50-200 microns, 100-500 microns, 200-500 microns, 150 -400 microns and/or a second filter with pores 20-200 microns in diameter, e.g., 20-100 microns, 40-200 microns, 100-200 microns or 20-150 microns in diameter. [0288] 96. The cartridge of any of embodiments 59-95, wherein the filtration unit comprises a first filter with pores that are 50 to 500 microns in diameter, e g., 50-200 microns, 100-500 microns, 200-500 microns, 150 -400 microns and/or a second filter with pores 5-80 microns in diameter, e.g., 5-50 microns, 20 - 80 microns, 10-80 microns.

[0289] 97. The cartridge of any of embodiments 59-96, wherein the filtration unit comprises a first filter with pores that are 50 to 500 microns in diameter, e g., 50-200 microns, 100-500 microns, 200-500 microns, 150 -400 microns and/or a second filter comprising pores that are 200-300 microns in diameter, 150-400 microns in diameter, or 200-600 microns in diameter.

[0290] 98. The cartridge of any of embodiments 59-97, wherein the filtration unit is fluidically connected a source comprising wash buffer.

[0291] 99. The cartridge of any of embodiments 59-98, wherein the magnetic processing unit comprises paramagnetic beads.

[0292] 100. The cartridge of any of embodiments 59-99, wherein the magnetic or paramagnetic particles are 0.1 nm- 100 pm in diameter (e.g., 0.1 nm- 10 nm; .1 nm - 1 pm; 10 pm - 100 pm; 50 pm- 100 pm).

[0293] 101. The cartridge of any of embodiments 59-100, wherein the cartridge comprises a plurality of magnetic processing units.

[0294] 102. The cartridge of any of embodiments 59-101, wherein the tangential flow filtration unit comprises two tangential flow filters connecting with upper and lower buffer regions functioning as a concentration gradient.

[0295] 103. The cartridge of any of embodiments 59-102, wherein the tangential flow filtration unit comprises an optical interrogation region.

[0296] 104. The cartridge of any of embodiments 59-103, wherein the at least one functional unit is fluidly connected to another functional unit or stand-alone unit via tubing, capillary, microcapillary, or cannula.

[0297] 105. The cartridge of any of embodiments 59-104, wherein the cartridge comprises a macrofluidic or microfluidic device.

[0298] 106. The cartridge of any of embodiments 59-105, wherein the at least on functional unit or stand-alone unit is embedded in a microfluidic or microfluidic device.

[0299] 107. The cartridge of any of embodiments 59-106, wherein the attachment features are present at a top surface of the cartridge, a bottom surface of the cartridge, or a lateral surface of the cartridge.

[0300] 108. A system comprising an instrument engaged with the cartridge of any of embodiments 59-107.

[0301] 109. The system of embodiment 108, wherein the system is engaged with the cartridge via tubing or cannulae. [0302] 110. The system of embodiment 108-109, wherein the cartridge comprises fluidic ports in communication with fluid ports situated in the instrument.

[0303] 111. The system of any of embodiments 108-110, wherein the instrument comprises a fluidics subassembly comprising: a. at least one pressure source of positive and/or negative pressure, wherein the at least one pressure source optionally comprises separate vacuum source and pump (e.g., syringe pump); b. at least one reagent container for containing liquids, fluidically connected to the at least one pressure source, optionally contained in a reagent cassette; or c. at least one cannula communicating with the at least one pressure source and, directly or indirectly through one or more fluidic lines, with at least one port of a functional unit, or of the processing chamber, when the at least one cartridge is engaged with the at least one cartridge interface.

[0304] 112. The system of any of embodiments 108-111, wherein the instrument comprises an actuator subassembly comprising: a. a disruptor actuator configured to engage the grinder assembly; or b. at least one valve actuator (e.g., a pinch valve actuator) configured to actuate one or more valves to control fluid flow in fluidic conduits in the at least one cartridge.

[0305] 113. The system of any of embodiments 108-112, wherein the instrument comprises magnetic separation assembly(s) comprising a source of magnetic force that is reversibly engageable with a functional unit engaged with a cartridge when the cartridge is engaged with the cartridge interface;

[0306] 114. The system of any of embodiments 108-113, wherein the system comprises a thermal assembly configured to regulate temperature of functional units engaged with the plurality of vessel bays or frame.

[0307] 115. A kit comprising the cartridge of any of embodiments 59-107 and a plurality of stand-alone vessels.

[0308] 116. The kit of embodiment 115, wherein the plurality of stand-alone vessels comprises: a. at least one processing chamber comprising an internal surface functionalized with grinding features; b. at least one filtration unit comprising a filter or strainer; c. at least one magnetic processing unit comprising magnetic particles; d. an output unit comprising a separable output tube; e. a flowcell unit; f. a tangential flow filtration unit; or g. a combination thereof.

[0309] 117. A method for purifying cells or subcellular organelles from a tissue comprising: a. providing a system of any of embodiments 108-114, wherein the system comprises a cartridge engaged therewith, wherein the cartridge comprises a tissue sample or cell sample within the at least one processing chamber or dissociation chamber; b. using a disruptor actuator to operate a grinder assembly to grind the tissue within the processing chamber, thereby releasing cells and/or subcellular organelles; and c. using a pressure source to move the released cells and/or subcellular organelles from the processing chamber directly or indirectly to an output receptacle of the output unit.

[0310] 118. The method of embodiment 117, further comprising, before moving the released cells and/or subcellular organelles into the output receptacle, performing at least one of the following operations: a. using the pressure source to move the released cells and/or subcellular organelles from the processing chamber into the filtration unit to separate debris from the cells and/or subcellular organelles; b. using the pressure source to move the released cells and/or subcellular organelles from either the processing chamber or the filtration unit into a magnetic processing unit, capturing cells and/or subcellular organelles in the magnetic processing unit by using the magnetic force to attract cells and/or subcellular organelles that are associated with magnetic or paramagnetic particles, and, optionally, moving on attracted material of the magnetic processing chamber, and optionally washing the attracted cells and/or subcellular organelles using the pressure source to move liquid into the magnetic processing chamber; c. using the pressure source to move at least some of the released cells and/or subcellular organelles into a flowcell and detecting living and/or dead cells and/or subcellular organelles using the measurement module; d. using the pressure source to move the cells and/or subcellular organelles into a tangential flow device to remove contaminants; or e. using the pressure source to move waste from a functional unit into the waste unit.

[0311] 119. The method of embodiment 117 or 118, further comprising using a first magnetic processing chamber to reduce a volume of the sample and a second magnetic processing chamber to perform reactions. [0312] 120. The method of any one of embodiments 117-119, further comprising using the first magnetic processing chamber to reduce a volume of a sample to 0.001, 0.005, 0.01, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, or 0.5 of its initial volume.

[0313] 121. The method of any of embodiments 117-120, wherein the cartridge comprises a tissue fragment and wherein the cartridge comprises a filtration unit with a pore size of 100-500 microns, e.g., 100-400 microns, 200-500 microns, 100-300 microns.

[0314] 122. The method of any of embodiments 117-121, wherein the method produces a single cell suspension.

[0315] 123. The method of any of embodiments 117-122, wherein the method produces a single cell suspension and the cartridge comprises a first filter with pores that are 50 to 500 microns in diameter, e.g., 50-200 microns, 100-500 microns, 200-500 microns, 150 -400 microns and/or a second filter with pores 20-200 microns in diameter, e.g., 20-100 microns, 40-200 microns, 100-200 microns or 20-150 microns in diameter.

[0316] 124. The method of any of embodiments 117-123, wherein the method produces a single sub-cellular organelle suspension (e.g., a single nuclei suspension).

[0317] 125. The method of any of embodiments 117-124, wherein the method produces a single sub-cellular organelle suspension (e.g., a single nuclei suspension) and the filtration unit comprises a first filter with pores 50 to 500 microns in diameter, e.g., 50-200 microns, 100-500 microns, 200-500 microns, 150 -400 microns in diameter; and/or a second filter comprising pores that are 5-80 microns in diameter, e g , 5-50 microns, 20 - 80 microns, 10-80 microns.

[0318] 126. The method of any of embodiments 117-125, wherein the method produces a single cardiomyocyte suspension.

[0319] 127. The method of any of embodiments 117-126, wherein the method produces a single cardiomyocyte suspension and the filtration unit comprises a first filter with pores 50 to 500 microns in diameter, e.g., 50-200 microns, 100-500 microns, 200-500 microns, 150 -400 microns in diameter; and/or a second filter comprising pores that are 200-300 microns in diameter, 150-400 microns in diameter, or 200-600 microns in diameter.

[0320] 128. A method of preparing a cartridge for use in an instrument that produces single cell suspensions, single nuclei suspensions or single subcellular organelle suspensions, the method comprising: a. providing a cartridge of any of embodiments 59-107; and b. attaching one or more of the plurality of the stand-alone units to the cartridge via the attachment features. [0321] 129. A method of preparing a cartridge for use in an instrument that produces single cell suspensions, single nuclei suspensions or single subcellular organelle suspensions, the method comprising: a. providing a first and second cartridge of any of embodiments 59-107; b. attaching a first set of the stand-alone units to the attachment features in the vessel bays of the first cartridge; c. attaching a second set of the stand-alone units to the attachment features in the vessel bays of the second cartridge, wherein the first set and the second set of stand-alone units comprise different combinations of stand-alone units.

[0322] 130. The method of embodiment 129, wherein the first and second cartridges each comprise a common region situated a common location on the first and second cartridge that interfaces with an integrated feature of the instrument.

[0323] 131. The method of preparing a cartridge of any of embodiments 59-107, wherein the integrated feature is a thermal controller, a magnetic field, an optical detector, a measurement device, fluidics subassembly, actuators, cannulae, tubing, pressure source, positive or negative pressure, reagent source, wash buffer source, fluorescent detector, regulatable magnetic field; fluid dispenser, or fluid aspirator.

EXAMPLES

Example 1: Production of filtered single cell suspensions with modular cartridges

[0324] In some embodiments the modular cartridge 205 is used to produce filtered single cell suspensions 1102 or filtered nuclei suspensions 1103. These applications could use the modular cartridge 205 as shown in Figure 6A and Figure 6B or could use a cartridge without the Magnetic Processing Chamber 905 and Waste Chamber 430, as shown in Figure 7A and Figure 7B. In some embodiments, a RFID tag or barcode or related device can be read from the cartridge 205 by the instrument to determine the appropriate processing or the location of different chambers. [0325] In this example, the cap 210 of the Dissociation Chamber 441 is removed and a tissue specimen 120 is added into the bottom of Dissociation Chamber 441. The cap 210 is replaced and the cartridge 205 is inserted into the receiver of the instrument where it is engaged with the lower cartridge interface 1502. In some embodiments, one or more cannula 1416 such as for the input port into the Dissociation Chamber 441 or other chambers can engage upon insertion or move after insertion to connect any cannulas 1416 engaging from the front or side of cartridge 205. The temperature regulating subsystem 1475 in the lower cartridge interface 1502 can set the temperature of the cartridge 205 as appropriate, typically 37°C for production of single cells and preferrably in the range of 4-10°C for the production of nuclei. [0326] The upper cartridge interface 1501 can then be lowered to dock the cannula 1416 with the appropriate cannula seats 2916 and position the instrument’s rotary motor 2120 to engage with the top of the cap 210 to raise and lower the rotor 353. The spring-loaded rotor in the cap 210 is engaged by the upper cartridge interface 1501 to raise, lower, and rotate disruption rotor 353 for selectable modes of mixing and mechanical tissue disaggregation. An auto-mince routine eliminates manual mincing for many tissue types, increasing reproducibility and convenience.

[0327] For filtered single cell suspensions, the sample is typically incubated at 37°C in a tissue-specific enzyme formulation (e.g., collagenase, elastase, protease, DNase, etc.) with incubation and mixing for 10-60 or 20-60 min followed by one or two mechanical disruption cycles. The mechanical tissue disruption results from slow rotation of the rotor in the cap, which has 500 mm rounded ‘teeth’ 355 on the bottom and the complementary teeth on the bottom of the Dissociation Chamber 441, and from dissociation by displacement of the dissociated sample through a ‘side gap’ 221 (e g., 250 mm ) between the side of the rotor 353 and the Dissociation Chamber 441 wall. The descent of rotor 353 as it dissociates a sample can be controlled by realtime force sensor 2115 feedback. Different disaggregation protocols can be preprogrammed for many standard tissues and can be customized for additional tissues.

[0328] After dissociation, the single cell suspension can be pulled through fdtration tubing 2929 by vacuum. The vacuum can be applied by filtration cannula 2928 on cannula input port 2921 or applied downstream, e.g., through MPC cannula 2938 to apply a vacuum on port 2923. The single cell suspension 1000 can then be filtered through one or more filters 341, e.g., 145, 70, and 50 mm pore sizes.

[0329] The filtered single cell suspension 1102 can then be pulled by vacuum into Output Chamber 850 and centrifuge tube 855 by applying vacuum to Output Chamber cannula 2945 on Output Chamber output port 2942.

[0330] The Dissociation Chamber 441 can then washed one or more times with buffer to facilitate complete recovery of the dissociated single cells, with each wash pulled successively through the Filtration Chamber 450 and into the Output Chamber 850.

Example 2: Production of filtered single nuclei suspensions with modular cartridges

[0331] For filtered nuclei suspensions, after adding the solid tissue specimen 120 to the Dissociation Chamber 441, replacing cap 120, and inserting into the cartridge interface 1500, the specimen 120 is mechanically disrupted in a nuclei isolation solution 120, e.g., Nuclei Isolation Reagent (#100-063-396, S2 Genomics), containing a proprietary formulation with 0.1% NP-40, at ~6°C using rotor 353 with a 150 mm side gap. About 90% of tissues can be processed with a single mechanical disruption cycle using a single protocol; additional protocols include a second disruption cycle for fibrous and other difficult tissues.

[0332] After dissociation, the single nuclei suspension is pulled through filtration tubing 2929 by vacuum. As described above for cell preparation, the vacuum can be applied by filtration cannula 2928 on cannula input port 2921 or applied downstream through port 2923. The single nuclei suspension 1050 can then be filtered through one or more filters 341, e.g., 145, 70, and 40 mm pore sizes to create a filtered single nuclei suspension 1103.

[0333] The filtered single nuclei suspension 1103 can then be pulled by vacuum into Output Chamber 850 and centrifuge tube 855 by applying vacuum to Output Chamber cannula 2945 on Output Chamber output port 2942.

[0334] The Dissociation Chamber 441 can be washed none, one, or more times with an osmoprotecting nuclei storage solution 413, e.g., Nuclei Storage Reagent (#100-063-623, S2 Genomics) without detergent to facilitate complete recovery of the nuclei, with each wash pulled successively through the Filtration Chamber 450 and into the Output Chamber 850.

Example 3 : Tangential flow processing of filtered single cell or nuclei suspensions.

[0335] To remove small debris or change buffers from single cell or nuclei suspensions, a tangential flow module 2700 (Figure 16) can be incorporated into a modular cartridge 205. This module can be located in many positions, e.g., after the Dissociation Chamber 441, or after the Filtration Chamber 450, or after the Magnetic Processing Chamber 905, and before the Output Chamber 850.

[0336] In some embodiments, the tangential flow module 2700 is located after the Filtration Chamber 450 and before the Output Chamber 850. In certain aspects, a workflow is to dissociate the solid tissue into single cell or nuclei suspensions, filter through the Filtration Chamber 450, and pull the sample from filtration chamber output port 2923 through tubing into tangential flow input port 2703 and into tangential flow region 2706 by applying vacuum on Output Chamber output port 2942 using Output Chamber cannula 2945. Buffer is circulated in upper buffer region 2707 and lower buffer region 2708 separated from tangential flow region 2706 by tangential flow filters 2710. The suspensions can be pulled continuously through tangential flow region 2706 or can pause or stop in tangential flow region 2706.

[0337] In some embodiments, the titer and viability of the tangentially purified single cell or nuclei suspensions can be interrogated by optical module 2600 in tangential flow module 2700 as illustrated in Figure 16B. In these embodiments, the titer of the sample can be adjusted by dilution in the Output Chamber 850 or other chambers. [0338] In some embodiments, the tangential flow filtration module 2700 can be incorporated in many different embodiments to produce samples in a buffer of choice with small debris removed.

Example 4: Magnetic processing to purify single cell suspensions wtih a modular cartridge.

[0339] Magnetic processing with paramagnetic bead handling can enable the purification of cells or nuclei, the enrichment of specific cell types, or depletion of specific cell types or dead cells or removal of myelin debris by immunomagnetic separation (IMS). The resulting purified cells with potentially a normalized titer can be eluted into a buffer of choice for the next workflow step, potentially eliminating the need for centrifugation. Tumors can be dissociated and purified tumor infiltrating lymphocytes (TILs) 1106 produced. TILs 1106 are of particular interest for adoptive cellular therapies and immunotherapy.

[0340] One embodiment begins with adding a solid tissue specimen 130 containing a tumor to the modular cartridge 205 as described in the previous examples. The solid tissue specimen 130 is then dissociated into single cells and filtered as described. Using a modular cartridge 205 as shown in Figure 6, the filtered single cell suspension 1102 at the bottom of the Filtration Chamber 450 containing cells from any normal tissue and cancerous tissue with TILs 1106 can be pulled into the Magnetic Processing Chamber 905 through filtration output tubing 2924 and MPC sample input dip tube 2934 by closing filtration pinch valve 2918 with filtration valve actuator 2961 and applying vacuum to MPC cannula 2938.

[0341] With the filtered single cell suspension 1102 containing cells from any normal tissue and cancerous tissue with TILs 1106 at the bottom of the Magnetic Processing Chamber 905, paramagnetic beads 685 to capture the cells of interest can either be added through MPC reagent input reagent port 2932 or can be predispensed into the Magnetic Processing Chamber 905 at the factory. The paramagnetic beads 685 can be functionalized such as comprised of antibody- functionalized magnetic beads 686 directed at a cell type of interest or an antigen found on all cells of a given species, or be functionalized with aptamers, or a fragment of an antibody, or a lectin, e.g., conconavalin A, or other moeities well known to one skilled in the art to bind the surface of cells. In some embodiments the amount of binding by paramagnetic beads 685 can be limiting and used to normalize the amount of cells bound.

[0342] The antibody-functionalized magnetic beads 686 or other functionalized magnetic beads 685 are then mixed with the filtered single cell suspension 1102. The mixing can be done by several methods. [0343] One favored mixing embodiment is by back and forth mixing by pulling the antibody- functionalized magnetic beads 686 and filtered single cell suspension 1102 into filtration output tubing 2924 and back into or towards Filtration Chamber 450. MPC pinch valve 2962 is opened using actuator 2963, filtration pinch valve 2918 is closed by filtration valve actuator 2961 (to prevent vacuum bleeding into the Dissociation Chamber 441), and filtration cannula 2928 applies vacuum to Filtration Chamber 450 to move the antibody -functionalized magnetic beads 686 and filtered single cell suspension 1102 into Filtration Chamber 450. The antibody-functionalized magnetic beads 686 and filtered single cell suspension 1102 can then be moved back into Magnetic Processing Chamber 905 by closing MPC pinch valve 2962 using actuator 2963, filtration pinch valve 2918 is opened by filtration valve actuator 2961, and Magnetic Processing Chamber cannula 2938 applies vacuum to pull the mixture back into Magnetic Processing Chamber 905. The process can be repeated until the desired mixing is achieved. With modular cartridge 205 other back and forth mixing embodiments can be used with pressure used to push the mixture or vacuum applied at different locations.

[0344] Another mixing embodiment is bubble mixing. In bubble mixing, air or other gases are pushed through a dip tube to produce bubbles that in turn mix the sample and beads. For example, with MPC pinch valve 2962 opened using actuator 2963 and filtration pinch valve 2918 closed by filtration valve actuator 2961, pressured air can be introduced through filtration cannula 2928 which will then move through filtration output tubing 2924 and through MPC sample input dip tube 2934 to produce bubbles with MPC cannula 2938 withdrawing an equal volume of air through MPC input reagent port 2932.

[0345] After the functionalized paramagnetic beads 685 and filtered single cell suspension 1102 are mixed, the mixture can be incubated with temperature control when required and then movable magnet 910 is moved close or against Magnetic Processing Chamber 905 in an engaged position to pull the beads and any attached cells to the bottom or side of of the Magnetic Processing Chamber 905.

[0346] To wash the mixed magnetic beads 685 and filtered single cell suspension 1102, with all valves open, the unbound material pulled into the Waste Chamber 430 using vacuum on waste chamber cannula 2955. Magnet 910 can then be moved away from Magnetic Processing Chamber 905 in an unengaged position and wash buffer, e.g., HBSS, PBS containing 2% fetal bovine serum plu2 1% EDTA, TE, MojoSort™, autoMACS® Running Buffer™, added using MPC cannula 2938. The sample, beads, and buffer can mixed as described and with the sample, beads, and buffer in the bottom of the Magnetic Processing Chamber 905, the unbound material can be pulled into the Waste Chamber 430 using vacuum on waste chamber cannula 2955. None, one, or more washes can be performed. [0347] When the washes are complete, the purified single-cell suspensions 1100 can be pulled into a centrifuge tube 855 attached to Output Chamber 850 by applying vacuum to output chamber cannula 2945 with waste chamber cannula 2955 closed to prevent flow from Waste Chamber 430.

[0348] In proof-of-concept experiments, single-cell suspensions were prepared on the Singulator 100 from a human lung adenocarcinoma and then post-processed off-cartridge with paramagnetic beads 685 to enrich for T-cells. A fresh human lung adenocarcinoma was obtained under Development of Tissue Processor project (PR6733) and dissociated into a filtered single cell suspension 1102 (Singulator 100, Mouse Lung Reagent/Mouse Lung Protocol). The filtered single cell suspension 1102 was spun (300 g/5 min) the pellet resuspended red blood cell lysis buffer for 4 min, quenched with DMEM buffer, and spun at 300 g for 5 min. The pellet was resuspended, filtered (40 mm Flowmi). counted (Nexcelom K2), and titer adjusted. The single cell suspension was then processed using immunomagnetic separation (EasySep™ Human T Cell Isolation Kit, StemCells) with two washes. Standard single-cell 3’ v3.1 gene expression libraries (lOx Genomics) were generated from -10,000 cells of the purified TILs 1106. Libraries were sequenced (NovaSeq, Illumina) to a depth of 20,000 reads. 1,408 median genes per cell were obtained with 85.3% fraction of reads per cell.

[0349] Figure 22 shows the clustering of scRNA-Seq data into 16 subtypes of T and B cells after the positive selection of TILs 1106 from the human lung adenocarcinoma, dissociated and double-filtered by the Singulator 100. This experiment confirms that Singulator dissociation for lung preserves the surface epitopes of many immune cells and demonstrates immunomagnetic separation of TILs 1106. In this instant invention, TILs 1106 may be purified on modular cartridges 205 with paramagnetic bead processing to purify TILs 1106 from human or animal tumors.

[0350] Single-cell suspensions from tumors can be produced and commercial beads coated with antibodies (e.g., Cell Therapy Systems; Dynabeads™ CD3/CD28, ThermoFisher) added to the Magnetic Processing Chamber to bind T-cells for TILs 1106 for positive selection. After mixing, the T-cells attached to the paramagnetic beads 685 can be captured by a magnet 910, which can be movable or electromagnetic or other types, and after capture the paramagnetic beads 685 with capture T- cells or other target can be washed twice or more, and released from the paramagnetic beads 685. The cells can be characterized by microscopy, FACS, and scRNA-Seq. The strength of the magnet 910, mixing methods, amounts of paramagnetic beads 685, selection of antibodies, ratio of beads to cells, and capture and wash protocols can be optimized. On- cartridge red blood cell lysis before paramagnetic beads 685 purification can be performed by adding RBC lysis mixture to the MPC 905 , mixing, incubating, and stopping the lysis by dilution with media before magnetic capture and purification.

[0351] In addition to purifying cell types using functionalized paramagnetic beads 685 as an enrichment step, functionalized paramagnetic beads 685 can be used to deplete targets from the cells. In this workflow, after mixing the functionalized paramagnetic beads 685 and the filtered single cell suspension 1102, the magnet 910 is moved to capture the functionalized paramagnetic beads 685 which pull down the target cells to be depleted. The supernatant, in the Magnet Processing Chamber 905, now depleted of the target, is then moved into the Output Chamber 850. One example would be to deplete TILs from a tumor sample to avoid sequencing the TILs if the tumor cells were of interest in the experiment. Another example is to use paramagnetic beads 685 functionalized to remove dead cells such as with antibodies to annexan V; annexan V binds to a phospholipid phosphatidylserine normally found on the inside of the plasma membrane. For dead cells and debris, the phospholipid phosphatidylserine is accessible to annexan V or another binding agents attached to functionalized paramagnetic beads 685 and the beads can remove dead cells selectively. These two examples are not intended to limit the applications but instead demonstrate to one skilled in the art the breadth of this instant disclosure.

[0352] The method can also encompass the capture of cells from the filtered single cell suspension 1102 by paramagnetic beads 685 to concentrate the filtered single cell suspension 1102, or to change buffers, or to normalize the number of cells. In these applications, the capture or depletion of dead cells or debris can be used to replace downstream centrifugation or FACS sample preparation. In some embodiments, an antibody or binding modality common to all cells from a given tissue or organism is used to capture the filtered single cell suspension 1102 on paramagnetic beads 685, still in the enzymatic cocktail from the solid tissue dissociation. The wash solution can be in a buffer compatible with downstream analysis. In some embodiments with a reversible crosslink to the functional capture antibody or other agent, after appropriate washing, the crosslink can be reversed to release the purified single cells, now in a new buffer.

[0353] A binding modality that is non-specific and binds single cells and nuclei is paramagnetic beads 685 that have been functionalized with concanavalin A, e.g., Product 93569 Cell Signaling Technology, or CUT ANA™ Concanavalin A Conjugated Paramagnetic Beads, SKU:21-1401, Epicypher Technologies. Activated Concanavalin A-functionalized paramagnetic beads 687 can be added to a filtered single cell suspension 1102 to bind the single cells to the concanavalin A-functionalized paramagnetic beads 687. The bound single cells can then be washed to remove debris such as cytosolic material including ribosomes, and a buffer of choice introduced before release of the single cells bound to the concanavalin A-functionalized paramagnetic beads 687 by unengaging magnet 910. Single cells in suspension 1000 or nuclei 1050 in suspension can be released from Concanavalin A-functionalized paramagnetic beads 687 by competing with the Concanavalin A with compounds such as methyl-glucoside or methylmannoside.

Example 5: Magnetic processing to purify single nuclei suspensions with a modular cartridge

[0354] Single nuclei can be purified using magnetic processing using antibodies or other binders specific to nuclei membrane proteins and other components. Examples of antibodies to nuclear envelope are comprised of TAPI monoclonal antibody (IQ224, ImmuQuest), lamin A/C monoclonal antibody (IQ251, ImmuQuest), nesprin 1 monoclonal antibody (IQ568, ImmuQuest), nuclear membrane antibody (NM97, Novus Biologicals). Concanavalin A-functionalized paramagnetic beads 687 can also purify nuclei.

[0355] The workflow process using modular cartridges 205 with a Magnetic Processing Chamber 905 is essentially as described above in the Example: Magnetic processing to purify single cell suspensions with a modular cartridge.

Example 6: Preparation of titered purified single cell or nuclei suspensions.

[0356] In some embodiments, titered purified single cells 1310 or titered purified nuclei 1320 can be created using an optics module 2600 to determine titer and the titer adjusted as required with the appropriate buffers.

[0357] In some embodiments, a cannula 1416 is used to withdraw samples from the Magnetic Processing Chamber 905 or other chamber; the cannula 1416 can be designed to either directly reach to the bottom of the Magnetic Processing Chamber 905 or other chamber, or can contact cannula seats 2916 on cartridge ports 2915 now connected to a dip tube 2917 that reaches the bottom of the Magnetic Processing Chamber 905 or other chamber. In this embodiment, vacuum is applied to cannula 1416 to withdraw a sample aliquot. In some embodiments, the cannula 1416 is a pipettor that may be controlled by a 2- or 3 -axis robot integrated or separate from the instrument. A known volume of the aliquot is then mixed with a stain, e.g., Acridine Orange/Propidium Iodine (AO/PI), trypan blue, or other live/dead or other stains, and then moved to Measurement System 500 where an optics module 2600 measures the titer and viability of single cells or nuclei. Following the measurement and analysis for single cells or nuclei or debris, the user can input via the user interface 740 a desired titer. If the measured titer is above the desired titer, the system could dispense the appropriate amount of buffer to the Magnetic Processing Chamber 905 or other chamber, and mix the sample as described. The measurement could be optionally repeated before moving the titered and diluted sample into the Output Chamber 850. If the desired titer is higher than the measured titer, the user can have the option to perform a paramagnetic bead 685 concentration of the sample before measurement and dilution to the proper titer.

[0358] In some embodiments, the modular cartridge 205 further comprises a flow cell 2620 as a module (Figure 23). In some embodiments, flow cell 2620 can be located on an outside surface of the modular cartridge 205 and be imaged from above or below. In this embodiment, the flow cell 2620 can be connected downstream of Magnetic Processing Chamber 905 or the Tangential Flow module 2700 or any other chamber. An aliquot of the sample is pulled by vacuum or pushed by pressure, for example, through MPC sample output dip tube 2936 to flowcell 2620 with pinch valve 491 closed, for example, by vacuum on waste chamber 430 using cannula 2955. Stain is added to cannula loading area 2623 and is pulled by vacuum through line 2622 and mixes with the sample in the flow cell 2620. The optic module 2600 images or detects single cells or nuclei or debris in optical area 2720. After measurement, the sample can then be diluted to the proper titer as described or further concentrated before reanalysis and dilution.

Example 7: Sample-to-answer determination of surface proteins

[0359] In some embodiments, the surface proteins of freshly dissociated specimens can be measured using a modular cartridge 205 and Tissue Processing System 80. As described above, a filtered single cell suspension 1102 can be produced from a solid tissue specimen 130 and moved into the Magnetic Processing Chamber 905. Affinity functionalized paramagnetic beads 692 can be added and specific or non-specific cells captured on the beads. One or more fluorescent-labeled antibody target surface epitopes of interest, e.g., CD4 or CD8, can be added and incubated. Following incubation, the magnet 910 can be engaged to capture the cells bound to the affinity functionalized paramagnetic beads 692 and the supernatant removed to the Waste Chamber 430. A wash solution can be added, the beads released by unengaging the magnet 910, and the solution mixed. The magnet 910 can be engaged and the wash removed. This process can be repeated until the residual unincorporated fluorescent label is at a low level. The cells bound to the affinity functionalized paramagnetic beads 692 can then be resuspended in a media or buffer of choice and the sample moved to an optical interrogation region 2720 for readout with a fluorescent optical module 2600 for one or more fluors. This can determine the presence of surface proteins.

Example 8: Cell health assays of freshly dissociated solid tissue

[0360] In some embodiments, the cell health of freshly dissociated specimens can be measured using a modular cartridge 205 and Tissue Processing System 80. As described above, a filtered single cell suspension 1102 can be produced from a solid tissue specimen 130 and moved into the Magnetic Processing Chamber 905. Affinity functionalized paramagnetic beads 692 can be added and specific or non-specific cells captured on the beads. In a wash step, Annexin V Binding Buffer can be added followed by Annexin V-FITC conjugated protein, e g., NEB Annexin V- FITC Early Apoptosis Detection Kit #6592, which binds to cell surfaces expressing phosphatidylserine, an early apotosis marker. Following incubation, the magnet 910 can be engaged to capture the cells bound to the affinity functionalized paramagnetic beads 692 and the supernatant removed to the Waste Chamber 430. A wash solution can be added, the beads released by unengaging the magnet 910, and the solution mixed. The magnet 910 can be engaged and the wash removed. This process can be repeated until the residual unincorporated FITC label is at a low level with the last wash containing propidium iodine. The cells bound to the affinity functionalized paramagnetic beads 692 can then be resuspended in in cold, IX Annexin V Binding Buffer and the sample moved to an optical interrogation region 2720 and imaged with a fluorescent optical module 2600 for FITC and propidium iodine. Cells stained with propidium iodide (PI), a non-cell-permeable DNA dye, indicate necrotic cells. Cells stained with both PI and annexin V-FITC demonstrate later stage apoptosis and early necrosis. The sample can be interrogated over time to measure the change in apoptosis and necrosis in the dissociated single cell suspension.

Example 9: Magnetic processing to purify prepare bulk DNA or RNA-Seq libraries on a modular cartridge

[0361] In some embodiments, the modular cartridge 205 can be applied to prepare nucleic acids or sequencing libraries from tissue specimens 120. To begin the workflows, tissue specimen 120 is added to Disocciation Chamber 441 of modular cartridge 205 and a chaotroph, e.g., 7 M guanidine, or sodium isocyanate, or other chaotrophs, added instead of enzymes or detergent, and mechanical disruption performed. The resultant lyzed cells and freed nucleic acid 1072 is then pulled through the Filtration Chamber 450 and into the Magnetic Processing Chamber 905 as described.

[0362] In the Magnetic Processing Chamber 905, paramagnetic beads 685 such as SPRI beads (Beckman Coulter), Dynabeads (Thermo Fisher), or many other beads with COOH or other surface coatings or no coatings are added as described, and then the freed nucleic acid precipitated onto the beads using salt and polyethylene glycol or other chemistries. The amount of beads can be chosen to be limit the amount of nucleic acids 1072 that can bind, thereby normalizing the nucleic acid concentration. The nucleic acid 1072 can be washed, such as with 70% ethanol, one or more times. Example 10: DNA Library Production using polishing, end repair, and ligation.

[0363] One embodiment of the workflow to produce bulk DNA libraries from tissues is illustrated in Figure 24. The bead purified double-stranded DNA can be fragmented enzymatically, e g. Fragmentase® (New England Biolabs, M0348), with restriction enzymes, nucleases, or other enzymes, or chemically. Enyzmes or chemicals can be added to Magnetic Processing Chamber 905 and incubated. Following fragmentation, the now fragmented DNA 1082 can be precipitated onto paramagnetic beads 685, the magnetic beads captured, and the nucleic acid 1072 purified by washes to produce purified fragmented DNA 1084.

[0364] The purified fragmented DNA 1084 can be end-polished in Magnetic Processing Chamber 905 by addition of reaction mix and enzymes, for example, the NEBNext® End Repair Module (NEB E 6050S) reagents, from a syringe pump 2130 to generate end-polished DNA product 810, an end-polished, blunt-ended double-stranded DNA having 5 -phosphates and 3 '-hydroxyls; other kits such as Agilent PCR polishing kit 200409 and other enzymology can perform the same function. Following end polishing, a magnetic separation, as described above, is performed in Magnetic Processing Chamber 905 to remove reactants and enzymes from end-polished DNA product 810.

[0365] Following polishing, A-tailing is used to generate fragments ready to ligate with a primer with a complementary T overhang and to prevent concatamer formation during ligation. A-tailing can be performed using commercially available kits such as the NEBNext® dA- Tailing Module (NEB E6053S) with enzyme and master mix added from a syringe pump 2130 to Magnetic Processing Chamber 905 containing end-polished DNA product 810 and incubating the reaction to produce blunt-ended double-stranded DNA having 5 '-phosphates with an A residue overhang on the 3’ end, A-tailing DNA product 815. Following A tailing, a magnetic separation is performed in Magnetic Processing Chamber 905 to remove reactants and enzymes from A-tailing DNA product 815.

[0366] A double stranded second primer 611 with a complementary T overhang can be ligated by DNA ligase onto the 3’ end of A-tailing DNA product 815. DNA ligase, DNA ligase reaction mix, and second primer 611 (such as NEB Next Adapter) are added by syringe pump 2130 to Magnetic Processing Chamber 905 and incubating the reaction. DNA ligation can be performed using commercially available kits or reactions, e.g., NEBNext® Quick Ligation Module, NEB E6056S. Following DNA ligation, a magnetic separation is performed in Magnetic Processing Chamber 905 to remove reactants and enzymes. The product is now a double stranded DNA product 820 that has incorporated second sequencing primer 611 or can have two adapters attached depending on the workflow. The product of the ligation is a bulk nucleic acid library 1205. By adjusting the crowding agent concentration, e.g., PEG and NaCl, the fragment sizes for the downstream NGS analysis can be selected by a two-step ‘heart cut’ precipitation onto beads, with one cut selecting for fragments longer than a lower cutoff, e.g., 250 bases, and the second cut selecting for fragments shorter than a high cutoff, e.g., 500 bases using the Magnetic Processing Chamber 905 to produce sized DNA bulk library 1220.

Example 11: DNA Library Production using Tagmentation

[0367] Bulk NGS DNA sequencing libraries can also be prepared using tagmentation with transposons including the Nextera Tagmentation (http://www.epibio.com/docs/default- source/protocols/nextera-dna-sample-prep-kit-(illumina— compatible).pdf?sfyrsn=4). In this embodiment, referring to Figure 25, the nucleic acid 1072 is produced in Magnetic Processing Chamber 905 as described and transposons, e.g., Nextera enzyme, reaction mix, and water are added by syringe pump 2130. The reaction is incubated for example at 55°C for 5 min. A bead purification is performed using Magnetic Processing Chamber 905 to remove reactants and purify the double stranded product with transposon inserted into the DNA on the paramagnetic beads 685. Syringe pump 2130 is used to add Nuclease-Free Water, Nextera Adaptor 2 (or other barcoded adapters), Nextera PCR Enzyme, PCR Buffer, and Nextera Primer Cocktail. If the Magnetic Processing Chamber 905 also has a thermal cycling capability in the instrument with cartridge Peltier 1440, nine cycles of PCR can be performed. A bead purification is performed to remove reactants and purify the double stranded DNA product before elution into buffer or water or two bead purifications used to perform a heart cut size selection. The sized DNA bulk library 1220 is now ready to QC and bridge amplification on the flow cell of the sequencer. Many variations of the method described here are within the instant disclosure and are obvious to one skilled in the art.

Example 12: Production of single-cell libraries from polyadenylated mRNA in singlecell or nuclei suspensions.

[0368] In some embodiments after disruption of tissue in a chaotroph and filtration, the lysate is moved into the Magnetic Processing Chamber 905, and mixed with COOH magnetic beads 685 which have poly-T containing primers to capture mRNA 1080. The total nucleic acid is first captured by precipitation, e.g., . with ethanol and PEG, to produce purified nucleic acid 1080 and then eluted into hybridization buffer. The poly-T magnetic beads 688 are then incubated to hybridize polyadenylated mRNA to the beads’ poly-T sequences to produce purified mRNA 1090. A reverse transcriptase reaction is performed in Magnetic Processing Chamber 905 to produce cDNA 1092 formed from the mRNA 1090. As required, fragmentation of the RNA or cDNA 1092 can be performed using methods comprised of chemical, biochemical, and physical methods. The produced cDNA can then be used in the library preparation as described above for bulk nucleic acid library preparation to produce a bulk RNA-Seq library 1225 and then sized to produce a sized bulk RNA-Seq library 1230.

Example 13: Preparation of single nuclei suspensions from FFPE on a modular cartridge

[0369] Single nuclei RNA sequencing (snRNA-Seq) is a powerful technique that allows for the analysis of gene expression and genetic variation amongst individual cellular nuclei within a tissue sample. One application of particular interest to researchers is the use of snRNA-Seq on formalin-fixed, paraffin-embedded (FFPE) tissue samples, which are commonly generated in clinical settings as a means of preserving patient samples for histological examination.

[0370] FFPE tissue samples have traditionally been difficult to study with molecular techniques due to the chemical modifications caused by the formalin fixation, which can lead to degradation of RNA and DNA However, recent advances in snRNA-Seq technology have made it possible to overcome these challenges and generate high-quality genomic data from FFPE samples at the single nucleus level.

[0371] The sample preparation challenge is to automatically prepare the FFPE sample for the production of nuclei. This is currently a manual labor intensive process. In this instant invention, the workflow is adopted to the modular cartridge 205. There are multiple configurations of the modular cartridge 205 which can be used.

[0372] Figure 27 illustrates one example of a modular cartridge 205 configuration. To use the modular cartridge 205, one or more FFPE curls 151, preferably two 50 □m FFPE curls 151, are added to Dissociation Chamber 441 as described. In some embodiments, the curls are first placed in a tissue ring 2300 (Figure 28) and then the tissue ring 2300 is placed in Dissociation Chamber 441. The FFPE curls 151 are then deparaffinized by addition of two mL of xylene or a xylene replacement such as CitriSolv or other chemistry and incubated with the rotor 353 mixing. The incubation can be held at room temperature or an elevated temperature, e.g., 37°C to accelerate dissolution of the paraffin into the xylene or xylene replacement or at a lower temperature. Following the incubation, with the rotor 353 lowered below the Dissociation Chamber output port 445, to prevent loss of the tissue, the xylene or xylene replacement is pulled by vacuum into the waste chamber 430 with Filtration Chamber input pinch valve 2918 closed and Waste chamber input pinch valve 2919 open. A second xylene or xylene replacement deparaffinization can be optionally performed.

[0373] The now deparaffinized FFPE curls 151 are rehydrated by addition of 2 mL of 100% ethanol with mixing by rotor 353, and after incubation for 1 min, removal of the ethanol to waste with the pinch valves in the same positions. This can be repeated for another wash with 100% ethanol, followed by sequential washes with 70%, 50%, and 30% ethanol, and then a wash with a buffer such as PBS with 2% fetal bovine serum and 1 U/ml RNase inhibitor using the same workflow.

[0374] The now deparaffinized and rehydrated FFPE curls 151 can optionally be enzymatically treated such as with Accutase, protein K, or other enyzmes or heat using the workflow immediately above with the appropriate incubation time and temperature. After removal of any enzymatic solution, a nuclei isolation reagent 412 preferably with RNase inhibitor is added, and the FFPE curls 151 are mechanically disrupted by rotor 353 at 4°C to produce a nuclei suspension 1050. Alternatively, a cell isolation reagent 405 is added and disrupted to produce a single cell suspension 1000 at 37°C as described above. The single cells or nuclei can then be pulled into the filtration chamber 450 with Filtration Chamber input pinch valve 2918 opened and Waste chamber input pinch valve 2919 closed. After filtering, the sample can be moved into Output Chamber 850 as described.

[0375] The times and volumes for the process can be adjusted as desired for optimal yields and RNA quality. The produced nuclei or cells can be used for bulk RNA or DNA sequencing or for scRNA-Seq or snRNA-Seq or other applications.

Some Advantages of this Disclosure

[0376] In some embodiments, this disclosure provides devices, instruments, and methods to standardize, optimize, and scale the dissociation of solid tissues into single cells or nuclei. The devices, instruments, and methods provided herein may provide for improved and less variable data comparisons among labs that previously may have been burdened by process disparities and variable dissociation-related cell-type representation.

[0377] In some embodiments, this disclosure also provides for the production of single cell suspensions from solid tissues using a more streamlined workflow, often with a single automated system. The system, devices and methods provided herein, in some embodiments, provide improved performance, including higher cell yields and/or viabilities, less distorted cell-type representations, improved performance with samples less than ~20 mg in size, and/or the ability to successfully process a large variety of cell types.

[0378] With the innovative system proposed here, a research site may be able to dissociate multiple solid tissues in a convenient, standardized, and reproducible process without dissociation domain expertise.

[0379] In addition to increasing throughput, in some embodiments, the devices, systems and methods provided herein integrate key disclosures to dramatically expand the range of applications, research capabilities, and future clinical applications of the system. First, in some cases, modular cartridges 205 are designed with reconfigurable processing chambers and on- cartridge pinch valves 491 to direct fluids within the cartridge. Second, in some embodiments, the system may have magnetic processing to dispense, capture, and rinse paramagnetic beads 685, capture cells or other biological materials, wash, and elute purified cells or processed biological materials from paramagnetic beads 685 within the modular cartridge 205. Third, in some embodiments, the modular cartridge 205 can have tangential flow filtration module 2700 to remove debris, concentrate the sample, and change buffer. Fourth, an optics module 2600 can interrogate samples to determine titer and viability or measure fluorescence, enabling the production of purified titered single cell suspensions 1310 or purified titered single cell suspensions 1320, or sample-to-answer assays.

[0380] Incorporating these new technologies with the existing Singulator infrastructure may extend the automated sample processing workflows possible with a cartridge. For example, in some embodiments, the applications include: a single-use disposable tissue dissociation cartridge that enables complex process workflows with multiple reagents; sequential reagent additions to any chamber; flow diversion to waste or sample collection chambers; the ability to dispense, capture, and wash paramagnetic beads; cell-specific capture/rinsing/release purification; and/or nucleic acid capture and linked molecular biology reactions as illustrated for bulk Next Generation Sequencing (NGS) library preparation.

[0381] This disclosure builds on the existing Singulator architecture to scale, for example, to eight sample capacity, optimizes dissociation and workflows in novel cartridges, and develops and integrates magnetic processing workflows.

[0382] In some cases, the systems, methods and devices provided herein include:

[0383] 1) Systems to process one to two (Figure 4) to eight (Figure 5) or more tissue samples from solid tissues to reproducible, standardized single cell and nuclei suspensions with optional paramagnetic bead purification using novel modular cartridges 205.

[0384] 2) Workflows and methods to dissociate tissues into single-cells or nuclei and enrich or deplete specific cell-types. Mechanical disruption hardware and methods, enzyme/chemical dissociation formulations, and protocols can be integrated with downstream workflows for scRNA-Seq, snRNA-Seq, ATAC-Seq, CITE-Seq, and other single cell genomic libraries.

[0385] In some cases, this disclosure provides the added ability to enrich or deplete specific cell types using paramagnetic bead processing.

Single cell and nuclei sequencing and other applications.

[0386] Many workflows and protocols for scRNA-Seq, CITE-Seq, snRNA-Seq, and ATAC- Seq can be developed, integrated, and optimized for cell type heterogeneity, specific cell type recovery, and minimization of adverse tissue dissociation effects (Figure 29) for the instant disclosure. Downstream library preparation can be by nanodroplet (lOx Genomics), SMART-Seq (SeqWell), combinatorial (Parse), or other methods. Quality metrics to assess dissociation and workflows can include viability, RT-qPCR, RNA distribution, and single cell and nuclei sequencing metrics including cell type representation, unmapped reads, and mitochondrial contamination. scDNA-Seq and single nuclei FFPE sample processing application can also be implemented on the system.

[0387] Figure 29 shows an overview of the workflows and applications for the system. The process variables can have broad operating ranges without ‘cliffs’ for yield and viability. With this disclosure, the dissociation and post-processing conditions may be optimized by single-cell and nuclei sequencing for maximal cellular heterogeneity and minimal gene expression alterations, including by testing cell dissociations at 4°C. Existing Singulator workflows, reagents, and protocols can be used to generate single cell or nuclei sequencing data for scRNA- Seq, snRNA-Seq, and ATAC-Seq.

[0388] It is important to note that a single protocol may not yield, in some cases, all cell-types from a tissue: tissue and cell-type specific protocols and reagent formulations may be needed for the broadest cell heterogeneity, or to preferentially produce specific cell -type subpopulations. The systems described in this disclosure may be able to execute protocols developed for any population and apply magnetic purifications to enrich or deplete for subpopulations.

[0389] The process optimization may first focus on effects of dissociation using existing downstream processing.

[0390] Dissociation. An important aspect of the dissociation is the mechanical tissue disruption. The effect of the side gap 221 of the rotor 353 has been tested for many mouse tissues, human tissues, and other tissues. The side gap 221 is set by the manufacturing of the disruptor (Figure 3). The bottom gap 222, between the rotor 353 and bottom of the Dissociation Chamber 441, can controlled by the force sensor 2115 in each instrument bay to minimize pressure on the tissue in real-time. The processing time, rotor speed, enzymes or chemical formulations, and buffer components can be evaluated, e.g., for each tissue type. The dissociation process can be optimized to balance yields and minimize single cell and nuclei damage, measured by visual observation, RT-qPCR of 0.2 mm filtrate, flow cytometry (scatter and fluorescence modes for nuclei integrity), and sc/snRNA-Seq metrics for candidate configurations.

[0391] Library construction. After purification of the single cell and/or nuclei suspensions, nanodroplet, combinatorial, and plate-based single cell and nuclei library construction methods can be performed on the single cell and nuclei suspensions to generate sequencing libraries. [0392] Metrics. The system output may be characterized by multiple metrics including yield, viability, RT-qPCR analysis, RNA sizing, and single cell sequencing quality. Cell or nuclei yields and viability measurements can be determined by automated counting (AO/PI, Nexcelom K2); microscopy of stained preparations to visualize cell/nuclei morphology, and FACS using laser scatter and fluorescence to assess cell and nuclei integrity, and distribution. One step RT-qPCR of single cell or nuclei suspensions or supernatants by ACTB, GAPDH, and fos with 3’ and 5’ primers, and by electrophoresis (TapeStation) can assess RNA quality and cell or nuclei damage. scRNA/snRNA-Seq or other sequencing libraries can be analyzed for metrics of cell type representation, gene count, unmapped genes, mitochondrial reads, and dissociation-induced gene expression changes.

Optimization of cell dissociation processes for scRNA-Seq and scCITE-Seq.

[0393] Generally, the first step in scRNA-Seq analysis of solid tissues is tissue dissociation. Protocols for dissociation of mouse (e.g., 6 mouse), human, or other tissues can be evaluated for cell heterogeneity and other metrics by scRNA-Seq.

[0394] Figure 30 shows an example of scRNA-Seq analysis of human lung at S2 using current mouse protocols and reagents. scRNA-Seq analysis of human lung was performed on cells isolated from a normal lung biopsy from a human patient with squamous cell carcinoma. Fresh human lung (CHTN) was obtained under Development of Tissue Processor project (PR6733) and dissociated into a single cell suspension (Singulator 100, Mouse Lung Reagent/Mouse Lung Protocol). The single cell suspension was spun (300 g/5 min) the pellet resuspended RBC lysis buffer for 4 min, quenched with DMEM, and spun at 300 g for 5 min. The pellet was resuspended, filtered (40 mm Flowmi). counted (Nexcelom K2), and titer adjusted. Standard single-cell 3’ v3.1 gene expression libraries (lOx Genomics) were generated from -10,000 cells. Libraries were sequenced (NovaSeq, Illumina) with bioinformatic analysis as described. Libraries were sequenced to a depth of 20,000 reads. 1,408 median genes per cell were obtained with 85.3% fraction of reads per cell.

[0395] The single cell dissociation process can be optimized for enzyme formulations, tissue incubation time, mixing method, mechanical disruption variables (speed, duration, disruptor profile, side and bottom disruption gaps), post-processing workflows, buffer composition, and other process variables for scRNA-Seq. Higher purity enzymes can improve cell-type heterogeneity.

[0396] For CITE-Seq, fluorescent and barcoded antibodies can be used with standard dissociation formulations and enzyme formulations re-optimized to preserve of surface markers. The re-optimization can include the deletion or substitution of an offending enzyme, use of higher purity enzymes, or adjustment of the enzyme incubation time. Conditions can be screened using fluorescently-labeled antibodies with flow cytometry followed by CITE-Seq to quantify and optimize conditions.

Optimization of nuclei dissociation processes for snRNA-Seq and ATAC-Seq.

[0397] Existing snRNA-Seq and ATAC-Seq workflows starting from dissociation of solid tissues may be further developed. snRNA-Seq is now a complementary analysis method to scRNA-Seq using nuclei suspensions. Frozen tissues can be processed into nuclei. For some tissues, a wider range of cell types is found with snRNA-Seq relative to scRNA-Seq. ATAC-Seq is a powerful application using nuclei to detect chromatin accessibility and active transcription regions

[0398] With the methods, systems and devices of the present disclosure, the preparation of nuclei suspensions is sometimes simpler than for single cells: a single reagent formulation works well for over 42 human, mouse, rat, insect, and other tissues on the Singulator. The activity of RNases in tissues varies greatly from a low level in brain tissue to over 180,000-fold higher in pancreas. Optimal concentrations RNase inhibitors can be determined for each tissue.

[0399] Figure 31 shows snRNA-Seq analysis of human liver tissue using the Singulator 100 with 18 annotated cell type clusters. 149 mg fresh human liver sample (CHTN) was dissociated (Singulator 100 with S2 Nuclei Isolation Reagent and S2 Nuclei Storage Reagent (NSR) with lU/mL RNase inhibitor (Sigma Protector)). The dual-filtered single nuclei suspension was removed from the cartridge, spun (500 g/ 5 min), the pellet resuspended in 20% Percoll solution, spun (700 g/10 min); the pellet resuspended in NSR with RNase inhibitor, filtered (40 mm Flowmi), counted (Nexcelom K2) and adjusted to 1,000 nuclei/mL. Single-cell gene expression libraries were generated (NextGEM v3.1, lOx) Genomics to capture and profile -10,000 nuclei. Libraries were sequenced (NovaSeq, Illumina) with a depth of 20,000 reads. Bioinformatic analysis obtained 2,030 median genes per nuclei for this sample with 72.6% fraction of reads per nuclei. The production of nuclei suspensions can be optimized for detergent composition and concentration, incubation time, mechanical disruption variables (speed, duration, disruptor height and rotation profile, side and bottom disruption gaps, etc.), reagent additives, buffer components, and post-processing methods.

[0400] A range of detergent concentrations centered on 0.1% NP-40 and/or 0.1% Triton-20 can be tested in proprietary formulations. Each component (detergents, osmoprotectants, salts, buffers) can be individually optimized. Because nuclei yield and quality can be tuned for different tissues by the side gap between the rotor and Dissociation Chamber, different side gaps, selfcentering rotors, and other designs as described above can be tested to achieve optimal processing for both yield and nuclei quality. The best methods may be tested by sequencing. 10401] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, section headings, the materials, methods, and examples are illustrative only and not intended to be limiting.

-SI-

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A system comprising:

(a) an instrument comprising:

(ii) an actuator subassembly comprising:

(1) a disruptor actuator configured to engage the grinder assembly; and

(2) at least one valve actuator (e.g., a pinch valve actuator) configured to actuate one or more valves to control fluid flow in fluidic conduits in the at least one cartridge;

(iii) a fluidics subassembly comprising:

(3) at least one cannula communicating with the at least one pressure source and, directly or indirectly through one or more fluidic lines, with at least one port of a functional unit, or of the processing chamber, when the at least one cartridge is engaged with the at least one cartridge interface; (vi) optionally, one or more magnetic separation assembly(s) comprising a source of magnetic force that is reversibly engageable with a functional unit engaged with a cartridge when the cartridge is engaged with the cartridge interface;

(vii) optionally, a thermal assembly configured to regulate temperature of functional units engaged with cartridge bays;

(b) optionally, at least one cartridge engaged with the at least one cartridge interface.

2. The system of claim 1, further comprising at least one motor configured to operate the disruptor actuator, the valve actuator, and the optional magnetic assembly.

3. The system of any of the preceding claims, further comprising a flowcell fluidically connected with the at least one pressure source and at least one fluid line fluidically connected with the processing chamber and/or at least one functional unit in the cartridge, wherein the flowcell is configured for optical interrogation by the optical detector.

4. The system of any of the preceding claims, further comprising a waste chamber fluidically connected with the at least one pressure source.

5. The system of any of the preceding claims, wherein the disruptor actuator comprises a linear driver (e.g., a stepper motor or a pneumatic driver) that drives the actuator in an up-down (Z axis) direction, and a rotary motor that rotates the actuator around a Z axis.

6. The system of any of the preceding claims, wherein the valve actuator is configured to close or open a valve by releasably pressing a pinching element against a flexible tube in the cartridge, which flexible tube fluidically connects (A) the processing chamber with a functional unit or (B) functional units with each other.

7. The system of any of the preceding claims, wherein the at least one pressure source comprises a vacuum source communicating with a vacuum accumulator, which vacuum accumulator is fluidically connected to one or more cannulae through one or more vacuum valves.

8. The system of any of the preceding claims, wherein the at least one pressure source comprises a pump fluidically connected through at least one valve to at least one of the reagent containers and at least one cannula, wherein the pump is configured to draw liquid from the reagent container and push liquid to the cannula.

9. The system of any of the preceding claims, wherein the at least one reagent container containing one or more of a reagent for dissociating tissue, paramagnetic beads, and wash reagents.

10. The system of any of the preceding claims, wherein the at least one cannula is configured to move from a disengaged position to an engaged position in which the cannula meets with a port in a functional unit or the processing chamber.

11. The system of any of the preceding claims, wherein the source of magnetic force is a magnet or electromagnet.

12. The system of any of the preceding claims, wherein reversibly engaging the source of magnetic force comprises moving the source toward or away from the functional unit.

13. The system of any of the preceding claims, wherein the thermal assembly comprises a Peltier device.

14. The system of any of the preceding claims, wherein the thermal assembly comprises temperature regulatory elements configured to reversibly engage the processing chamber or at least one functional unit, e.g., by moving the elements toward or away from the processing chamber or functional unit.

15. A method for purifying cells or subcellular organelles from a tissue comprising:

(I) providing a system of any of the preceding claims, wherein the system comprises a cartridge engaged therewith, wherein the cartridge comprises a tissue sample and dissociation reagent in the processing chamber;

(II) using the disruptor actuator to operate the grinder assembly to grind the tissue, thereby releasing cells and/or subcellular organelles; and (III) using the pressure source to move the released cells and/or subcellular organelles from the processing chamber directly or indirectly to an output receptacle of the output unit.

16. The method of claim 15, further comprising, before moving the released cells and/or subcellular organelles into the output receptacle, performing at least one of the following operations:

- using the pressure source to move waste from a functional unit into the waste unit.

17. The method of claim 15-16, wherein the tangential flow device comprises two tangential flow fdters connecting with upper and lower buffer regions functioning as a concentration gradient.

18. The method of any of claims 15-17, wherein grinding the tissue comprises executing a computer script indicating one or more of: a number of times the grinder assembly is moved in a Z direction, a pressure with which the grinder assembly presses the tissue in the Z direction, a number of rotations of the grinder assembly in the clockwise or counterclockwise directions.

19. The method of any of claims 15-18, wherein the tissue is fresh tissue or preserved tissue (e.g., FFPE tissue).

20. The method of any of claims 15-19, further comprising isolating and sequencing nucleic acids from the purified cells and/or subcellular organelles.

21. A method for preparing titered, purified single cells and/or subcellular organelles performed in a system of any of claim 1-14, comprising:

(b) staining the cells and/or subcellular organelles in a known volume;

(d) using the measurement module, determining titer and/or viability of the stained cells and/or subcellular organelles;

22. The method of claim 21, wherein cells and/or subcellular organelles are concentrated by immobilizing the cells and/or subcellular organelles in the magnetic processing unit by capture on magnetic or paramagnetic particles and immobilization using the magnetic force, and removing liquid from the magnetic processing unit to achieve the desired titer, optionally adding liquid as necessary.

23. The method of claim 21-22, wherein immobilizing comprises capturing cells and/or subcellular organelles on magnetic or paramagnetic particles derivatized with antibodies specific for the cells and/or subcellular organelles; and, immobilizing the particles by apply magnetic force to the magnetic processing unit; separating waste from the immobilized particles by withdrawing the waste from the magnetic processing unit; optionally, releasing the captured cells and/or subcellular organelles from the antibodies (e.g., by adjusting pH); and resuspended the cells and/or subcellular organelles in a liquid.

24. The method of any of claims 21-23, wherein using a measurement module comprises moving the cells and/or subcellular organelles into a flowcell and interrogating the flowcell with the optical detector.

25. A method for detecting surface proteins on cells or subcellular organelles from a tissue comprising:

(I) providing a system of any of claims 1-14, wherein the system comprises a cartridge engaged therewith, wherein the cartridge comprises a tissue sample and dissociation reagent in the processing chamber;

(VII) using the optical detector to detect the labeled proteins.

26. A method for determining health of cells or subcellular organelles from a tissue comprising: (I) providing a system of any of claims 1-14, wherein the system comprises a cartridge engaged therewith, wherein the cartridge comprises a tissue sample and dissociation reagent in the processing chamber;

(V) labeling the cells and/or subcellular organelles with a marker that differentiates healthy cells and/or organelles from necrotic, late stage apoptosis, and/or early necrotic cells and/or subcellular organelles;

27. A method for purifying DNA or RNA from cells and tissue, comprising:

(III) using the pressure source to move the released cells and/or subcellular organelles from the processing chamber directly or indirectly to a magnetic processing unit; (IV) using the magnetic separation assembly, immobilizing the released to DNA or RNA using particles that capture nucleic acids (e.g., SPRI beads), removing unbound material and, optionally, washing the particles;

28. The method of claim 27, further comprising:

29. The method of claim 27 or 28, further comprising performing PCR on DNA in the magnetic processing chamber or other chamber.

30. The method of claim 28, further comprising performing a sizing cut on a library.

31. A cartridge for dissociating tissue, comprising:

(b) a grinder assembly comprising a plunger comprising a rotor having a side, the grinder assembly slidably positioned in the processing chamber through the top orifice; wherein:

(i) the rotor comprises a side that comprises a plurality of gap setting features on the side, wherein, when the rotor is positioned in the processing chamber, the gap setting features maintain a gap between the side of the rotor and the side wall of the processing chamber.

32. The cartridge of claim 31, wherein the rotor has a thickness between about one millimeters and about fifty millimeters.

33. The cartridge of claim 31-32, wherein the gap setting features are configured as bumps, swirls or stripes.

34. The cartridge of any of claims 31-33, wherein the gap setting features are positioned at a top of the side of the rotor.

35. The cartridge of any of claims 31-34, wherein the gap setting features are configured as stripes oriented obliquely with respect to the side of the rotor.

36. The cartridge of any of claims 31-35, wherein the rotor comprises between three and thirty-six gap setting features.

37. The cartridge of any of claims 31-36, wherein each gap setting feature is separated from an adjacent gap setting feature by between 10 degrees and 120 degrees, e.g., between 30 degrees and 60 degrees.

38. The cartridge of any of claims 31-37, wherein comprising a gap between the rotor and the side wall of about 1 micron and 1,000 microns.

39. A cartridge for dissociating tissue, comprising:

(e) either the rotor comprises a center post and the stator comprises a center hole, or the stator comprises a center post and the rotor comprises a center hole; and

(f) wherein, when the rotor contacts the stator:

(1) the center post mates with the center hole, and

(2) the rings in the stator and the rings in the rotor are positioned such that rings of teeth in the stator mesh with the one or more central teeth and rings of teeth in the rotor.

40. The cartridge of claim 39, wherein the center post has a height of between about

0.5 mm and about 3 mm.

41. The cartridge of claim 39 or 40, wherein the center post and hole set a gap between a grinding feature of the stator and a grinding feature of the rotor of between about 0.005 mm and about 0.5 mm.

42. The cartridge of any of claims 39-41, wherein a plurality of the teeth have a trapezoidal cross-section.

43. The cartridge of any of claims 39-42, wherein the stator and the rotor each comprise three rings of teeth.

44. The cartridge of any of claims 39-43, wherein the rotor comprises an inner ring comprising six teeth, a middle ring comprising six teeth and an outer ring comprising 11 teeth.

45. The cartridge of any of claims 39-44, wherein the rotor comprises teeth at a density of about 1 tooth per 0.0025 mm² to about 1 tooth per 0.10 mm², e.g., about 1 tooth per 0.05 mm²

46. The cartridge of any of claims 39-45, wherein the stator comprises an inner ring comprising four teeth, a middle ring comprising six teeth and an outer ring comprising 10 teeth.

47. The cartridge of any of claims 39-46, wherein the stator comprises teeth at a density of about 1 tooth per 0.002 mm² to about 1 tooth per 0.08 mm², e.g., about 1 tooth per 0.04 mm²

48. The cartridge of any of claims 39-47, wherein one or more teeth have a height of about 500 microns and a width of about 1 mm to 2 mm.

49. The cartridge of any of claims 39-48, wherein the processing chamber has a volume between 0.05 mb and 100 m , e.g., between 2 mb and 5 mL, between 10 mb and 50 mL, or between 10 mL and 20 mL.

50. The cartridge of any of claims 39-49, wherein the processing chamber has a cross-sectional area of between about 78 mm² (e.g., radius of about 5 mm) and about 1256 mm² (e.g., radius of about 20 mm), e.g., about 452 mm² (e.g., radius of about 12 mm).

51. The cartridge of any of claims 39-50, comprising a tissue sample no greater than 20 mg, no greater than 10 mg, no greater than 5 mg, no greater than 2 mg, or no greater than 1 mg.

52. The cartridge of any of claims 39-51, comprising a gap between the rotor and the sidewall of about 1 micron and 500 microns.

53. The cartridge of any of claims 39-52, wherein the first processing chamber port is positioned above a top of the rotor when the rotor is fully depressed.

54. The cartridge of any of claims 39-52, wherein the grinder assembly further comprises a cap attached to the plunger and configured to cover the orifice and position the grinder assembly in the processing chamber.

55. The cartridge of any of claims 39-54, wherein the plunger is spring-biased toward the cap.

56. The cartridge of any of claims 39-55, wherein the cap comprises a key slot to engage an actuator.

57. The cartridge of any of claims 39-56, wherein the rotor of the plunger is biased toward the cap (e.g., spring biased).

58. The cartridge of any of claims 39-57, wherein the rotor has sufficient clearance from the processing chamber walls to allow liquid, cells and nuclei to pass around the rotor during depression, and the first processing port is positioned above the rotor when fully depressed.

59. A cartridge adapted to interface with an instrument wherein a. the cartridge comprises a frame comprising (i) at least one functional unit and (ii) a plurality of vessel bays; b. the at least one functional unit comprises:

(ii) at least one filtration unit comprising a filter or strainer; (iii) at least one magnetic processing unit comprising paramagnetic or magnetic particles;

(iv) an output unit comprising a separable output tube;

(v) a flowcell unit;

(vi) a tangential flow filtration unit; or

60. The cartridge of claim 59, wherein the vessel bay is not engaged with or attached to a stand-alone unit.

61. The cartridge of claim 59 or 60, wherein the frame, the at least one functional unit and the plurality of vessel bays are molded together as a single unit.

62. The cartridge of any of claims 59-61, wherein the frame, the at least one functional unit and the plurality of vessel bays are injected-molded as a single unit.

63. The cartridge of any of claims 59-62, wherein the frame, the at least one functional unit and the plurality of vessel bays comprise a polymeric material.

64. The cartridge of any of claims 59-63, wherein the at least one functional unit is an independent unit that is attached to and locked into, clipped into, or snapped into the frame via the attachment features.

65. The cartridge of any of claims 59-64, wherein the at least one functional unit is an independent unit that is attached to and locked into, clipped into, or snapped into the frame via the attachment features and is removable.

66. The cartridge of any of claims 59-65, wherein the at least one functional unit is an independent unit that is attached to and locked into, clipped into, clicked into or snapped into the frame via the attachment features and is not manually removable without a tool or is irreversibly attached to the frame.

67. The cartridge of any of claims 59-66, wherein the at least one functional unit is an independent unit that is removably attached to, removably snapped into, or removably twisted into the attachment feature in the frame.

68. The cartridge of any of claims 59-67, wherein the attachment features comprise: a. a press-fit comprising a recess or hole with a shape (optionally conical or cylindrical), that can receive a complementary-shaped projection (optionally, conical or cylindrical, where applicable) that is situated on a standalone unit; b. a press-fit comprising a recess or aperture functionalized with ridges or threads that can receive a projection situated on a stand-alone unit that comprises complementary ridges, grooves, or threads, where applicable; c. a press-fit comprising a projection with a shape that is complementary to a recess or hole present in a stand-alone unit, wherein the shape is optionally conical or cylindrical; d. a press-fit comprising a projection with functionalized with ridges or threads that are complementary to ridges, grooves, or threads present within a hole or recess within a stand-alone unit; e. a click-in function that can click into a click-in feature present on a stand-alone unit; f. a click-in function that can click into a snap-in feature present on a stand-alone unit; g. a twist function that can twist into or receive a twist feature on a stand-alone unit, wherein the twist function or feature comprises complementary ridges, grooves or threads; h. an adhesive, epoxy adhesive or glue; or i. a flange.

69. The cartridge of any of claims 59-68, wherein a plurality of stand-alone units are attached to the vessel bays via the attachment features and the plurality of stand-alone units comprise: a. at least one processing chamber comprising an internal surface functionalized with grinding features; b. at least one filtration unit comprising a filter; c. at least one magnetic processing unit comprising magnetic particles; d. an output unit comprising a separable output tube; e. a flowcell unit; f. a tangential flow filtration unit; g. a waste unit; or h. a combination thereof.

70. The cartridge of any of claims 59-69, wherein the at least one functional unit comprises: at least one processing chamber comprising an internal surface functionalized with grinding features.

71. The cartridge of any of claims 59-70, wherein the at least one functional unit comprises at least one processing chamber comprising an internal surface functionalized with grinding features and wherein the plurality of stand-alone units comprises an output unit comprising a separable output tube, wherein the stand-alone unit is attached to a vessel bay.

72. The cartridge of any of claims 59-71, wherein the grinding features comprise a plurality of teeth or ridges.

73. The cartridge of any of any of claims 59-72, the cartridge further comprising apertures designed to engage with tubing or cannulae.

74. The cartridge of any of claims 59-73, wherein the cartridge comprises tubing or cannulae.

75. The cartridge of any of claims 59-74, wherein the cartridge further comprises a tissue disruptor within the at least on processing chamber.

76. The cartridge of any of claims 59-75, wherein at least one of the at least one functional unit is fluidically connected to at least one of the stand-alone units.

77. The cartridge of any of claims 59-76, wherein the least one processing chamber comprises a stator and a rotor, with, optionally, each comprising teeth.

78. The cartridge of any of claims 59-77, wherein the rotor comprises teeth at a density of about 1 tooth per 0.0025 mm² to about 1 tooth per 0.10 mm², e.g., about 1 tooth per 0.05 mm²

79. The cartridge of any of claims 59-78, wherein the stator comprises teeth at a density of about 1 tooth per 0.002 mm² to about 1 tooth per 0.08 mm², e.g., about 1 tooth per 0.04 mm²

80. The cartridge of any of claims 59-79, wherein one or more teeth have a height of about 500 microns and a width of about 1 mm to 2 mm.

81. The cartridge of any of claims 59-80, wherein at least one of the functional units or stand-alone units has a volume between 1 ul, 20 ul, 50 ul, 100 ul, 500 ul, 1 mL, 2 mL, 5 mL, 5 mL and 100 mL, e g., between 10 mL and 50 mL, e.g. between 10 mL and 20 mL.

82. The cartridge of any of claims 59-81, wherein the processing chamber has a volume between 1 ul, 20 ul, 50 ul, 100 ul, 500 ul, 1 mL, 2 mL, 5 mL, 5 mL and 100 mL, e.g., between 10 mL and 50 mL, e.g. between 10 mL and 20 mL.

83. The cartridge of any of claims 59-82, wherein: a. at least one of the functional units or stand-alone units comprises a flow cell that, optionally, has a volume between 1 ul and 1 mL, e g., 1 ul- 0.5 ml; 10 ul-. l ml; 1 ul -,2 mL; 1 ul - 100 ul; b. at least one of the functional units or stand-alone units comprises a microfluidic device, microfluidic chip, laminar or microcapillary with a volume 1 ul - 1 mL, e.g., 1 ul-.l mL, 10 ul-.l mL, 50 ul-.2 mL, 10 ul- 50 ul, .1 mL- 1 mL, or 5 ul - 500 ul; c. the cartridge comprises a microfluidic device attached to the cartridge that facilities transfer of fluids from or within the functional units or stand-alone units; d. the cartridge comprises a microfluidic device or chip; e. the cartridge comprises a flow cell with channels that are 10 - 1000 uM in width, length or depth, e.g., 10-100, SO- SOO, 50-1000 uM; f. the cartridge comprises a magnetic processing chamber that is situated within the flowcell; g. the cartridge comprises a magnetic processing chamber comprising a detection unit; h. the cartridge comprises a polymer frame or polypropenol frame and, optionally, a functional unit that is glass, a window, optically transparent, quartz or translucent; or i. the cartridge comprises a functional unit that is glass, a window, optically transparent, quartz or translucent, and, optionally, the frame is a polymer frame.

84. The cartridge of any of claims 59-83, wherein the processing chamber, at least one functional unit, or at least one of the stand-alone units, has a cross-sectional area of between about 12.6 mm² (e.g., radius about 2 mm), 78 mm² (e.g., radius of about 5 mm) and about 1256 mm² (e g., radius of about 20 mm), e.g., about 452 mm² (e g., radius of about 12 mm).

85. The cartridge of any of claims 59-84, wherein the output tube comprises a single-cell, single-nuclei, or single-sub-cellular organelle suspension.

86. The cartridge of any of claims 59-85, wherein the filtration unit comprises a filter or strainer having pores no greater than about 40 microns (e.g., no greater than about 30 microns, no greater than about 20 microns), and an optional second strainer having pores 40 - 200 microns, less than 40 microns, greater than 5 microns, or 100-500 microns.

87. The cartridge of any of claims 59-86, wherein the filtration unit comprises two or more filters or strainers.

88. The cartridge of any of claims 59-87, wherein the two or more filters or strainers comprise different pore sizes.

89. The cartridge of any of claims 59-88, wherein a pore size of one filter is 100 to 500 microns and a pore size of a different filter is less than 50 microns, or between 2 microns and 40 microns.

90. The cartridge of any of claims 59-89, wherein the two or more filters or strainers comprise a first filter with first pores and a second filter with second pores, wherein the first pores comprise pores with a larger diameter than at least one of the second pores.

91. The cartridge of any of claims 59-90, wherein the two or more filters or strainers further comprise a third filter with third pores that comprise pores with a diameter smaller than a diameter of at least one of the second pores.

92. The cartridge of any of claims 59-91, wherein the first filter is situated upstream of the second filter, which is, optionally upstream of the third filter if present.

93. The cartridge of any of claims 59-92, wherein the filtration unit comprises two or more filters in a stacked configuration, vertical stacked configuration, or horizontal stacked configuration.

94. The cartridge of any of claims 59-93, wherein the filtration unit comprises a first filter with pores that are 50 to 500 microns in diameter, e.g., 50-200 microns, 100-500 microns, 200-500 microns, 150 -400 microns.

95. The cartridge of any of claims 59-94, wherein the filtration unit comprises a first filter with pores that are 50 to 500 microns in diameter, e.g., 50-200 microns, 100-500 microns, 200-500 microns, 150 -400 microns and/or a second filter with pores 20-200 microns in diameter, e.g., 20-100 microns, 40-200 microns, 100-200 microns or 20-150 microns in diameter.

96. The cartridge of any of claims 59-95, wherein the filtration unit comprises a first filter with pores that are 50 to 500 microns in diameter, e.g., 50-200 microns, 100-500 microns, 200-500 microns, 150 -400 microns and/or a second filter with pores 5-80 microns in diameter, e.g., 5-50 microns, 20 - 80 microns, 10-80 microns.

97. The cartridge of any of claims 59-96, wherein the filtration unit comprises a first filter with pores that are 50 to 500 microns in diameter, e.g., 50-200 microns, 100-500 microns, 200-500 microns, 150 -400 microns and/or a second filter comprising pores that are 200-300 microns in diameter, 150-400 microns in diameter, or 200-600 microns in diameter.

98. The cartridge of any of claims 59-97, wherein the filtration unit is fluidically connected a source comprising wash buffer.

99. The cartridge of any of claims 59-98, wherein the magnetic processing unit comprises paramagnetic beads.

100. The cartridge of any of claims 59-99, wherein the magnetic or paramagnetic particles are 0.1 nm- 100 pm in diameter (e.g., 0.1 nm- 10 nm; .1 nm - 1 pm; 10 pm - 100 pm; 50 pm-100 pm).

101. The cartridge of any of claims 59-100, wherein the cartridge comprises a plurality of magnetic processing units.

102. The cartridge of any of claims 59-101, wherein the tangential flow filtration unit comprises two tangential flow filters connecting with upper and lower buffer regions functioning as a concentration gradient.

103. The cartridge of any of claims 59-102, wherein the tangential flow filtration unit comprises an optical interrogation region.

104. The cartridge of any of claims 59-103, wherein the at least one functional unit is fluidly connected to another functional unit or stand-alone unit via tubing, capillary, microcapillary, or cannula.

105. The cartridge of any of claims 59-104, wherein the cartridge comprises a macrofluidic or microfluidic device.

106. The cartridge of any of claims 59-105, wherein the at least on functional unit or stand-alone unit is embedded in a microfluidic or microfluidic device.

107. The cartridge of any of claims 59-106, wherein the attachment features are present at a top surface of the cartridge, a bottom surface of the cartridge, or a lateral surface of the cartridge.

108. A system comprising an instrument engaged with the cartridge of any of claims 59-107.

109. The system of claim 108, wherein the system is engaged with the cartridge via tubing or cannulae.

110. The system of claim 108-109, wherein the cartridge comprises fluidic ports in communication with fluid ports situated in the instrument.

111. The system of any of claims 108-110, wherein the instrument comprises a fluidics subassembly comprising: a. at least one pressure source of positive and/or negative pressure, wherein the at least one pressure source optionally comprises separate vacuum source and pump (e g., syringe pump); b. at least one reagent container for containing liquids, fluidically connected to the at least one pressure source, optionally contained in a reagent cassette; or c. at least one cannula communicating with the at least one pressure source and, directly or indirectly through one or more fluidic lines, with at least one port of a functional unit, or of the processing chamber, when the at least one cartridge is engaged with the at least one cartridge interface.

112. The system of any of claims 108-111, wherein the instrument comprises an actuator subassembly comprising: a. a disruptor actuator configured to engage the grinder assembly; or b. at least one valve actuator (e.g., a pinch valve actuator) configured to actuate one or more valves to control fluid flow in fluidic conduits in the at least one cartridge.

113. The system of any of claims 108-112, wherein the instrument comprises magnetic separation assembly(s) comprising a source of magnetic force that is reversibly engageable with a functional unit engaged with a cartridge when the cartridge is engaged with the cartridge interface;

114. The system of any of claims 108-113, wherein the system comprises a thermal assembly configured to regulate temperature of functional units engaged with the plurality of vessel bays or frame.

115. A kit comprising the cartridge of any of claims 59-107 and a plurality of standalone vessels.

116. The kit of claim 115, wherein the plurality of stand-alone vessels comprises: a. at least one processing chamber comprising an internal surface functionalized with grinding features; b. at least one filtration unit comprising a filter or strainer; c. at least one magnetic processing unit comprising magnetic particles; d. an output unit comprising a separable output tube; e. a flowcell unit; f. a tangential flow filtration unit; or g. a combination thereof;

117. A method for purifying cells or subcellular organelles from a tissue comprising: a. providing a system of any of claims 108-114, wherein the system comprises a cartridge engaged therewith, wherein the cartridge comprises a tissue sample or cell sample within the at least one processing chamber or dissociation chamber; b. using a disruptor actuator to operate a grinder assembly to grind the tissue within the processing chamber, thereby releasing cells and/or subcellular organelles; and c. using a pressure source to move the released cells and/or subcellular organelles from the processing chamber directly or indirectly to an output receptacle of the output unit.

118. The method of claim 117, further comprising, before moving the released cells and/or subcellular organelles into the output receptacle, performing at least one of the following operations: a. using the pressure source to move the released cells and/or subcellular organelles from the processing chamber into the filtration unit to separate debris from the cells and/or subcellular organelles; b. using the pressure source to move the released cells and/or subcellular organelles from either the processing chamber or the filtration unit into a magnetic processing unit, capturing cells and/or subcellular organelles in the magnetic processing unit by using the magnetic force to attract cells and/or subcellular organelles that are associated with magnetic or paramagnetic particles, and, optionally, moving on attracted material of the magnetic processing chamber, and optionally washing the attracted cells and/or subcellular organelles using the pressure source to move liquid into the magnetic processing chamber; c. using the pressure source to move at least some of the released cells and/or subcellular organelles into a flowcell and detecting living and/or dead cells and/or subcellular organelles using the measurement module; d. using the pressure source to move the cells and/or subcellular organelles into a tangential flow device to remove contaminants; or e. using the pressure source to move waste from a functional unit into the waste unit.

119. The method of claim 117 or 118, further comprising using a first magnetic processing chamber to reduce a volume of the sample and a second magnetic processing chamber to perform reactions.

120. The method of any one of claims 117-119, further comprising using the first magnetic processing chamber to reduce a volume of a sample to 0.001, 0.005, 0.01, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, or 0.5 of its initial volume.

121. The method of any of claims 117-120, wherein the cartridge comprises a tissue fragment and wherein the cartridge comprises a filtration unit with a pore size of 100-500 microns, e.g., 100-400 microns, 200-500 microns, 100-300 microns.

122. The method of any of claims 117-121, wherein the method produces a single cell suspension.

123. The method of any of claims 117-122, wherein the method produces a single cell suspension and the cartridge comprises a first filter with pores that are 50 to 500 microns in diameter, e.g., 50-200 microns, 100-500 microns, 200-500 microns, 150 -400 microns and/or a second filter with pores 20-200 microns in diameter, e.g., 20-100 microns, 40-200 microns, 100-200 microns or 20-150 microns in diameter.

124. The method of any of claims 117-123, wherein the method produces a single sub-cellular organelle suspension (e.g., a single nuclei suspension).

125. The method of any of claims 117-124, wherein the method produces a single sub-cellular organelle suspension (e.g., a single nuclei suspension) and the filtration unit comprises a first filter with pores 50 to 500 microns in diameter, e.g., 50-200 microns, 100-500 microns, 200-500 microns, 150 -400 microns in diameter; and/or a second filter comprising pores that are 5-80 microns in diameter, e.g., 5-50 microns, 20 - 80 microns, 10-80 microns.

126. The method of any of claims 117-125, wherein the method produces a single cardiomyocyte suspension.

127. The method of any of claims 117-126, wherein the method produces a single cardiomyocyte suspension and the filtration unit comprises a first filter with pores 50 to 500 microns in diameter, e.g., 50-200 microns, 100-500 microns, 200-500 microns, 150 -400 microns in diameter; and/or a second filter comprising pores that are 200-300 microns in diameter, 150-400 microns in diameter, or 200-600 microns in diameter.

128. A method of preparing a cartridge for use in an instrument that produces single cell suspensions, single nuclei suspensions or single subcellular organelle suspensions, the method comprising: a. providing a cartridge of any of claims 59-107; and b. attaching one or more of the plurality of the stand-alone units to the cartridge via the attachment features.

129. A method of preparing a cartridge for use in an instrument that produces single cell suspensions, single nuclei suspensions or single subcellular organelle suspensions, the method comprising: a. providing a first and second cartridge of any of claims 59- 107; b. attaching a first set of the stand-alone units to the attachment features in the vessel bays of the first cartridge; c. attaching a second set of the stand-alone units to the attachment features in the vessel bays of the second cartridge, wherein the first set and the second set of standalone units comprise different combinations of standalone units

130. The method of claim 129, wherein the first and second cartridges each comprise a common region situated a common location on the first and second cartridge that interfaces with an integrated feature of the instrument.

131. The method of preparing a cartridge of any of claims 59-107, wherein the integrated feature is a thermal controller, a magnetic field, an optical detector, a measurement device, fluidics subassembly, actuators, cannulae, tubing, pressure source, positive or negative pressure, reagent source, wash buffer source, fluorescent detector, regulatable magnetic field; fluid dispenser, or fluid aspirator.