HK1163569B - Processing blood - Google Patents

Description

Treating blood

Cross reference to related patent application

This application claims priority to U.S. provisional application No. 61/140,196, filed on 23/12/2008, which is hereby incorporated by reference in its entirety.

Technical Field

The present invention relates generally to methods and devices for processing blood, and more particularly, to methods and apparatus for leukocyte isolation.

Background

Blood cells are continuously produced during the life of an individual and are derived from the most primitive blood cells, the so-called Hematopoietic Stem Cells (HSCs). Hematopoietic stem cells are capable of producing Hematopoietic Progenitor Cells (HPCs) and blood cells of different cell types, such as Red Blood Cells (RBCs) and white blood cells or White Blood Cells (WBCs), and are often found in the bone marrow. More mature blood cell types are present in blood and lymphoid tissues. Hematopoiesis is the continuous production of blood cells from HSCs and HPCs in an individual. This will generate peripheral blood with a variety of different types of blood cells belonging to different myeloid and lymphoid lineages and having different degrees of maturity. These blood cells are responsible for physiological processes such as oxygen transport by red blood cells, immune function by dendritic cells, B and T lymphocytes, and inflammatory responses by granulocytes and macrophages.

Apheresis is a medical procedure in which an individual's blood is passed through a device to obtain the principal components (e.g., monocytes) and return the other components to the blood circulation. The isolation is typically a three-step process comprising: (1) taking blood from an individual; (2) separating blood components (e.g., based on density); and (3) returning certain components of the blood to the individual. Blood is generally divided into three parts: RBC (about 45% of total blood), "buffy coat" (less than 1% of total blood), and plasma (about 55% of total blood). Different types of blood apheresis procedures can be employed depending on the blood component to be removed. For example, "plasmapheresis" generally refers to the separation and collection of blood plasma; "platelet apheresis" refers to the separation and collection of platelets; whereas "leukapheresis" generally refers to the isolation and collection of White Blood Cells (WBCs).

As a result of the development of medical science, the isolation can be connected to the patient and performed in a closed loop continuous flow manner. The apparatus used for this purpose comprises, for example, the following dissociation systems: COBE (chip on Board)Spectra, Trima, Spectra Optia systems (all sold by Gambro BCT) and Amicus and CS-3000+ (sold by Fenwal/Baxter).

Recently, leukapheresis has also been used to collect certain fractions of blood mononuclear cells (MNC) for use in bone marrow transplantation and other disease areas. For example, patients who have undergone resection to treat malignancy can be infused with large quantities of donor mononuclear cells (those present in peripheral blood, also known as peripheral blood progenitor cells or PBPCs) containing HSCs and HPCs to subsequently reconstitute their hematopoietic system. In this example, the buffy coat (containing the majority of WBCs (granulocytes, lymphocytes, monocytes), PBPCs, and a portion of platelets) is first collected while the remaining components of the blood (including plasma, RBCs, platelets, and a portion of WBCs) are returned to the individual. PBPC was then enriched and isolated, while the remainder of the buffy coat (constituting approximately 99% of the buffy coat) was discarded. This process of cell enrichment (i.e., cell isolation and purification) is today performed in a patient-free manner, using equipment separate from those of the isolation machines. Devices for this purpose include, for example, Baxter Isolex 300i and Miltenyi CliniMACS, which enrich PBPCs on the cell surface based on specific ligands (CD34, both available and CD133 available in Miltenyi). Such as Gambro COBE 2991 blood cell processor or Baxter Cytomate^TMOther self-contained devices such as cell washing systems are commonly used to wash, concentrate or place cells in a suitable growth or immersion medium.

In another application, leukapheresis may be used to treat WBCs in an individual in a process known as photopheresis (Edelson et al, Yale J Biol Med.1989 Nov-Dec; 62 (6): 565-77). In this procedure, the subject first receives a dose of photoactivatable substance (e.g., 8-methoxy bone supplement)Lipin). And then performing an isolation process in which WBCs of the individual are irradiated with ultraviolet a (uva) light to activate the above substances and inhibit metabolic processes of the WBCs. Apparatus for this purpose include, for example, UVAR and UVARXTS^TMA photopheresis system (sold by Therakos).

In addition to the enrichment process described above, the collected PBPC can be modified in another process prior to re-injection back into the subject. This is typically achieved by using a variety of techniques in cell culture. Finally, the modified cells (e.g., altered phenotype, genotype, or activity) can be reintroduced into the patient to achieve certain therapeutic benefits. Examples of modification procedures include the preparation of HSC/HPC containing anti-HIV genes (R.G.Amado. et al, Human Gene Therapy 15(2004), 251-262) and the production of cytotoxic T lymphocytes that are "educated" to target and kill specific tumors.

FIG. 1 shows a schematic block diagram of one prior art method of taking blood from a patient, processing, and returning it to the patient. Here, the arrangement 100 of the apparatus may be used sequentially to perform cell collection and cell enrichment techniques using leukapheresis, such as cell washing, purification. The block diagram also lists optional cell modifications. Exemplary equipment that can be used in the present method includes Cobe Spectra equipment. Such devices 110 are used for leukapheresis (collection), in turn with a cell washing device 120 (enrichment), such as a Baxter CytoMate device; the cell washing apparatus is in turn used with a cell purification (enrichment) apparatus 130 such as a Baxter Isolex 300i apparatus. In addition, cell manipulation (modification) devices 140 can be used, including but not limited to: electroporation, liposome infection, viral transduction, light (UVA, UVB, etc.), drug addition, cell activation, pressurization, heating functions, and the like. The processed blood cell packets 150 output by the devices 110, 120, 130 and 140 are provided to the patient 160 for return of the processed blood.

Fig. 2 shows a specific example of a method 300 for cell collection, enrichment, and modification. This example is a method for introducing an anti-HIV gene into CD34+ HSC/HPC, wherein over a 5 day cycle:

in step 310, monocytes are collected, i.e. harvested, by leukapheresis. In this step 310, the other blood cell components, i.e., red blood cells, platelets, plasma, and polymorphonuclear cells, are returned to the patient.

In step 320, the mononuclear cell fraction is washed (day 2) using, for example, CytoMate (see above); target CD34+ cells are enriched (day 2) using, for example, an Isolex 300i device, and non-CD 34+ cells are discarded.

In step 330, CD34+ cells were cultured in the presence of cytokines (day 2) and anti-HIV genes (ribozymes against conserved regions of the tat/vpr gene) were introduced using murine retroviruses (day 4).

After step 330, a product release test is performed (day 5) and the cells are injected into the same individual that was originally leukapheresis.

However, the separation has inherent drawbacks and disadvantages, e.g., the separation is simply a liquid component collection process. Despite the advances in technology, the complex steps of collection, enrichment, and (optionally) modification of target blood cells are still performed by using separate continuous or discontinuous devices, as described previously. Among these steps, only collections are connected to the patient today; in only one example, the collection and modification (photopheresis) is patient-attached. These present non-continuous processes are time consuming, wasteful of materials, labor and cost (J.Gryn et al, Journal of Heamatotherpy & Stem Cell Research 11(2002), 719-. These procedures also cause serious concerns such as the following: (i) safety issues arising from potential microbial infections; and (ii) chain of custody issues arising from cell selection and modification assurance systems (i.e., ensuring that the correct cells are returned to the patient and maintaining cell integrity). For example, hemolysis is a rare complication due to kinking of the edge line of the apheresis collection cassette (r. reddy, transfer and apheresis Science 32(2005) 63-72).

To further illustrate, thrombocytopenia (platelet depletion) is a well-known adverse consequence of leukocyte depletion and is the most frequently reported side effect of leukocyte depletion in children (J. Sevilla et al, transfer and Apheresis Science 31(2004) 221-. Thrombocytopenia is of paramount importance because patients often exhibit thrombocytopenia due to their underlying disease and produce additional platelet loss upon leukapheresis. Ideally, in individuals with an insufficient number of platelets due to a disease state, the platelets isolated in the buffy coat should be isolated and returned to the individual. However, in practice the isolated platelets are simply discarded as waste. In addition, the reduction of platelets in the buffy coat has the additional beneficial effect of increasing the efficiency of immunoaffinity selection of CD34+ progenitor cells (a PBPC) by an average of 1.8-fold (R. Moog, transfer and Apheresis science 31(2004) 207-220).

Another drawback of the leukapheresis process is the loss of useful lymphocytes for some patients. As previously mentioned, HSCs and HPCs (particularly CD34+ progenitor cells) are typically selected for reconstitution of an individual's hematopoietic system. For individuals infected with Human Immunodeficiency Virus (HIV), the use of leukapheresis to select for CD34+ progenitors lost a small number of useful lymphocytes (e.g., CD3+ and CD4+ cells) that they already have in their body. About 1.3% of CD34+ progenitor cells after mobilization are among the smallest cellular components collected during PBPC leukocyte isolation, while lymphocytes and monocytes account for up to 70% of the isolation products (V.Witt et al, Journal of Clinical Apheresis 16(2001) 161-168).

There is a need for an apparatus that overcomes or at least ameliorates one or more deficiencies in existing systems, including those described above.

Disclosure of Invention

According to one aspect of the present invention, a device for treating blood is provided. The device includes: an inlet interface for connecting to a patient to receive blood directly from the patient's blood circulation; a leukocyte isolation module interfaced with the inlet for collecting a batch of mononuclear blood cells from the received blood; an enrichment module coupled to the leukapheresis module for simultaneously enriching target cells separated from non-target cells in the bulk of mononuclear blood cells; an outlet interface connected to at least one of the leukapheresis module and enrichment module for connecting the patient to return enriched target cells to the patient's blood circulation; the device and patient, when connected together, form a closed circuit; and a controller for automatic control of the operation of the inlet and outlet interfaces, the leukocyte isolation module and the enrichment module.

According to another aspect of the present invention, a method of treating blood is provided. The method comprises the following steps: obtaining blood from a patient connected to a single blood processing apparatus to form a closed circuit between the patient and the blood processing apparatus; collecting a batch of mononuclear blood cells by leukapheresis performed by the blood processing apparatus in the closed circuit; and simultaneously enriching target cells separated from non-target cells in the batch of mononuclear blood cells using the blood processing apparatus in the closed loop.

According to yet another aspect of the present invention, a system for processing blood is provided. The system comprises: a device for obtaining blood from a patient, and a single blood processing apparatus; the blood processing apparatus is connected to the blood access device and the patient to form a closed circuit between the patient and the blood processing apparatus. The blood processing apparatus includes: a module for collecting a bulk mononuclear blood cell from blood by leukapheresis performed using said blood processing apparatus in said closed circuit; and a module for simultaneously enriching target cells separated from non-target cells in the batch of mononuclear blood cells using the blood processing apparatus in the closed loop.

These and other aspects of the invention will be described in more detail below.

Drawings

Embodiments of the invention will now be described with reference to the accompanying drawings, in which:

FIG. 1 is a block diagram of an apparatus for cell collection by leukapheresis and enrichment by cell washing and purification;

FIG. 2 is a schematic diagram depicting a specific example of an existing method for cell collection, enrichment, and modification;

FIG. 3 is a flow diagram of a method of processing blood cells according to one embodiment of the present invention;

FIG. 4 is a schematic diagram depicting the simultaneous collection of cells, enrichment of target cells, and return of non-target cells;

FIG. 5 is a schematic diagram depicting the simultaneous collection of cells, enrichment of target cells, and return of target cells;

FIG. 6 is a schematic diagram depicting a method of simultaneous cell collection from a patient, enrichment of target cells, modification of target cells, and return of modified target cells to the patient;

FIG. 7 is a schematic diagram of an apparatus for performing the collection and enrichment according to one embodiment of the present invention; the symbols represent the following components: a saline water bag is arranged on the upper portion of the bag,a peristaltic pump is arranged on the upper portion of the shell,the clamping device is used for clamping the workpiece,the air detector is provided with a plurality of air inlets,a pressure gauge for measuring the pressure of the gas,the clamping device is used for clamping the workpiece,

FIG. 8 is a schematic view of an apparatus for performing the collection, enrichment, and modification according to another embodiment of the present invention; the symbols represent the following components: a saline water bag is arranged on the upper portion of the bag,a peristaltic pump is arranged on the upper portion of the shell,the clamping device is used for clamping the workpiece,the air detector is provided with a plurality of air inlets,a pressure gauge for measuring the pressure of the gas,the clamping device is used for clamping the workpiece,

FIG. 9 is a perspective view of the elements of a blood processing apparatus containing a collection, enrichment and (optionally) modification unit or module;

FIG. 10 is a perspective view of the elements of the blood processing apparatus of FIG. 9, but with emphasis on the cell enrichment module/process;

FIG. 11 is a perspective view of the elements of the blood processing apparatus of FIG. 9 or FIG. 10, but with emphasis on an optional cell modification step within the apparatus; and

FIG. 12 is a schematic view of an apparatus for performing collection and enrichment according to yet another embodiment of the present invention. The symbols represent the following components: a saline water bag is arranged on the upper portion of the bag,a peristaltic pump is arranged on the upper portion of the shell,the clamping device is used for clamping the workpiece,the air detector is provided with a plurality of air inlets,a pressure gauge for measuring the pressure of the gas,the clamping device is used for clamping the workpiece,a magnetic body which is provided with a magnetic body,

Detailed Description

Methods, devices and systems for processing blood cells are described below. In particular, the disclosed methods, devices and systems are used for leukapheresis, enabling simultaneous collection and enrichment of specific target cells from the peripheral blood of an individual, and returning the remaining blood components to the individual. In addition, the collected target cells may be modified and returned to the individual during the isolation process, or may be returned to the individual at a later time. Embodiments of the present invention relate to a closed loop device that enables the simultaneous collection and enrichment of specific target cells from the peripheral blood of an individual and the return of non-target cells to the individual. The target cell may be modified to alter its phenotype, genotype or activity and returned to the individual in an expanded portion of the closed loop. Embodiments of the present invention can effectively perform the apheresis procedure in a closed loop, continuous flow fashion connected to the patient, thereby enriching only the target component of the blood (e.g., CD34+ progenitor cells), while returning all other remaining components to the patient. In addition, with the option of returning the modified cells to the patient, certain other functions (e.g., changing phenotype) may be carried on the target cells. The provision of said device allows to significantly reduce the operating costs (without the need for a plurality of apparatuses and consumables) and to guarantee the stability of the product. Enabling the isolation process to be carried out at a single location in a single apparatus also reduces the risk of product damage or loss.

However, it will be apparent to those skilled in the art from this disclosure that modifications and/or substitutions may be made thereto without departing from the scope and spirit of the invention.

Definition of

A multi-function device having a closed circuit and method of use thereof may be patient-connected and include the following:

a) collecting: performing leukapheresis collection on a bulk mononuclear blood cell population containing a target cell population of interest; and at the same time

b) Enrichment: the target cell population from the bulk monocyte population is enriched.

The enriched target cell population can be returned to the patient or removed for subsequent off-line applications including modification, which can also include subsequent injection of the modified cells into the patient. Off-line application of the target cell population may include use for research or monitoring. The non-target cell population may be returned to the patient at the same time, removed off-line for use, or optionally discarded. In addition, the method may modify the target blood cell population in an extension of the closed loop process and then return the modified target blood cell population to the patient.

FIG. 3 shows, at a high level, a method 300 of treating blood, the method 300 including steps 310 and 316 and 330 (shown by solid line boxes). Although not shown in fig. 3, steps 312-316 may be repeated. The method 300 may optionally include one or more of steps 320 and 326 (illustrated by the dashed boxes of FIG. 3). Again, one or more of these steps 320-326 may be repeated (not shown in FIG. 3). Although the steps of method 300 are shown as occurring sequentially and in a particular order, method 300 is not limited to all of the steps being performed (some of which are optional), or to a particular order, sequential processing. Those skilled in the art will appreciate that the order of the steps may be varied in light of the teachings of the present invention. Additionally, one or more steps may be in parallel. For example, steps 312 to 316 may be in parallel. Also, step 326 may be concurrent with steps 314 and 316. The method 300 of treating blood will be described in more detail below.

Processing begins at step 310. In step 312, blood is obtained from a patient connected to a single blood processing apparatus to form a closed circuit between the patient and the blood processing apparatus.

In step 314, a batch of mononuclear blood cells is collected from the blood by leukapheresis performed using the blood processing apparatus in the closed circuit. The collecting step 314 may include collecting mononuclear blood cells using differential centrifugation. The differential centrifugation may be performed by a continuous flow system.

In step 316, the blood processing apparatus in the closed loop is simultaneously used to enrich the target cells separated from the non-target cells in the batch of mononuclear blood cells. The target cell can be a B cell, T cell, dendritic cell, monocyte, neutrophil, Natural Killer (NK) cell, T regulatory cell, T helper cell, Cytotoxic T Lymphocyte (CTL), Hematopoietic Stem Cell (HSC), hematopoietic progenitor cell, endothelial cell, epithelial cell, mesenchymal cell, lymphocyte, lymphokine-activated killer cell (LAK), or Tumor Infiltrating Lymphocyte (TIL). The T cells may be enriched. The T cells may be CD8+ or CD4 +. The hematopoietic progenitor cells and the hematopoietic stem cells may be enriched. The hematopoietic stem cells and the hematopoietic progenitor cells may be positive for one or more of CD34, CD133, and CD 143. Alternatively, the target cell may be at least one of a malignant tumor cell from blood, a malignant tumor cell from tissue, a virally infected cell, a bacterially infected cell, at least one virus, at least one bacterium, a parasite, a fetal cell, and a pathogenic effector cell.

The enrichment step 316 can include ligand capture to enrich the target cells. The ligand may be an antibody specific for a cell surface ligand. The cell surface ligand may be an epithelial cell adhesion molecule (EpCAM), a selectin, an adhesion molecule receptor, a homing receptor, a cytokine receptor, a chemokine receptor, or an enzyme. The cell surface ligand may be a cluster marker (CD) antigen. The CD antigen may be CD1a, CD4, CD8, CD14, CD25, CD34, CD133, or CD 143. The enrichment of target cells in step 316 can be achieved by magnetic, fluorescence activated cell sorting, microfluidics, solid supports, acoustics, bioluminescence, antibody labeling, and enzyme substrates. The solid support may comprise particles. The particles may be at least one of magnetic particles and density-modifying particles.

The collection and enrichment steps 314, 316 may be performed in different parts of the blood processing apparatus.

In step 320, the enriched target cells can be modified. The modification step 320 may involve modifications that include one or more of activation, swelling, induction of apoptosis, genetic modification, and induction of antigen specificity. The modification step 320 may involve modification by at least one of: cross-linking cell surface receptors, irradiation, and treatment with at least one of cytokines, chemokines, antigenic stimulation, hormones, drugs, pressure, and heat. The irradiation may be at least one of gamma, beta, alpha, and light irradiation. The light radiation may be ultraviolet a (uva), ultraviolet b (uvb) and visible light radiation. Alternatively, the modification step 320 may involve genetic modification by transfection or transduction of genetic material into at least a portion of the target cells. Transfection of genetic material may be performed by one of electroporation and lipofection. Transduction of genetic material may be performed by viral vector transduction. In another alternative, the modifying step 320 may involve modification of at least one of genetically modified blood cells that include Cytotoxic T Lymphocyte (CTL) activation, T regulatory cell (Treg) activation, and protection from Human Immunodeficiency Virus (HIV).

In step 322, non-target cells may be modified. Additionally, in step 324, the non-target cells can be returned to the patient. The non-target cells may be returned to the patient connected in the closed circuit, or to the patient disconnected from the closed circuit. In addition, the non-target cells may be discarded.

In step 326, the number of cells in the collecting and enriching steps can be monitored simultaneously. This will allow the collection to be completed once enough cells have been collected and enriched, and allow the collection to be tailored to the patient.

The method may include maintaining a continuous connection to the patient in a closed loop while processing the target cells, or disconnecting the patient from the closed loop for a period of time while processing the target cells. The process terminates (ends) in step 330. These and other aspects will be described in more detail below.

Simultaneous cell collection and targeting for off-line application or disposal including modification Cell enrichment

Fig. 4 illustrates, at a high level, a method 400 of simultaneously performing cell collection 410, target cell enrichment 420, and return of non-target cells 452 from a patient 450 (all shown in loop 402). The enriched target cells 430 can be taken offline for study/testing 462, 470 or discarded 482. Alternatively, the enriched target cells 430, 462 are modified for subsequent injection 464 of the target cells into the patient 450. This aspect includes simultaneous cell collection 410, target cell enrichment 420, and return 452 of non-target cells to the patient 450 in a closed loop. The leukapheresis collection step 410 produces a monocyte population. An online enrichment step 420 of the target cells is performed. The non-target cells are returned 452 to the patient 450. The enriched target cells 430 can be used offline and can optionally be modified for study, testing, etc. 470 or for subsequent injection 464 of the target cells into the patient 450. Target cell 462 can be used with or without modification. The cells may also be discarded 482. Where the target cells are modified and returned 464 to the patient, the non-target cells 452 may not necessarily be returned to the patient 450. If desired, the simultaneous cell collection 410, target cell enrichment 420, and off-line cell modification 400 can be repeated multiple times, for example, to achieve a certain number of target cells. Target cell enrichment 420 may be performed on one or several cell types. Target cell modification can be performed on one or several cell types. The closed loop is connected to the patient 450 at cell collection 410 and cell return 452.

Simultaneous cell collection and target cell enrichment for return and off-line use or discarding of non-target cells

Fig. 5 illustrates, at a high level, a method 500 of cell collection 510 from a patient 550, target cell enrichment 520, and return of target cells 552 to the patient 550. The enriched non-target cells 530 are then used offline, with or without modification, for studies/tests 562, 570, or discarded 582. Furthermore, the leukapheresis collection step 510 produces a monocyte population. An online enrichment step 520 of the target cells is performed. The target cells 552 are returned to the patient 550. This aspect includes simultaneous cell collection 510, target cell enrichment 520, and return 552 of the target cells to the patient 550. The non-target cells 562 can be used offline for research, testing, etc. 570 (with or without modification steps), or discarded 582. The closed circuit is connected to the patient 550 at cell collection 510 and cell return 552.

Simultaneous cell collectionEnrichment and modification of target cells

Fig. 6 shows, at a high level, a method 600 of simultaneous cell collection 610 from a patient 650, enrichment 620 of target cells, modification 660 of target cells 630, and return 670 of modified target cells to the patient 650. Non-target cells 652 may also be returned to the patient 650. This aspect includes simultaneous cell collection 610, target cell enrichment 620, modification 660 of the target cells, and return of the modified target cells 670 to the patient 650 in a closed loop process. Alternatively, non-target cells 652 may be returned to the patient. The closed circuit is connected to the patient 650 as the cells are collected 610 and returned 652, 670.

Cell collection

One embodiment of the invention includes cell collection and simultaneous cell enrichment. The collection is a leukapheresis collection of a batch of mononuclear blood cells from which the target blood cells are enriched. This step may be accomplished by obtaining monocytes from the patient using any method known in the art, including, but not limited to, using differential centrifugation. The apparatus used for this purpose comprises a COBESpectra, the Trima Spectra Optia System (all sold by Gambro BCT), and Amicus or CS-300 (sold by Fenwal/Baxter), Gambro Cobe Spectra or Optia, Fenwal Amicus or CS-3000. Preferably, the differential centrifugation is carried out by a continuous flow system. In a preferred embodiment, the batch blood cell collection uses Therakos CellEx technology due to its superior collection efficiency and lower extracorporeal volume compared to other devices including those listed above. In leukapheresis, a non-monocyte population is reinjected into the individual.

Fig. 9 shows a blood treatment apparatus according to an embodiment of the present invention. The patient 940 is connected to the apparatus 900 in a closed loop, the input conduit 950 of the apparatus 900 is connected to the patient 950 to provide blood as input to the apparatus 900, and the output conduit 952 thereof serves as a return path from the apparatus 900 to the patient. The device 900 has an inlet interface to receive blood directly from the patient's blood circulation. The device 900 also has an outlet interface to return enriched target and/or non-target cells to the patient's blood circulation. The device 900 and the patient form a closed circuit when connected together. The apparatus 900 includes a leukocyte isolation collection unit 910 and an enrichment unit 920. (leukapheresis) the collection unit or module 910 collects a batch of mononuclear blood cells from the received blood. An enrichment unit or module 920 simultaneously enriches target cells separated from non-target cells in the batch of mononuclear blood cells. The device 900 has an operator interface 960 for receiving input or providing output to an operator (not shown). The apparatus 900 also includes a pump/valve seat 964. The apparatus 900 may also include an optional modification unit 930. The device 900 includes a centrifuge 962 for processing blood cells as described below. A controller (not shown) is coupled to the operator interface 960 and other modules for automatically controlling the operation of the apparatus 900.

In the Therakos CellEx system, blood is separated into red blood cells and a "buffy coat" by a centrifuge cup such as the Latham cup shown in U.S. Pat. No. 4,303,193 (incorporated herein by reference in its entirety) entitled "Apparatus for separating blood into components of blood", issued to Latham Jr on 12.1.1981. The Latham cup is a blood component separator that has been used for some time in the medical leukapheresis market and in medical procedures such as extracorporeal photopheresis (ECP). U.S. Pat. No. 5984887, "Photopheresis treatment of Leucocyte" (Photopheresis therapy) provides a description of the method of Photopheresis in vitro and of cell separation and centrifugation thereof.

Fig. 7 is a more detailed schematic diagram of the blood processing apparatus shown in fig. 9 (and the system of fig. 10 described below). In fig. 7, the blood processing apparatus 700 is shown connected to a patient 736. The collection node 702 and the return node 756 are connected to the patient in the manner shown in FIG. 9. Collection node 702 is part of an input interface, including conduit 704, which in turn is connected to a pressure ("collection") sensor 720. In turn, the collection pressure sensor 720 is connected to an air detector 722, and the air detector 722 is connected to a collection valve 724. Collection valve 724 is connected to a "collection" peristaltic pump 726, which peristaltic pump 726 is in turn connected to a cup pressure sensor 728. Pressure sensor 720 affects the operation of collection pump 726. The cup pressure sensor 728 is connected to the centrifuge cup 730. One output of the centrifuge cup 730 is connected to a red blood cell pump (RBC)732 and a conduit 734, the conduit 734 in turn being connected to a return path, which is described in more detail below.

An Anticoagulant (AC) bag 710 is connected to an anticoagulant peristaltic pump 712 and appropriate tubing. The pump 712 is in turn connected to a valve 714, which valve 714 is in turn connected to an air detector 716. Air detector 716 is connected to input conduit 704 and collection pressure sensor 720 by suitable conduits. This configuration allows anticoagulant to be administered to blood input into the device 700 from the patient 736.

Another conduit 770 provides an output from the centrifuge cup 730 and is connected to a valve 772. Also connected to conduit 770 is conduit 782 which is connected to valve 784. Valve 784 is in turn connected to return bag 740. The return bag 740 is connected to an air detector 742, which air detector 742 in turn is connected to a valve 744. The valve 744 is in turn connected with the valve 798, the saline valve 760, and the saline valve 760 is in turn connected with the saline bag 762, the conduit 734, and the return pump 746. The return bag 740, air detector 742, valve 744, and return pump 746 together form a return path. The pump 746 is connected to a return valve 748, and the return valve 748 is connected to an air detector 750. The air detector 750 is coupled to a return pressure sensor 752, and the return pressure sensor 752 is coupled to a conduit 754 and a return node 756.

The valve 772 is connected to a sensor 788 capable of detecting red blood cells. The conduit 774 is also connected to a valve 772 and, in turn, to a white blood cell (buffy) pump 776. A leukocyte pump 776 is attached to plate 778. The output of the plate 778 is connected to a conduit 797, which conduit 797 in turn is connected to a valve 798. Valve 798 is connected to return pump 746. The HCT sensor 788 is connected to valves 790 and 791 arranged in parallel. The valve 790 is connected to a collection bag 786. The valve 791 is connected to a processing bag 737 to which the enriching reagent is added. The processing bag 737 is coupled to a valve 793, which valve 793 is in turn coupled to an air detector 794. Air detector 794 is connected to valve 798.

The optional buffer bag 795 is connected to a valve 796, which valve 796 in turn is connected to an air detector 794.

Cell enrichment

Fig. 10 shows the device 900 of fig. 9, renumbered as device 1000. Patient 1040 is connected to device 1000 in a closed loop manner through input conduit 1050 and output conduit 1052. In this embodiment, the monocyte population 1010 is enriched 1020 by, for example, antibody-coated particle capture (e.g., magnetic particle capture). The output of the enrichment 1020 is enriched target cells 1060 that can be returned to the patient. Additionally, non-target cells from enrichment 1020 can be returned to patient 1040 by device 1000. The enriched cells may be used for specific purposes including, but not limited to:

1. elimination from the bloodstream (e.g., leukemia lymphoma, myeloma cells); these cell types can be selected as cells to be eliminated from the blood and discarded.

2. Modified for return to the patient for positive benefit; some examples include:

a. inducing an immune response by enrichment and modification of leukemia cells or metastatic cancer cells;

b. modifying to generate cytotoxic T lymphocytes against a particular cancer; and

modification of hsc/HPC to include genes to affect disease progression, such as anti-HIV genes to affect HIV/AIDS.

3. For research or testing, etc., which may include an optional modification step.

The target cells are cells enriched from peripheral blood after collection of a batch of monocytes. Cell types that can be enriched in the leukapheresis batch include, but are not limited to: b lymphocytes, T lymphocytes, CD4 and CD8T lymphocytes, dendritic cells, monocytes, Natural Killer (NK) cells, T regulatory cells, T-helper cells, Cytotoxic T Lymphocytes (CTL), Hematopoietic Stem Cells (HSC), hematopoietic progenitor cells, endothelial cells, epithelial cells, lymphokine-activated killer cells (LAK), Tumor Infiltrating Lymphocytes (TIL), mesenchymal stem cell epithelial cells-see Table 1(fundamental immunology By William E.Paul 2003Lippincott Williams & Wilkins ISBN 0781735149; Essential Haematology, Hoffbrand, Pettit and Moss).

TABLE 1

Other cell types targeted for disposal may be any known in the art including, but not limited to, cancer/leukemia cells from blood or other tissues, virally or bacterially infected cells, virally or bacterially or parasites, fetal cells, or pathogenic effector cells. These cells can be enriched by using appropriate surface antigens. These latter cells may also be directed to modification for the purpose of #2 above, i.e., cell modification and returning of the modified cells for a therapeutic immune response.

In the enrichment step, more than one target cell type can be enriched. The system can be enriched for multiple cell types using a variety of methods, for example, cell types can be separately enriched in different chambers of the apparatus (900 and 1000 of fig. 9 and 10). Different cell types can be disposed of together (e.g., all returned or discarded or modified), or cell types can be disposed of separately (e.g., a set returned, a set discarded, a set modified, or a set modified in a different way for all), or variations of the foregoing.

Enrichment of target cells can be used to eliminate target cells from peripheral blood (e.g., leukemia cells), or to enrich to a desired percent purity for therapeutic use, or for research/testing, etc.

The target cells may be enriched, e.g., captured, by chemical or physical means, and isolated, i.e., enriched from a bulk blood cell population. The enrichment process may employ one or more methods known in the art including, but not limited to, antigen capture, beads (beads), magnetic, fluorescence activated cell sorting, microfluidics, solid supports, acoustics, bioluminescence, antibody labeling, or enzyme substrates. Suitable solid supports include particles, including but not limited to ferromagnetic particles and density-modifying particles. These particles are available, for example, from Miltenyi Biotec and Dynal (Curr Opin Immunol.1991, 4.3 (2): 238-. The existing method is adopted; the method for releasing the trapped cells comprises: i) competition with excess ligand; ii) enzymatic digestion; iii) change in pH, iv) change in ionic strength, v) removal of magnetic field, vi) physical agitation.

The ligand specific for one or more target cell populations may be any known in the art, preferably an antibody specific for a cell surface ligand. The cell surface ligand may be a cluster-labeled (CD) antigen including, but not limited to, CD1a, CD4, CD8, CD14, CD25, CD34, and CD133, which typically use specific antibodies to capture/select target cells. The cell surface ligand can be, but is not limited to, EpCAM (epithelial cell adhesion molecule), selectins, adhesion molecule receptors, homing receptors, cytokine receptors, chemokine receptors, and enzymes including aldehyde dehydrogenase and other intracellular enzymes. Various surface markers are shown in table 1.

As an example, one method of enriching cells is to use antibodies or nucleic acid ligands. The term antibody refers to an immunoglobulin molecule capable of binding to an epitope present on an antigen. The term antibody as used in this application refers to a cell binding molecule. The term is intended to include intact immunoglobulin molecules such as monoclonal and polyclonal antibodies, as well as bispecific antibodies, humanized antibodies, chimeric antibodies, anti-idiopathic (anti-ID) antibodies, single chain antibodies, Fab fragments, F (ab') fragments, fusion proteins, and variants of the foregoing antibodies that comprise a ligand recognition site of the desired specificity. As used herein, a nucleic acid ligand is a non-naturally occurring nucleic acid or peptide that has a desired effect on a target. The desired effect includes, but is not limited to, binding to the target, altering the target in a catalytic manner, reacting with the target in a manner that modifies/alters the target or the functional activity of the target, covalently binding to the target in a manner that is in the case of suicide inhibitors, facilitating reactions between the target and other molecules.

HSC/HPC can be enriched by a variety of methods, including the use of cell surface markers CD34 or CD133 or higher levels of alcohol dehydrogenase (ALDH). In one embodiment of the invention, the CD34+ HSC/HPC cells are enriched and subsequently modified by the step of introducing an anti-HIV gene. The introduction can be performed by a variety of methods, for example, by retroviral transduction.

The target cells may be returned to the patient. In certain diseases, it is advantageous that the committed cell population can be discarded or preserved for diagnostic/monitoring purposes. For example, the cell search was developed by Immunicon Inc. under the name CellSearch^TMThe diagnostic procedure of (1) can be used to measure rare tumor cells in blood by means of a magnetic bead isolation system (ref.). This larger scale collection procedure may improve the sensitivity of such diagnostic methods. It may be beneficial to discard specific target tumor cells or pathogenic cells such as Th17 cells in autoimmune diseases (reference). Finally, lymphopenia induction has had better outcomes for some therapies for reasons such as providing room for cell therapy (Dudley, ME et al, science.2002, 10/25; 298 (5594): 850-4.Epub, 2002, 9/19). The cell population for discarding may be any known in the art, including but not limited to malignant cells from blood or other tissues, virally or bacterially infected cells, virally or bacterially or parasitically, fetal cells, or pathogenic effector cells such as Th1, Th17, CTLs, and the like. This enrichment is performed and the percentage purity required for treatment is achieved. In some cases, a certain percentage of enrichment is required (see below). The efficiency of clearance from the blood is more important in removing pathogenic cells (e.g., cancer cells or leukemia cells) than the actual final percent purity. These cells can also be targeted for modification as described above for # 2.

The two steps of cell collection and enrichment are carried out in the same device in a closed loop manner; the above steps may be performed in the same or different parts of the apparatus. Non-target cells may be returned to the patient or discarded, or used off-line for research/testing, as therapeutically needed. In the case of an immune disabling condition such as HIV or a lymphopenia condition, for example, non-target cells may be returned in a closed loop system to return essential cells, the absence of which may harm the patient. In other cases where there is no benefit in returning non-target cells or non-target cells, the non-target cells may be discarded or used off-line for other purposes. For example, such benefits may arise from exposure of a patient to lymphopenia, which may improve the efficacy of certain cell therapies (Dudley, ME et al, science.2002, 25.10; 298 (5594): 850-4.Epub, 2002, 19.9).

Cell modification

Fig. 11 shows the device 900 or 1000 of fig. 9 or 10, and renumbered as device 1100. The patient 1140 is connected to the apparatus 1100 in a closed loop manner through an input conduit 1150 and an output conduit 1152. In this embodiment, an additional modification step of the target cells in a closed loop and connected to the patient is provided. This step represents an extension of the closed loop system connecting the patient for cell collection and enrichment. Modification of target cells in a patient-connected closed loop system can serve as an extension of the collected and enriched patient-connected closed loop system. As shown in fig. 11, the enriched target cells 1160 are modified in a container 1170 to provide modified target cells 1180. Optional modifications may be one or more of electroporation, lipofection, viral transduction, light (ultraviolet a (uva), ultraviolet b (uvb), etc.), addition of drugs, cell activation, pressure, heat, etc.

Fig. 8 is a more detailed schematic view of the blood processing apparatus shown in fig. 11. Fig. 8 illustrates the connection of a blood processing apparatus 800 to a patient 836. Elements of the apparatus 700 shown in fig. 7 that are identical to the apparatus 800 of fig. 8 have the same/corresponding reference numerals, except that the first digit is changed to the corresponding figure number (7XX and 8XX), so the collection node 702 of fig. 7 is the collection node 802 of fig. 8. For the sake of brevity, the description of the corresponding features is not repeated in the description of fig. 8, as those elements in fig. 7 that are the same as in fig. 8 have the same function and configuration. Instead, only the differences between fig. 7 and 8 will be described below. The collection bag 886 is connected to a reforming pump 831, which in turn is connected to a reforming unit or module 833. The modified module 833 is in turn connected to a modified cell bag 835. The configuration of the apparatus 800 of fig. 8 is otherwise identical to that of fig. 7.

Modification may also be performed in a non-continuous ex vivo modification of cells to alter cell phenotype, genotype, or activity. This can be done by addition of cytokines, cross-linking of specific receptors, addition of antigens, transfection of DNA, RNA or proteins, induction of apoptotic cells, gene binding including viral transduction. In this example, the enriched target cell population 1160 is removed for a separate discontinuous modification step to alter cell phenotype/genotype/activity. The modified cells can then be used for research or for treatment by injection back into the patient. The degree of enrichment is that required for research/testing or therapeutic use.

The enriched target cell population may be modified by any method known in the art including, but not limited to, activation, expansion, induction of apoptosis, genetic manipulation, induction of antigen specificity, and the like. This can be achieved by, for example, addition of cytokines, cross-linking of specific receptors, addition of antigens, introduction of DNA, RNA or proteins, viral transduction, electroporation, lipofection, treatment with light of various wavelengths, addition of drugs, trapping of cells or cell components, pressure, heat, etc.

The cells can be modified by a variety of means in all cases except for photopheresis (see below), with independent processing or equipment performed in a procedure without patient attachment. There exist a number of examples of procedures involving ex vivo cell modification of unconnected patients to alter cell phenotype/genotype/activity; this can be done, for example, by adding cytokines, cross-linking specific receptors, adding antigens, transfecting DNA or RNA, introducing proteins, apoptotic cell induction, or by gene binding, for example, by viral transduction. Methods for performing this process include, but are not limited to, electroporation, lipofection, viral transduction, irradiation, incubation with drugs, cell trapping, cell activation, pressurization, heating, cross-linking of cell surface receptors, treatment with cytokines, chemokines, hormones, and the like. For example, electroporation or electro-permeabilization is a method for introducing extracellular compounds such as genetic material (DNA or RNA) into cells by causing the permeability of the cell membrane to increase by an applied electric field. This technology is now commonly used for research purposes and has been subjected to clinical trials which show potential utility in human therapy.

Cells for modification include, but are not limited to, B lymphocytes, T lymphocytes, CD4 and CD8T lymphocytes, dendritic cells, monocytes, Natural Killer (NK) cells, T regulatory cells, T helper cells, Cytotoxic T Lymphocytes (CTL), Hematopoietic Stem Cells (HSC), hematopoietic progenitor cells, endothelial cells, epithelial cells, lymphokine-activated killer cells (LAK), Tumor Infiltrating Lymphocytes (TIL), and epithelial cells-see Table 1 (functional Immunology by William E.Paul 2003Lippincott Williams & Wilkins ISBN 0781735149; Essential Haematology, Hoffbrand, Pettit, and Moss).

These modified cells can be used to treat a variety of diseases or conditions. For example, c.h.june.j.clin.invest.117, (2007)1466-1476 describes adoptive T cell therapy. In this example, peripheral blood lymphocytes are collected from the patient, enriched in a separate step and incubated using an activation system to increase anti-tumor CTL activity. HSCs have been used in bone marrow transplantation for many years and are finding increasing use in other applications such as cardiovascular therapy and wound healing.

Modification can be accomplished using any method known in the art, including but not limited to transfection or transfer of genetic material into at least a portion of the target cell population, cross-linking of specific receptors, or treatment with cytokines. Transfection or transduction of genetic material may be performed by any method known in the art, including, but not limited to, by vector transduction, electroporation, or lipofection. The modification may be any known in the art including, but not limited to, Cytotoxic T Lymphocyte (CTL) activation, T regulatory cell (Treg) activation, induction of apoptosis, or genetic modification of blood cells for protection from Human Immunodeficiency Virus (HIV).

Treatment of HIV with genetically modified hematopoietic progenitor/stem cells is described in International (PCT) patent publication No. WO 03/006691 to Amado et al (2004). In this system, HSC/HPC are collected from the patient as a mononuclear fraction by leukapheresis, enriched by a separate Baxter device and transduced and incubated prior to injection into the patient (see fig. 2). In embodiments of the invention where the patient is leukapheresis for a shorter period of time, the cells will be safely enriched in a closed loop and, more importantly, non-target cells can be returned to the lymphopenia patient (see fig. 4).

There are a variety of other target cells that can be enriched by the device and used for therapy, and some examples are given herein. Dendritic cells are used for the treatment of cancer, infectious diseases and immunodeficiency diseases (Nature.2007, 9.27 days; 449 (7161): 419-26. Review). NK cells are used for the treatment of cancer. T regulatory cells are being tested for the treatment of graft versus host disease (GvHD) (Semin Immunol.2006, 4 months; 18 (2): 78-88), immunodeficiency, atopic dermatitis compounds, and asthma (Curr Opin AllergyClin Immunol.2006, 2 months; 6 (1): 12-6. Review). CTLs are used to treat cancer, infectious diseases, and allergies. Endothelial cells are used for cell regeneration therapy of bladder, vasculature, etc.

In embodiments where all three steps of collection, enrichment and modification are combined in a closed circuit connecting the patient, the extent of enrichment and modification is determined by the value required for the treatment. For example, in HIV gene therapy, it is desirable to enrich HSC/HPC to > 20% and more preferably > 80% so that large amounts of HSC/HPC can be transduced using anti-HIV gene constructs. There is a need to optimize transduction so that large amounts of genetically modified HSC/HPC can be re-injected into patients. The foregoing is described by way of example only.

In another example, T regulatory cells may be enriched and subsequently expanded; purity is usually required to be > 75% and preferably > 90% in order to limit the overgrowth of effector T cells during the modification/stimulation step. Thus, the enrichment and modification parameters vary with disease and medical needs. Again, the foregoing is described by way of example only.

In another embodiment, embodiments of the present invention allow for monitoring of the steps as they are performed, i.e., in real time, such as measurements of hematocrit, cell number, cell phenotype, cell activation, cell size, and the like. For example, in terms of HSC/HPC enrichment and modification, this allows for the determination of parameters of the treatment as it is performed, such as measuring the number of CD34+ cells and the number of transduced CD34+ cells.

All references cited herein are incorporated by reference. These documents include U.S. Pat. No. 7211037 ("Apparatus for continuous separation of biological fluids into components and methods of use thereof") issued to Briggs et al on day 5 and 1 of 2007 and U.S. Pat. No. 7186230 ("Method and Apparatus for continuous separation of biological fluids into components") issued to Briggs et al on day 3 and 6 of 2007. The following examples are intended to illustrate but not limit embodiments of the invention.

EXAMPLE 1 Collection of monocytes and CD4+ T lymphocytes from peripheral blood

Peripheral blood bags were prepared to represent pseudo-patients. 4 units of ABO matched whole blood from healthy donors were collected into ACD-a anticoagulant 1-2 days prior to use. The multiple units of blood were white blood cell depleted by filtration through a Sepacell white blood cell reduction filter and pooled into a 2L blood bag. Leukapak buffy coats were added to achieve a physiological concentration for white blood cell counting, and the pseudo-patient bags were kept on a rocking platform (r) at room temperature to ensure a homogeneous cell suspension. 10mL samples were taken from the pseudo-patient bags and baseline cellular composition was determined by electron cell counting and automated identification on a Beckman Coulter AcT counter and immunophenotyping was evaluated by flow cytometry using monoclonal antibody panels (panels) including CD45-FITC, CD3-PECy7, CD4-APC, CD8-PECy5, CD14-PECy7, CD15-PE, CD20-APC, CD 34-PE.

An example of the cellular composition within a pseudo-patient bag is:

blood processing system

The Therakos CellEx photopheresis system is used as the basis of the blood treatment system. As shown in fig. 9, the system 900 includes several components, including a centrifugal chamber 962, a pump block 964, a photoactivation chamber, and a user-friendly software driven operational interface 960. Additional clamps and pumps were added as necessary in this example and used to modify the single use disposable specific to the CellEx photopheresis procedure. In the present example of collecting monocytes and enriching for CD4+ cells from peripheral blood, the photoactivation chamber is not required. The CellEx photopheresis system uses a 1-omega 2-omega centrifugal technology and is connected with a Latham cup connected with a driving pipe of a three-interface inner cavity to realize continuous whole blood processing. Collection of a similar number of monocytes can be achieved by a lower in vitro volume compared to other leukocyte isolation devices. The CellEx photopheresis system can be run in either single-needle (batch return) or dual-needle (continuous return) mode access, which provides flexibility to the patient. In this example, the dual needle mode is used to send blood from the pseudo-patient bag to the pseudo-patient return bag in a single pass.

The Therakos CellEx photopheresis system requires loading and perfusion of a disposable process kit (procedural kit) prior to collection of monocytes. The kit is a single-use, unitary disposable set that is made up of multiple elements including a Latham centrifuge cup, a pump tube manager, and a light-activated module. In this example, the process kit is modified to include additional bags and clamps. The modified process kit was installed and primed according to the Therakos CellEx photopheresis System operating Manual. Once the kit is loaded, the system performs an automated 7-minute perfusion procedure to ensure proper kit loading, test kit integrity and instrument integrity, and perfuse the sterile fluid path with anticoagulant. The anticoagulant used in this example was ACD-A.

After perfusion, the system is ready for a pseudo-patient connection. The 2L pseudo-patient blood bag was connected to the inlet of the CellEx System disposable kit or at the "kit collection access" line. A 2L empty blood bag is connected to the outlet or "kit return access" line to represent the other arm of the dummy patient and is referred to as the "return bag". After connecting the two donor access lines, the CellEx system is configured to operate in a double-needle mode. Other system parameters are used under default settings. The system parameters are:

1) 1500mL of whole blood was processed and,

2) blood collection rate 50 mL/min, and

3) the anticoagulant ratio was 10: 1.

The system automatically processed whole blood in a volume of 1500mL by pressing a start button on the operator interface to initiate blood collection.

As blood is continuously drawn from the dummy patient into the Latham cup, red blood cells and plasma are continuously removed and returned through a second intravenous line, represented in this example by a "return bag". In the single needle mode, red blood cells and plasma are returned through the same line in a batch fashion. The CellEx system pump mount drives multiple pumps and directs and transfers blood components throughout the blood processing. Monocytes were retained as a buffy coat between red blood cells and plasma in the cup. The position of the "buffy coat" is monitored using a laser beam.

When processing 1500mL of whole blood, the CellEx system enters the "buffy coat collection" mode. Collection of monocytes is accomplished by stopping the pump that controls the flow of blood to the "return bag". This allows the red blood cells to enter the cup and displace the "buffy coat" (despite some perturbation of the buffy coat) up and out through the open valve via the plasma port at the top of the cup. The plasma and "buffy coat" are introduced into a "processing bag" previously filled with anticoagulant. When the system hematocrit optical sensor detects a hematocrit of 3%, the collection pump is temporarily stopped and the cup is rotated to allow the white blood cell band to reform. And then collected into a processing bag until the optical sensor detects a hematocrit of 24%. This triggers the valve to close and divert fluid from the cup to the return line. The "treatment bag" now contains the collected monocyte preparation. The "treatment bag" consists essentially of mononuclear cells, together with platelets and a low concentration of granulocytes and red blood cells with a hematocrit of about 1-2%. Cell "processing bags" are connected to additional bags for enrichment via modified process tools.

Examples of monocyte collections from 1570mL of anticoagulated whole blood (pseudo-patient) are:

enrichment of CD4+ target cells from harvested monocytes

After completing the CellEx monocyte collection, a portion of the monocyte product was washed with cell enrichment buffer and CD4+ selection beads (Dynal) were introduced at a concentration via a needle-free access port of the "processing bag". The monocyte and bead mixture was incubated for 30 minutes while being recirculated through the spiral channel of the photoactivation module of the CellEx disposable kit. The incubation was terminated by transferring the cells via peristaltic pump into a bag placed in a magnetic particle concentrator. CD4+ target cells were retained in the "enrichment cell bag" and the magnetic beads (Dynabeads) were removed by adding dismantling beads (detechabeads). The enriched target CD4+ and non-target cell fractions were collected in separate collection bags. Samples were taken and cell number, yield and purity were determined using a Coulter cytometer and a flow cytometer with associated cell surface markers.

The numbers shown below are from 2mL small aliquots of collected monocytes.

EXAMPLE 2 Collection of monocytes from peripheral blood and enrichment of CD8+ cells

SUMMARY

Fig. 12 shows an improved system 1200 in relation to the system 700 of fig. 7. For purposes of brevity, features in fig. 7 that are the same as in the system 1200 of fig. 12 retain the same reference numerals (e.g., the anticoagulant pump 712 in fig. 7 and 12). Additionally, these same numbered features in the system 1200 of FIG. 12 retain the same configuration unless explicitly described below. The system 1200 of fig. 12 is a blood processing apparatus that involves collection and enrichment (type 2) and contains 3 new bags, 6 new clamps, and 2 magnets. As shown in fig. 12, a standard CellEx process kit was modified. Instead of a photoactivation chamber, a CLINIcell25 bag 1278 was used. The patient 1200 is represented in fig. 12 as patient bag # 11206 and patient bag #2, with patient bag #1 connected to the return node 756 and patient bag #2 not shown in fig. 12 but instead of bag 1206 and connected to the return node 756 at a different stage of the process to achieve cell counts in collection and enrichment.

The system of fig. 12 is modified as follows. Magnets 1276 are positioned adjacent to plate 778 and engage or disengage plate 778. In fig. 7, the output of the HCT sensor 788 is connected in parallel to valves 790 and 791(NEW1 and NEW2), which valves 790 and 791 are in turn connected to a collection bag 786 and a treatment bag 737, respectively. This structure is maintained in fig. 12, but an additional parallel channel is added to the output of HCT sensor 788. Valve (NEW6)1240 is connected to the output of HCT sensor 788 and in turn to waste bag 1242. Another valve (NEW5)1250 is connected to the output of HCT 788 and conduit 1252 is connected between valve 1250 and valve (NEW3)793 and air detector 794. Additionally, the output of valve (NEW4)796 is connected between valve 793 and air detector 794 in FIG. 12, rather than between air detector 794 and valve 798 as shown in FIG. 7. Finally, a second magnet 1254 is disposed adjacent to the passageway between the valve (NEW1)790 and the collection bag 786.

The 4 whole blood units were combined to create a "pseudo-patient" 1204 connected to collection junction 702 and sampled for coulter counter and flow cytometry analysis. The modified kit was loaded onto the CellEx apparatus and valves NEW 1790, NEW4796, NEW 51250 and NEW61240 were closed and valves NEW 2791 and NEW 3794 were opened. As an initial state, this provides an open access for passage of the processing bag 737. The kit was primed using standard CellEx software, and diagnostic software running on a laptop computer was connected to the IR port of CellEx to run other user-configured operations on pumps, valves, and centrifuges.

Perfusion

Closing valve NEW 2791 and opening valve NEW 1790 creates a channel to the collection bag 786, which is perfused with buffer by circulating the leukocyte/recirculation pump 776 clockwise. Once the line is primed, the pump 776 is stopped, the valve NEW 3793 is closed, and the valve NEW4796 is opened to allow the selection buffer 795 to be aspirated to the various locations of the kit. The leukocyte/recirculation pump 776 is turned on counterclockwise. After priming the line to the selection buffer 795, the pump 776 is stopped, the valve NEW 1790 is closed, and the valve NEW61240 is opened. This will open a passage to the waste bag 1242. The line to the waste bag 1242 may be primed by turning on the leukocyte/recirculation pump 776 in a clockwise direction. When the line to the waste bag 1242 has been primed, the pump 776 is stopped, the valves NEW61240 and NEW4796 are closed, and the valve NEW 51252 is opened to prime the line 1252 of the shunt processing bag 737 by running the leukocyte/recirculation pump counter-clockwise. Once the lines 1252 and 1250 are primed, the pump 776 is stopped, valve NEW 51250 is closed and valves NEW 2791 and NEW 3793 are opened; and (5) completing perfusion.

And "Patient attachment and Collection

The "pseudo-patient" 1204 is connected to the collection lines 702 and 704, and the patient bag # 11206 is connected to the return lines 756 and 754. A standard CellEx double needle program was run using default settings to collect the buffy coat (as described in example 1 above). Immediately after collection of the buffy coat, the "stop" button was pressed, pausing the auto-run CellEx software. The CellEx pump, NEW valve and centrifuge were then controlled by the operator and using diagnostic software on the notebook computer.

Enrichment of target cells

All valves in system 1200 were closed except for valve (NEW2)791, (NEW4)796, (blue-plasma bottom) 744, (powder-plasma top) 784 and (return) 748. This creates an open channel for pumping the remaining material in the cup 730 and the return bag 740 to the patient bag # 11206. This is accomplished by rotating red blood cell pump 732 clockwise and return pump 746 counterclockwise. Thus, a channel is formed from the centrifuge cup through the pumps 732 and 746 to the patient bag # 11206 via elements 748, 750, 752, 754, and 756 and through the return bag 740. The pumps 732 and 746 are stopped, the valve (blue-plasma bottom) 744 is closed, and the saline valve 764 is opened. To clean the cup 730, the red cell pump 732 is operated in a counterclockwise direction and saline is drawn from the saline bag 762 into the cup 730. When the cup 730 is about half full, the pump 732 is stopped and the brine valve 764 is closed. Centrifuge 730 is then pulsed and blood is drawn into patient bag # 11206 by rotating red blood cell pump 732 clockwise and return pump 746 counter clockwise. When cup 730 is empty, the red blood cell pump 732 is stopped and the speed of the return pump 746 is briefly increased to flush the remaining blood from the line into patient bag # 11206. The pump 746 is stopped and the patient bag # 11206 is replaced with a patient bag #2 (not shown in fig. 12), which patient bag #2 is connected to the return node 756. Samples from patient bag #1 were analyzed on a coulter counter and cell composition was obtained by flow cytometry. A total of 1800ml of blood was treated, with a total nucleated cell count of 6.6X 10⁶and/mL. CD8 cells constituted 8.1% of the starting material. After enrichment, nbuffy was 139mL, which was always presentNuclear cell count 24.2X 10⁶mL, with 22.4% being positive for CD 8. (recovery rate 78%)

The remaining cells in the tubing are aspirated into the processing bag 737 by running the leukocyte/recirculation pump 776 at 100 ml/min clockwise for several seconds. The pump 776 is then stopped, the valve NEW4796 is closed and the valve NEW 3793 is opened and the volume of the collected buffy coat is determined by weight. The treatment bag 737 is agitated to mix the contents and samples are collected for coulter counting and flow cytometry analysis.

In this example, the number of cells in the treatment bag 791 was adjusted to 1X 10⁹This number is the number recorded that a 5mL vial using dynabeads is capable of capturing. Dynabeads were injected into the treatment bag 737 and the bead/cell mixture was circulated through the plate 778 and treatment bag 737 by running the leukocyte/recycle pump 776 clockwise. In this mode, valves 1240, 1250, 790, 796, 772, and 798 are closed. Valves 791 and 793 are opened. Thus, the cycle passes through processing bag 737 via members 793, 794, and 780 to plate 778. The magnets 1276 do not mate with the plate 778. Circulation continues from the plate 778 through the leukocyte pump 776, the HCT sensor 788 and the valve 791 to the treatment bag 737. Thus, in this mode, the cycle through this channel is counter-clockwise. During this incubation period, cells expressing a specific cellular antigen (CD8 in this example) were bound to antibody-coated dynabeads. This incubation and cycle continues for at least 30 minutes while the treatment bag 737 and plate 778 are mixed or stirred.

At the completion of the antigen/antibody cycling step, plate 778 was placed in a Dynal clinexixvo MPC (8k gauss magnet) 1276 with magnet 1276 working. The leukocyte/recirculation pump 776 continues to aspirate for several minutes to remove the dynabeads from the tubing between the plate 778 and the top of the treatment bag 737.

Once the line between plate 778 and treatment bag 737 is emptied, pump 776 is stopped, valve (NEW2)791 is closed, valve (NEW6)1240 is opened, and then leukocyte/recirculation pump 776 is run in a clockwise direction. Fluid communication into the processing bag 737 is interrupted by closing valve 791. With magnet 1276 active, circulation flows from the treatment bag through elements 793, 794 and 780 to plate 778. All cells in the processing bag 737 are aspirated through plate 778. The Dynabead-cell complex (CD8 positive pool fraction) was trapped in plate 778 by magnet 1276. With valve (NEW6)1240 open, circulation continues from plate 778 through leukocyte pump 776 and HCT sensor 788 to waste bag 1242. Thereby allowing the remaining cells (negative fraction) to flow into waste bag 1242.

When the processing bag 737 is empty, the leukocyte/recirculation pump 776 is stopped, the valve (NEW3)793 is closed, the valve (NEW4)796 is opened, and the pump 776 is re-run in the same direction to allow the selection buffer from the bag 795 to flush the line from the bottom of the processing bag 737, through the plate 778, and to the waste bag 1242, thereby ensuring that most of the cells remaining in the line are processed.

When the line has been flushed with buffer for several minutes, the leukocyte/recirculation pump 776 is stopped, valve (NEW6)1240 is closed, valve (NEW2)791 is opened and plate 778 is moved out of magnet 1276. Buffer from bag 795 was added to plate 778 and treatment bag 737 by circulating leukocyte/recirculation pump 776 clockwise. Upon addition of sufficient buffer, the pump 776 is stopped, valve (NEW4)796 is closed, valve (NEW3)793 is opened and the pump 776 is then restarted. The cell-bead mixture was passed through plate 778 and treatment bag 737 for a number of cycles of minutes to resuspend the Dynabead-cell complex. The cycle passes through processing bag 737 via members 793, 794 and 780 to plate 778. Circulation continues from the plate 778 through the leukocyte pump 776, the HCT sensor 788 and the valve 791 to the treatment bag 737. Thus, in this mode, the cycle through the channel is counter-clockwise. This step can be repeated and is equivalent to washing the positive part to remove impurities.

After washing, 2ml of Dynal's detachhabead was injected into the treatment bag 737 and incubated with Dynabead-cell complex by running the leukocyte/recirculation pump 776 clockwise for at least 45 minutes. After incubation, plate 778 is placed in magnet 1276. The leukocyte/recirculation pump 776 is rotated clockwise for a few minutes to clear the dynabeads from the tubing between the plate 778 and the top of the treatment bag 737. Once the dynabeads are cleared, the pump 776 is stopped, the valve (NEW2)791 into the treatment bag 737 is closed, the valve (NEW1)790 into the collection bag 786 is opened, and the leukocyte/recirculation pump 776 is run in a clockwise direction. Circulation is from the treatment bag 737 via members 793, 794 and 780 to the plate 778. Circulation continues from the plate 778 through the leukocyte cell pump 776, the HCT sensor 788, and the valve 790 to the collection bag 786. The magnets 1276 remain engaged with the plate 778.

The fluid and cells in the treatment bag 737 are aspirated through plate 778, dynabeads (now separated from the cells) are captured by magnet 1276, and the cells (positive selection) flow into collection bag 786. Any dynabeads that are not captured by the primary magnet 1276 should then be captured by the secondary magnet 1254 before entering the collection bag 786. When the processing bag 786 is empty, the leukocyte/recirculation pump 776 is stopped, the valve (NEW3)793 is closed, the valve (NEW4)796 is opened, and then the pump 776 is re-run in the same direction. The buffer from the selection buffer bag 795 flushes the line from the bottom of the processing bag 737, through the plate 778, and to the collection bag 786, thereby ensuring that most of the cells in the line are processed.

When the line has been flushed with buffer for several minutes, the leukocyte/recirculation pump 776 is stopped. Waste bag 1240 and collection bag 786 are weighed to determine the collection volume, and waste bag 1240 (the unwanted portion) is sampled for coulter counting, flow cytometry and pH analysis. The enriched fraction in the collection bag 786 is concentrated and then sampled for coulter counting, flow cytometry and pH analysis. The yield of CD8 positive cells was 33% with a purity of 92%.

Return of non-target cells

The cells in the waste bag 1242 are concentrated for return to the patient represented by patient bag #2 (not shown in fig. 12 but replaceable bag 1206). All valves in system 1200 were closed except for valves (NEW5)1250, (NEW6)1240, (green-buffy bottom) 798, (pink-plasma top) 784, and (return) 748, which were open. This opens a path to transfer the contents of the waste bag 1242 into the cup 730 and the overflow is collected in the return bag 740. Thus, circulation from the waste bag 1242 passes through valves 140 and 1250, air detector 794, valve 798 and pump 732 to centrifuge cup 730. From the cup 730, the cycle enters the return bag 740 via 784. This is accomplished by rotating the red blood cell pump 732 counterclockwise.

Once waste bag 1240 is empty, pump 732 is stopped and all valves are closed, but valve (blue-plasma bottom) 744, (powder-plasma top) 784, and (return) 748 are opened. The cup 730 is purged of all air at 20 ml/min by running the red blood cell pump 732 in a counter-clockwise direction while the centrifuge 730 is turned on to a speed of 600 and 1000RPM for several seconds, and then the centrifuge 730 is turned off. The process of turning on and off the centrifuge 730 while the red blood cell pump 732 continues to aspirate is repeated a number of times until no more air bubbles are observed to remain in the cup 730. Once completed, centrifuge 730 is slowly raised to full speed while pump 732 is still running, thereby allowing the contents of cup 730 to separate for several minutes.

After separation, the red cell pump 732 is stopped, valves (NEW2)791 and (yellow-white cell top) 772 are opened, and valve (powder-plasma top) 784 is closed. The counterclockwise suction of the red blood cell pump 732 is resumed at 20 ml/min. Circulation is from the return bag 742 to the centrifuge cup 730 via the air detector 742, valve 774 and pump 732. From cup 730, the cycle continues through valve 772, HCT sensor 788 and valve 791 to the processing bag. This process results in the removal of salt from the top of the cup 730 while retaining most of the non-target cells in the blood product within the cup 730.

When the return bag 740 is empty, the centrifuge 730 is stopped and all of the contents of the cup 730 are returned to the patient bag #2 (not shown in fig. 12) by the red blood cell pump 732 rotating clockwise and the return pump 746 rotating counterclockwise through the valve 748, the air detector 750, the pressure sensor 752, and the return node 756. When cup 730 is emptied, pumps 732 and 746 are stopped, valve (blue-plasma bottom) 744 is closed, saline valve 764 is opened and the return pump 746 is run at 100 ml/min for an additional few seconds to flush the remaining non-target blood cells into patient bag #2 (not shown in fig. 12). The pump 746 is stopped, patient bag #2 is weighed to determine total volume, and samples are taken for coulter counting and flow cytometric analysis. Non-target cells contained only 2.7% CD8 cells.

EXAMPLE 3 Collection of monocytes from peripheral blood and enrichment of CD34+ cells

Collection and enrichment of CD34+ cells can be performed as described in examples 1 and 2, using a substance that specifically binds to CD 34.

Enrichment of CD34+ cells from harvested monocytes

In one embodiment of the invention, the subject may be mobilized using granulocyte colony stimulating factor (G-CSF). After completion of standard CellEx monocyte collection, washing was performed with cell enrichment buffer PBS/edta (Miltenyi) supplemented with HSA and CD34+ selection beads (Miltenyi) introduced through the needle-free access port of the "collection bag". The monocyte and bead mixture was incubated for 30 minutes while being recirculated through the capture module of the modified CellEx disposable kit and the incubation was terminated by transferring the cells to the Miltenyi CliniMACS magnetic system via a peristaltic pump at about the manufacturer's suggested flow rate. CD34+ target cells can be enriched from non-target cells, and the target cells and non-target cell fractions can be collected in separate collection bags and further modified or returned to the patient.

Results

The Therakos CellEx photopheresis system is capable of collecting higher yields of monocytes and can be connected in a single fluid path to a cell enrichment system for additional enrichment of target cells. Further improvements in the connections and interfaces between the collection and enrichment modules of the combined system can improve target cell recovery and yield.

EXAMPLE 4 Collection of monocytes, enrichment of CD4+ cells and modification from peripheral blood

The cells of examples 1 and 2 can be modified in the closed fluidic path shown in fig. 8.

The enriched cells were transferred to the modification chamber using a pump. Agents such as growth factors (e.g., interleukin-2), peptide and/or gene delivery agents (e.g., viral vectors) are introduced and the cells are maintained at a constant temperature (culture). This enables the cell to change phenotype and/or genotype and to have different physical properties and functions. The modified cells may be further cultured and used as therapeutic agents.

Hardware and software requirement specification

The pump mount remains substantially the same as the existing CellEx and there is room for an additional pump head to be added-the lower left corner of the pump mount.

If the spheres and plates for photopheresis are removed from the CellEx, there is sufficient space to add the items needed for collection or even modification. Additional bag hooks may be added on the left side of the instrument.

In the foregoing manner, the present application discloses various methods, devices and systems for processing blood cells. Although only a few embodiments have been disclosed, it will be apparent to those skilled in the art from this disclosure that various changes and substitutions can be made herein without departing from the scope and spirit of the invention.

Claims (21)

1. A device for processing blood, the device comprising:

an inlet interface for connecting a patient to receive blood directly from the patient's blood circulation;

a leukocyte isolation module coupled to the inlet interface for collecting a bulk mononuclear blood cells from the received blood;

an enrichment module coupled to the leukocyte isolation module for simultaneously enriching target cells isolated from non-target cells in the bulk of mononuclear blood cells;

a target cell modification module adapted to provide a modification selected from the group consisting of Cytotoxic T Lymphocyte (CTL) activation, T regulatory cell (Treg) activation, and genetic modification to protect blood cells from Human Immunodeficiency Virus (HIV), the target cell modification module linked to at least one of the leukocyte isolation module and the enrichment module, the target cell modification module modifying the enriched target cells;

an outlet interface connected to at least one of the leukocyte isolation module and the enrichment module for connecting the patient to return at least the enriched target cells to the patient's blood circulation such that the device and the patient form a closed circuit when connected together; and

a controller for automated control of the operation of the inlet and outlet interfaces, the leukocyte isolation module, the enrichment module, and the target cell modification module.

2. The apparatus of claim 1, wherein the controller comprises:

a memory for storing data and instructions for automatically controlling the operation of the inlet and outlet interfaces, the leukocyte isolation module, the enrichment module, and the target cell modification module; and

a processor coupled to the memory and having access to the data and the instructions, the processor adapted to execute the instructions for automatically controlling the operation of the inlet and outlet interfaces, the leukocyte isolation module, the enrichment module, and the target cell modification module.

3. The device of claim 1, further comprising means for returning the modified target cells to the patient.

4. The device of claim 1, further comprising a non-target cell modification module coupled to at least one of the leukocyte isolation module and the enrichment module, the non-target cell modification module modifying the non-target cells.

5. The device of claim 4, further comprising means for returning said modified non-target cells to said patient.

6. The device of claim 1, further comprising at least one pump for circulating at least a portion of the blood in the device.

7. The apparatus of claim 6, further comprising a first pump and at least one first valve connected to the inlet interface for providing at least a portion of the received blood to the leukocyte isolation module; and a second pump and at least one second valve interfaced with the outlet for returning at least a portion of the blood from the device.

8. The device of claim 1, wherein the leukocyte isolation module comprises a centrifuge cup that collects the mononuclear blood cells using differential centrifugation.

9. The apparatus of claim 8, wherein the differential centrifugation is performed by a continuous flow system.

10. A system for processing blood, the system comprising:

means for obtaining blood from a patient; and

a unitary blood processing apparatus connected to the access device and the patient to form a closed circuit between the patient and the blood processing apparatus, the blood processing apparatus comprising:

means for collecting a bulk mononuclear blood cell from said blood by leukapheresis performed using said blood processing apparatus in said closed circuit;

means for simultaneously enriching target cells separated from non-target cells in the batch of mononuclear blood cells using the blood processing apparatus in the closed circuit; and

a device for target cell modification adapted to provide a modification selected from the group consisting of Cytotoxic T Lymphocyte (CTL) activation, T regulatory cell (Treg) activation, and genetic modification to protect blood cells from Human Immunodeficiency Virus (HIV), said target cell modification device being linked to at least one of said collection device and said enrichment device, said target cell modification device modifying said enriched target cells.

11. The system of claim 10, further comprising means for discarding the non-target cells.

12. The system of claim 10, further comprising means for simultaneously monitoring the collection and enrichment of the collection means and the enrichment means, respectively.

13. The system of claim 10, wherein the collection device collects the mononuclear blood cells using differential centrifugation, the enrichment device enriches the target cells using ligand capture.

14. The system of claim 13, wherein the ligand is an antibody specific for a cell surface ligand.

15. The system of claim 14, wherein the cell surface ligand is selected from the group consisting of epithelial cell adhesion molecule (EpCAM), selectins, adhesion molecule receptors, homing receptors, cytokine receptors, chemokine receptors, enzymes, and CD antigens.

16. The system of claim 13, wherein the differential centrifugation is performed by a continuous flow system.

17. The system of claim 15, wherein the CD antigen is selected from the group consisting of: CD1a, CD4, CD8, CD14, CD25, CD34, CD133, and CD 143.

18. The system of claim 10, wherein the target cell is selected from the group consisting of: dendritic cells, monocytes, neutrophils, Hematopoietic Stem Cells (HSCs), hematopoietic progenitor cells, endothelial cells, epithelial cells, mesenchymal cells, and lymphocytes.

19. The system of claim 18, wherein said hematopoietic progenitor cells and said hematopoietic stem cells are enriched and optionally positive for at least one of CD34, CD133, and CD 143.

20. The system of claim 10, wherein the target cell is selected from the group consisting of: b cells, T cells, Natural Killer (NK) cells, lymphokine-activated killer cells (LAKs), and Tumor Infiltrating Lymphocytes (TILs).

21. The system of claim 10, wherein the target cell is selected from the group consisting of: t regulatory cells, T helper cells and Cytotoxic T Lymphocytes (CTLs).