WO2025173010A1 - Device and method for cell harvesting - Google Patents

Abstract

A device for lifting and collecting cells and a method of using the same are disclosed. The device comprises a cell collection head; an arm connected to the cell collection head, and extending proximally from the cell collection head to form a device body; and at least two flow channels, wherein a first inlet of a first one of the at least two flow channels is proximally disposed at the arm, and a first outlet of the first one of the at least two flow channels is distally disposed at the cell collection head, and an outlet of a second one of the at least two flow channels is proximally disposed at the arm, and an inlet of the second one of the at least two flow channels is distally disposed at the cell collection head.

Description

DEVICE AND METHOD FOR CELL HARVESTING

CROSS REFERENCE

[0001] This application claims the benefit of priority of Israeli Patent Application No. 310822, filed on February 13, 2024, the content of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention generally relates to cell harvesting devices and methods. More particularly, the present invention relates to devices and methods for detaching and collecting cells from a cell culture surface.

BACKGROUND

[0003] Many laboratory procedures require the cultivation of tissues for subsequent analysis and diagnostic tests. The tissues are cultivated in tissue culture vessels, such as flasks or petri dishes. Tissue culture vessels are employed by depositing a controlled amount of a liquid growth medium in the vessel. A small sample of the tissue that is to be cultivated then is deposited into the vessel. The vessel is closed by placing the cap over the tubular neck or by placing the top wall across the open top defined by the side walls. The vessel then is stored in an environment that is conducive to tissue growth. Tissue growing in the vessel must be removed and analyzed periodically. In some cell types growing tissue is likely to attach itself to the bottom wall of the vessel, and hence must be scraped from the bottom wall for analysis.

[0004] Cell scrapers are employed for removing tissue from the bottom surface of a tissue culture vessel. The typical cell scraper includes a long thin handle unitarily molded from a rigid plastic material connected to a scraper blade molded unitarily from plastic which includes a planar scraping edge. The dimensions of the blade will vary depending upon the intended application, and specifically in accordance with the size of the tissue culture vessel. [0005] Employing cell scrapers for harvesting of the cell culture from the inner surface of the flask requires gaining access through what is generally a narrow flask opening. Further, there is a need to remove the cells from the surface in such a way that minimal damage occurs to them. Therefore, this process is time consuming and normally therefore requires the expertise of a skilled technician.

[0006] Cell disassociation is typically achieved using proteolytic enzymes such as trypsin. Nevertheless, incubating cells with too high a trypsin concentration for a too long time period, damages cell membranes and may kill the cells. Furthermore, the use of trypsin and other proteolytic enzymes may leave residues in the harvested cells and thus may be forbidden in some use cases. Thus, there is a need for a device and method for harvesting cells from a cell culture surface, without the use of proteolytic enzymes and without damaging the culture.

[0007] The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the figures.

SUMMARY

[0008] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods which are meant to be exemplary and illustrative, not limiting in scope.

[0009] One embodiment provides a device comprising: a device body comprising: a cell collection head; and at least two flow channels, wherein a first inlet of a first one of the at least two flow channels is connectable to a fluid source, and a first outlet of the first one of the at least two flow channels is distally disposed at the cell collection head, wherein an outlet of a second one of the at least two flow channels is proximally disposed at a fluid collection reservoir, and an inlet of the second one of the at least two flow channels is distally disposed at the cell collection head, and wherein the fluid source is at least one of: an internal fluid reservoir and an external fluid reservoir.

[0010] According to some embodiments, the device body is shaped as an arm connected to the cell collection head, extending proximally from the cell collection head to form an integral piece.

[0011] According to some embodiments, the at least two flow channels may be disposed internally within the device body.

The device, according to some embodiments, may further comprise at least one pump fluidly connected to at least one of: said first inlet of said first flow channel and said second outlet of said second flow channel.

[0012] The device may further comprise a control system to control a movement of said cell collection head.

[0013] According to some embodiments, the at least one pump has at least a first operation state, and at least a second operation state, wherein in the first operation state the at least one pump ejects fluid from the fluid source, via the collection head to the cell culture surface, and in the at least second operation state the at least one pump draws fluid from the cell culture surface, via the cell collection head to the fluid collection reservoir.

[0014] The device may further include a control system to control the operation state of the at least one pump.

[0015] According to some embodiments, the device may further comprise a filter configured to separate drawn fluid from cells in the drawn fluid.

[0016] Some aspects of the present invention include a method for harvesting cells, the method may include ejecting a fluid from a first fluid source via a first fluid channel onto a cell culture surface, to detach cells from the cell culture surface; and, drawing the ejected fluid and at least a portion of the detached cells, via a second fluid channel, thereby harvesting cells from the cell culture surface. [0017] The method according to some embodiments may further include controlling an operation of one or more pumps according to at least one of: a predefined protocol, and a criteria, measured in real time.

[0018] According to some embodiments, the predefined protocol comprises one or more of a predetermined flowrate, a predetermined time duration, and the type of fluid.

[0019] According to some embodiments, the criteria measured in real time are selected from a list consisting of: the ejected volume of fluid, the drawn volume of fluid, collected cells count, and a collected cells mass.

[0020] In some embodiments, the predefined flowrate is between 0.3ml/sec and 50ml/sec.

[0021] In some embodiments, the fluid is a liquid, and the predetermined flowrate is selected from a list consisting of: 0.3ml/sec, 0.6ml/sec and Iml/sec. In other embodiments, the fluid is air, and the predetermined flowrate is selected from a list consisting of: 2.5 Iml/sec, 8ml/sec, and 11.41 ml/sec.

[0022] According to some embodiments, the method may further include scraping the cell culture surface with a cell collection head of a cell harvesting device, to further detach cells from the cell culture surface.

[0023] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the figures and by study of the following detailed description.

BRIEF DESCRIPTION OF THE FIGURES

[0024] Exemplary embodiments are illustrated in referenced figures. Dimensions of components and features shown in the figures are generally chosen for convenience and clarity of presentation and are not necessarily shown to scale. The figures are listed below. [0025] Figs. 1 is a schematic illustration of a device for harvesting cells, in accordance with an embodiment; and

[0026] Figs. 2A and 2B are flow charts of methods for harvesting cells, in accordance with some embodiments.

DETAILED DESCRIPTION

[0027] Disclosed herein, are a device and methods for harvesting cells. The devices and methods described herein may be utilized to lifting, retrieving, and In some embodiments, collecting cells and /or fluids (e.g., liquids) from a cell culture surface. Advantageously, the device and method may facilitate lifting of cells by applying a suitable shear force to maintain acceptable cell viability. The acceptable cell viability may be regarded as at least 70%. According to some embodiments, the device and method may facilitate lifting cells without contact between the device and the cell culture surface, by applying a fluid (e.g., air, water, etc.) within a predefined flowrate to the cell culture surface and collecting cells/fluids therefrom.

[0028] As used herein, a cell is defined as the basic structural and functional unit of all forms of life.

[0029] The device and method disclosed herein may offer significant advantages over prior cell harvesting techniques. By utilizing controlled fluid dynamics and mechanical action, this approach may enable efficient cell detachment and collection without relying on chemical agents such as trypsin or other proteolytic enzymes. Omitting the use of such chemicals eliminates potential risks associated with residual enzymatic activity, which may damage cell membranes, alter cellular properties or damage to the administrated tissue. This chemical-free method may preserve cell viability and maintain the native characteristics of harvested cells, which is particularly crucial for sensitive cell types or downstream applications requiring unaltered cellular states. Additionally, the absence of chemical reagents may simplify regulatory compliance and reduce costs associated with specialized reagents. The precise control over fluid flow rates and mechanical forces provided by this device may allow for gentler cell harvesting, potentially increasing yield and reducing cellular stress compared to traditional enzymatic methods. This approach may be especially beneficial for applications in regenerative medicine, cell therapy, and other fields where maintaining cellular integrity and function is paramount

[0030] As used herein, the term "cell culture surface" refers to any type of surface to which cells are at least partially being attached or adhered (i.e. a cell binding / attachment to the surface) while maintaining vitality of the adherent and/or growing cell. Suitable cell culture surfaces allow proliferation and growth of adherent cells. Non-limiting examples of suitable cell culture surfaces include plastic or coated plastic (e.g., coated with collagen), methylcellulose, organic and inorganic polymers and nano particles surfaces of: slides, cell culture plates, multi-well plate (e.g., 2-well plates, 6-well plates, 24-well plates, 48- well plates, 96-well plates, etc.), petri dishes and flasks.

[0031] The term "adherent cell” refers to a cell, such as a prokaryotic or eukaryotic cell, that remains associated with, immobilized on, or in certain contact with the cell culture surface. Such type of cells after culturing can withstand or survive washing and medium exchanging process, a process that is prerequisite to many cell-based cultures and /or assays.

[0032] The device of the invention may advantageously be used in automated systems, such as an element (either integral or detachable) within cell culture systems/bioreactors.

[0033] Reference is now made to Fig. 1 which shows a device 100 that may be used for lifting and collecting cells and /or liquids from a cell culture surface, in accordance with an embodiment. The cells/liquids may be collected from flat surfaces such e.g. in tissue culture flasks, chambers or trays as well as from the surface of 3-dimentional particles such as beads and scaffolds. In a nonlimiting example, the scaffolds can be printed, chemically designed etc. [0034] To facilitate the description of device 100, three orthogonal axes are indicated in Fig. 1. The axis labelled 'longitudinal axis' refers to a central axis that runs along the length of an arm 102 of device 100 from a proximal end 104 to a distal end 106 of a cell collection head 108 extending from arm 102. The axis labelled 'vertical axis' runs along the width of cell collection head 108 from a first side 110 to a second side 111 of cell collection head 108. The axis labelled 'lateral axis' indicates the “thickness” (also "depth") of device 100 and is perpendicular to both the vertical and the longitudinal axes.

[0035] Device 100 may include at least two flow channels 112, and cell collection head 108 (e.g., blade). In some embodiments, device 100 may further include arm 102 connected to and extending proximally from cell collection head 108 to form a device body 101, such that a movement by arm 102 translates to a corresponding motion of cell collection head 108. In some embodiments, arm 102 is rigidly connected to and extending proximally from cell collection head 108. In some embodiments, the connection is a detachable connection. In some embodiments, arm 102 is equipped with a joint (not shown) to allow flexibility of arm 102, e.g., with respect to collection head 108. In some embodiments, arm 102 is flexible and an orientation of a distal portion of arm 102 may be adjusted relative to an orientation of a proximal portion of arm 102 along the longitudinal and/or vertical and/or lateral axes thereby affecting the orientation of distally disposed cell collection head 108 relative to the proximal portion of arm 102.

[0036] In some embodiments, at least one of cell collection head 108 and arm 102 houses at least a portion of at least two flow channels 112. In some embodiments, at least a portion of at least two flow channels 112 is disposed internally within device body 101. In some embodiments, at least two flow channels 112 are disposed internally within device body 101.

[0037] In some embodiments, at least two flow channels 112 may include a first flow channel 112a and second flow channel 112b. In the nonlimiting example illustrated in Fig. 1 first flow channel 112a is for providing fluid to the cell culture, and second flow channel 112b is for collecting fluid/cells from the cell culture. A first inlet 114 of first flow channel 112a of at least two flow channels 112 may be proximally disposed at arm 102, and a first outlet 116 of first flow channel 112a may be distally disposed at cell collection head 108. A second outlet 118 of a second flow channel 112b of at least two flow channels 112 may be proximally disposed at arm 102, and a second inlet 120 of second flow channel 112b may be distally disposed at cell collection head 108.

[0038] In some embodiments, first outlet 116 and second inlet 120 may have a size (e.g., orifice) of between 15G to 34G, for example, 15G, 20G, 21G, 24G, 25G, 28G, 30G, 33G, 34G, and any value or range in between. In some embodiments, at least second inlet 120, may have at least 20% larger than a size of a cell, for example, at least 20% larger than 25 pm. In a nonlimiting example, size (e.g., internal diameter may be between 30 pm, to 200 pm.

[0039] In some embodiments, outlet 116 of first flow channel 112a and/or inlet 120 of second flow channel 112b may be disposed on a distal collection surface 122 of cell collection head 108. Distal collection surface 122 may be designed for lifting cells from a cell culture surface. In some embodiments, distal collection surface 122 may be configured to scrape the culture surface. Therefore, cell collection head 108 may be a scraper configured to physically detach cells from a cell culture surface, with or without the use of fluids, such as air, saline, and the like.

[0040] According to some embodiments, head 108 may not require actual contact with the cell culture surface in order to detach cells from the surface and collect them, solely by applying a fluid in a predefined flowrate, such as, for example, liquid (e.g., saline) at a flowrate of 0.3ml/sec, 0.6ml/sec, Iml/sec, 2ml/sec, 4ml/sec or even up to 10 ml/sec or air in a flowrate of 2 ml/sec, 2.5 Iml/sec, 8ml/sec, 11.41 ml/sec, 13ml/sec, 15ml/sec, 41 ml/sec, and up to 50ml/sec, for a predefined duration, such as for example, 15 seconds, 30 seconds, 45 seconds, 60 seconds, 90 seconds, 120 seconds, etc.

[0041] In a first nonlimiting example, the fluid is a liquid (e.g., an aqueous solution, such as, saline (e.g., Phosphate buffered saline (PBS), serum free medium, etc.), and the predetermined flowrate is between 0.3ml/sec to 4 ml/sec, and any value or range in between. In a second nonlimiting example, the fluid is air, and the predetermined flowrate is between 1 ml/sec to 50 ml/sec, and any value or range in between.

[0042] According to some embodiments fluid may be applied from one or more first outlets 116 and may be collected from one or more second inlets 120. According to some embodiments, fluid may be applied from the center of collection head 108 and/or from one or more sides of collection head 108 (e.g., from outlets 116 disposed at the sides of collection head 108). According to some embodiments, the controller may control from which outlets 116 fluid may be applied, and from which inlets 120 fluid and cells will be collected. According to some embodiments, this may be done based on the type of cells, the required flow rate and the like.

[0043] In some embodiments, two or more first outlets 116 and two or more of second inlets 120 may be arranged in an alternating arrangement. This configuration may allow for more efficient and uniform fluid distribution and cell collection across the cell culture surface. By alternating first outlets 116 and second inlets 120, for fluid ejection and collection, the device may create a more balanced and effective harvesting process. The alternating arrangement may help minimize unreachable zones or areas of uneven fluid flow, potentially improving the overall efficiency of cell detachment and collection. Additionally, this configuration may enable more precise control over the fluid dynamics at the cell culture surface, allowing for optimization of shear forces and fluid exchange rates. [0001] In some embodiments, device 100 may further include one or more fluid reservoirs 124 for storing fluids collected via first flow channel 112a and/or fluids intended for distribution via second flow channel 112b. In some embodiments, one or more fluid reservoirs 124 include a first fluid reservoir 124a for storing fluid (e.g., washing fluids and feeding fluids) intended for distribution onto the cell culture surface via first flow channel 112a; and a second fluid reservoir 124b for storing fluids collected via second flow channel 112b. One or more fluid reservoirs 124 are fluidly connected to at least one of at least two flow channels 112. In some embodiments, one or more fluids reservoirs 124 are fluidly connected to proximally disposed inlet 114 and/or proximally disposed outlet 118. Fluids may enter and/or exit one or more fluids reservoirs 124 sequentially and/or simultaneously. In some embodiments, the fluids are selected from liquid, gas, and a combination thereof. In some embodiments, the fluids further comprise particles. In some embodiments, the fluids further comprise cells and/or components thereof.

[0002] In some embodiments, device 100 may further include at least one pump 130 fluidly connected to at least one of: proximally disposed inlet 114 and proximally disposed outlet 118 for pumping fluids into and/or from device 100, respectively. In a non-limiting example fluids pumped from first fluids reservoir 124a are distributed onto a cell culture surface via distally disposed outlet 116 of first fluid channel 112a. In another non-limiting example, fluids pumped from a cell culture surface via distally disposed inlet 118 of second fluid channel 112b are collected into second fluids reservoir 124b. In some embodiments, second reservoir 124b may be equipped with a filter (not shown) to separate collected cells from drawn fluid, e.g., to prevent cells from entering second fluid reservoir 124b. It should be appreciated by those skilled in the art, that the cells separated from the fluid by the filter, may be returned to a new cell culture surface to grow a new line of cells. It should be further appreciated that the fluid separated from the cells may be analyzed and/or may be reused for further cycles of cell harvesting from the same or another cell culture surface.

[0044] In some embodiments, device 100 may further include a control system 140. The control system may regulate movement of arm 102 to induce a corresponding movement of cell collection head 108. In some embodiments, the control system may control the pump to regulate pumping of fluids into and/or from one or more fluids reservoirs 124 via one or more flow channels 112.

[0045] In some embodiments, at least one pump 130 may have at least a first operation state, and at least a second operation state, wherein in the first operation state at least one pump 130 ejects fluid from fluid source (e.g., first fluids reservoir 124a), via collection head 108 to the cell culture, and in the at least second operation state at least one pump 130 may draw fluid from the cell culture, via the cell collection head to a fluid collection reservoir (e.g., second fluids reservoir 124b). In some embodiments, the pump may work in one or more operation cycles. Each operation cycle may include first operation state and the second operation state. In some embodiments, a controller 140 may control the number of operation cycles. For example, 2, 3, 4, 5, 6, or more operation cycles may be conducted by pump 130 at each harvesting session.

[0046] In some embodiments, controller 140 may control the operation state of at least one pump 130. For example, controller 140 may control the operation duration of the at least second operation state. In some embodiments, the duration may be between 5 to 30 seconds and any value or range in between, for example, 5 sec, 10 sec, 15 sec, 20 sec, 25 sec, 30 sec, or more, as discussed in the Examples section, hereinbelow.

[0047] According to some embodiments, control system 140 may comprise a controller or processor (not shown), in active communication with one or more pumps 130, and may control the operation of one or more pumps 130 according to a predefined protocol (e.g., for a predefined time period and/or in a predefined flowrate), or may control the operation of one or more pumps 130 according to measured criteria, in real time, such as, for example, an expelled volume of fluid, a collected volume of fluid, collected cells count, a collected mass of cells, and the like.

[0048] Reference is now made to Fig. 2A, which is a flow chart of a method for using the device of Fig. 1 for lifting and collecting cells (also referred to as harvesting cells) from a cell culture surface in accordance with an embodiment of the invention.

[0049] In step 2010, a fluid, such as air, saline, or any other suitable liquid or gas, may be ejected from a fluid source to a cell culture surface, from which cells are to be harvested. The fluid source may be a fluid reservoir internal to a cell harvesting device or may be an external fluid source or reservoir. According to some embodiments, the fluid source may be connected or connectable to a fluid channel having an outlet at a cell collection head (108 in Fig. 1) of a cell harvesting device such as device 100 in Fig. 1.

[0050] According to some embodiments, the ejection of fluid from the fluid source to the cell culture surface, may include controlling, by a controller, one or more pumps (130 in Fig. 1). According to some embodiments, the control of the one or more pumps may be according to at least one of: a predefined protocol, and a criteria, measured in real time. [0051] According to some embodiments, the predefined protocol may include one or more of a predetermined flowrate, a predetermined time duration, and the type of fluid. According to some embodiments, the predefined flowrate is between 0.3ml/sec and 50ml/sec. According to other embodiments, the fluid is a liquid, and the predetermined flowrate is between 0.3ml/sec and lOml/sec. For example, selected from a list consisting of: 0.3ml/sec, 0.6ml/sec and Iml/sec. In yet other embodiments, the fluid is air, and the predetermined flowrate is between 2ml/sec and 50ml/sec. For example, selected from a list consisting of: 2 ml/sec, 2.51ml/sec, 8ml/sec, 11.41 ml/sec, and 41 ml/sec.

[0052] According to some embodiments, the criteria measured in real time may be selected from a list consisting of: the ejected volume of fluid, the drawn volume of fluid, collected cells count, and a collected cells mass.

[0053] According to some embodiments, the protocol may further include scraping the cell culture surface with a cell collection head of a cell harvesting device, to further detach cells from the cell culture surface.

[0054] In step 2020, the ejected fluid may be drawn back, e.g., through a fluid inlet at cell collection head and via a fluid channel, the drawn fluid, including cells detached from cell culture surface, by the ejected fluid and/or by the scraping of the surface by the cell collection head, may be collected to a reservoir such as reservoir 124b.

[0055] According to some embodiments, drawing of fluid from the cell culture surface, may include controlling, by a controller, one or more pumps (130 in Fig. 1). According to some embodiments, the control of the one or more pumps may be according to at least one of: a predefined protocol, and a criteria, measured in real time, mutatis mutandis, as described above for ejected fluids.

[0056] For example, the predefined protocol for drawing fluid from the cell culture surface may include, starting drawing a predefined time after ejection of fluid has initiated. According to some embodiments, the drawing may be initiated after a predefined volume of fluid has been ejected or may start and operate in parallel with the ejection of fluid. The drawing of fluid and cells may be ceased when a stop condition has been reached, such as for example, a predefined volume of fluid and cells have been drawn, the collected fluid reservoir (124b) is full, a predefined cell mass has been collected or the like.

[0057] Reference is now made to Fig. 2B which illustrates a method according to some embodiments. As seen in Fig. 2B a distal portion of distally disposed cell collection head 108 of device body 101 is placed onto a cell culture surface allowing distal collection surface 122 to contact the cell culture surface (250) or is brought to proximity with the surface 122 without actual contact. A fluid is introduced into first inlet 114 of first fluid channel 112a disposed with proximally disposed arm 102 of device body 101 (252). In some embodiments, the fluid is introduced from first fluid reservoir 124a. The fluid is ejected from first fluid channel 112a via first outlet 116 disposed on distally disposed cell collection head 108 of device body 101 (254). In some embodiments, the ejected fluid may induce a shear force suitable for maintaining cell viability and loosening cell adherence. In some embodiments, the ejected fluid may loosen cells adherence to a cell culture surface. Proximally disposed arm 102 which is rigidly connected to cell collection head 108 may be moved in a direction parallel to the plane of the cell culture surface (256). The connection between arm 102 and cell collection head 108 is used to transfer the movement of arm 102 to a corresponding motion of cell collection head 108 on or in proximity to the cell culture surface to lift the cells from the cell culture surface (258). In some embodiments, the motion of cell collection head 108 is suitable for lifting cells while maintaining cell viability. In some embodiments, the motion facilitates the lifting of the cells from the cell culture surface by distal collection surface 122. In some embodiments, the lifted cells assimilate in the fluid. In some embodiments, the motion is across the lateral axis, the vertical axis, and/or a rotational movement. The fluid is drawn into second inlet 120 of second fluid channel 112b disposed with distally disposed cell collection head 108 of device body 101 (260). The drawn fluid departs from second fluid channel 112b via second outlet 118 proximally disposed at arm 102 (262). In some embodiments, the drawn fluid is collected into second reservoir 124b (262).

[0058] In some embodiments, steps 254 and 260 may be performed in a sequential manner and/or at least partially simultaneously. In some embodiments, applying steps 254 and 260 simultaneously may result in circulation motion of the fluid which may facilitate a release of the cells from the cell culture surface. In some embodiments, each of steps 254, 258 and 260, may be performed independently. In some embodiments, steps 254, 258 and 260, may be performed simultaneously or in any order. In some embodiments, steps 258 and 260 may be performed in a sequential manner and/or at least partially simultaneously. In some embodiments, each of steps 254 and 260 may be performed one or more times in any order prior to step 258.

[0059] In some embodiments, the control system may regulate performance of each of the steps. In some embodiments, the control unit may regulate ejection and drawing of fluid as well as the motion of cell collection head to maintain cell viability.

[0060] In some embodiments, control unit may operate the first and second flow channels to apply an optimal shear force suitable for maintaining cells viability. A person skilled in the art would appreciate that the optimal shear force depends on the cell type. Further the optimal shear force applied may depend on the type of the desired biological function (e.g., differentiation, migration, etc.).

[0061] The present invention may be a system, a method, and/or a computer program product. The computer program product may include a computer readable storage medium (or media) having computer readable program instructions thereon for causing a processor to carry out aspects of the present invention.

[0062] The computer readable storage medium can be a tangible device that can retain and store instructions for use by an instruction execution device. The computer readable storage medium may be, for example, but is not limited to, an electronic storage device, a magnetic storage device, an optical storage device, an electromagnetic storage device, a semiconductor storage device, or any suitable combination of the foregoing. A non- exhaustive list of more specific examples of the computer readable storage medium includes the following: a portable computer diskette, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), a static random access memory (SRAM), a portable compact disc read-only memory (CD-ROM), a digital versatile disk (DVD), a memory stick, a floppy disk, a mechanically encoded device such as punch-cards or raised structures in a groove having instructions recorded thereon, and any suitable combination of the foregoing. A computer readable storage medium, as used herein, is not to be construed as being transitory signals per se, such as radio waves or other freely propagating electromagnetic waves, electromagnetic waves propagating through a waveguide or other transmission media (e.g., light pulses passing through a fiber-optic cable), or electrical signals transmitted through a wire.

[0063] Computer readable program instructions described herein can be downloaded to respective computing/processing devices from a computer readable storage medium or to an external computer or external storage device via a network, for example, the Internet, a local area network, a wide area network and/or a wireless network. The network may comprise copper transmission cables, optical transmission fibers, wireless transmission, routers, firewalls, switches, gateway computers and/or edge servers. A network adapter card or network interface in each computing/processing device receives computer readable program instructions from the network and forwards the computer readable program instructions for storage in a computer readable storage medium within the respective computing/processing device.

[0064] Computer readable program instructions for carrying out operations of the present invention may be assembler instructions, instruction-set-architecture (ISA) instructions, machine instructions, machine dependent instructions, microcode, firmware instructions, state-setting data, or either source code or object code written in any combination of one or more programming languages, including an object oriented programming language such as Java, Smalltalk, C++ or the like, and conventional procedural programming languages, such as the "C" programming language or similar programming languages. The computer readable program instructions may execute entirely on the user's computer, partly on the user's computer, as a stand-alone software package, partly on the user's computer and partly on a remote computer or entirely on the remote computer or server. In the latter scenario, the remote computer may be connected to the user's computer through any type of network, including a local area network (LAN) or a wide area network (WAN), or the connection may be made to an external computer (for example, through the Internet using an Internet Service Provider). In some embodiments, electronic circuitry including, for example, programmable logic circuitry, field- programmable gate arrays (FPGA), or programmable logic arrays (PLA) may execute the computer readable program instructions by utilizing state information of the computer readable program instructions to personalize the electronic circuitry, in order to perform aspects of the present invention.

[0065] Aspects of the present invention are described herein with reference to flowchart illustrations and/or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of the invention. It will be understood that each block of the flowchart illustrations and/or block diagrams, and combinations of blocks in the flowchart illustrations and/or block diagrams, can be implemented by computer readable program instructions.

[0066] These computer readable program instructions may be provided to a processor of a general-purpose computer, special purpose computer, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, create means for implementing the functions/acts specified in the flowchart and/or block diagram block or blocks. These computer readable program instructions may also be stored in a computer readable storage medium that can direct a computer, a programmable data processing apparatus, and/or other devices to function in a particular manner, such that the computer readable storage medium having instructions stored therein comprises an article of manufacture including instructions which implement aspects of the function/act specified in the flowchart and/or block diagram block or blocks.

[0067] The computer readable program instructions may also be loaded onto a computer, other programmable data processing apparatus, or other device to cause a series of operational steps to be performed on the computer, other programmable apparatus or other device to produce a computer implemented process, such that the instructions which execute on the computer, other programmable apparatus, or other device implement the functions/acts specified in the flowchart and/or block diagram block or blocks.

[0068] The flowchart and block diagrams in the Figures illustrate the architecture, functionality, and operation of possible implementations of systems, methods, and computer program products according to various embodiments of the present invention. In this regard, each block in the flowchart or block diagrams may represent a module, segment, or portion of instructions, which comprises one or more executable instructions for implementing the specified logical function(s). In some alternative implementations, the functions noted in the block may occur out of the order noted in the figures. For example, two blocks shown in succession may, in fact, be executed substantially concurrently, or the blocks may sometimes be executed in the reverse order, depending upon the functionality involved. It will also be noted that each block of the block diagrams and/or flowchart illustration, and combinations of blocks in the block diagrams and/or flowchart illustration, can be implemented by special purpose hardware -based systems that perform the specified functions or acts or carry out combinations of special purpose hardware and computer instructions.

Examples

[0069] Several cell harvesting experiments were performed using a device, such as, device 100 and the methods disclosed hereinabove. In all the cell harvesting experiments, the control included harvesting using scrapping only, i.e., mechanical scraping of the cultured cells surface. Two parameters were tested, yield (%) in comparison to the control and viable cells (%).

[0070] In all experiments the cell culture surface includes Microglia mesenchymal model Cell-line, and DMEM medium, Pen-strep (wt.1%) and Fetal bovine serum (10 wt.%). The harvesting assays were conducted using two types of vessels, a 6-well plate, and a T75 flask, as known in the art. Several needle’s Gauges were applied ranging from 21G to 30G. Each tested process was done by applying repeating short pulses wherein the duration of each pulse was 5, 10, 15 and 20 seconds to a total of 5, 10, 30, 45, 60 and 120 seconds where liquid was used and short pulses of 5, 10, 15 and 20 seconds to a total of 15, 30, 45, 60, 120 and 240 seconds.

[0071] Cells that were cultured in the 6-well plate were collected to a 15 ml conical tube, and cells that were cultured in the T75 flask were collected to a 50 ml conical tube.

Example 1- Liquid aided harvesting

[0072] Three setups were tested, A) control- scrapping at different time durations, B) injecting PBS liquid and drawing the ejected liquid, and C) combining scrapping and liquid injection and drawing.

[0073] Table 1 summarizes the results of Example No. 1:

[0074] Where N in the number of conducted tests.

[0075] The three yield categories are defined in comparison to the control-based. 1) below 75% was defined as insufficient yield, 2) between 75% to 125%, was defined as comparable to the control, and 3) above 125% was defined as better than the control. [0076] With respect to viability a value of 80%, was defined as a cutoff value, wherein below this cutoff value was considered insufficient, while above 80% was considered as sufficient.

[0077] As shown, the combination of liquid injection and scraping in both G21 and G25 needles but also liquid only at 25G needle gave the best yield results. In some experiments, the yield was higher than the yield of the control, having yield higher than 125% of the control. With respect to viability of collected cells, all tested methods provided sufficient results (e.g., between 80% and 100%).

[0078] When the duration of each harvesting act was tested, at least 30 second of injection and drawing of liquids were required when only liquid was used for harvesting. The yield was increased when the injection and drawing lasted for 45 and 50 sec, in both the 6-well plate, and the T75 flask. When combined with mechanical scrapping, the optimal duration was found to be between 15 to 45 sec.

Example 2- Air aided harvesting

[0079] Three scenarios were tested, A) control- scrapping at different time durations, B) injecting air and drawing the cells, and C) combining scrapping and air injection and drawing.

[0080] Table 2 summarizes the results Example No. 2.

Where N in the number of conducted tests.

[0081] The three yield categories are defined in comparison to the control -based. 1) below 75% was defined as insufficient yield, 2) between 75% to 125%, was defined as substantially the same as the control, and 3) above 125% was defined as better than the control.

[0082] With respect to viability a value of 80%, was defined as a cutoff value, wherein below this cutoff value was considered insufficient, while above 80% was considered as sufficient. When using air, the best yield results were obtained when only air and syringe 25G were used in harvesting cells from 6 wells. The combination of air and scrapping using both 25G and 30G needles also had acceptable yields and acceptable Viability.

[0083] When the duration of the air injection was tested, the optimal duration was found to be between 15 to 45 sec. Applying the air for 120 sec or more using 25G needle, resulted in poorer yields and viability.

[0084] The descriptions of the various embodiments of the present invention have been presented for purposes of illustration but are not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terminology used herein was chosen to best explain the principles of the embodiments, the practical application or technical improvement over technologies found in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

CLAIMS What is claimed is:

1. A device for harvesting cells from a cell culture surface comprising: a device body comprising: a cell collection head; and at least two flow channels, wherein a first inlet of a first flow channel of the at least two flow channels is connectable to a fluid source, and a first outlet of the first flow channel is distally located at the cell collection head, wherein an outlet of a second flow channel of the at least two flow channels is proximally located at a fluid collection reservoir, and a second inlet of the second channel is distally located at the cell collection head, and wherein the fluid source is at least one of: an internal fluid reservoir and an external fluid reservoir.

2. The device of claim 1, wherein the body is shaped as an arm connected to the cell collection head, extending proximally from the cell collection head to form an integral piece.

3. The device of any one of claims 1 or 2, wherein the at least two flow channels are disposed internally within the device body.

4. The device of any one of the preceding claims, comprising two or more first outlets and two or more of second inlets arranged in an alternating arrangement.

5. The device of any one of the preceding claims, wherein the second inlet has a size of between 15G to 34G.

6. The device of any one of the preceding claims, wherein the fluid is selected from, air, and an aqueous solution.

7. The device of any one of the preceding claims, further comprising at least one pump fluidly connected to at least one of: said first inlet of said first flow channel and said second outlet of said second flow channel.

8. The device of any one of claims 2 to 7, further comprising a control system to control a movement of said cell collection head.

9. The device according to claim 7, wherein the at least one pump has at least a first operation state, and at least a second operation state, wherein in the first operation state the at least one pump ejects fluid from the fluid source, via the collection head to the cell culture surface, and in the at least second operation state the at least one pump draws fluid from the cell culture surface, via the cell collection head to the fluid collection reservoir.

10. The device of claim 9 further comprising a control system to control the operation state of the at least one pump.

11. The device of claim 10, wherein the control system controls the operation duration of the at least second operation state and the number of operation cycles.

12. The device of claim 11, wherein the duration is between 5 to 30 seconds.

13. The device of any one of the preceding claims, wherein the fluid is a liquid, and the predetermined flowrate is selected from a list consisting of: 0.3ml/sec, 0.6ml/sec and Iml/sec.

14. The device of any one of claims 1 to 12, wherein the fluid is air, and the predetermined flowrate is selected from a list consisting of: 2.5 Iml/sec, 8ml/sec, and 11.41 ml/sec.

15. The device according to any one of the preceding claims, further comprising a filter configured to separate drawn fluid from cells in the drawn fluid.

16. A method for harvesting cells, comprising: ejecting a fluid from a first fluid source via a first fluid channel onto a cell culture surface, to detach cells from the cell culture surface; and, drawing the ejected fluid and at least a portion of the detached cells, via a second fluid channel, thereby harvesting cells from the cell culture surface.

17. The method according to claim 16, wherein ejecting the fluid comprises controlling an operation of one or more pumps according to at least one of: a predefined protocol, and one or more criteria, measured in real time.

18. The method according to claim 17, wherein the predefined protocol comprises one or more of a predetermined flowrate, a predetermined time duration, and the type of fluid.

19. The method according to claim 17, wherein the one or more criteria measured in real time is selected from a list consisting of: the ejected volume of fluid, the drawn volume of fluid, a collected cells count, and a collected cells mass.

20. The method according to claim 18, wherein the predefined flowrate is between 0.3ml/sec and 50ml/sec.

21. The method according to claim 18, wherein the fluid is a liquid, and the predetermined flowrate is selected from a list consisting of: 0.3ml/sec, 0.6ml/sec and Iml/sec.

22. The method according to claim 18, wherein the fluid is air, and the predetermined flowrate is selected from a list consisting of: 2.51ml/sec, 8ml/sec, and 11.41 ml/sec.

23. The method according to any one of claims 16-22 further comprising scraping the cell culture surface with a cell collection head of a cell harvesting device, to further detach cells from the cell culture surface.