Method for preparing therapeutically active cells using microfluidics

Abstract

To provide methods of collecting and rapidly processing cells that have therapeutic uses.SOLUTION: A method of engineering a population of target cells comprises: a) isolating the target cells from a crude fluid composition, where the isolation procedure comprises performing Deterministic Lateral Displacement (DLD) on a microfluidic device, where the device comprises an array of obstacles arranged in rows in a channel bounded by a first wall and a second wall opposite to the first wall, where the obstacles are disposed in a manner such that, when the composition is fluidically passed through the channel, target cells flow to one or more collection outlets where an enriched product is collected and contaminant cells or particles that are of different sizes than the target cells flow to one more waste outlet separate from the collection outlets; b) genetically engineering the target cells obtained from the collection outlets to have a desired phenotype.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 553,723, filed September 1, 2017; the benefit of U.S. Provisional Patent Application No. 62 / 567,553, filed October 3, 2017; the benefit of Provisional Patent Application No. 62 / 635,304, filed February 26, 2018; and the benefit of Provisional Patent Application No. 62 / 656,939, filed April 12, 2018. In addition, this application is a continuation-in-part of PCT / US2017 / 057876, filed October 23, 2017. All of these prior applications are incorporated herein by reference in their entirety.

[0002] Statement on Federally Sponsored Research This invention was made with government support under Grant No. CA174121 and No. HL110574 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] The present invention is primarily directed to methods for preparing therapeutic cells and compositions that use microfluidic devices to separate cells based on size. [Background technology]

[0004] Cell therapy, especially CAR-T cell therapy, has shown remarkable efficacy in treating B-cell diseases such as B-cell acute lymphoblastic leukemia (B-ALL) and B-cell lymphoma. As a result, the demand for autologous therapy has increased dramatically, and development efforts have expanded to focus on cancers characterized by solid tumors, such as glioblastoma (Vonderheide, et al., Immunol. Rev. 257:7-13 (2014); Fousek, et al., Clin. Cancer Res. 21:3384-3392 (2015); Wang, et al., Mol. Ther. Oncolytics 3:16015 (2016); Sadelain, et al., Nature 545:423-431 (2017)). Targeted gene editing using CRISPR / Cas-9 in focused populations of autologous cells, such as stem cells, may further stimulate demand (Johnson, et al., Cancer Cell Res. 27:38-58 (2017)).

[0005] The preparation of cells for personalized therapy is typically a labor-intensive process that relies on procedures adapted from blood banking or protein bioprocessing procedures that are less suitable for therapeutic applications. In part, cell-specific separation is achieved (Powell, et al., Cytotherapy 11:923-935 (2009); TerumoBCT. ELUTRA Cell Separation System. Manufacturer recommendations for the enrichment of lymphocytes from apheresis residues), but this comes at the expense of cell viability and yield (Chiche-Lapierre, Cytotherapy 18(6):S47 (2016)). Cell loss associated with the processing steps is typically significant (Hokland, et al., Scand. J. Immunol. 11:353-356 (1980); Stroncek, et al., J. Transl. Med. 12:241 (2014)). Therefore, there is a need for more efficient processes. Summary of the Invention

[0006] The present invention is directed to methods for collecting and rapidly processing cells, particularly cells with therapeutic applications. Many of these methods rely on deterministic lateral displacement (DLD), a process that involves flowing a sample through a specially designed microfluidic device containing a micropost array that is inclined at a slight angle from the direction of fluid flow (Davis, et al., Proc. Natl. Acad. Sci. USA 103:14779-14784 (2006); Inglis, et al., Lab Chip 6:655-658 (2006); Chen, et al., Biomicrofluidics. 9(5):054105 (2015)). Cells larger than the target size of the micropost array can be gently deflected ("bumped") by the microposts into a clean buffer stream, effectively separating them from smaller, undeflected cells and particles, while simultaneously washing the cells in a non-invasive process. The advantageous features of DLD with respect to cell processing are listed in Table 1.

[0007] [Table 1]

[0008] Methods for manipulating target cells In its first aspect, the present invention is directed to a method for genetically manipulating a target cell population. This is accomplished by isolating target cells from a crude fluid composition by performing deterministic lateral displacement (DLD) on a microfluidic device. The device is characterized by the presence of at least one channel extending from a sample inlet to one or more fluid outlets and bounded by a first wall and a second wall opposite the first wall. An array of obstacles is arranged in rows in the channel, with each successive row of obstacles being laterally shifted relative to the previous row. The obstacles are arranged in such a manner that, when a crude fluid composition is applied to the device's inlet and passes through the channel, the target cells flow to one or more collection outlets where a concentrated product is collected, while contaminating cells or particles flow to another waste outlet that is separate from the collection outlets. Once the target cells have been purified using the device, they are transfected or transduced with nucleic acids designed to confer a desired phenotype to the cells, for example, to express a chimeric molecule (preferably a protein that makes the cells therapeutically valuable). The cell population can then be expanded by in vitro culture. When cultured and expanded, the yield of recombinantly engineered target cells exhibiting the desired phenotype is preferably at least 10% higher, and more preferably at least 20%, 30%, 40%, or 50% higher, than identical cells that have not been subjected to DLD (particularly cells that have been exposed to Ficoll centrifugation but not DLD).

[0009] In a preferred embodiment, the crude fluid composition is a blood preparation, more preferably a white blood cell preparation obtained by performing apheresis or leukapheresis on the patient's blood. Preferred target cells include T cells, B cells, NK cells, monocytes, and progenitor cells, with T cells (particularly natural killer T cells) being most preferred. Aside from white blood cells, other cell types, such as dendritic cells or stem cells, can also serve as target cells.

[0010] Generally, crude fluid compositions containing target cells are processed without freezing (at least until they are genetically engineered) and at the site of collection. The crude fluid composition is preferably a patient's blood, more preferably a composition containing leukocytes obtained as a result of performing apheresis or leukapheresis on such blood. However, the term "crude fluid composition" also includes body fluids such as lymph or synovial fluid, as well as fluid compositions prepared from bone marrow or other tissues. Crude fluid compositions may also be derived from tumors or other abnormal tissues.

[0011] Although it is not essential that target cells be bound to a carrier before being genetically engineered, it is preferable to bind one or more carriers to them either before or after (preferably before) DLD is first performed. The precise means by which this occurs is not critical to the invention, but the binding should be performed "in a manner that facilitates DLD separation." As used in this context, this term means that the method must ultimately result in binding that exhibits specificity for a particular target cell type, provides an increase in the size of the complex relative to unbound cells of at least 2 μm (and alternatively, when expressed as a percentage, at least 20%, 50%, 100%, 200%, 500%, or 1000%), and allows the target cells to be released from the complex by chemical or enzymatic cleavage, chemical lysis, digestion, competition with other binding agents, shear stress generation, physical shear, e.g., with a pipette, or other means, if the therapeutic or other application requires free target cells.

[0012] In a preferred embodiment, the carrier has an affinity substance (e.g., an antibody, activator, hapten, aptamer, nucleic acid sequence, or other compound) on its surface that enables the carrier to bind specifically directly to the target cell. Alternatively, an intermediate protein, cell, or other agent may be present that specifically binds both the target cell and the carrier. For example, an antibody may be used that recognizes a surface antigen on the target cell and also specifically binds to the carrier (e.g., through the presence of a second antibody on the carrier surface, avidin / biotin binding, or some other similar interaction). In addition, target cells may specifically interact with other cells to form complexes; in doing so, the other cells may serve as biological carriers, i.e., they may increase the effective size of the target cell, thereby facilitating its separation from uncomplexed cells. For example, human T cells may interact with sheep red blood cells or autologous human red blood cells to form rosettes of cells, which can then be purified as complexes. Alternatively, other carriers may specifically bind cells in such rosettes to further facilitate size-based separation.

[0013] As used in this context, the term "specificity" means that in the crude fluid composition, at least 100 (preferably at least 1000) target cells will be bound by the carrier for every 1 non-target cell bound. If the carrier is bound after DLD, the binding can occur before or after the target cells are genetically engineered.

[0014] Binding of carriers can serve to stabilize cells, activate them (e.g., to divide), or facilitate the isolation of one type of cell from another. As suggested above, binding of carriers to cells can occur at various times in the method, including during the time the cells are being harvested. To improve separation, carriers can be selected so that binding of a single carrier to a cell results in a carrier-cell complex that is substantially larger than the size of the cell alone. Alternatively, carriers smaller than the target cells can be used. In this case, it is preferred that several carriers specifically bind to a single cell, thereby forming a complex containing a single cell and multiple carriers. During DLD, complexed target cells can be separated from uncomplexed cells of similar size, providing purification that would not otherwise occur.

[0015] To achieve such separation, the diameter of the complex should preferably be at least 20% longer than that of the uncomplexed target cell, more preferably at least 50% longer, at least twice as long, or at least 10 times longer. As mentioned above, this increase in size can be achieved either by binding a single large carrier to the target cell, or by binding several smaller carriers. This can be achieved using: a) only carriers with a diameter at least the same as (or in other embodiments at least twice or at least 10 times) the diameter of the target cell; b) only carriers with a diameter 50% or less (or in other embodiments, 25% or less or 15% or less) of the diameter of the target cell; or c) a mixture of large and small carriers with these size characteristics (e.g., there can be one group of carriers with a diameter at least the same as (or at least 2 times or 10 times) the diameter of the target cell and a second group of carriers with a diameter 50% or less (or 25% or less or 15% or less) of the diameter of the target cell). Typically, carriers have diameters of 1-1000 μm (often in the range of 5-600 μm or 5-400 μm). Ideally, complexes would be separated from other cells or contaminants by DLD on a microfluidic device with an array of obstacles with a critical size lower than the size of the complexes but higher than the size of uncomplexed non-target cells or contaminants.

[0016] In addition, carriers can serve to "complement DLD separation" rather than directly facilitate DLD separation with this technology. For example, carriers (e.g., Janus or strawberry-like particles) can contain two or more different chemical properties that support ready-to-use differential, size-independent secondary properties, such as chemical, electrochemical, or magnetic properties, and impart to the cells to which they bind, properties that can be used in downstream processes. Thus, the particles can be used to facilitate magnetic separation, electroporation, or gene transfer. They can also impart beneficial changes in cellular properties, such as metabolic or regenerative properties.

[0017] In particularly important embodiments, carrier binding can be used as a means of separating specific leukocytes, particularly T cells, including natural killer T cells, from other leukocytes, such as granulocytes and monocytes, and / or from other cells. This can be done, for example, in a two-step process in which DLD is performed on target cells not bound to a carrier using an array of obstacles having a critical size smaller than the target cells, and also on a complex comprising target cells and a carrier using an array of obstacles having a critical size smaller than the complex but larger than the uncomplexed cells. The DLD steps can be performed in either order; i.e., DLD can be performed on the complex before or after being performed on the uncomplexed target cells.

[0018] From the time when the crude fluid composition is obtained, no more than 4 hours (preferably, no more than 3 hours, no more than 2 hours, or no more than 1 hour) should elapse between the time when the target cells are first bound to the carrier. Additionally, from the time when the crude fluid composition is obtained, no more than 5 hours (preferably, no more than 4 hours, no more than 3 hours, or no more than 2 hours) should elapse between the time when the crude fluid composition is obtained and the time when the target cells are first transfected or transduced.

[0019] In a particularly preferred embodiment, the target cells in the above methods are T cells (particularly natural killer T cells and memory T cells), which are engineered to express chimeric antigen receptors on their surface. The procedures for generating these CAR T cells are described in more detail below.

[0020] Methods for generating CAR T cells The present invention includes methods for producing CAR T cells by obtaining a crude fluid composition comprising T cells (particularly natural killer T cells and memory T cells) and performing DLD on said composition using a microfluidic device. Typically, the crude fluid composition comprising T cells is an apheresis or leukapheresis product derived from a patient's blood and containing white blood cells.

[0021] A microfluidic device must have at least one channel extending from a sample inlet to one or more fluid outlets, the channel surrounded by a first wall and a second wall opposite the first wall. An array of obstacles is arranged in rows in the channel, with each successive row of obstacles shifted laterally relative to the previous row. The obstacles are arranged in such a way that when a crude fluid composition containing T cells is applied to the device's inlet and fluidly passes through the channel, the T cells flow to one or more collection outlets where a concentrated product is collected, while other cells (e.g., red blood cells and platelets) or other particles of a different (typically smaller) size than the T cells flow to another waste outlet separate from the collection outlet. Once obtained, the T cells are genetically engineered to produce chimeric antigen receptors (CARs) on their surface using procedures well established in the art. These receptors generally bind antigens present on the surface of cells associated with a disease or abnormal condition. For example, the receptor may bind an antigen unique to or overexpressed on the surface of cancer cells. In this regard, CD19 may be such an antigen.

[0022] Genetic engineering of CAR-expressing T cells generally involves transfecting or transducing T cells with a nucleic acid; once generated, CAR T cells can be expanded in number by growing the cells in vitro. Activators or other factors can be added during this process to promote growth; among the agents that can be used are IL-2 and IL-15. After DLD, recombinant engineering, and expansion, the yield of T cells expressing a chimeric receptor on their surface should, in some embodiments, be at least 10% higher, and preferably at least 20%, 30%, 40%, or 50% higher, than T cells prepared in the same manner but not subjected to DLD. Similarly, in some embodiments, the yield of T cells expressing a chimeric receptor on their surface should, in some embodiments, be at least 10% higher, and preferably at least 20%, 30%, 40%, or 50% higher, than T cells isolated by Ficoll centrifugation and not subjected to DLD.

[0023] Chimeric receptors typically have a) an extracellular region containing an antigen-binding domain, b) a transmembrane region, and c) an intracellular region. The cells may also be recombinantly engineered with sequences that provide the cells with a molecular switch that, when triggered, reduces the number or activity of CAR T cells. In a preferred embodiment, the antigen-binding domain is a single-chain variable fragment (scFv) derived from the antigen-binding regions of both the heavy and light chains of a monoclonal antibody. Preferably, a hinge region of 2-20 amino acids connecting the extracellular and transmembrane regions is also present. The transmembrane region may have a CD3ζ, CD4, CD8, or CD28 protein sequence, and the intracellular region should have a signaling domain, typically derived from CD3ζ, CD137, or CD28. Other signaling sequences that act to regulate or stimulate activity may also be included.

[0024] After obtaining a crude fluid composition containing T cells, or while they are being collected, the T cells can be bound to one or more carriers to facilitate DLD separation, for the reasons discussed above. This is preferably done before DLD is performed. However, it can also occur after DLD is performed, either before or after the cells are first transfected or transduced. In a preferred embodiment, the carrier should include an affinity substance (e.g., an antibody, activator, hapten, or aptamer) on its surface that specifically binds T cells, preferably natural killer T cells. As used in this context, the term "specificity" means that the carrier preferentially binds the desired T cells compared to any other cells in the composition. For example, a carrier may bind 100 or 1000 CD8+ T cells for each instance in which it binds a different type of cell.

[0025] In some embodiments, the support may be spherical in shape and made of either biological or synthetic materials, including collagen, polysaccharides including polystyrene, acrylamide, alginate, and magnetic materials. Additionally, the support may serve to complement DLD separations.

[0026] To help achieve separation, the diameter of the complex formed between the T cell and the carrier should preferably be at least 20% longer, preferably at least 50% longer, at least twice as long, or at least 10 times longer than the uncomplexed T cell. This increase in size can be due to the binding of a single large carrier to the target cell or the binding of several smaller carriers. Binding can involve using: a) only carriers having a length diameter at least the same as (or in other embodiments at least twice, or at least 10 times) the diameter of the T cell; b) only carriers having a length diameter 50% or less (or in other embodiments, 25% or less, or 15% or less) of the diameter of the T cell; or c) a mixture of large and small carriers with these size characteristics (e.g., there can be one group of carriers having a length diameter at least the same as (or at least 2 or 10 times) the diameter of the T cell and a second group of carriers having a length diameter 50% or less (or 25% or less, or 15% or less) of the diameter of the T cell). Typically, the carriers have diameters of 1-1000 μm (often in the range of 5-600 μm or 5-400 μm). Ideally, complexes would be separated from uncomplexed cells or contaminants by DLD on a microfluidic device with an array of obstacles with a critical size lower than the size of the complexes but higher than the size of the uncomplexed non-target cells or contaminants.

[0027] As discussed above with respect to target cells, purification of T cells can involve a two-step process. For example, DLD can be performed on unbound T cells using an array of obstacles with a critical size smaller than the T cells. The composition containing the separated T cells, along with other cells or particles, can then be collected and bound to one or more carriers to facilitate DLD separation, to which the T cells specifically bind. The complexes formed thereby can then be separated on an array of obstacles with a critical size smaller than the complexes but larger than the uncomplexed cells. In principle, the DLD steps can be performed in either order; i.e., they can be performed first on the complexes or first on the uncomplexed T cells.

[0028] Preferably, no more than 4 hours (more preferably, no more than 3 hours, no more than 2 hours, or no more than 1 hour) should elapse between the time the crude fluid composition comprising T cells is obtained (e.g., from the time apheresis or leukapheresis is completed) and the time the T cells are bound to the carrier. In addition, no more than 5 hours (preferably, no more than 4 hours, no more than 3 hours, or no more than 2 hours) should elapse between the time the T cells are obtained and the time the T cells are transfected or transduced for the first time. Ideally, all steps in generating CAR T cells are performed in the same facility where the crude fluid composition comprising the T cells is obtained, and all steps are completed within 4 hours (preferably, no more than 3 hours), and without freezing the cells.

[0029] Treating cancer, autoimmune diseases, or infectious diseases with CAR T cells In another aspect, the present invention is directed to a method of treating a patient for cancer, autoimmune disease, or infectious disease by administering CAR T cells engineered to express a chimeric antigen receptor that recognizes a cancer cell antigen or an antigen on cells that cause or contribute to the autoimmune disease or infectious disease. CAR T cells can be produced using the methods discussed in the above section, i.e., by obtaining a crude fluid composition containing T cells (preferably a leukocyte-containing apheresis or leukapheresis product drawn from a patient) and then performing DLD on the composition using a microfluidic device. CAR T cells (preferably natural killer T cells and memory T cells) recovered in this manner are then expanded by growing the cells in vitro. Finally, the cells are administered to the patient, which should generally be the same patient who provided the blood from which the T cells were isolated.

[0030] Preferably, the yield of T cells expressing a chimeric receptor on their surface after DLD, recombinant engineering, and expansion is at least 10% higher, and more preferably at least 20%, 30%, 40%, or 50% higher, than T cells prepared in the same manner but not subjected to DLD. For example, the yield of T cells expressing a chimeric receptor on their surface may be at least 10% higher, and preferably at least 20%, 30%, 40%, or 50% higher, than T cells isolated by Ficoll centrifugation and not subjected to DLD.

[0031] Chimeric receptors typically have at least a) an extracellular region containing an antigen-binding domain, b) a transmembrane region, and c) an intracellular region. The cells may also be recombinantly engineered with sequences that provide the cells with a molecular switch that, when triggered, reduces the number or activity of CAR T cells. In a preferred embodiment, the antigen-binding domain is a single-chain variable fragment (scFv) derived from the antigen-binding regions of both the heavy and light chains of a monoclonal antibody. A hinge region of 2-20 amino acids is also present, connecting the extracellular and transmembrane regions. The transmembrane region itself may have CD3ζ, CD4, CD8, or CD28 protein sequences, and the intracellular region has signaling domains, typically derived from the CD3ζ and / or CD28 intracellular domains. Other signaling sequences that act to regulate or stimulate activity may also be included.

[0032] After obtaining the crude fluid composition, or during the time the crude fluid composition is being collected, one or more carriers can be attached to the T cells present in the composition to facilitate or complement DLD isolation. This is preferably done before DLD is performed. However, it can also occur after DLD is performed, either before or after the cells are genetically engineered. Preferably, the attachment facilitates DLD isolation, and the carrier includes on its surface an antibody, activator, or other agent that specifically binds to T cells, particularly natural killer T cells. As used in this context, the term "specificity" means that the carrier preferentially binds to the desired T cells compared to any other cells in the composition. For example, a carrier may bind 100 or 1000 CD8+ T cells per carrier that binds other types of cells.

[0033] The diameter of the complex formed between the T cell and the carrier should preferably be at least 20% longer than that of the uncomplexed T cell, more preferably at least 50% longer, at least twice as long, or at least 10 times longer. This increase in size can be due to the binding of a single large carrier to the cell, or to the binding of several smaller carriers. Binding can involve using: a) only carriers having a length diameter at least the same as (or in other embodiments at least twice, or at least 10 times) the diameter of the T cell; b) only carriers having a length diameter 50% or less (or in other embodiments, 25% or less, or 15% or less) of the diameter of the T cell; or c) a mixture of large and small carriers with these size characteristics (e.g., there can be one group of carriers having a length diameter at least the same as (or at least 2 or 10 times) the diameter of the T cell and a second group of carriers having a length diameter 50% or less (or 25% or less, or 15% or less) of the diameter of the T cell). Typically, the carriers have diameters of 1-1000 μm (often in the range of 5-600 μm or 5-400 μm). Ideally, complexes would be separated from uncomplexed cells or contaminants by DLD on a microfluidic device with an array of obstacles with a critical size lower than the size of the complexes but higher than the size of the uncomplexed non-target cells or contaminants.

[0034] T cell purification can involve a two-step process. For example, DLD can be performed on unbound T cells using an array of obstacles with a critical size smaller than the T cells. The composition containing the separated T cells, along with other cells or particles, can then be collected and bound to one or more carriers to facilitate DLD separation, to which the T cells specifically bind. The complexes formed thereby can then be separated on an array of obstacles with a critical size smaller than the complexes but larger than the uncomplexed cells. In principle, the DLD steps can be performed in either order; i.e., they can be performed first on the complexes or first on the uncomplexed T cells.

[0035] Preferably, no more than 4 hours (more preferably, no more than 3 hours, no more than 2 hours, or no more than 1 hour) should elapse from the time T cell acquisition is completed (e.g., from the time apheresis or leukapheresis is completed) until the T cells are bound to the carrier. In addition, no more than 5 hours (preferably, no more than 4 hours, no more than 3 hours, or no more than 2 hours) should elapse from the time T cell acquisition is completed until the T cells are transfected or transduced for the first time. Ideally, all steps in generating CAR T cells are performed in the same facility where the crude fluid composition containing T cells is obtained, and all steps are completed within 4 hours (preferably, no more than 3 hours).

[0036] The CAR T cells thus produced can be used to treat patients with leukemia, such as acute lymphoblastic leukemia, using procedures well established in the field of clinical medicine. In these cases, the CAR can recognize CD19 or CD20 as a tumor antigen. The method can also be used for solid tumors, in which case the recognized antigens can include CD22; RORI; mesothelin; CD33 / IL3Ra; c-Met; PSMA; glycolipid F77; EGFRvIII; GD-2; NY-ESO-1; MAGE A3; and combinations thereof. Regarding autoimmune diseases, CAR T cells can be used to treat rheumatoid arthritis, lupus, multiple sclerosis, ankylosing spondylitis, type 1 diabetes, or vasculitis.

[0037] In some embodiments, target cells produced by the above methods will be available for administration to a patient sooner than if the cells were produced using a method that does not involve DLD. These cells can be administered one or more days sooner, preferably two, three, four, five, or more days sooner. The cells can be administered within 8 to 10 days from the time the crude fluid composition is obtained.

[0038] Cell collection and processing The present invention is also directed to protocols for collecting and processing patient-derived cells that are designed to rapidly process the cells and can generally be performed at the site where the cells are collected. The protocols can be used as part of the methods for preparing target cells and CAR T cells described above. Some aspects of these protocols are illustrated in Figures 13 and 14 and can be compared to the protocol shown in Figure 12. In the particular procedure illustrated, a composition obtained by apheresis of whole blood is obtained, and T cells in the composition are then selected. The term "selected" in this context means that the T cells are bound by an agent that recognizes T cells with specificity (as defined above). The selected T cells are then isolated using a DLD and transferred to a selected fluid medium.

[0039] More generally, the present invention relates to a method for collecting target cells by: a) obtaining a crude fluid composition comprising target cells from a patient; and b) performing deterministic lateral displacement (DLD) on the fluid composition to obtain a composition enriched in target cells, wherein either before or after DLD, a carrier is attached to the target cells to facilitate DLD isolation. For example, a carrier having an affinity substance (e.g., an antibody, activator, hapten, or aptamer) on its surface that binds with specificity (as defined above) to target cells can be used.

[0040] If desired, the carriers may be bound to the target cells for the time that the target cells are being collected from the patient, and no more than 5 hours (preferably, more than 4 hours, more than 3 hours, more than 2 hours, or more than 1 hour) should elapse between the time that the crude fluid composition containing the target cells is obtained and the time that the carriers are bound to the target cells.

[0041] The diameter of the complex formed between the target cell and one or more carriers should preferably be at least 20% longer than that of the uncomplexed cell, preferably at least 50% longer, at least twice as long, or at least 10 times longer. This increase in size can be due to the binding of a single large carrier to the target cell, or to the binding of several smaller carriers. Binding can involve using i) only carriers having a length diameter at least the same as (or in other embodiments at least twice, or at least 10 times) the diameter of the target cell; ii) only carriers having a length diameter 50% or less (or in other embodiments, 25% or less, or 15% or less) of the diameter of the target cell; or iii) a mixture of large and small carriers having these size characteristics (e.g., there can be one group of carriers having a length diameter at least the same as (or at least 2 or 10 times) the diameter of the target cell and a second group of carriers having a length diameter 50% or less (or 25% or less, or 15% or less) of the diameter of the target cell). Typically, carriers have diameters of 1-1000 μm (often in the range of 5-600 μm or 5-400 μm). Ideally, complexes would be separated from other cells or contaminants by DLD on a microfluidic device with an array of obstacles with a critical size lower than the size of the complexes but higher than the size of the uncomplexed cells or contaminants.

[0042] In a preferred embodiment, the crude fluid composition containing target cells is obtained by performing apheresis or leukapheresis on blood from a patient. The composition may contain one or more additives that act as anticoagulants or prevent platelet activation. Examples of such additives include ticlopidine, inosine, protocatechuic acid, acetylsalicylic acid, and tirofiban, alone or in combination.

[0043] The microfluidic device must have at least one channel extending from a sample inlet to one or more fluid outlets, the channel being bounded by a first wall and a second wall opposite the first wall. There must also be an array of obstacles arranged in rows in the channel, with each successive row of obstacles being laterally shifted relative to the previous row, so that when a crude fluid composition containing the target cells is applied to the device inlet and fluidly passes through the channel, the target cells flow to one or more collection outlets where an enriched product is collected, and contaminating cells or particles present in the crude fluid composition and that are a different size from the target cells flow to another waste outlet that is separate from the collection outlet.

[0044] In particularly preferred embodiments, the target cell is a T cell selected from the group consisting of natural killer T cells, central memory T cells, helper T cells, and regulatory T cells, with natural killer T cells being most preferred. In alternative preferred embodiments, the target cell is a stem cell, B cell, macrophage, monocyte, dendritic cell, or progenitor cell.

[0045] In addition to steps a) and b), the method of the present invention may include c) genetically engineering the cells by transducing them with a viral vector (alternatively, the cells may be transfected and / or expanded in number electrically, chemically, or using nanoparticles), and / or d) treating the same patient from which the target cells were obtained with the collected target cells. In addition, the collected cells may be cultured and / or cryopreserved. When the target cells are T cells, the culture should generally be carried out in the presence of an activator, preferably an activator bound to a carrier. Among the factors that may be included in T cell culture are IL-2 and IL-15.

[0046] In some embodiments, target cells produced by the above methods will be available for administration to a patient sooner than if the cells were produced using a method that does not involve DLD. These cells can be administered one or more days sooner, preferably two, three, four, five, or more days sooner. The cells can be administered within 8 to 10 days from the time the crude fluid composition is obtained.

[0047] In addition to the methods discussed above, the present invention includes target cells produced by the methods, and methods of treatment in which the target cells are administered to a patient.

[0048] Altering white blood cell characteristics using DLD Reducing platelet levels in leukocyte preparations has advantages both for generating CAR T cells and for preparing leukocytes for other therapeutic uses. In this regard, the present invention is based, in part, on the concept that DLD reduces the total number of platelets in an apheresis sample more effectively than when using Ficoll separation in general (see Figures 19-21), and when using buffers that do not promote platelet aggregation in particular (see Figures 22-23). ​​When used in combination with separation based on magnetic beads that specifically bind to T cells, DLD results in preparations of cells that can expand more rapidly than when using such beads either alone or in combination with Ficoll centrifugation (see Figure 24). This effect is due, in part, to a reduction in platelet count and factors independent of the number of platelets present (see Figure 24). Furthermore, results obtained using the DLD / magnetic bead procedure are more consistent (see Figure 25), and expanded T cells from this procedure contain a higher percentage of T cells with a central memory phenotype when compared to populations prepared using the Ficoll / magnetic bead approach (see Figure 26). The high initial cell recovery from DLD combined with a) faster T cell expansion and b) a higher percentage of central memory cells means that therapeutically effective levels of T cells can be made available to patients more quickly.

[0049] In one embodiment, the present invention is directed to a method for reducing the platelet to leukocyte ratio in an apheresis sample by performing deterministic lateral displacement on the sample in the absence of centrifugation or elutriation to obtain a product in which the platelet to leukocyte ratio is at least 20% (preferably 50% or 70%) lower than the ratio obtained when the same procedure is performed using centrifugation (including gradient centrifugation or counterflow centrifugation) or elutriation instead of DLD. Preferably, this procedure does not include a separation step performed on the apheresis sample prior to DLD, and DLD is performed in a buffer that does not contain intercalators or other agents that alter platelet size and do not promote platelet aggregation. Agents to avoid include dextran and other highly charged polymers. In addition to lowering the platelet to leukocyte ratio, the total platelet count in the DLD-derived product should be at least 70% lower, preferably at least 90% lower, than in the apheresis sample.

[0050] In another aspect, the present invention is directed to a method for purifying T cells from an apheresis sample by performing DLD on the sample, followed by an affinity separation step and T cell expansion by culturing in the presence of an activator. This process should result in at least twice the number of T cells produced by the same procedure performed using Ficoll centrifugation instead of DLD. A preferred affinity method involves the use of magnetic beads that specifically bind to T cells via an antibody that recognizes at least CD3, and may include CD3 together with CD28 or other costimulatory molecules. The number of T cells obtained after 14 days in culture should be at least two-fold (preferably at least four-fold or six-fold) higher than the number produced by the same procedure performed using Ficoll centrifugation instead of DLD. The percentage of memory T cells in the product produced by this method should be at least 10% (preferably at least 20%) higher than the percentage produced using the same procedure using Ficoll centrifugation instead of DLD.

[0051] This method is particularly suitable for producing T cells for CAR T cell therapy.By using DLD instead of Ficoll centrifugation, the time required to produce a sufficient number of cells for patient treatment is reduced by at least 5% (preferably at least 10% or at least 20%), and CAR T cells can be prepared without freezing.In a preferred method, cells are collected from patient and processed by DLD, optionally by affinity method at the same place.Genetic transformation can also occur at the same place, and preferably, within 1 hour from the completion of apheresis to the start of DLD.

[0052] The present invention also encompasses a method for reducing the platelet to leukocyte ratio in an apheresis sample by performing deterministic lateral displacement (DLD) on the sample. DLD is performed without centrifugation or elutriation to obtain a product in which the total number of platelets is at least 70% (preferably at least 90%) lower than in the apheresis sample. Preferably, DLD should be performed in a buffer that does not contain intercalators and does not promote platelet aggregation. Preferably, the buffer does not contain dextran or other highly charged polymers.

[0053] The present invention is not limited to leukocytes, but also includes other therapeutically valuable cells, particularly cells that may be present in apheresis preparations, including circulating stem cells. The benefits of DLD, including the benefits of removing unwanted platelets, should be applicable to a variety of processes.

[0054] A method for using DLD for large volumes of leukapheresis material One advantage of DLD is that it can be used to process relatively large amounts of material as well as small amounts of material with little increase in volume. The procedure can be used not only to process large volumes of material, but also for leukapheresis products, which are small in volume due to leukocyte enrichment by centrifugation.

[0055] Thus, in another aspect, the present invention is directed to a system for purifying cells from a large-volume leukapheresis process, in which at least one microfluidic device is used to separate materials by DLD. The objective is to obtain leukocytes that can be used therapeutically or that secrete therapeutically usable agents. Of particular importance, the present invention involves binding one or more carriers to specific types of leukocytes to facilitate, and optionally complement, DLD separation, and then performing DLD on the complexes. In this way, specific types of leukocytes can be separated from cells that are approximately the same size and would not be able to be divided by DLD in the absence of complex formation. In this regard, a two-step procedure, as discussed above, in which one DLD procedure separates unbound leukocytes from smaller material and another DLD procedure separates carrier-leukocyte complexes from uncomplexed cells may be advantageous. Essentially the same technique can be used in other contexts as well, for example, for cultured cells, provided that cell-specific carriers are available. In all cases, the cells may be recombinantly engineered to alter the expression of one or more of their genes.

[0056] For leukapheresis material, a microfluidic device must have at least one channel extending from a sample inlet to both a "collection outlet" for collecting white blood cells (WBCs) or specific leukapheresis-carrier complexes and a "waste outlet" through which material of a different size (typically smaller) than WBCs or uncomplexed leukapheresis flows. The channel is bounded by a first wall and a second wall opposite the first wall and contains an array of obstacles arranged in rows, with each successive row laterally shifted relative to the previous row. The obstacles are arranged in such a way that, when leukapheresis material is applied to the device's inlet and fluidly passes through the channel, cells or cell complexes are deflected toward the collection outlet(s) where the enriched product is collected, while material of a different (typically smaller) size flows toward one or more separate waste outlets.

[0057] To facilitate rapid processing of large volumes of starting material, the obstacles in the microfluidic device can be designed in a diamond or triangular shape, and each device can have 6 to 40 channels. Additionally, the microfluidic device can be part of a system containing 2 to 20 microfluidic devices (see Figure 7). Individual devices can be operated at flow rates of 14 ml / h, but flow rates of at least 25 ml / h (preferably at least 40 ml / h, 60 ml / h, 80 ml / h, or 100 ml / h) are preferred, allowing large sample volumes (at least 200 ml, preferably 400-600 ml) to be processed within an hour.

[0058] Viable cell isolation In another aspect, the present invention is directed to a method for separating viable cells from non-viable cells, comprising the steps of: (a) obtaining a sample comprising viable and non-viable cells, wherein the viable cells may have a first predetermined size and the non-viable cells may have a second predetermined size, and wherein the first predetermined size may be equal to or greater than a critical size and the second predetermined size may be smaller than the critical size; (b) applying the sample to a device, wherein the device may include an array of obstacles arranged in a number of rows, wherein the rows may be shifted laterally relative to each other and wherein the rows may be configured to deflect particles equal to or greater than the critical size in a first direction and particles smaller than the critical size in a second direction; and (c) flowing the sample through the device, wherein the viable cells may be deflected in the first direction by the obstacles and the non-viable cells may be deflected in the second direction, thereby separating the viable cells from the non-viable cells. The critical size can be about 1.1 times the second predetermined size, and in some embodiments, the viable cells can be actively dividing cells. In some embodiments, the device can include at least three zones with progressively smaller obstacles and gaps.

[0059] Isolation of adherent cells The present invention also includes a method for obtaining adherent target cells, preferably therapeutically valuable cells, e.g., adherent stem cells, by: a) obtaining a crude fluid composition containing adherent target cells from a patient; and b) performing deterministic lateral displacement (DLD) to obtain a composition enriched in adherent target cells. During this process, the adherent target cells may be bound to one or more carriers to facilitate or complement DLD isolation. For example, the carrier may have on its surface an affinity substance (e.g., an antibody, activator, hapten, or aptamer) that binds with specificity (as defined above) to the adherent target cells, and may be transfected or transduced with a nucleic acid designed to confer a desired phenotype to the cells, e.g., to express a chimeric molecule (preferably a protein that renders the cells more therapeutically valuable).

[0060] The carriers can be added while the crude fluid composition is being collected, or alternatively, after collection is complete but before DLD is performed for the first time. In a second alternative, DLD can be performed for the first time before the carriers are added. For example, if the adherent cells have a size less than the critical size, the crude fluid composition can be applied to the device before the carriers are added, the adherent cells can be collected, and the cells can then be attached to one or more carriers to form complexes larger than the critical size of the device, after which a second DLD step can be performed and the carrier-adherent cell complexes can be collected.

[0061] Preferably, no more than 3 hours (more preferably, no more than 2 hours or no more than 1 hour) elapses from the time the crude fluid composition is obtained from the patient until the adherent cells are first bound to the carrier. In another preferred embodiment, no more than 4 hours (preferably, no more than 3 hours or no more than 2 hours) elapses from the time the crude fluid composition is obtained from the patient until the adherent cells or carrier-adherent cell complexes are first recovered from the device.

[0062] The above methodology can be used to separate adherent target cells, such as adherent stem cells, from a plurality of other cells, comprising: a) contacting a crude fluid composition comprising adherent target cells and a plurality of other cells, wherein the adherent target cells at least partially associate with one or more carriers to facilitate DLD separation, forming carrier-associated adherent target cell complexes, the complexes comprising an increased size relative to the plurality of other cells, the size of the carrier-associated adherent cell complexes preferably being at least 50% larger than a critical size, and other uncomplexed cells comprising a size less than the critical size; b) applying the crude fluid composition containing the carrier-associated adherent cell complexes to a device, wherein the device is configured to separate the adherent target cells from a plurality of other cells in a row. The device comprises an array of arranged obstacles, the rows of which are shifted laterally relative to each other and are configured to deflect cells or complexes of a critical size or greater in a first direction and cells or complexes of a critical size or less in a second direction; c) flowing the crude fluid composition containing the carrier-associated adherent target cell complexes through the device, wherein the complexes are deflected in the first direction by the obstacles and uncomplexed cells are deflected in the second direction, thereby separating the carrier-associated adherent cell complexes from the other uncomplexed cells; d) collecting the fluid composition containing the separated carrier-associated adherent target cell complexes.

[0063] The diameter of the complex formed between the adherent target cells and one or more carriers should preferably be at least 20% longer than that of the uncomplexed cells, preferably at least 50% longer, at least twice as long, or at least 10 times longer. This increase in size can be due to the binding of a single large carrier to the adherent target cells or the binding of several smaller carriers. The binding can involve using a) only carriers having a length diameter at least the same as (or in other embodiments at least twice, or at least 10 times) the diameter of the adherent target cells; b) only carriers having a length diameter 50% or less (or in other embodiments, 25% or less, or 15% or less) of the diameter of the adherent target cells; or c) a mixture of large and small carriers having these size characteristics (e.g., there can be one group of carriers having a length diameter at least the same as (or at least 2 or 10 times) the diameter of the adherent target cells and a second group of carriers having a length diameter 50% or less (or 25% or less, or 15% or less) of the diameter of the adherent target cells). Typically, the carrier has a diameter of 1 to 1000 μm (often in the range of 5 to 600 μm or 5 to 400 μm).

[0064] The carriers can be made of any of the materials known in the art for culturing adherent cells, including polypropylene, polystyrene, glass, gelatin, collagen, polysaccharides, plastics, acrylamide, and alginate. They can be uncoated or coated with a material that promotes adhesion and growth (e.g., serum, collagen, protein, or polymer), and can have an agent attached to the surface (e.g., an antibody, antibody fragment, substrate, activator, or other material). In some embodiments, the diluent can be growth medium, and the steps can be performed sequentially, with buffer exchange performed after step (e).

[0065] Examples of specific adherent cells that can be isolated in the above-described methods include: MRC-5 cells; HeLa cells; Vero cells; NIH 3T3 cells; L929 cells; Sf21 cells; Sf9 cells; A549 cells; A9 cells; AtT-20 cells; BALB / 3T3 cells; BHK-21 cells; BHL-100 cells; BT cells; Caco-2 cells; Chang cells; Clone 9 cells; Clone M-3 cells;COS-1 cells;COS-3 cells;COS-7 cells;CRFK cells;CV-1 cells;D-17 cells;Daudi cells;GH1 cells;GH3 cells;HaK cells;HCT-15 cells;HL-60 cells;HT-10 80 cells; HEK cells, HT-29 cells; HUVEC cells; I-10 cells; IM-9 cells; JEG-2 cells; Jensen cells; Jurkat cells; K-562 cells; KB cells; KG-1 cells; L2 cells; LLC-WRC 256 cells; McCoy cells; MCF7 cells; WI-38 cells; WISH cells;

[0066] Isolation of activator-bound cells The present invention also includes methods for purifying activation-competent cells using the above procedures. In a preferred embodiment, the present invention is directed to a method for separating activated cells from a plurality of other cells by: a) contacting a crude fluid composition comprising activation-competent cells and a plurality of other cells with one or more carriers in a manner that promotes DLD separation, wherein one or more of the carriers comprises a cellular activator, wherein the one or more carriers at least partially associate with the activation-competent cells by the cellular activator upon or after contact to produce carrier-associated cells, wherein the at least partial association of the cellular activator with the activation-competent cells activates the activation-competent cells, and wherein the carrier-associated cell complexes comprise an increased size relative to other cells, and wherein the carriers a) applying the crude fluid composition to a device comprising an array of obstacles arranged in rows, the rows being laterally shifted relative to each other and configured to deflect particles of a critical size or greater in a first direction and particles of a critical size or less in a second direction; b) flowing the sample through the device, the carrier-associated cell complexes being deflected in the first direction by the obstacles and the cells of the plurality of other cells being deflected in the second direction, thereby separating the activated cells from the plurality of other cells. The fluid composition containing the separated carrier-associated cell complexes can then be collected. During this process, the cells can be optionally transfected or transduced with a nucleic acid designed to confer a desired phenotype to the cells, for example, to express a chimeric molecule (preferably a protein that renders the cells more therapeutically valuable).

[0067] The cells capable of activation may be selected from the group consisting of T cells, B cells, macrophages, dendritic cells, granulocytes, innate lymphoid cells, megakaryocytes, natural killer cells, thrombocytes, synoviocytes, beta cells, liver cells, pancreatic cells, DE3 lysogenized cells, yeast cells, plant cells, and stem cells.

[0068] The cellular activator may be selected from the group consisting of an antibody or antibody fragment, CD3, CD28, an antigen, a helper T cell, a receptor, a cytokine, a glycoprotein, and any combination thereof. In other embodiments, the activator may be a small molecule compound and may be selected from the group consisting of insulin, IPTG, lactose, allolactose, a lipid, a glycoside, a terpene, a steroid, an alkaloid, and any combination thereof.

[0069] In a preferred embodiment, the activation-competent cells are collected from a patient as part of a crude fluid composition comprising the activation-competent cells and a plurality of other cells, and no more than 4 hours (preferably no more than 3 hours, no more than 2 hours, or no more than 1 hour) elapses between the time the crude fluid composition is obtained from the patient and the time the carrier is attached to the activation-competent cells. It is also preferred that no more than 4 hours elapses between the time the crude fluid composition is obtained from the patient and the time step c) is completed. Alternatively, the method can be modified by attaching the activator before collection of the cells begins.

[0070] Preferably, the diameter of the complex formed between the activation-competent cells and one or more carriers should be at least 20% longer than that of the uncomplexed cells, more preferably at least 50% longer, at least twice as long, or at least 10 times longer. This increase in size can be due to the binding of a single large carrier to the activation-competent cells, or the binding of several smaller carriers. The binding can involve using: a) only carriers having a diameter at least the same as (or in other embodiments at least twice, or at least 10 times) the diameter of the activation-competent cells; b) only carriers having a diameter 50% or less (or in other embodiments, 25% or less, or 15% or less) the diameter of the activation-competent cells; or c) a mixture of large and small carriers with these size characteristics (e.g., there can be one group of carriers having a diameter at least the same as (or at least 2 or 10 times) the diameter of the activation-competent cells and a second group of carriers having a diameter 50% or less (or 25% or less, or 15% or less) the diameter of the activation-competent cells). Typically, the carrier has a diameter of 1 to 1000 μm (often in the range of 5 to 600 μm or 5 to 400 μm).

[0071] Isolation of compounds from cells In another embodiment, the present invention includes a method for removing a compound from cells, comprising: (a) obtaining a fluid composition comprising cells and a compound, wherein the cells have a predetermined size greater than the predetermined size of the compound, and the predetermined size of the cells is equal to or greater than a critical size, and the predetermined size of the compound is less than the critical size; (b) applying the sample to a device comprising an array of obstacles arranged in rows, the rows being shifted laterally relative to each other and configured to deflect particles equal to or greater than the critical size in a first direction and particles less than the critical size in a second direction; and (c) flowing the sample through the device, during which the cells are deflected in the first direction by the obstacles and the compounds are deflected in the second direction, thereby removing the compounds from the cells. In some embodiments, the method can further include culturing the cells after step (c) or recycling the cells to the culture from which the fluid composition of step (a) was obtained.

[0072] The compound may be a toxic compound and may be selected from the group consisting of antibiotics, antifungals, toxic metabolites, sodium azide, metal ions, endotoxins, plasticizers, pesticides, and combinations thereof. In other embodiments, the compound may be a spent chemical moiety.

[0073] Continuous purification of secretory cell products The present invention also includes a method for continuously purifying a secretory product from cells, comprising: (a) obtaining a fluid composition (which may be a cell culture composition) comprising cells, wherein the cells are suspended in the fluid composition (or the cells are attached to one or more carriers to facilitate DLD separation and form carrier-cell complexes), the cells secrete a secretory product into the fluid composition, the cells (or the carrier-cell complexes) having a predetermined size greater than the predetermined size of the secretory product, the predetermined size of the cells (or the carrier-cell complexes) being equal to or greater than a critical size, and the predetermined size of the secretory product being less than the critical size; (b) applying the fluid composition comprising the cells (or the carrier-cell complexes) to a DLD device, wherein the device comprises an array of obstacles arranged in a number of rows, the rows being laterally spaced relative to each other. (c) flowing a fluid composition comprising the cells or carrier-cell complexes through the device, wherein the cells or carrier-cell complexes are deflected by the obstacle in the first direction and the secretory product in the second direction, thereby separating the secretory product from the cells; (d) collecting the secretory product, thereby producing a fluid composition of the secretory product that has been purified; (e) collecting the recovered fluid composition comprising the separated cells or carrier-cell complexes; (f) reapplying the cells (or the carrier-cell complexes) to the fluid composition; and repeating steps (a) through (e), thereby continuously purifying the secretory product from the cells.

[0074] The secreted product can be a protein, an antibody, a biofuel, a polymer, a small molecule, or any combination thereof, and the cells can be bacterial cells, algal cells, mammalian cells, and tumor cells. In one preferred embodiment, the secreted product is a protein, an antibody, a polymer, or a small molecule of high therapeutic value. Additionally, the fluid composition of step (a) can be obtained from a culture in which cells are growing on a carrier and the cells are growing thereon.

[0075] Use of the microfluidic sizing device More broadly, the present invention is directed to a method for manipulating a population of target cells prepared by any size-based microfluidic separation method. Size-based cell sorting differences can result from the use of bump arrays as discussed herein, or from inertial forces generated by controlling the flow rate during separation, or from the design of the microfluidic device itself (see U.S. Pat. Nos. 9,895,694 and 9,610,582, which are incorporated herein by reference in their entireties). Single or multiple separation procedures can be used. For example, target cells can be separated from small particles and cells using one microfluidic procedure and from large particles and cells using a second procedure.

[0076] Once the target cells are isolated, they are genetically engineered to have the desired phenotype. This can be achieved using standard recombinant methodologies to transfect or transform cells. For example, cells can be transfected with a vector to express the recombinant phenotype. Avoiding centrifugation prior to genetic engineering should result in at least a 20% increase in cells with the desired characteristics.

[0077] Preferred target cells are leukocytes (especially T cells) or stem cells, and preferred crude liquid compositions are blood or apheresis preparations obtained from patients. The central goal is to reduce the platelet to target cell ratio in these preparations by at least 50%, preferably at least 80% or 90%. Target cell isolation should be performed under conditions where the total number of platelets is at least 70% (preferably at least 90%) lower than in the starting apheresis preparation so that a product can be obtained.

[0078] During or after genetic manipulation, the cells are expanded in cell culture. Using the above procedure, the number of T cells obtained after 14 days in culture should be at least 2-fold (preferably at least 5- or 10-fold) higher than in procedures in which cells are isolated using centrifugation. Furthermore, the percentage of memory T cells in the culture relative to the total number of T cells should be at least 10% (preferably 20% or 30%) higher than in procedures in which T cells are isolated by centrifugation.

[0079] No more than one hour should elapse from the time apheresis collection is completed until DLD is performed, and no more than four hours should elapse from the time apheresis sample acquisition is completed until target cells are isolated and genetically manipulated.

[0080] In a particularly preferred embodiment, the above method is used to produce CAR T cells. This involves first obtaining a crude composition containing T cells by apheresis, then isolating the cells on a microfluidic device using one or more procedures that separate T cells from platelets based on size differences. As a result, a product enriched in T cells and depleted of platelets is obtained. In the next step, the isolated T cells are genetically engineered to express a chimeric antigen receptor (CAR) on their surface. These cells are cultured to expand their numbers and then collected. T cells should not be centrifuged or water-treated at any stage before being genetically engineered; in a preferred embodiment, the reagents used for genetic engineering are separated from the cells by size using a microfluidic device. In an additional preferred embodiment, the T cells are collected by transferring them into a pharmaceutical composition for administration to a patient.

[0081] The T cells should not be frozen before collection or transfer into the pharmaceutical composition, and preferably at least 90% of the platelets are removed. Before or during culture, the cells may be exposed to a T cell activator or carrier. This may help stabilize the cells and facilitate size-based microfluidic separation. However, it should be noted that neither the activator nor the carrier necessarily needs to be bound to magnetic beads or particles.

[0082] Compared to procedures in which cells are isolated or concentrated by centrifugation, CAR T cells obtained by microfluidic separation should be available for patient use at least one day (preferably at least 3, 5, or 10 days) earlier. Overall, the time required to generate a sufficient number of CAR T cells for processing should be at least 10% (preferably at least 20% or 30%) shorter than when cells are separated by the same method using Ficoll centrifugation. This is, in part, because by using size-based microfluidic separation, the number of CAR T cells obtained after 14 days in culture is typically at least two-fold (preferably four-fold or eight-fold) higher than the number in cultures using cells obtained by Ficoll centrifugation. Furthermore, when cells prepared by this method are administered to patients, they should exhibit at least 10% less senescence than cells isolated from apheresis compositions by centrifugation.

[0083] The present invention also encompasses treating a patient for a disease or condition by administering a therapeutically effective amount of cells prepared by the methods discussed above, including any disease or condition that responds to engineered white blood cells or stem cells, and at least in the case of CAR T cells, cancer is among the diseases that may be treated. [Brief explanation of the drawings]

[0084] [Figure 1]Figures 1A-1C show the different operating modes of the DLD. These include i) separation (Figure 1A), ii) buffer exchange (Figure 1B), and iii) concentration (Figure 1C). In each mode, essentially all particles larger than a critical diameter are deflected from the point of entry toward the array, resulting in size selection, buffer exchange, or concentration as a function of the device geometry. In all cases, particles smaller than the critical diameter pass straight through the device under laminar flow conditions and then exit the device. Figure 1D shows the 14-lane DLD design used for separation mode. The depicted array and microchannels have a total length of 75 mm and a width of 40 mm, with each individual lane being 1.8 mm wide. Figures 1E-1F show close-up views of the plastic diamond post array and the integrated collection port for the outlet. Figure 1G depicts a photograph of leukapheresis product being processed using the prototype device at 10 PSI.

[0085] [Figure 2] Figure 2A is a scatter plot showing the range of platelet and WBC cell counts for the normal donors used in this study. The mean WBC count was 162.4 × 106 / mL and the mean platelet count was 2718 × 103 / µL (+), respectively. Outlier samples (▲) clogged the 20 µm prefilter and were excluded from the dataset. Input samples are shown (Figures 2C and 2D). Representative 24-hour leukapheresis input from a normal donor (Figure 2B) and PBMC product processed with either a 14-lane diamond post array DLD (Figure 2E) or Ficoll-Hypaque (Figure 2F) at 10 PSI. Representative DLD products (Figure 2G) and Ficoll (Figure 2H) from the same leukapheresis donor (#37). Input (Figures 2B, 2C, 2D) and product fractions (Figures 2E and 2F) were fixed and stained on slides with CD41-FITC (platelets) and CD45-Alexa647 (WBCs), and counterstained with DAPI (nuclear DNA).

[0086] [Figure 3]This figure shows the consistency of cell activation in DLD vs. Ficoll and direct magnet approaches (CD4, CD8 vs. CD25 at day 8). Cell activation and phenotypic profiles demonstrate a shift during expansion toward a classical central memory T cell-associated phenotype (day 8). Cells were counted and de-beaded as previously described. At each time point, approximately 100,000 cells were stained with CD3-BV421, CD45RA-BV605, CD95-FITC, CD279-PE, CD25-APC, CD4-Alexa 700, and CD8-APC-Cy7, incubated in the dark for 30 minutes at room temperature, washed with 10 volumes of PBS, then centrifuged and fixed in 1.0% paraformaldehyde in PBS. Samples were acquired on a BD FACSAria and analyzed using FlowLogic software, gated on CD3, forward scatter, and side scatter.

[0087] [Figure 4] Figure 4 is a graph depicting the rapid acquisition of memory cell phenotype and consistent activation of samples with DLD compared to Ficoll and direct magnet. Plots of % CD45RA-, CD25+ cells are shown, measuring conversion to T cell activation and conversion via CD45 RO status. Because the experiment was designed to address the initial ability to proliferate, cells were given 200 units IL-2 / mL culture only on day 3 and again on day 8.

[0088] [Figure 5]These figures relate to the fold expansion (×106) of CD3 cells from DLD, Ficoll, and direct magnet. Aliquots of DLD product and Ficoll cells were incubated with CD3 / CD28 beads according to the Thermo-Fisher CTS protocol, using a T cell density of 1×107 T cells / mL. Cells and beads were incubated on a rotary mixer for 60 minutes before magnetic separation, using a ratio of approximately 2.5 beads / T cell and, for direct magnet, approximately 5.0 beads / T cell. Either stimulated or unstimulated (unseparated PMBCs) cells were diluted to 0.5×106 cells / mL in complete medium (RPMI-1640 + 10% FBS + antibiotics without IL-2) and plated in timed reactions to avoid any disturbance of the culture at intermediate time points. On day 3, 200 IU of IL-2 / mL was added to the stimulated and separated arms as recommended by the manufacturer. After de-beading using the manufacturer's protocol (pipetting), cell counts were determined on days 3, 8, and 15 using a Coulter counter (Scepter) and verified by bead-based absolute counting using flow cytometry on a BD FACSCalibur using a no-wash approach with a fluorescence threshold for CD45 and staining with CD3-FITC, CD45-PerCP, and the DNA stain DRAQ5 to ensure effective discrimination of doublets and any cells with beads still attached. Correlation between counting methods was acceptable, with a slope of 0.95 and R2 = 0.944. Medium was added to cultures on days 6 and 9 to maintain cell density within an acceptable range (<3.0 × 106 cells / mL). The day 15 data point for donor 21 was lost due to contamination. Means or %CV are shown as horizontal bars, as indicated (Figure 5A). FIG. 5B shows the percentage of central memory T cells (day 15), and FIG. 5C shows the number of central memory T cells (day 15).

[0089] [Figure 6]Figures 6A-6B show cytometric analysis of central memory T cells and the number of central memory cells generated. Figure 6A: Central memory T cells: CD3+ T cells were gated on singlets, followed by CD3 vs. side scatter, and central memory phenotyped using a four-parameter gate of CD45RO, CCR7, CD28, and CD95 to define the central memory population. The population was backgated, and central memory cells were displayed in red in the color plot as a fraction of T cells. All non-red cells in the color plot represent non-central memory T cells. Figure 6B: Phenotype conversion and key metrics (day 15): Key metrics show the number of donors with central memory cell counts >50%, along with the mean and %CV associated with central memory expansion.

[0090] [Figure 7] Figure 7 is a schematic diagram showing how the individual chips are designed to be stackable in layers to achieve the throughput required by any particular application using established manufacturing approaches: injection molding layers are planned as the system is developed.

[0091] [Figure 8]These are supplemental figures showing WBC enrichment by DLD. Figure 8A: DLD product drawn from whole blood: Whole blood was passed through the first DLD to remove red blood cells. A second in-line enrichment DLD, designed to achieve an enrichment factor of 12, was connected to the product output of the separation DLD. Equal volumes of product and waste, along with an equal number of absolute count beads, were added to tubes and analyzed by flow cytometry. The resulting relative cell:bead ratios for the waste (Figure 8B) and concentrate (Figure 8C) were calculated relative to the input material to determine the enrichment fold. White blood cells were stained with CD45 PerCP and 1 mM DRAQ5 (which was used as the fluorescence threshold) to acquire both beads and white blood cells. 5,000 bead events were acquired. (All reagents from eBioscience). Designed enrichment factor: 12.0×; observed relative enrichment: 15.714 / 1.302 = 12.07×.

[0092] [Figure 9] Figure 9 is a supplemental figure showing CD25 and CD4 expression in unstimulated CD3+ T cells (day 8) purified by either DLD or Ficoll methods. Cells were prepared as described and analyzed as in Figure 3. Mean CD4+ 25+: Ficoll: 20.25%; DLD: 8%.

[0093] [Figure 10] This is a supplemental figure showing the allocation of IL-2-expanded central memory T cells by major subset. First panel: CD8 (green), CD4 (blue). CD4+CD8+ (red) central memory cells were sequentially gated for: CD3+, CD45RO+CCR7+, CD28+CD95+. The relative abundance of IL-2-activated CD4 subsets is evident.

[0094] [Figure 11]FIG. 11 is a supplemental figure depicting the yield in this study and estimates of the number of central memory T cells after expansion with IL-2, assuming a typical leukapheresis collection from a donor with 50×10 WBC cells / mL and containing 50% CD3 lymphocytes in 250 mL.

[0095] [Figure 12] Figure 12 shows a protocol that could in principle be used to generate CAR T cells and administer them to a patient. It is included to contrast with the other procedures discussed herein and does not represent the actual work performed.

[0096] [Figure 13] Figure 13 shows a proposed protocol for generating CAR T cells that differs from the protocol in Figure 12 in the first step of the procedure. The steps in the central portion of the figure are included for comparison. The diagram is intended to illustrate the inventive concept and does not represent the actual work performed.

[0097] [Figure 14] Figure 14 shows a second proposed protocol for generating CAR T cells that differs from the protocol of Figure 12 in the first step of the procedure. The steps in the central portion of the figure are included for comparison. As with Figures 12 and 13, the diagram is intended to illustrate the inventive concept and does not represent the actual work performed.

[0098] [Figure 15] FIG. 15 shows a schematic diagram of a device for removing secretory products from spent cells.

[0099] [Figure 16] FIG. 16 shows a schematic diagram of a device for the continuous removal of toxic compounds from actively growing cells.

[0100] [Figure 17]FIG. 17 shows a schematic diagram of a device for the continuous removal of toxic compounds from actively growing cells, including the option of adding carrier between each repetition.

[0101] [Figure 18] FIG. 18A shows an example of a mirrored array of obstacles with a downshift. A central channel is between the array of obstacles on the left and the array of obstacles on the right. The central channel can be a collection channel for particles of at least a critical size (i.e., particles of at least a critical size can be deflected by the array into the central channel, while particles below the critical size can pass through the channel with the bulk flow). A change in channel width can be achieved for a mirrored array with a downshift by downshifting rows. The amount of downshift can vary based on the size and / or cross-sectional shape of the obstacles. FIG. 18B shows a mirrored array of obstacles without a downshift. The left and right arrays can deflect particles of at least a critical size into the central channel.

[0102] [Figure 19] These figures show platelets remaining in an apheresis sample processed using DLD (FIG. 19A) and Ficoll centrifugation (FIG. 19B). Platelets are the smaller, brighter cells (some indicated by arrows), while white blood cells are the larger, darker cells. It can be seen that the relative number of platelets is substantially lower in the apheresis sample processed by DLD.

[0103] [Figure 20]Figure 20 graphically illustrates the percentage of platelets retained in apheresis samples processed by Ficoll centrifugation (white bars) and DLD (striped bars) using three different buffers. The x-axis is the percentage of platelets retained. Results for the 1% BSA / PBS buffer are shown on the far left of the figure; the percentage for the buffer containing F127 poloxamer intercalator is in the middle, and the percentage for the elutriation buffer is on the far right.

[0104] [Figure 21] Figure 21 shows the results from Figure 22, with the Y-axis representing the number of platelets per white blood cell, i.e., the platelet to white blood cell ratio. The triple-striped bars represent the ratio present in the initial apheresis sample, the open bars are the ratios after processing with Ficoll centrifugation, and the widely spaced single-striped bars are the ratios after processing with DLD.

[0105] [Figure 22] Figure 22 is a bar graph showing T cell proliferation resulting from processing of apheresis samples with Ficoll centrifugation (solid white bars) and DLD (bars with a single stripe adjacent to a white bar). Moving from left to right, the bars show the effect of adding 10% (double-striped bars), 50% (bars covered with small specks), and 100% (bars with dark, widely spaced bars adjacent to a speckled bar) of the platelets originally present in the apheresis starting material back to cells processed with DLD before expansion. The left side of the figure shows cells processed through DLD without EDTA on day 0 and then stimulated with CD3 / CD28, with CD3-positive cells counted on days 3, 7, and 14. The right side of the figure shows cells treated with 2 mM EDTA on day 0 and then stimulated with CD3 / CD28, with CD3-positive cells counted on days 3, 7, and 14.

[0106] [Figure 23]Figure 23 compares the variability in proliferation of T cells obtained using three methods of processing: separation using magnetic beads only (leftmost bar in the figure), separation using Ficoll centrifugation followed by magnetic beads (middle bar), and separation with magnetic beads followed by DLD (rightmost bar).

[0107] [Figure 24] FIG. 24 shows the percentage of cells present in the expanded T cell population of FIG. 23 that are central memory T cells.

[0108] Overview of Workflow Diagrams: Figures 25-32 are all included to illustrate concepts and are not related to the actual experiments performed. The diagrams illustrate the basic workflow typically involved in obtaining cells from a patient that are then processed and used therapeutically, typically treating the same patient from whom the cells were obtained. While the diagrams, for the most part, refer specifically to the production of CAR T cells, it is understood that the process is generally applicable to the production of white blood cells and other cells for therapeutic purposes. For many such processes, including CAR T cells, cells collected from a patient are recombinantly engineered to express a gene of therapeutic interest. Any type of such engineering may be part of the illustrated process. The diagrams separate steps into those performed in the clinic and those performed at another location, referred to for purposes of illustration as the "manufacturing site." Figure 25 shows the expected workflow for a typical process used, for example, in the production of CAR T cells. After a cryopreserved sample is thawed, washed, debulked, and concentrated, cell transformation typically occurs at the manufacturing site. Figure 26 shows several places (small speckled boxes) in a prototype CAR T cell process where DLD may be used while maintaining the same basic workflow. Figures 27-32 then show a number of illustrative examples of how DLD may be used to modify the workflow and improve the prototype process. The primary goals are to produce cells of better overall quality, reduce the time required to obtain a sufficient number of cells to treat a patient, and automate steps that are currently more labor-intensive.

[0109] [Figure 25] Figure 25 illustrates the basic workflow typically involved in current methods of processing cells from a patient for therapeutic use. As illustrated, the process begins with collection of cells at the clinic ("apheresis collection"), proceeds to a manufacturing site where the cells may be expanded and manipulated, and ends with administration of the cells to the patient at the clinic ("reinfusion").

[0110] [Figure 26]Figure 26 shows some steps (small dotted boxes) in the CAR T workflow where DLD can be used. In this example, the basic workflow remains essentially unchanged.

[0111] [Figure 27] Figure 27 illustrates that a DLD may be used immediately after apheresis to clean up cells before freezing (box covered with small specks). The advantage of using a DLD at this point is that it removes platelets from the cells before transport, thereby eliminating the deleterious effects of platelet activation, clot formation, any storage-related degranulation, and overall cell loss. In this way, the DLD improves the quality of the cell composition being transported and replaces one of the hands-on labor steps in the process with an automated counterpart.

[0112] [Figure 28] In this scenario, DLDs are used (boxes covered with small spots) during the initial steps in which cells are cleaned, activated, DLD-isolated, and transferred into the desired medium, such as growth medium. Importantly, freezing and cryopreservation in the clinic and subsequent thawing at the manufacturing site are avoided, which should reduce the time required to complete the process by, for example, about 2-3 days. Although not preferred, the cryopreservation step can still be performed if necessary. Other steps that may be avoided are indicated in underlined text.

[0113] [Figure 29] In this scenario, DLD is used in combination with magnetic beads that bind to cell surface CD3 on T cells. This scenario has the same platelet reduction benefits as described above, but may be used to activate cells and initiate growth much more quickly than the prototype procedure. Cells may also be shipped at a warm temperature compatible with growth. As a result, corresponding steps performed at the manufacturing site are eliminated, correspondingly reducing overall processing time.

[0114] [Figure 30] The most significant difference in the scenario shown in Figure 30 compared to that of Figure 29 is that the cells are genetically transformed with the virus immediately after exposure to the activator (see the box covered with small dots). Rather than waiting several days before activation and transformation, the cells are purified, activated, transformed, and grown in culture medium within 24 hours of completing apheresis collection. Preferably, the time from completion of apheresis to exposure to the vector should be within 12 hours, more preferably within 6 or 3 hours. Most preferably, exposure occurs within 2 hours of completing apheresis, and in all cases before the cells are frozen. The diagram illustrates many processing steps at the manufacturing site that may be eliminated, and the time required to obtain enough cells to treat a patient is expected to be reduced by at least 3 or 4 days.

[0115] [Figure 31] The scenario shown in Figure 31 is similar to that of Figure 30, but an affinity separation procedure (exemplified by magnetic beads that recognize CD3-containing cells) is added earlier to aid in the isolation of T cells. DLDs are also used to purify and / or enrich cells.

[0116] [Figure 32] In the scenario shown in Figure 32, T cells collected by apheresis are purified, manipulated, expanded, and concentrated for reinfusion without having to be frozen, all steps that can be performed at the location where the cells are collected, without the need for transportation, if desired. DETAILED DESCRIPTION OF THE INVENTION

[0117] definition Apheresis: As used herein, this term refers to a procedure in which blood from a patient or donor is separated into its components, e.g., plasma, white blood cells, and red blood cells. More specific terms are "plateletpheresis" (referring to the separation of platelets) and "leukapheresis" (referring to the separation of white blood cells). In this context, the term "separation" refers to obtaining a product enriched in a particular component compared to whole blood, and does not imply that absolute purity has been achieved.

[0118] CAR T cells: The term "CAR" is an acronym for "chimeric antigen receptor." Thus, a "CAR T cell" is a T cell that has been genetically engineered to express a chimeric receptor.

[0119] CAR T cell therapy: This term refers to any procedure in which a disease is treated with CAR T cells. Diseases that can be treated include hematological and solid tumor cancers, autoimmune diseases, and infectious diseases.

[0120] Carrier: As used herein, the term "carrier" refers to an agent, e.g., a bead or particle, made of either biological or synthetic material, added to a preparation for the purpose of binding directly or indirectly (i.e., through one or more intermediate cells, particles, or compounds) to some or all of the compounds or cells present. Carriers can be made from a variety of different materials, including DEAE-dextran, glass, polystyrene, plastic, acrylamide, collagen, and alginate, and typically range in size from 1 to 1000 μm. They may be coated or uncoated and may have surfaces modified to contain affinity substances (e.g., antibodies, activators, haptens, aptamers, particles, or other compounds) that recognize antigens or other molecules on the surface of cells. Carriers can also be magnetized, which can provide an additional means of purification to complement DLD, and they can contain particles (e.g., Janus or strawberry-like particles) that impart secondary properties to cells or cell complexes that are not size-related. For example, the particles may result in chemical, electrochemical, or magnetic properties that can be used in downstream processes such as magnetic separation, electroporation, gene transfer, and / or certain analytical chemistry processes. The particles may also cause metabolic changes in cells, activate cells, or promote cell division.

[0121] Carriers that "promote DLD separation" binding: This term refers to carriers and methods of binding carriers that, depending on the context, affect how cells, proteins, or particles behave during DLD. Specifically, "binding in a manner that promotes DLD separation" means that a) the binding must exhibit specificity for a particular target cell type, protein, or particle, and b) must result in a complex that provides an increase in size of the complex compared to the unbound cell, protein, or particle. For binding to target cells, there must be an increase of at least 2 μm (or, expressed as a percentage, at least 20%, 50%, 100%, 200%, 500%, or 1000%). If a therapeutic or other application requires that target cells, proteins, or other particles be released from the complex to fulfill their intended use, the term "so as to facilitate DLD separation" also requires that the complex permit such release, e.g., by chemical or enzymatic cleavage, chemical dissolution, digestion, by competition with other binding agents, or by physical shear (e.g., using a pipette to create shear stress), and that the released target cells, proteins, or other particles remain active; e.g., therapeutic cells after release from the complex must still maintain the biological activity that makes them therapeutically useful.

[0122] Carriers can also be bound "in a manner that complements DLD separation": this term refers to carriers and methods of binding carriers that alter the chemical, electrochemical, or magnetic properties of cells or cell complexes, or alter one or more biological activities of the cells, regardless of whether they increase their size sufficiently to facilitate DLD separation. Carriers that complement DLD separation also do not necessarily bind specifically to target cells; i.e., they may have to be combined with some other agent that makes them specific, or they may simply be added to a cell preparation and bind nonspecifically. The terms "in a manner that complements DLD separation" and "in a manner that promotes DLD separation" are not mutually exclusive. Binding can both complement DLD separation and also promote DLD separation. For example, a polysaccharide carrier may have an activator on its surface that increases cell growth rate, and binding of one or more of these carriers may also promote DLD separation. Alternatively, binding can only promote or only complement DLD separation.

[0123] Target cell: As used herein, a "target cell" is a cell that is required by the various procedures described herein or that is designed to be purified, collected, manipulated, etc. The identity of the particular cell depends on the context in which the term is used. For example, if the purpose of a procedure is to isolate a particular type of stem cell, then that cell is the target cell for that procedure.

[0124] Isolate, purify: Unless otherwise indicated, these terms, as used herein, are synonymous and refer to the enrichment of a desired product relative to undesired materials. The terms do not necessarily mean that the product is completely isolated or completely pure. For example, if a starting sample had target cells that made up 2% of the cells in the sample, and a procedure was performed that resulted in a composition in which the target cells were 60% of the cells present, the procedure would have been successful in isolating or purifying the target cells.

[0125] Bump Array: The terms "bump array" and "obstacle array" are used synonymously herein to describe an ordered array of obstacles placed in a flow channel through which a fluid having cells or particles can pass.

[0126] Deterministic Lateral Displacement: As used herein, the term "deterministic lateral displacement" or "DLD" refers to a process in which particles are deterministically deflected in a path through an array based on their size, which is related to some parameter of the array. This process can be used to separate cells, which is the general context in which this process is discussed herein. However, it is important to recognize that DLD can also be used to concentrate cells and for buffer exchange. The process is generally described herein with respect to continuous flow (DC conditions, i.e., bulk fluid flow in only one direction). However, DLD can also function with oscillatory flow (AC conditions, i.e., bulk fluid flow that alternates between two directions).

[0127] Critical size: The "critical size" or "predetermined size" of a particle passing through an obstacle array describes the size limit of a particle that can follow the laminar flow of a fluid. Particles larger than the critical size may be "bumped" out of the flow path of the fluid, while particles having a size smaller than the critical size (or predetermined size) are not necessarily so removed. If the fluid flow profile through the gap is symmetrical about a plane that bisects the gap in the direction of bulk fluid flow, the critical size may be the same for both sides of the gap; however, if the profile is asymmetrical, the critical size on the two sides of the gap may be different.

[0128] Fluid Flow: As used herein in the context of a DLD, the terms "fluid flow" and "bulk fluid flow" refer to macroscopic movement in a general direction across an array of obstacles. These terms do not take into account temporary displacements of the fluid flow, where the fluid moves around obstacles to keep the fluid moving in the general direction.

[0129] Tilt angle ε: In a bump array device, the tilt angle is the angle between the direction of bulk fluid flow and the direction defined by the alignment of successive (in the direction of bulk fluid flow) rows of obstacles in the array.

[0130] Array direction: In a bump array device, the "array direction" is the direction defined by the alignment of successive rows of obstacles in the array. A particle is "bumped" in the bump array if, when it passes through the gap and encounters a downstream obstacle, the particle's overall trajectory follows the array direction of the bump array (i.e., moves at an oblique angle ε relative to the bulk fluid flow). A particle is not bumped if its overall trajectory follows the direction of the bulk fluid flow under those circumstances.

[0131] The present invention is primarily concerned with the use of DLD in preparing therapeutically valuable cells. The following text provides guidance regarding the methods disclosed herein, as well as information that may be useful in making and using devices involved in carrying out those methods.

[0132] 1. Microfluidic Plate Design Cells, particularly cells in compositions prepared by apheresis or leukapheresis, can be isolated by performing DLD using a microfluidic device containing channels through which fluid flows from an inlet at one end of the device to an outlet at the opposite end. The basic principles of size-based microfluidic separation and the design of obstacle arrays for separating cells have been provided elsewhere (see US 2014 / 0342375; US 2016 / 0139012; 7,318,902, and US 7,150,812, which are incorporated herein by reference in their entireties) and are summarized in the following sections.

[0133] During DLD, a fluid sample containing cells is introduced into the device at an inlet and carried with the fluid flowing through the device to an outlet. As cells in the sample traverse the device, they encounter posts or other obstacles, which are positioned in rows and form gaps or pores through which the cells must pass. Each successive row of obstacles is offset relative to the previous row to form an array direction that differs from the direction of fluid flow in the flow channel. The width of the gaps between obstacles, the shape of the obstacles, and the orientation of the obstacles that form the gaps, along with the "tilt angle" defined by these two directions, are key factors in determining the "critical size" for the array. Cells with sizes larger than the critical size move in the array direction rather than in the direction of bulk fluid flow, while particles with sizes smaller than the critical size move in the direction of bulk fluid flow. In devices used with compositions derived by leukapheresis, array features can be selected that result in white blood cells being deflected in the array direction, while red blood cells and platelets continue in the direction of bulk fluid flow. To separate a selected type of white blood cell from others of similar size, a carrier can then be used that binds to the cells to facilitate DLD separation, thereby resulting in a complex that is larger than the uncomplexed white blood cells. It may then be possible to perform the separation on a device that has a critical size that is smaller than the complex but larger than the uncomplexed cells.

[0134] The obstacles used in the device may be cylindrical in shape, or may be triangular, square, rectangular, diamond, trapezoidal, hexagonal, or teardrop shaped. In addition, adjacent obstacles may have a geometry such that the portion of the obstacle that defines the gap is either symmetric or asymmetric with respect to the axis of the gap that extends in the direction of bulk fluid flow.

[0135] II. Microfluidic Device Fabrication and Operation The general procedure for making and using the microfluidic device that can separate cells based on size is well known in the art.Such device includes those described in US 5,837,115; US 7,150,812; US 6,685,841; US ​​7,318,902; 7,472,794; and US 7,735,652 (all of which are incorporated herein by reference in their entirety). Other references that provide guidance that may be useful in making and using devices for the present invention include: US 5,427,663; US 7,276,170; US 6,913,697; US 7,988,840; US 8,021,614; US 8,282,799; US 8,304,230; US 8,579,117; US 2006 / 0134599; US 2007 / 0160503; US 20050282293; US 2006 / 0121624; US 2005 / 0266433; US 2007 / 0026381; US ​​2007 / 0026414; US 2007 / 0026417; 2007 / 0026415; US 2007 / 0026413; US 2007 / 0099207; US 2007 / 0196820; US 2007 / 0059680; US 2007 / 0059718; US 2007 / 005916; US 2007 / 0059774; US 2007 / 0059781; US ​​2007 / 0059719; US 2006 / 0223178; US 2008 / 0124721; US ​​2008 / 0090239; US 2008 / 0113358; and WO2012094642 (all of which are incorporated by reference herein in their entireties). Among the various references describing the construction and use of devices, US 7,150,812 provides particularly good guidance, and 7,735,652 is of particular interest with regard to microfluidic devices for separations performed on samples containing cells found in blood (see also US 2007 / 0160503 in this regard).

[0136] The devices can be fabricated using any of the materials from which microscale and nanoscale fluid handling devices are typically fabricated, including silicon, glass, plastic, and hybrid materials. A variety of thermoplastic materials suitable for microfluidic fabrication are available, offering a wide selection of mechanical and chemical properties that can be reinforced and further tailored to specific applications.

[0137] Techniques for fabricating devices include replica molding, soft lithography using PDMS, thermosetting polyester, embossing, injection molding, laser cutting, and combinations thereof. Further details can be found in "Disposable microfluidic devices: fabrication, function and application," Fiorini, et al. (BioTechniques 38:429-446 (March 2005)), which is incorporated herein by reference in its entirety. The book "Lab on a Chip Technology," edited by Keith E. Herold and Avraham Rasooly, Caister Academic Press Norfolk UK (2009), is another resource for fabrication methods and is incorporated herein by reference in its entirety.

[0138] High-throughput embossing methods, such as reel-to-reel processing of thermoplastics, are attractive methods for industrial microfluidic chip fabrication. The use of single-chip hot embossing can be a cost-effective technique for achieving high-quality microfluidic devices during the prototype manufacturing stage. A method for replicating microscale features in two thermoplastics, polymethyl methacrylate (PMMA) and / or polycarbonate (PC), is described in "Microfluidic device fabrication by thermoplastic hot-embossing," Yang, et al. (Methods Mol. Biol. 949: 115-23 (2013)), which is incorporated herein by reference in its entirety.

[0139] A flow channel can be constructed using two or more parts that, when assembled, form a closed cavity (preferably one with an opening for adding or withdrawing fluid) with an obstacle disposed therein. The obstacles can be fabricated in one or more of the parts that are assembled to form the flow channel, or they can be fabricated in the form of an insert sandwiched between two or more parts that define the boundaries of the flow channel.

[0140] An obstacle can be a solid object that spans the flow channel, possibly from one surface of the flow channel to the opposite surface of the flow channel. If the obstacle is integral with (or an extension of) one of the surfaces of the flow channel at one end of the obstacle, the other end of the obstacle can be blocked or pressed against the opposite surface of the flow channel. A small space (preferably too small to accommodate any particles of interest for the intended application) is permitted between one end of the obstacle and the surface of the flow channel, provided that the space does not adversely affect the structural stability of the obstacle or the associated flow properties of the device.

[0141] The number of obstacles present should be sufficient to achieve the particle separation properties of the array. Obstacles can generally be organized into rows and columns (Note: the use of the terms "rows and columns" does not mean or imply that the rows and columns are perpendicular to one another). Obstacles that are generally aligned transverse to fluid flow in a flow channel can be referred to as obstacles in a column. Obstacles adjacent to one another in a column can define gaps through which fluid can flow.

[0142] Obstacles in adjacent columns can be oblique from one another by an angle characterized by an inclination angle designated ε (epsilon). Thus, for several columns adjacent to one another (i.e., several columns of obstacles passed successively by fluid flow in a single direction generally transverse to the columns), corresponding obstacles in the columns can be oblique from one another such that the corresponding obstacles form a row of obstacles extending at an angle ε relative to the direction of fluid flow through the column. The inclination angle can be selected so that the columns are spaced apart from one another such that 1 / ε (when expressed in radians) is an integer, and the rows of obstacles repeat periodically. Obstacles in a single column can also be oblique from one another by the same or different inclination angles. As an example, the rows and columns can be arranged at a 90-degree angle relative to one another, with both the row and column being inclined at the same angle of ε relative to the direction of bulk fluid flow through the flow channel.

[0143] Surfaces can be coated to modify their properties and the polymeric materials used to fabricate devices, and can be modified in many ways. In some cases, functional groups such as amines or carboxylic acids, either native to the polymer or added using wet chemistry or plasma treatment, are used to crosslink proteins or other molecules. DNA can be attached to COC and PMMA substrates using surface amine groups. Surfactants such as Pluronic® can be used to make surfaces hydrophilic and protein-repellent by adding Pluronic® to PDMS formulations. In some cases, a layer of PMMA is spin-coated onto the device, e.g., a microfluidic chip, and the PMMA is "doped" with hydroxypropyl cellulose to alter its contact angle.

[0144] To reduce nonspecific adsorption of cells or compounds, such as those released by lysed cells or found in biological samples, to the channel walls, one or more walls can be chemically modified to be nonadhesive or repelling. The walls can be coated with a thin film coating (e.g., a monolayer) of a commercially available antifouling reagent, such as those used to form hydrogels. Additional examples of chemical species that can be used to modify the channel walls include oligoethylene glycols, fluorinated polymers, organosilanes, thiols, polyethylene glycols, hyaluronic acid, bovine serum albumin, polyvinyl alcohol, mucin, poly-HEMA, methacrylated PEG, and agarose. Charged polymers can also be used to repel oppositely charged species. The type of chemical species used for repulsion and the method of attachment to the channel walls can depend on the nature of the species to be repelled and the nature of the wall and the species to be attached. Such surface modification techniques are well known in the art. The walls can be functionalized before or after the device is assembled.

[0145] III.CAR T cells The method for producing and using CAR T cell is well known in the art.The procedure is described in, for example, US 9,629,877; US 9,328,156; US 8,906,682; US 2017 / 0224789; US 2017 / 0166866; US 2017 / 0137515; US 2016 / 0361360; US 2016 / 0081314; US 2015 / 0299317; and US 2015 / 0024482 (each of which is incorporated herein by reference in its entirety).

[0146] IV. Separation Processes Using DLD The DLD devices described herein can be used to purify cells, cell fragments, cell adducts, or nucleic acids. As discussed herein, these devices can also be used to separate a cell population of interest from multiple other cells. Separation and purification of blood components using the devices can be found, for example, in U.S. Patent Application Publication No. 2016 / 0139012, the teachings of which are incorporated herein by reference in their entirety. A brief discussion of a few illustrative separations is provided below.

[0147] A. Viable cells In one embodiment, the device is used in a procedure designed to separate viable cells from nonviable cells. The term "viable cells" refers to cells capable of growth, actively dividing, capable of reproduction, etc. If viable cells have a larger size than nonviable cells, the DLD device can be designed to include a critical size that is larger than the predetermined size of nonviable cells and smaller than the predetermined size of viable cells. The critical size can be small, such as 1.1 times larger (or smaller) than the predetermined size of nonviable cells, but is generally preferred to be larger (or smaller), e.g., about 1.2 to 2 times, preferably 3 to 10 times.

[0148] B. Adherent cells In another embodiment, the DLD device can be used in a procedure for isolating adherent cells. As used herein, the term "adherent cells" refers to cells that have the ability to adhere to a surface. Adherent cells include immortalized cells used in cell culture and can be derived from a mammalian host. In some cases, adherent cells can be trypsinized prior to purification. Examples of adherent cells include MRC-5 cells; HeLa cells; Vero cells; NIH 3T3 cells; L929 cells; Sf21 cells; Sf9 cells; A549 cells; A9 cells; AtT-20 cells; BALB / 3T3 cells; BHK-21 cells; BHL-100 cells; BT cells; Caco-2 cells; Chang cells; Clone 9 cells; Clone 10 cells; M-3 cells;COS-1 cells;COS-3 cells;COS-7 cells;CRFK cells;CV-1 cells;D-17 cells;Daudi cells;GH1 cells;GH3 cells;HaK cells;HCT-15 cells;HL-60 cells;HT -1080 cells; HT-29 cells; HUVEC cells; I-10 cells; IM-9 cells; JEG-2 cells; Jensen cells; Jurkat cells; K-562 cells; KB cells; KG-1 cells; L2 cells; LLC-WRC 256 cells; McCoy cells; MCF7 cells; WI-38 cells; WISH cells; G2 cells; BAE-1 cells; SH-SY5Y cells; and any derivatives thereof, including engineered and recombinant strains.

[0149] In some embodiments, the procedure can include separating the cells from a diluent, such as growth medium, which can provide for efficient maintenance of the culture of adherent cells. For example, a culture of adherent cells in growth medium can be exchanged into a transfection medium containing a transfection reagent, into a second growth medium designed to induce changes in the adherent cells, such as stem cell differentiation, or into successive wash buffers designed to remove compounds from the culture.

[0150] In a particularly preferred procedure, adherent cells are purified through association with one or more carriers that are bound to facilitate DLD separation. The carriers can be of the type described herein, and the binding can stabilize and / or activate the cells. Carriers are typically in the 1-1000 μm range, but can be outside this range.

[0151] The association between the carrier and the cells should result in a complex of increased size relative to other materials not associated with the carrier. Depending on the specific size of the cells and carrier, and the number of cells and carriers present, the complex may range from a few percent larger than the uncomplexed cells to many times the size of the uncomplexed cells. To facilitate separation, an increase of at least 20% is desirable, with higher percentages (50; 100; 1000 or more) being preferred.

[0152] C. Activated cells The DLD device can also be used in procedures to separate activated cells or cells capable of activation from multiple other cells. While activated cells can be grown on a large scale, in a preferred embodiment, the cells are derived from a single patient and DLD is performed within at least a few hours of collection. The terms "activated cells" and "cells capable of activation" refer to cells that have been activated or can be activated, respectively, through association, incubation, or contact with a cell activator. Examples of cells capable of activation include cells that play a role in immune or inflammatory responses, such as T cells and B cells; regulatory T cells, macrophages, dendritic cells, granulocytes, innate lymphoid cells, megakaryocytes, natural killer cells, thrombocytes, synoviocytes, etc.; cells that play a role in metabolism, such as beta cells, liver cells, and pancreatic cells; and recombinant cells capable of inducible protein expression, such as DE3-lysogenized E. coli cells, yeast cells, plant cells, etc.

[0153] Typically, one or more carriers have an activator on their surface. Examples of cell activators include proteins, antibodies, cytokines, CD3, CD28, antigens against specific proteins, helper T cells, receptors, and glycoproteins; hormones such as insulin and glucagon; IPTG, lactose, allolactose, lipids, glycosides, terpenes, steroids, and alkaloids. The activatable cells should at least partially associate with the carrier through the interaction between the activatable cells and the cell activator on the surface of the carrier. The complexes formed may be only a few percent larger than uncomplexed cells, or may be many times the size of uncomplexed cells. To facilitate separation, an increase of at least 20% is desirable, with higher percentages (40, 50, 100, 1000, or more) being preferred.

[0154] D. Separation of Cells from Toxic Material DLDs can also be used in purification procedures designed to remove compounds that may be toxic to cells or to keep cells free of contamination by toxic compounds. Examples include antibiotics, cryopreservatives, antifungals, toxic metabolites, sodium azide, metal ions, metal ion chelators, endotoxins, plasticizers, pesticides, and combinations thereof. The device can be used to remove toxic compounds from cells to ensure consistent production of materials from the cells. In some cases, the cells may be log-phase cells. The term "log-phase cells" refers to actively dividing cells in a period of growth characterized by exponential logarithmic growth. In the log phase, a cell population can double at a constant rate, such that plotting the natural logarithm of cell number against time yields a straight line.

[0155] The ability to isolate toxic materials can be important for a wide variety of cells, including bacterial strains such as BL21, Tuner, Origami, Origami B, Rosetta, C41, C43, DH5α, DH10β, or XL1Blue; yeast strains such as strains of the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, and Yarrowia; algae; and MRC-5 cells; HeLa cells; Vero cells; NIH 3T3 cells; L929 cells; Sf21 cells; Sf9 cells; A549 cells; A9 cells; AtT-20 cells; BALB / 3T3 cells; BHK-21 cells; BHL-100 cells; BT cells; Caco-2 cells; Chang cells; Clone 9 cells; Clone 10 cells; M-3 cells;COS-1 cells;COS-3 cells;COS-7 cells;CRFK cells;CV-1 cells;D-17 cells;Daudi cells;GH1 cells;GH3 cells;HaK cells;HCT-15 cells;HL-60 cells;HT -1080 cells; HT-29 cells; HUVEC cells; I-10 cells; IM-9 cells; JEG-2 cells; Jensen cells; Jurkat cells; K-562 cells; KB cells; KG-1 cells; L2 cells; LLC-WRC 256 cells; McCoy cells; MCF7 cells; WI-38 cells; WISH cells; Mammalian cell cultures, including cultures of G2 cells; BAE-1 cells; SH-SY5Y cells; stem cells and any derivatives thereof, including engineered and recombinant strains.

[0156] E. Purification of secreted material from cells DLD separation can also be used to purify materials secreted from cells. Examples of such secreted materials include proteins, peptides, enzymes, antibodies, fuels, biofuels such as those derived from algae, polymers, small molecules such as simple organic molecules, complex organic molecules, drugs and prodrugs, carbohydrates, and combinations thereof. Secreted products can include therapeutically useful proteins such as insulin, imatinib, T cells, T cell receptors, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic agents.

[0157] Figure 15 is a schematic diagram depicting the use of DLD in the purification of secreted products. In some cases, cells may be in aqueous suspension, such as a buffer or growth medium, so that the cells secrete the product into the suspension. Examples of such secreted products include proteins, peptides, enzymes, antibodies, fuels, biofuels such as those derived from algae, polymers, small molecules such as simple organic molecules, complex organic molecules, drugs and prodrugs, carbohydrates, and combinations thereof. Secreted products may include therapeutically useful proteins such as insulin, imatinib, T cells, T cell receptors, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic agents.

[0158] Purification can be performed, for example, in situations where cells have a predetermined size greater than the predetermined size of a secreted compound, where the predetermined size of the cells is equal to or greater than a critical size, and the predetermined size of the secreted product is less than a critical size. In such a configuration, when applied to a DLD device, the cells can be deflected in a first direction, while the secreted compound can be deflected in a second direction, thereby separating the secreted compound from the cells. Alternatively, the secreted protein can be captured by a large carrier to which it binds to facilitate DLD separation. DLD can then be performed, after which the carrier-protein complex can be processed to further purify or release the protein.

[0159] Such a process can be performed in an iterative manner, such that populations of separated particles can be continuously looped back into the device for further separation. In this regard, Figures 16 and 17 are schematic diagrams of an iterative process in which separated cells are looped back into a DLD device after separation. In some cases, cells can be looped from a first device to a second, different device with obstacles containing different critical sizes. Such a system can enable systematic separation of multiple size ranges by manipulating the critical size range. In other cases, cells can be looped back into the same device previously used to separate the separated particles. This system can be advantageous for the continuous purification of actively dividing cells or compounds that are actively expressing. For example, such a method could be combined with a method for purifying secretory products that collects both the secretory product from one flow stream and the cells producing the secretory product from another flow stream. Because cells can continuously produce secretory products, the purified cells can be reapplied to the device to continuously collect secretory products from the cells.

[0160] F. Purity and Yield The purity, yield, and viability of cells produced by the DLD methods discussed herein will vary based on several factors, including the nature of the starting material, the exact procedure used, and the characteristics of the DLD device. Preferably, a purification, yield, and viability of at least 60% should be obtained, with higher percentages, at least 70%, 80%, or 90%, being more preferred. In preferred embodiments, the methods can be used to isolate leukocytes from whole blood, apheresis products, or leukocyte apheresis products with a purity, yield, and viability of at least 70%, with higher percentages (at least 80%, 85%, or 90%) being preferred.

[0161] V. Technical background Without being bound by any particular theory, a general discussion of some technical aspects of microfluidics can be helpful in understanding the factors that influence separations performed in this field. A variety of microfabricated sieving matrices have been disclosed for separating particles (Chou, et al., Proc. Natl. Acad. Sci. 96:13762 (1999); Han, et al., Science 288:1026 (2000); Huang, et al., Nat. Biotechnol. 20:1048 (2002); Turner et al., Phys. Rev. Lett. 88(12):128103 (2002); Huang, et al., Phys. Rev. Lett. 89:178301 (2002); U.S. Patent No. 5,427,663; U.S. Patent No. 7,150,812; U.S. Patent No. 6,881,317). Bump array (also known as "obstacle array") devices have been described, and their basic operation is explained, for example, in U.S. Patent No. 7,150,812 (incorporated herein by reference in its entirety). Bump arrays essentially operate by segregating particles that pass through an array of obstacles (typically a periodically ordered array), with separation occurring between particles that follow an "array direction" that is oblique from the direction of bulk fluid flow or from the direction of an applied electric field (U.S. Patent No. 7,150,812).

[0162] A. Bump Array In some arrays, the geometry of adjacent obstacles is such that the portion of the obstacles defining the gap is symmetrical about the gap's axis, which extends in the direction of bulk fluid flow. The velocity or volume profile of fluid flow through such a gap is approximately parabolic across the gap, with fluid velocity and flow rate being zero (assuming no slip flow) at the surface of each gap-defining obstacle and reaching a maximum at the center of the gap. A parabolic profile means that a fluid layer of a given width adjacent one of the gap-defining obstacles contains an equal proportion of fluid flow rate as a fluid layer of the same width adjacent any other gap-defining obstacle, and the critical size of a particle to be "bumped" during passage through the gap is the same regardless of which obstacle the particle moves near.

[0163] In some cases, the particle size separation performance of an obstacle array can be improved by shaping and arranging the obstacles so that the portions of adjacent obstacles that deflect fluid flow into the gaps between them are not symmetrical with respect to the axis of the gap extending in the direction of bulk fluid flow. Such a lack of flow symmetry into the gaps can result in asymmetric fluid flow profiles within the gaps. Concentration of fluid flow on one side of the gap (i.e., the result of an asymmetric fluid flow profile through the gaps) can lower the critical size of particles induced to move in the array direction rather than in the direction of bulk fluid flow. This is because the asymmetry in the flow profile causes a difference between the width of the flow layer adjacent to one obstacle containing a selected percentage of fluid flow through the gap and the width of the flow layer adjacent to another obstacle containing the same percentage of fluid flow and defining the gap. The different widths of the fluid layers adjacent to the obstacles define gaps exhibiting two different critical particle sizes. A particle crossing a gap may be bumped (i.e., move in the array direction rather than in the bulk fluid flow direction) if it exceeds a critical size of the fluid layer in which it is carried. Thus, a particle crossing a gap with an asymmetric flow profile may be bumped if it moves in the fluid layer adjacent to one obstacle, but not bumped if it moves in the fluid layer adjacent to another obstacle that defines the gap.

[0164] In another aspect, reducing the rounding of the edges of the obstacles that define the gaps can improve the particle size separation performance of an obstacle array. For example, an array of obstacles with triangular cross sections with sharp vertices can exhibit a lower critical particle size than an array of triangular obstacles of the same size and spacing with rounded vertices.

[0165] Thus, by sharpening the edges of obstacles that define gaps in an obstacle array, the critical size of particles deflected toward the array under the influence of bulk fluid flow can be reduced without necessarily reducing the size of the obstacles. Conversely, obstacles with sharper edges can be spaced farther apart than obstacles of the same size with less sharp edges, yet still produce particle separation properties equivalent to those of obstacles with less sharp edges.

[0166] B. Fractionation Range The objects that are separated by size in microfluidic devices include cells, biomolecules, inorganic beads, and other objects. Typical sizes that are separated range from 100 nanometers to 50 micrometers. However, larger and smaller particles may also be separated.

[0167] C. Capacity Depending on the design, a device, or combination of devices, can be used to process between about 10 μl and at least 500 μl of sample, between about 500 μl and about 40 mL of sample, between about 500 μl and about 20 mL of sample, between about 20 mL and about 200 mL of sample, between about 40 mL and about 200 mL of sample, or at least 200 mL of sample.

[0168] D. Channel The device may contain one or more channels with one or more inlets and one or more outlets. The inlets may be used for samples or crude (i.e., unpurified) fluid compositions, for buffers, or to introduce reagents. The outlets may be used to collect product or as waste outlets. The channels may be about 0.5-100 mm wide and about 2-200 mm long, although different widths and lengths are also possible. The depth may be 1-1000 μm, and there may be anywhere from one to 100 or more channels. The volume may vary over a very wide range, from a few μl to many ml, and the device may have multiple zones (stages or sections) with different configurations of obstacles.

[0169] E. Gap size (edge-to-edge distance between posts or obstacles) The gap size (edge-to-edge distance between posts or obstacles) in the array of obstacles can vary from about a few micrometers (e.g., 1-500) or can be greater than 1 millimeter. In some embodiments, the obstacles have diameters of 1-3000 micrometers and can have various shapes (spherical, triangular, teardrop, diamond, square, rectangular, etc.). The first row of posts can be located close to (e.g., within 5 μm of) the inlet or can be farther apart than 1 mm.

[0170] (F) Stackable chips The device may include multiple stackable chips. The device may include about 1 to 50 chips. In some cases, the device may have multiple chips arranged in series, in parallel, or both.

[0171] VI. Concept of the invention The following numbered paragraphs present inventive concepts that are part of this application. These concepts are expressed in the form of example claims E1-E234. E1. A method for manipulating a population of target cells, comprising the steps of: a) isolating target cells from a crude fluid composition, the isolation procedure comprising performing deterministic lateral displacement (DLD) on a microfluidic device, the device comprising: i) at least one channel extending from a sample inlet to one or more fluid outlets, said channel being bounded by a first wall and a second wall opposite said first wall; ii) an array of obstacles arranged in rows in the channel, with each successive row of obstacles being laterally shifted relative to the previous row, the obstacles being positioned in such a manner that when the crude fluid composition is applied to an inlet of the device and fluidly passed through the channel, target cells flow to one or more collection outlets where an enriched product is collected, and contaminant cells or particles that are a different size from the target cells flow to another waste outlet that is separate from the collection outlets. a process comprising: b) genetically engineering the target cells obtained from the collection outlet to have a desired phenotype. E2. The method of claim 1, wherein the genetically manipulating step comprises transfecting or transducing target cells, and the genetically engineered target cells are propagated by culturing them in vitro. E3. The method of claim 2, wherein the yield of target cells exhibiting the desired phenotype is at least 10% higher than identical cells isolated by Ficoll centrifugation and not subjected to DLD. E4. The method of claim 1, wherein the crude fluid composition is blood or a composition obtained by performing apheresis or leukapheresis on blood. E5. The method of any one of claims 1 to 4, wherein the target cells are leukocytes. E6. The method of any one of claims 1 to 4, wherein the target cells are B cells, T cells, NK cells, monocytes, or progenitor cells. E7. The method of any one of claims 1 to 4, wherein the target cells are dendritic cells. E8. The method of any one of claims 1-7, wherein the crude fluid composition is obtained from a patient. E9. The method of claim 8, wherein the target cells in the crude fluid composition are not bound to a carrier before being transduced or transfected. E10. The method of claim 8, wherein the target cells are bound to one or more carriers prior to DLD to facilitate or complement DLD separation. E11. The method of claim 9, wherein the target cells are bound to one or more carriers after DLD has been performed and either before or after transduction or transfection thereof to facilitate or complement DLD separation. E12. The method of claim 10 or 11, wherein the one or more carriers comprise an affinity substance on the surface that specifically binds to the target cells. E13. The method of claim 12, wherein the agent is an antibody, an activator, a hapten, or an aptamer. E14. The method of any one of claims 10 to 13, wherein the diameter of the carrier is at least as long as the diameter of the target cell. E15. The method according to any one of claims 10 to 13, wherein the total diameter of the carrier is 50% or less of the diameter of the target cell. E16. The method of any one of claims 10 to 13, wherein the overall diameter of the carrier is at least twice the length of the diameter of the target cell. E17. The method according to any one of claims 10 to 13, wherein the total diameter of the carrier is 25% or less of the diameter of the target cell. E18. The method of any one of claims 10 to 13, wherein one group of carriers has a diameter at least as long as the target cells, and a second group of carriers has a diameter no greater than 50% of the diameter of the target cells. E19. The method of any one of claims 10 to 13, wherein one group of carriers has a diameter at least twice the length of the target cells, and a second group of carriers has a diameter no greater than 25% the length of the target cells. E20. The method of any one of claims 10 to 19, wherein the carrier is made of collagen or a polysaccharide. E21. The method of any one of claims 10 to 20, wherein the carrier is made of gelatin or alginate. E22. The method of any one of claims 10 to 21, wherein a crude fluid composition is obtained from a patient, and no more than 4 hours elapse between the time the crude fluid composition is obtained and the time the carrier is first bound to the target cells. E23. The method of any one of claims 10-21, wherein the crude fluid composition is an apheresis or leukapheresis product drawn from a patient's blood, and no more than four hours have elapsed between the completion of the apheresis or leukapheresis and the first binding of the carrier to the target cells. E24. The method of any one of claims 1 to 23, wherein a crude fluid composition is obtained from a patient and no more than 5 hours elapse between the time the crude fluid composition is obtained and the time the target cells are transfected or transduced for the first time. E25. The method of any one of claims 1-23, wherein the crude fluid composition is an apheresis or leukapheresis product drawn from a patient's blood, and no more than 5 hours have elapsed between the time the apheresis or leukapheresis is completed and the time the target cells are first transfected or transduced. E26. The method of claim 24 or 25, wherein no more than 4 hours have elapsed since the first time the target cells are transfected or transduced. E27. A method for producing chimeric antigen receptor (CAR) T cells, comprising the steps of: a) obtaining a crude fluid composition comprising T cells; b) i) at least one channel extending from a sample inlet to one or more fluid outlets, said channel being bounded by a first wall and a second wall opposite said first wall; ii) an array of obstacles arranged in rows in the channel, with each successive row of obstacles being laterally shifted relative to the previous row, the obstacles being arranged in such a manner that when the crude fluid composition is applied to an inlet of the device and fluidly passed through the channel, T cells in the composition flow to one or more collection outlets where an enriched product is collected, and cells or particles in the crude fluid composition that are a different size than the T cells flow to another waste outlet that is separate from the collection outlets. performing deterministic lateral displacement (DLD) on the crude fluid composition using a microfluidic device comprising: c) genetically engineering T cells in the enriched product obtained in step b) to produce chimeric antigen receptors (CARs) on their surface. E28. The method of claim 27, wherein the crude fluid composition is an apheresis product or leukapheresis product obtained from blood from a patient, and when the crude fluid composition is applied to an inlet of the device and fluidly passes through the channels, T cells in the composition flow to one or more collection outlets where an enriched product is collected, and red blood cells, platelets, or other particles present in the crude fluid composition and of a different size flow to another waste outlet separate from the collection outlets. E29. The method of claim 27 or 28, wherein the genetic manipulation comprises transfecting or transducing target cells, and the genetically manipulated target cells are further expanded by growing the cells in vitro. E30. The method of any one of claims 27-29, wherein the yield of T cells expressing the chimeric receptor on their surface is at least 10% higher than T cells isolated by Ficoll centrifugation from a crude fluid composition and not subjected to DLD. E31. The method of claim 30, wherein the yield of T cells expressing the chimeric receptor on their surface is at least 20% greater than T cells isolated by Ficoll centrifugation from a crude fluid composition and not subjected to DLD. E32. The method of claim 30, wherein the yield of T cells expressing the chimeric receptor on their surface is at least 50% greater than T cells isolated from the crude fluid composition by Ficoll centrifugation and not subjected to DLD. E33. The method of any one of claims 27-32, wherein the CAR comprises: a) an extracellular region comprising an antigen-binding domain; b) a transmembrane region; and c) an intracellular region, and the CAR T cell optionally comprises one or more recombinant sequences that provide the cell with a molecular switch that, when triggered, reduces the number or activity of CAR T cells. E34. The method of claim 33, wherein the antigen-binding domain is a single-chain variable fragment (scFv) derived from the antigen-binding regions of both the heavy and light chains of a monoclonal antibody. E35. The method of claim 33 or 34, wherein the CAR comprises a hinge region of 2 to 20 amino acids connecting the extracellular region and the transmembrane region. E36. The method of claim 35, wherein the transmembrane region comprises a CD8 or CD28 protein sequence. E37. The method of any one of claims 33 to 36, wherein the intracellular region comprises a signaling domain derived from the CD3ζ, CD137, or CD28 intracellular domain. E38. The method of any one of claims 27-37, wherein the crude fluid composition comprising T cells is obtained from a patient with cancer, an autoimmune disease, or an infectious disease. E39. The method of claim 38, wherein after obtaining a crude fluid composition comprising T cells, the T cells in the fluid composition are bound to one or more carriers to facilitate DLD isolation. E40. The method of claim 39, wherein the T cells are bound to one or more carriers prior to DLD to facilitate DLD separation. E41. The method of claim 39, wherein the T cells are bound to one or more carriers after DLD has been performed and either before or after they are genetically engineered to facilitate DLD separation. E42. The method of any one of claims 39-41, wherein the one or more carriers comprise on their surface an antibody or activator that specifically binds to the T cells. E43. The method of any one of claims 39 to 42, wherein the overall diameter of the carrier is at least as long as the diameter of the T cell. E44. The method of any one of claims 39 to 42, wherein the total diameter of the carrier is 50% or less of the diameter of the T cell. E45. The method of any one of claims 39-42, wherein the overall diameter of the carrier is at least twice the length of the diameter of the T cell. E46. The method of any one of claims 39 to 42, wherein the total diameter of the carrier is no more than 25% of the diameter of the T cell. E47. The method of any one of claims 39-42, wherein one group of carriers has a diameter at least as long as the T cells, and a second group of carriers has a diameter no greater than 50% of the diameter of the T cells. E48. The method of any one of claims 39-42, wherein one group of carriers has a diameter at least twice the length of the T cells, and a second group of carriers has a diameter no greater than 25% the length of the T cells. E49. The method of any one of claims 39-48, wherein the carrier is made of collagen or a polysaccharide. E50. The method of any one of claims 39 to 49, wherein the carrier is made of gelatin or alginate. E51. The method of any one of claims 39 to 50, wherein no more than 4 hours elapse between the time when the crude fluid composition containing T cells is obtained and the time when the carrier is bound to the T cells. E52. The method of any one of claims 39-50, wherein the crude fluid composition is an apheresis or leukapheresis product drawn from a patient's blood, and no more than four hours elapse between the completion of the apheresis or leukapheresis and the binding of the carrier to the target cells. E53. The method of any one of claims 27 to 50, wherein no more than 5 hours elapse between the time when the crude fluid composition containing T cells is obtained and the time when the T cells are transfected or transduced for the first time. E54. The method of any one of claims 27-50, wherein the crude fluid composition is an apheresis or leukapheresis product drawn from the patient's blood, and no more than 5 hours elapse between the time the apheresis or leukapheresis is completed and the time the T cells are first transfected or transduced. E55. The method of claim 53 or 54, wherein no more than 4 hours have elapsed since the first time the T cells were transfected or transduced. E56. The method of any one of claims 27-55, wherein all steps in generating the CAR T cells are performed in the same facility where the crude fluid composition comprising the T cells is obtained, and all steps are completed within a total of 4 hours. E57. A CAR T cell produced by the method of any one of claims 27 to 55. E58. A method of treating a patient for cancer, autoimmune disease, or infectious disease, comprising administering to the patient CAR T cells engineered to express a chimeric antigen receptor that recognizes an antigen on cancer cells, autoimmune cells, or infected cells from the patient, wherein the CAR T cells have been produced by a process comprising the steps of: a) obtaining a crude fluid composition comprising T cells from a patient; b) i) at least one channel extending from a sample inlet to one or more fluid outlets, said channel being bounded by a first wall and a second wall opposite said first wall; ii) an array of obstacles arranged in rows in the channel, with each successive row of obstacles being laterally shifted relative to the previous row, the obstacles being arranged in such a manner that when the crude fluid composition is applied to an inlet of the device and fluidly passed through the channel, T cells in the composition flow to one or more collection outlets where an enriched product is collected, and cells or particles in the crude fluid composition that are a different size than the T cells flow to another waste outlet that is separate from the collection outlets. performing deterministic lateral displacement (DLD) on the crude fluid composition using a microfluidic device comprising: c) genetically engineering the T cells obtained in step b) to express a chimeric antigen receptor (CAR) on their surface; d) expanding the number of engineered T cells by growing the cells in vitro; and e) administering the engineered T cells to the patient from whom the crude fluid composition was obtained. E59. The method of claim 58, wherein the crude fluid composition is an apheresis or leukapheresis product obtained from blood from the patient, and when the crude fluid composition is applied to an inlet of the device and fluidly passes through the channels, T cells in the composition flow to one or more collection outlets where an enriched product is collected, and red blood cells, platelets, or other particles present in the crude fluid composition and of a different size flow to another waste outlet separate from the collection outlets. E60. The method of claim 58 or 59, wherein the genetic manipulation comprises transfecting or transducing the target cell. E61. The method of claim 60, wherein the yield of cells expressing the chimeric receptor on their surface is at least 10% greater than T cells isolated by Ficoll centrifugation from a crude fluid composition and not subjected to DLD. E62. The method of claim 60, wherein the yield of target cells expressing the chimeric receptor on their surface is at least 50% greater than T cells isolated by Ficoll centrifugation from a crude fluid composition and not subjected to DLD. E63. The method of any one of claims 58-62, wherein the CAR comprises: a) an extracellular region comprising an antigen-binding domain; b) a transmembrane region; and c) an intracellular region, and the CAR T cell optionally comprises one or more recombinant sequences that provide the cell with a molecular switch that, when triggered, reduces the number or activity of CAR T cells. E64. The method of claim 63, wherein the antigen-binding domain is a single-chain variable fragment (scFv) derived from the antigen-binding regions of both the heavy and light chains of a monoclonal antibody. E65. The method of claim 63 or 64, wherein the CAR comprises a hinge region of 2 to 20 amino acids connecting the extracellular region and the transmembrane region. E66. The method of any one of claims 63-65, wherein the transmembrane region comprises a CD8 or CD28 protein sequence. E67. The method of any one of claims 63-66, wherein the intracellular region comprises a signaling domain derived from the CD3ζ, CD137, or CD28 intracellular domain. E68. The method of any one of claims 58-67, wherein the patient has leukemia. E69. The method of claim 68, wherein the leukemia is acute lymphoblastic leukemia. E70. The method of claim 68 or 69, wherein the CAR recognizes the antigen CD19 or CD20. E71. The method of any one of claims 58-67, wherein the patient has a solid tumor. E72. The method of claim 71, wherein the CAR recognizes an antigen selected from the group consisting of CD22; RORI; mesothelin; CD33 / IL3Ra; c-Met; PSMA; glycolipid F77; EGFRvIII; GD-2; NY-ESO-1 TCR; MAGE A3 TCR; and combinations thereof. E73. The method of any one of claims 58-72, wherein after obtaining a crude fluid composition containing T cells, the T cells in the fluid are bound to a carrier to facilitate DLD isolation. E74. The method of claim 73, wherein prior to performing DLD, the T cells are bound to one or more carriers to facilitate DLD separation. E75. The method of claim 73, wherein after DLD is performed and either before or after the T cells are genetically engineered to express the chimeric receptor, the T cells are coupled to one or more carriers to facilitate DLD separation. E76. The method of any one of claims 73-75, wherein the one or more carriers comprise an antibody or activator on the surface that specifically binds to the T cells. E77. The method of any one of claims 73 to 76, wherein the overall diameter of the carrier is at least as long as the diameter of the T cell. E78. The method of any one of claims 73 to 76, wherein the total diameter of the carrier is no more than 50% of the diameter of the T cell. E79. The method of any one of claims 73-76, wherein the overall diameter of the carrier is at least twice the length of the diameter of the T cell. E80. The method of any one of claims 73 to 76, wherein the total diameter of the carrier is no greater than 25% of the diameter of the T cell. E81. The method of claim 80, wherein one group of carriers has a diameter at least as long as the T cells, and a second group of carriers has a diameter no longer than 50% of the diameter of the T cells. E82. The method of any one of claims 73-81, wherein one group of carriers has a diameter at least twice the length of the T cells, and a second group of carriers has a diameter no more than 25% the length of the T cells. E83. The method of any one of claims 73-82, wherein the carrier is made of collagen or a polysaccharide. E84. The method of any one of claims 73-83, wherein the carrier is made of gelatin or alginate. E85. The method of any one of claims 73 to 84, wherein no more than 4 hours elapse between the time when the crude fluid composition containing T cells is obtained and the time when the carrier is bound to the T cells. E86. The method of any one of claims 73-84, wherein the crude fluid composition is an apheresis or leukapheresis product drawn from a patient's blood, and no more than four hours elapse between the completion of the apheresis or leukapheresis and the binding of the carrier to the target cells. E87. The method of any one of claims 73 to 84, wherein no more than 5 hours elapse between the time the crude fluid composition comprising T cells is obtained and the time the T cells are transfected or transduced for the first time. E88. The method of any one of claims 73-84, wherein the crude fluid composition is an apheresis or leukapheresis product drawn from the patient's blood, and no more than 5 hours have elapsed between the time the apheresis or leukapheresis is completed and the time the T cells are first transfected or transduced. E89. The method of claim 87 or 88, wherein no more than 4 hours have elapsed since the first time the T cells were transfected or transduced. E90. The method of any one of claims 58-89, wherein the T cells are available for administration to the patient at least one day earlier than for cells treated by a method that does not include DLD. E91. The method of any one of claims 58-89, wherein the target cells are available for administration to a patient at least 3 days earlier than for cells treated by a method that does not include DLD. E92. a) obtaining a crude fluid composition containing target cells from a patient; b) performing deterministic lateral displacement (DLD) on the crude fluid composition comprising said target cells using a microfluidic device to obtain a composition enriched in the target cells; a method for collecting target cells from a patient, comprising: binding one or more carriers to the target cells either before or after DLD to facilitate DLD isolation; and wherein no more than 5 hours elapse between the time a crude fluid composition containing the target cells is obtained from the patient and the time the carriers are bound to the target cells. E93. The method of claim 92, wherein the one or more carriers comprise an antibody or activator on the surface that specifically binds to the target cells. E94. The method of claim 92 or 93, wherein the overall diameter of the carrier is at least as long as the diameter of the target cell. E95. The method of claim 92 or 93, wherein the total diameter of the carrier is no greater than 50% of the diameter of the target cell. E96. The method of claim 92 or 93, wherein the overall diameter of the carrier is at least twice as long as the diameter of the target cell. E97. The method of claim 92 or 93, wherein the total diameter of the carrier is no greater than 25% of the diameter of the target cell. E98. The method of claim 92 or 93, wherein one group of carriers has a diameter at least as long as the target cells, and a second group of carriers has a diameter no longer than 50% of the diameter of the target cells. E99. The method of claim 92 or 93, wherein one group of carriers has a diameter at least twice the length of the T cells, and a second group of carriers has a diameter no more than 25% the length of the T cells. E100. The method of any one of claims 92 to 99, wherein the carrier is made of collagen or a polysaccharide. E101. The method of any one of claims 92 to 100, wherein the carrier is made of gelatin or alginate. E102. The method of any one of claims 92 to 101, wherein no more than 4 hours elapse between the time when the crude fluid composition containing the target cells is obtained and the time when the carrier is bound to the target cells. E103. The method of any one of claims 92 to 101, wherein no more than 3 hours elapse between the time when the crude fluid composition containing the target cells is obtained and the time when the carrier is bound to the target cells. E104. The method of any one of claims 92 to 103, wherein the crude fluid composition containing the target cells is obtained by performing apheresis or leukapheresis on blood from a patient. E105. The method of any one of claims 92-104, wherein the target cells in the composition enriched for target cells by DLD are transduced using a viral vector. E106. The method of claim 105, wherein the target cells are transfected electrically, chemically, or with nanoparticles. E107. The microfluidic device a) at least one channel extending from a sample inlet to one or more fluid outlets, said channel being bounded by a first wall and a second wall opposite said first wall; b) an array of obstacles arranged in rows in the channel, with each successive row of obstacles being laterally shifted relative to the previous row, the obstacles being positioned in such a manner that when a crude fluid composition comprising the target cells is applied to an inlet of the device and fluidly passes through the channel, the target cells flow to one or more collection outlets where an enriched product is collected, and contaminant cells or particles present in the crude fluid composition and that are a different size from the target cells flow to another waste outlet that is separate from the collection outlets. The method according to any one of claims 92 to 106, comprising: E108. The method of any one of claims 92 to 107, wherein the target cell is a T cell. E109. The method of claim 108, wherein the T cells are selected from the group consisting of natural killer T cells, central memory T cells, helper T cells, and regulatory T cells. E110. The method of any one of claims 92 to 107, wherein the target cell is a stem cell. E111. The method of any one of claims 92 to 107, wherein the target cell is a B cell, a macrophage, a dendritic cell, or a granulocyte. E112. The method of any one of claims 92-111, wherein the crude fluid composition containing the target cells comprises one or more additives that act as anticoagulants or prevent platelet activation. E113. The method of claim 112, wherein the additive is selected from the group consisting of ticlopidine, inosine, protocatechuic acid, acetylsalicylic acid, and tirofiban. E114. The method of any one of claims 92-113, wherein steps a) and b) are both performed on-site where a crude fluid composition containing target cells is obtained from a patient. E115. The method of any one of claims 92 to 114, wherein no more than 4 hours elapse between the time a crude fluid composition containing target cells is obtained from a patient and the time a carrier is bound to the target cells. E116. The method of any one of claims 92-114, wherein the crude fluid composition is an apheresis or leukapheresis product drawn from a patient's blood, and no more than four hours elapse between the completion of the apheresis or leukapheresis and the binding of the carrier to the target cells. E117. c) genetically manipulating and / or increasing the number of cells; and / or d) treating the same patient from whom said target cells were obtained with the collected target cells. The method of any one of claims 92 to 116, further comprising: E118. The method of claim 117, wherein after step e), the target cells are cryopreserved. E119. The method of claim 117 or 118, wherein the target cells cultured in step c) are T cells cultured in the presence of an activator. E120. The method of claim 119, wherein the activator is bound to a carrier. E121. The method of any one of claims 58-89, wherein the target cells are available for administration to a patient at least one day earlier than for cells treated by a method that does not include DLD. E122. The method of any one of claims 58-89, wherein the target cells are available for administration to a patient at least 3 days earlier than for cells treated by a method that does not include DLD. E123. A target cell produced by the method of any one of claims 92 to 122. E124. A method of treating a patient for a disease or condition comprising administering to said patient the target cells of claim 123. E125. A method for separating adherent cells from a plurality of other cells, comprising the steps of: a) contacting a crude fluid composition comprising a plurality of other cells and adherent cells with one or more carriers that are bound in a manner that promotes DLD separation, wherein the adherent cells at least partially associate with the carriers upon or after contact to form carrier-associated adherent cell complexes, the carrier-associated adherent cell complexes comprising an increased size relative to cells in the plurality of other cells, the size of the carrier-associated adherent cell complexes being equal to or greater than a critical size, and the cells in the plurality of other cells comprising a size less than the critical size; b) applying the crude fluid composition to a device comprising an array of obstacles arranged in rows, the rows being shifted laterally relative to one another, the rows being configured to deflect particles equal to or greater than the critical size in a first direction and particles less than the critical size in a second direction; and c) flowing the sample containing the carrier-associated adherent cell complexes through the device, wherein the carrier-associated adherent cell complexes are deflected in the first direction by the obstacle and cells in the plurality of other cells are deflected in the second direction, thereby separating the carrier-associated adherent cell complexes from the plurality of other cells; d) collecting the fluid composition comprising the separated carrier-associated adherent cell complexes. E126. The method of claim 125, wherein the adherent cells are collected from the patient as part of a crude fluid composition comprising the adherent cells and a plurality of other cells, and no more than 3 hours elapse between the time the crude fluid composition is obtained from the patient and the time the adherent cells are first bound to a carrier. E127. The method of claim 125, wherein no more than two hours elapse between the time the crude fluid composition is obtained from the patient and the time the adherent cells are first bound to a carrier. E128. The method of claim 125, wherein no more than one hour elapses between the time the crude fluid composition is obtained from the patient and the time the adherent cells are first bound to a carrier. E129. The method of claim 125, wherein no more than 4 hours elapse between the time the crude fluid composition is obtained from the patient and the time the adherent cells or carrier-adherent cell complexes are collected from the device for the first time. E130. The method of claim 125, wherein no more than four hours elapse between the time the crude fluid composition is obtained from the patient and the time the adherent cells or carrier-adherent cell complexes are collected from the device for the first time. E131. The method of any one of claims 125 to 130, wherein the carrier comprises on its surface an antibody or activator that specifically binds to the adherent cells. E132. The method of any one of claims 125 to 131, wherein the diameter of the carrier is at least as long as the diameter of the adherent cells. E133. The method of any one of claims 125-131, wherein the overall diameter of the carrier is at least twice the length of the diameter of the adherent cells. E134. The method of any one of claims 125-131, wherein the overall diameter of the carrier is at least 10 times the diameter of the adherent cells. E135. The method of any one of claims 125 to 131, wherein the total diameter of the carrier is 10 to 600 μm. E136. The adherent cells are MRC-5 cells; HeLa cells; Vero cells; NIH 3T3 cells; L929 cells; Sf21 cells; Sf9 cells; A549 cells; A9 cells; AtT-20 cells; BALB / 3T3 cells; BHK-21 cells; BHL-100 cells; BT cells; Caco-2 cells; Chang cells; Clone 9 cells; The method of any one of claims 125 to 135, wherein the cell is selected from the group consisting of M-3 cells; COS-1 cells; COS-3 cells; COS-7 cells; CRFK cells; CV-1 cells; D-17 cells; Daudi cells; GH1 cells; GH3 cells; HaK cells; HCT-15 cells; HL-60 cells; HT-1080 cells; HEK cells, HT-29 cells; HUVEC cells; I-10 cells; IM-9 cells; JEG-2 cells; Jensen cells; Jurkat cells; K-562 cells; KB cells; KG-1 cells; L2 cells; LLC-WRC 256 cells; McCoy cells; MCF7 cells; WI-38 cells; WISH cells; XC cells; Y-1 cells; CHO cells; Raw 264.7 cells; HEP G2 cells; BAE-1 cells; SH-SY5Y cells, and any derivatives thereof. E137. The method of any one of claims 125 to 135, wherein the adherent cells are stem cells. E138. A method for separating activated cells from a plurality of other cells, comprising the steps of: a) contacting a crude fluid composition comprising cells capable of activation and a plurality of other cells with one or more carriers, wherein at least one carrier comprises a cellular activator, wherein the cellular activator at least partially associates with cells capable of activation by the cellular activator upon or after contact to form carrier-associated cell complexes, wherein the association of the cellular activator with cells capable of activation by the cellular activator at least partially activates the activation-competent cells, and wherein the carrier-associated cell complexes have an increased size relative to cells in the plurality of other cells, wherein the size of the carrier-associated cell complexes is equal to or greater than a critical size, and wherein cells in the plurality of other cells have a size less than the critical size; b) applying the sample to a device comprising an array of obstacles arranged in rows, the rows being shifted laterally relative to one another, the rows being configured to deflect particles equal to or greater than the critical size in a first direction and particles less than the critical size in a second direction; and c) flowing the sample through the device, wherein the carrier-associated cell complexes are deflected in the first direction by the obstacle and cells in the plurality of other cells are deflected in the second direction, thereby separating the activated cells from the plurality of other cells; d) collecting the fluid composition containing the separated carrier-associated cell complexes. E139. The method of claim 0, wherein the cells capable of activation are selected from the group consisting of T cells, B cells, regulatory T cells, macrophages, dendritic cells, granulocytes, innate lymphoid cells, megakaryocytes, natural killer cells, thrombocytes, synoviocytes, beta cells, liver cells, pancreatic cells, DE3 lysogenized cells, yeast cells, plant cells, and stem cells. E140. The method of claim 138 or 139, wherein the cell activator is a protein. E141. The method of claim 140, wherein the protein is an antibody. E142. The method of claim 140, wherein the protein is selected from the group consisting of CD3, CD28, an antigen, a helper T cell, a receptor, a cytokine, a glycoprotein, and any combination thereof. E143. The method of claim 138, wherein the cellular activator is selected from the group consisting of insulin, IPTG, lactose, allolactose, lipids, glycosides, terpenes, steroids, alkaloids, and any combination thereof. E144. The method of any one of claims 138-143, wherein the activation-competent cells are collected from the patient as part of a crude fluid composition comprising the activation-competent cells and a plurality of other cells, and no more than four hours elapse between the time the crude fluid composition is obtained from the patient and the time the activation-competent cells are bound to a carrier. E145. The method of any one of claims 138-143, wherein no more than 3 hours elapse between the time the crude fluid composition is obtained from the patient and the time the activation-competent cells are bound to a carrier. E146. The method of any one of claims 138-143, wherein no more than two hours elapse between the time the crude fluid composition is obtained from the patient and the time the activation-competent cells are bound to a carrier. E147. The method of any one of claims 138-143, wherein no more than 4 hours elapse between the time the crude fluid composition is obtained from the patient and the time step c) is completed. E148. The method of any one of claims 138-143, wherein no more than 3 hours elapse between the time the crude fluid composition is obtained from the patient and the time step c) is completed. E149. The method of any one of claims 138-148, wherein the overall diameter of the carrier is at least as long as the activation-competent cells. E150. The method of any one of claims 138-148, wherein the overall diameter of the carrier is at least twice as long as the diameter of the activation-competent cells. E151. The method of any one of claims 138-148, wherein the overall diameter of the carrier is at least 10 times the length of the diameter of 138-148. E152. The method of any one of claims 138 to 148, wherein the diameter of the carrier is 10 to 600 μm. E153. A method for continuous purification of a secretory product from cells, comprising the steps of: a) obtaining a fluid composition comprising cells, the cells being suspended in the fluid composition, the cells secreting a secretory product into the suspension, the cells having a predetermined size greater than the predetermined size of the secretory product, the predetermined size of the cells being equal to or greater than a critical size, and the predetermined size of the secretory product being less than the critical size; b) applying the cell-containing fluid composition to a device comprising an array of obstacles arranged in rows, the rows being shifted laterally relative to one another, the rows being configured to deflect particles equal to or greater than the critical size in a first direction and particles less than the critical size in a second direction; b) flowing the sample through the device, wherein the cells are deflected in the first direction by the obstacle and the secretory products are deflected in the second direction, thereby separating the secretory products from the cells; c) collecting said secretory product, thereby resulting in a sample of said secretory product that is substantially pure; d) collecting the recovered fluid composition containing the separated cells; and e) reapplying the recovered fluid composition containing the separated cells to the device and repeating steps (a) through (e), thereby continuously purifying secretory products from the cells. E154. The method of claim 153, wherein the secreted product is selected from the group consisting of a protein, an antibody, a biofuel, a polymer, a small molecule, and any combination thereof. E155. The method of claim 153, wherein the cell is selected from the group consisting of a bacterial cell, an algal cell, a mammalian cell, and a tumor cell. E156. A method for reducing the platelet to leukocyte ratio in an apheresis sample, comprising performing deterministic lateral displacement (DLD) on the sample in the absence of centrifugation or elutriation, wherein the product has a platelet to leukocyte ratio that is at least 20% lower than the ratio obtained by the same procedure performed using centrifugation or elutriation instead of DLD. E157. The method of claim 156, wherein the product is obtained with a platelet to white blood cell ratio that is at least 20% lower than the ratio obtained by the same procedure performed using density gradient centrifugation or counterflow centrifugation. E158. The method of claim 156, wherein the product is obtained with a platelet to white blood cell ratio that is at least 20% lower than the ratio obtained by the same procedure performed using elutriation. E159. The method of claim 156, wherein a product is obtained with a platelet to white blood cell ratio that is at least 50% lower than that obtained using centrifugation or elutriation instead of DLD. E160. The method of any one of claims 156-159, wherein no separation step is performed on the apheresis sample prior to DLD. E161. The method of any one of claims 156-160, wherein DLD is performed in a buffer that does not contain intercalating agents that alter platelet size and that does not promote platelet aggregation. E162. The method of any one of claims 156-159, wherein DLD is performed in a buffer that does not contain dextran or other highly charged polymers. E163. The method of any one of claims 156-162, wherein the total number of platelets in the product is at least 70% lower than in the apheresis sample. E164. The method of any one of claims 156-163, wherein the total number of platelets in the product is at least 90% lower than in the apheresis sample. E165. A method for purifying T cells from an apheresis sample, comprising performing DLD on the sample, followed by an affinity separation step and expansion of the T cells by culturing in the presence of an activator, wherein the number of T cells obtained is at least twice the number produced by the same procedure performed using Ficoll centrifugation instead of DLD. E166. The method of claim 165, wherein the affinity separation step comprises the use of magnetic beads or particles containing antibodies that bind to CD3. E167. The method of claim 165, wherein the number of T cells obtained after 14 days of culture is at least twice the number produced by the same procedure performed using Ficoll centrifugation instead of DLD. E168. The method of claim 165, wherein the number of T cells obtained after 14 days of culture is at least four times the number produced by the same procedure performed using Ficoll centrifugation instead of DLD. E169. The method of any one of claims 156-168, wherein when the cells in the DLD product are transformed with a vector to express a recombinant phenotype, the yield of cells exhibiting the desired phenotype is at least 10% higher than identical cells isolated by Ficoll centrifugation and not subjected to DLD. E170. The method of any one of claims 156-169, wherein when the cells in the DLD product are transformed with a vector to express a recombinant phenotype, the yield of T cells exhibiting the desired phenotype is at least 20% higher than identical cells isolated by Ficoll centrifugation and not subjected to DLD. E171. The method of any one of claims 163-165, wherein the proportion of memory T cells relative to the total number of T cells in the DLD product is at least 10% higher than the proportion produced using the same procedure with Ficoll centrifugation instead of DLD. E172. The method of any one of claims 156-171, wherein the method is used to produce T cells for CAR T cell therapy and the time required to produce a sufficient number of cells to treat a patient is reduced by at least 20% using DLD instead of Ficoll centrifugation. E173. The method of claim 172, wherein the process for producing CAR T cells does not include a step in which the cells are frozen. E174. The method of either claim 172 or claim 173, wherein the treatment of T cells is performed at the same site where apheresis is performed. E175. The method of any one of claims 172-174, wherein the T cells are genetically transformed at the same site where apheresis is performed. E176. The method of any one of claims 156-169, wherein less than one hour elapses from the time apheresis sample collection is completed to the time DLD is performed. E177. A method for reducing the ratio of platelets to leukocytes in an apheresis sample, comprising performing deterministic lateral displacement (DLD) on the sample in the absence of centrifugation or elutriation, wherein a product is obtained in which the total number of platelets in the product is at least 90% lower than in the apheresis sample. E178. The method of claim 177, wherein DLD is performed in a buffer that does not contain intercalating agents that alter platelet size and that does not promote platelet aggregation. E179. The method of claim 177, wherein DLD is performed in a buffer that does not contain dextran or other highly charged polymers. E180. A method for engineering CAR T cells, comprising the steps of: a) obtaining a sample composition from a patient by apheresis, said sample composition comprising T cells; b) performing DLD on the sample composition so as to reduce the total number of platelets present by at least 70%, the DLD comprising: i) at least one channel extending from a sample inlet to one or more fluid outlets, said channel being bounded by a first wall and a second wall opposite said first wall; ii) an array of obstacles arranged in rows in the channel, with each successive row of obstacles being laterally shifted relative to the previous row, the obstacles being arranged in such a manner that when a crude fluid composition is applied to an inlet of the device and fluidly passed through the channel, T cells in the composition flow to one or more collection outlets where an enriched product is collected, and platelets and other materials smaller than T cells flow to another waste outlet that is separate from the collection outlets. a step carried out on a microfluidic device comprising: c) genetically engineering the T cells in the enriched product obtained in step b) to produce chimeric antigen receptors (CARs) on their surface; d) culturing the T cells to expand their numbers; e) transferring the T cells into a pharmaceutical composition for administration to a patient. E181. The method of claim 180, wherein at least 90% of the platelets are removed in step b). E182. The method of either claim 180 or claim 181, wherein all isolation and concentration steps are performed using DLD. E183. The method of any one of claims 180-182, wherein the cells are exposed to a T cell activator before or during culture. E184. The method of any one of claims 180-183, wherein during step b) and before step c), the cells are transferred to medium in the presence or addition of a T cell activator. E185. The method of claim 184, in which the medium containing the activator is treated with DLD to separate the activator from the cells, and the cells are transferred to medium in the presence or addition of a vector for recombinantly engineering the cells. E186. The method of any one of claims 180-185, wherein the cells are not frozen until they are transferred into a pharmaceutical composition for administration to a patient. E187. The method of any one of claims 180-185, wherein the cells are not frozen at any step. E188. The method of any one of claims 180-187, wherein the process is completed at least one day faster than in procedures in which cells are isolated or enriched by methods other than DLD. E189. The method of any one of claims 180-187, wherein the process is completed at least three days faster than in procedures in which cells are isolated or enriched by methods other than DLD. E190. The method of any one of claims 180-187, wherein the process is completed at least 5 days faster than in procedures in which cells are isolated or enriched by methods other than DLD. E191. The method of any one of claims 180-187, wherein the process is completed at least 10 days faster than in procedures in which cells are isolated or enriched by methods other than DLD. E192. The method of any one of claims 180-191, wherein in step d), the number of T cells obtained after 14 days of culture is at least two-fold higher than the number produced by the same procedure performed using Ficoll centrifugation instead of DLD. E193. The method of any one of claims 180-191, wherein in step d), the number of T cells obtained after 14 days of culture is at least four times higher than the number produced by the same procedure performed using Ficoll centrifugation instead of DLD. E194. A method for preparing CAR T cells, comprising the steps of: a) Collecting cells from a patient by apheresis; b) performing DLD on the cells obtained in step a) to separate the leukocytes from other cells and particles and transferring the leukocytes to a medium that supports their proliferation and that contains or is supplemented with T cell activating factors; c) performing DLD to separate T cells recombinantly engineered to express a chimeric antigen receptor (CAR) on their surface from the culture medium of step b) and transfer them to culture medium; d) DLD is performed to separate the T cells from the reagent and transfer them to growth medium; e) culturing the cells to increase cell numbers; f) DLD is performed to transfer the expanded T cells into culture medium for administration to the patient. E195. The method of claim 194, wherein the cells are not frozen until transferred into a pharmaceutical composition for administration to a patient. E196. The method of claim 194, wherein the cells have not been frozen at any stage. E197. The method of any one of claims 194-196, wherein the process is completed at least three days faster than in procedures in which cells are isolated or enriched by methods other than DLD. E198. The method of any one of claims 194-196, wherein the process is completed at least 5 days faster than in procedures in which cells are isolated or enriched by methods other than DLD. E199. The method of any one of claims 194-196, wherein the process is completed at least 10 days faster than in procedures in which cells are isolated or enriched by methods other than DLD. E200. A method for preparing CAR T cells for the treatment of a patient, comprising the steps of: a) collecting cells from a patient by apheresis; b) performing DLD on the cells obtained in step a) to separate the leukocytes from other cells and particles and transferring the leukocytes to a medium that supports their proliferation and that contains or is supplemented with T cell activating factors; c) separating T cells from the medium of step b) into a medium containing a vector for genetically engineering the T cells to produce a chimeric antigen receptor (CAR) on their surface; d) DLD is performed to separate the T cells from the reagent and transfer them into culture medium for administration to the patient. E201. The method of claim 200, wherein the cells are not frozen until transferred into a pharmaceutical composition for administration to a patient. E202. The method of claim 200, wherein the cells have not been frozen at any stage. E203. The method of any one of claims 200-202, wherein the process is completed at least three days faster than in procedures in which cells are isolated or enriched by methods other than DLD. E204. The method of any one of claims 200-202, wherein the process is completed at least 5 days faster than in procedures in which cells are isolated or enriched by methods other than DLD. E205. The method of any one of claims 200-202, wherein the process is completed at least 10 days faster than in procedures in which cells are isolated or enriched by methods other than DLD. E206. The method of any one of claims 180-205, wherein step b) produces a product having a platelet to leukocyte ratio that is at least 50% lower than the ratio obtained using centrifugation or elutriation instead of DLD. E207. The method of any one of claims 180-205, wherein the yield of T cells exhibiting the desired CAR T phenotype is at least 10% higher than identical cells isolated by Ficoll centrifugation and not subjected to DLD. E208. The method of any one of claims 180-205, wherein the yield of T cells exhibiting the desired CAR T phenotype is at least 20% higher than identical cells isolated by Ficoll centrifugation and not subjected to DLD. E209. The method of any one of claims 180-208, wherein the time required to produce a sufficient number of T cells for patient treatment is at least 5% shorter than if the same method were performed using Ficoll centrifugation rather than DLD to isolate cells from the apheresis starting material. E210. The method of any one of claims 180-209, wherein the time required to produce a sufficient number of T cells for patient treatment is at least 10% shorter than if the same method were performed using Ficoll centrifugation rather than DLD to isolate cells from the apheresis starting material. E211. The method of any one of claims 180-209, wherein when cells prepared by said method are administered to a patient, the cells exhibit at least 10% less senescence than cells processed from an apheresis composition using centrifugation or elutriation methods instead of DLD. E212. The method of any one of claims 180-209, wherein when cells prepared by said method are administered to a patient, the cells exhibit increased potency over cells processed from an apheresis composition using centrifugation or elutriation methods instead of DLD. E213. A method of treating a patient for a disease or condition comprising administering to said patient a therapeutically effective amount of cells prepared by the method of any one of claims 180-209. E214. The method of claim 213, wherein the disease or condition is cancer. E215. A method for producing therapeutically active cells, comprising the steps of: a) obtaining a sample composition from a patient comprising said cells; b) performing DLD on the sample to produce a composition enriched in therapeutically active cells, wherein DLD is performed in a microfluidic device comprising: i) at least one channel extending from a sample inlet to one or more fluid outlets, the channel being bounded by a first wall and a second wall opposite the first wall; ii) an arrangement of obstacles arranged in rows within the channel, with each subsequent row of obstacles being laterally shifted relative to the previous row, said obstacles such that when a crude fluid composition is applied to an inlet of the device and fluidly passed through the channel, therapeutically active cells in the composition flow to one or more collection outlets where a concentrated product is collected, and wherein cells and materials smaller than the therapeutically active cells flow to one or more waste outlets where they are separated from the collection outlet; c) optionally genetically engineering the therapeutically active cells in the enriched product obtained in step b); d) optionally culturing and expanding the therapeutically active cells; e) Transferring the therapeutically active cells into a pharmaceutical composition for administration to a patient. E216. The method of claim 215, wherein the therapeutically active cells are stem cells. E217. The method of claim 216, wherein the stem cells are found in the circulation and the sample composition is prepared by apheresis. E218. The method of claim 217, wherein at least 70% of the platelets are removed from the concentrated product of step b). E219. The method of claim 217, wherein at least 90% of the platelets are removed from the concentrated product of step b). E220. The method of any one of claims 215-219, wherein all isolation and concentration steps are performed using DLD. E221. The method of any one of claims 215-220, wherein the cells are not frozen until transferred into a pharmaceutical composition for administration to a patient. E222. The method of any one of claims 215-220, wherein the cells have not been frozen at any stage. E223. The method of any one of claims 215-222, wherein the process is completed at least one day faster than in procedures in which cells are isolated or enriched by methods other than DLD. E224. The method of any one of claims 215-222, wherein the process is completed at least three days faster than in procedures in which cells are isolated or enriched by methods other than DLD. E225. The method of any one of claims 215-222, wherein the process is completed at least 5 days faster than in procedures in which cells are isolated or enriched by methods other than DLD. E226. The method of any one of claims 215-222, wherein the yield of therapeutically active cells obtained from the sample composition is at least 25% greater than that obtained by a procedure in which the cells are isolated or enriched by a method other than DLD. E227. The method of any one of claims 215-222, wherein the process is completed at least three days faster than in procedures in which cells are isolated or enriched by methods other than DLD. E228. The method of any one of claims 215-222, wherein the process is completed at least 5 days faster than in procedures in which cells are isolated or enriched by methods other than DLD. E229. The method of any one of claims 215-222, wherein the time required to produce a sufficient number of therapeutically active cells for patient treatment is at least 5% less than if the same method were performed using Ficoll centrifugation rather than DLD to isolate cells from the sample composition. E230. The method of any one of claims 215-222, wherein the time required to produce a sufficient number of therapeutically active cells for patient treatment is at least 10% less than if the same method were performed using Ficoll centrifugation rather than DLD to isolate cells from the sample composition. E231. The method of any one of claims 215-230, wherein when the therapeutically active cells prepared by said method are administered to a patient, the cells exhibit at least 10% less senescence than cells processed from an apheresis composition using centrifugation or elutriation methods instead of DLD. E232. The method of any one of claims 215-230, wherein when therapeutically active cells prepared by said method are administered to a patient, the cells exhibit increased potency over cells processed from the sample composition using centrifugation or elutriation methods instead of DLD. E233. A method of treating a patient for a disease or condition comprising administering to said patient a therapeutically effective amount of therapeutically active cells prepared by the method of any one of claims 215-230. E234. The method of claim 233, wherein the therapeutically active cells are stem cells and the disease or condition is a genetic disease. E235. A method for manipulating a population of target cells, comprising: a) isolating target cells from a crude liquid composition, wherein the isolation is performed on a microfluidic device using one or more means for separating the target cells from other cells in the crude liquid composition based on differences in cell size, wherein the isolation process results in a target cell-enriched composition; b) genetically engineering the target cells obtained from step a) to have a desired phenotype. Including, A method in which the target cells are not centrifuged or eluted before being genetically engineered. E236. The method of claim 235, wherein said target cells are white blood cells or stem cells. E237. The method of claim 235, wherein said target cell is a T cell. E238. The method of any one of claims 235-237, wherein the crude liquid composition is blood or an apheresis preparation obtained from a patient. E239. The method of claim 238, wherein isolation of target cells is performed under conditions such that the target cell-enriched composition has a total number of platelets that is at least 70% lower than the apheresis preparation. E240. The method of claim 239, wherein the total number of platelets in the target cell-enriched composition is at least 90% lower than in the apheresis preparation. E241. The method of claim 240, wherein the platelet to target cell ratio is at least 50% lower than in the apheresis preparation. E242. The method of claim 238, wherein the target cells are T cells that are expanded in culture after isolation. E243. The method of claim 242, wherein the number of T cells obtained after 14 days of culture is at least two times greater than in a procedure in which the T cells are isolated by a process including a centrifugation step. E244. The method of claim 242, wherein the number of T cells obtained after 14 days of culture is at least four times greater than in a procedure in which the T cells are isolated by a process including a centrifugation step. E245. The method of claim 244, wherein the percentage of memory T cells relative to the total number of T cells in culture is at least 10% higher than in a procedure in which the T cells are isolated by a process including a centrifugation step. E246. The method of claim 244, wherein the percentage of memory T cells relative to the total number of T cells in culture is at least 20% higher than in a procedure in which the T cells are isolated by a process including a centrifugation step. E247. The method of any one of claims 235-246, wherein when cells in the composition enriched for target cells are transformed with a vector and exhibit a recombinant phenotype, the yield of target cells exhibiting the desired phenotype is at least 20% greater than the same cells isolated by centrifugation. E248. The method of any one of claims 238-247, wherein no more than one hour elapses between the time collection of the apheresis sample is completed and the time separation using the microfluidic device is performed. E249. The method of any one of claims 238-247, wherein the elapsed time between the completion of acquisition of the apheresis sample from the patient and the completion of isolation of the target cells is four hours or less. E250. The method of any one of claims 238-247, wherein no more than four hours elapse between the completion of the apheresis sample from the patient and the cells being genetically engineered. E251. A method for preparing CAR T cells, comprising: a) obtaining a crude liquid composition from a patient by apheresis, wherein the sample comprises T cells; b) isolating T cells from the crude liquid composition, wherein the isolation is performed on a microfluidic device using one or more procedures that separate T cells from platelets and other cells in the crude liquid composition based on size differences, resulting in a T cell-enriched, platelet-depleted composition; c) genetically engineering the target cells obtained in step a) to produce chimeric antigen receptors (CARs) on their surface, wherein the T cells are not centrifuged or eluted at any step prior to genetic engineering; d) expanding the number of genetically engineered T cells; e) harvesting the cultured cells obtained in step d). A method comprising: E252. The method of claim 251, wherein in step c), the T cells are collected by being transferred into a pharmaceutical composition for administration to a patient. E253. The method of claim 251 or 252, wherein the cells are not frozen prior to harvesting. E254. The method of any one of claims 251-253, wherein at least 90% of the platelets are removed in step b). E255. The method of any one of claims 251-254, wherein the cells are exposed to a T cell activator or carrier before or during culture. E256. The method of claim 255, wherein neither the activator nor the carrier is bound to a magnetic bead or particle. E257. The method of any one of claims 251-256, wherein the CAR T cells are available for administration to the patient at least one day earlier compared to a procedure in which the cells are isolated or enriched by a method not involving the use of a microfluidic device. E258. The method of any one of claims 251-256, wherein the CAR T cells are available for administration to the patient at least 3 days earlier compared to a procedure in which the cells are isolated or enriched by a method not involving the use of a microfluidic device. E259. The method of any one of claims 251-258, wherein in step d), the number of CAR T cells obtained after 14 days of culture is at least two times higher than the number obtained using Ficoll centrifugation. E260. The method of any one of claims 251-258, wherein in step d), the number of CAR T cells obtained after 14 days of culture is at least four times greater than the number obtained using Ficoll centrifugation. E261. The method of any one of claims 251-260, wherein the T cells and CAR T cells are never frozen prior to administration to the patient. E262. A method for preparing CAR T cells for the treatment of a patient, comprising: a) obtaining a crude liquid composition from a patient by apheresis, wherein the sample comprises T cells; b) isolating T cells from the crude liquid composition, wherein the isolation is performed on a microfluidic device using one or more procedures that separate T cells from platelets and other cells in the crude liquid composition based on size differences, resulting in a T cell-enriched, platelet-depleted composition; c) genetically engineering the target cells obtained in step a) to produce chimeric antigen receptors (CARs) on their surface, wherein the T cells are not centrifuged or eluted at any step prior to genetic engineering; d) expanding the number of genetically engineered T cells; e) separating T cells on the microfluidic device using one or more procedures that separate T cells from a reagent based on size differences. A method comprising: E263. The method of claim 262, wherein the cells are not frozen until transferred into a pharmaceutical composition for administration to a patient. E264. The method of claim 262, wherein the cells are not frozen at any step. E265. The method of any one of claims 262-264, wherein the process is completed at least three days faster compared to a procedure in which the cells are isolated or enriched by a method that does not involve the use of a microfluidic device. E266. The method of any one of claims 262-265, wherein the CAR T cells are available for administration to the patient at least one day earlier compared to a procedure in which the cells are isolated or enriched by a method not involving the use of a microfluidic device. E267. The method of any one of claims 262-265, wherein the CAR T cells are available for administration to the patient at least 3 days earlier compared to a procedure in which the cells are isolated or enriched by a method not involving the use of a microfluidic device. E268. The method of any one of claims 262-267, wherein step b) produces a product composition having a platelet to white blood cell ratio that is at least 50% lower than that obtained using centrifugation or elution processes. E269. The method of any one of claims 262-268, wherein the time required to produce sufficient numbers of CAR T cells for patient treatment is at least 5% shorter than when the same method is performed using cells isolated by Ficoll centrifugation. E270. The method of any one of claims 262-269, wherein the time required to produce sufficient numbers of CAR T cells for patient treatment is at least 10% shorter than when the same method is performed using Ficoll centrifugation to isolate cells from the apheresis starting material. E271. The method of any one of claims 262-270, wherein when cells prepared by said method are administered to a patient, they exhibit at least 10% less senescence than cells processed from an apheresis composition using centrifugation or elution processes. E272. A method of treating a patient for a disease or condition comprising administering to the patient a therapeutically effective amount of cells prepared by the method of any one of claims 235-271. E273. The method of claim 272, wherein said disease or condition is cancer. [Example]

[0172] The following examples are intended to illustrate, but not limit, the present invention.

[0173] Example 1 This study focuses on apheresis samples, which are essential for CAR-T cell manufacturing. Inherent variability related to donor health, disease status, and prior chemotherapy all affect the quality of leukapheresis collection and, potentially, the efficiency of various steps in the manufacturing protocol (Levine, et al., Mol. Therapy: Meth. Clin. Dev. 4:92-101 (2017)). To stress-test the automated DLD leukapheresis, residual leukapheresis (LRS chamber fraction) was collected from plateletapheresis donations. These plateletapheresis samples typically have near-normal red blood cell counts, 10-20 times more lymphocytes and monocytes, and few granulocytes. They also have approximately 10 times higher platelet counts than normal peripheral blood.

[0174] Twelve donors were processed to compare yields of major blood cell types and processing capacity by DLD versus Ficoll-Hypaque density gradient centrifugation (the "gold standard"). Four of these donors were also evaluated for "T cell expansion capacity" over a 15-day period. Each donor sample was processed with both DLD and Ficoll, and for the four donors studied for T cell expansion capacity, samples were processed using direct magnetic extraction.

[0175] material and method Microchip design and fabrication: The DLD arrays used in this study consisted of a single-zone, mirrored diamond-shaped post design (see D'Silva, J., "Throughout Microfluidic Capture of Rare Cells from Large Volumes of Blood"; A Dissertation Presented to the Faculty of Princeton University in Candidacy for the Degree of Doctor of Philosophy (2016)). There were 14 parallel arrays per chip, resulting in a 14-lane DLD device (Figure 1D). The devices were designed with a 16 μm gap between posts and a 1 / 42 slope, resulting in a critical diameter of approximately 4 μm. Plastic DLD devices were fabricated using a process called soft embossing. First, a silicon (Si) master for the plastic DLD microchip was fabricated using standard photolithographic deep reactive ion etching techniques (PRISM, Princeton University). The features on the silicon master were then transferred to a soft elastomeric mold (Edge Embossing, Medford, MA) by pouring and curing an elastomer over the Si features. The elastomer was peeled off, resulting in a reusable negative imprint of the silicon master. A blank plastic sheet was placed between the elastomeric molds, and then, using a combination of pressure and temperature, the plastic was extruded into the features (wells) of the soft elastomeric negative mold, replicating the positive features and depth of the original silicon master. The soft tool was then peeled off from the plastic device, producing a flat piece of surface-embossed plastic to a depth of approximately 100 μm, with a pattern of flow channels and grooves around the array of microposts (Figure 1D, inset). Ports were created for fluidic access to the inlet and outlet ends of the microchip.After cleaning by sonication, the device was covered with a heat-sensitive, hydrophilic adhesive (ARFlow Adhesives Research, Glen Rock, PA). The entire chip was 40 × 75 mm and 1 mm thick, smaller than the size of a credit card.

[0176] DLD Microchip Operation: The microfluidic device was assembled inside an optically transparent, pressure-resistant manifold with fluidic connections. Fluids were flowed through the DLD microchip using a constant air pressure controller (MFCS-EZ, Fluigent, Lowell, MA). Two different pressure controls were used: one for the buffer and one for the sample. The flow path for the buffer line included tubing connecting a buffer reservoir (60 mL syringe), an in-line degasser (Biotech DEGASi, Minneapolis, MN), and the buffer inlet port of the manifold. The flow path for the sample included tubing connecting a sample reservoir (20 mL syringe), a 25 mm diameter, 20 μm PureFlow nylon filter to retain aggregates larger than the microchip's nominal pore size (16 μm), and the sample inlet port of the manifold. The manifold's outlet ports were connected by tubing to collection reservoirs for the waste and product fractions.

[0177] The microchip, filter, and tubing were primed and blocked with running buffer for 15 minutes before loading the sample. The DLD setup was primed by loading running buffer into the buffer reservoir (60 mL syringe) and then pressurizing; the fluid then passed through the tubing and into the manifold "buffer in" port (Figure 1). Air in the manifold port was vented through a separate port at the inlet, which was then sealed. Buffer was then flowed into the microchip and out both the product and waste outlets to remove all air from the micropost array. Simultaneously, buffer was backflowed through the manifold "sample in" port and in-line filter to expel any air. This preparation process required approximately 5 minutes of hands-on time and removed all air from the microchip, manifold, and tubing. After the preparation process, the setup was continued to be flushed with buffer for an additional 15 minutes to block all internal surfaces; this process was automated and required no hands-on effort.

[0178] After the blocking step, the system was depressurized and the sample was loaded into the sample container (20 mL syringe). The sample (see below) was diluted with 1 part sample per 4 parts running buffer (0.2x) before loading into the DLD. First the buffer source, then the sample source, was repressurized, resulting in both buffer and sample entering their respective ports on the manifold and the microchip, and flowing into the parallel microchip (separation mode, see Figure 1Ai). Once the sample was loaded and under running pressure, the system automatically processed the entire sample volume. Both the product and waste fractions were collected in pre-weighed sterile conical 50 mL tubes and weighed after collection to determine the collected volume.

[0179] Buffer Systems. Three different EDTA-free buffer formulations were tested in the DLD: Phosphate Buffered Saline [Ca ++ / Mg ++0.5% F127 (Pluronic F-127, Sigma Aldrich, St. Louis, MO) in phosphate-buffered saline [Ca-free] (Quality biological, Gaithersburg, MD). ++ / Mg ++ 1% bovine serum albumin (BSA) (Affymetrix, Santa Clara, CA) in [free] and an isotonic elutriation buffer (EB) composed of 50% Plasmalyte A (Baxter, Deerfield, IL) and 50% of a mixture containing 1.0% BSA (Affymetrix, Santa Clara, CA), 1.0 mM N-acetyl-cysteine, 2% dextrose, and 0.45% NaCl (all from Sigma-Aldrich, St. Louis, MO). The buffer was prepared fresh daily and sterile filtered through a 0.2 μm filter flask before use in the DLD. Although better DLD performance has been established with the addition of poloxamer (Johnson, et al., Cancer Cell Res. 27:38-58 (2017)), all samples in the expansion group were processed with an isotonic elutriation buffer and were best conditioned with this CAR-T cell manufacturing approach.

[0180] Biological samples. Leukocyte reduction system (LRS) chamber samples from platelet apheresis donations of screened normal donors using the Trima system (Terumo, Tokyo, Japan) were obtained from a local blood bank. Cell counts were performed at the time of collection by the blood bank. Counts were validated in our laboratory using a Beckman Coulter AcT2 Diff2 clinical hematology analyzer and ranged from 76 to 313.3 × 10. 3 WBC / µL and between 0.8 and 4.87 x 10 6Platelets / μL ranged between 0.1 and 0.2. All samples were kept overnight at room temperature on an orbital shaker (Biocotek, China) to mimic overnight transport and then processed the following day (approximately 24 hours later). Each donor sample was processed with both DLD and Ficoll, and for the four donors used for T cell proliferation and immunophenotyping studies, samples were also processed using direct magnetic extraction.

[0181] Ficoll-Hypaque. Peripheral blood mononuclear cells (PBMCs) were obtained by diluting the LRS sample 0.5x in RPMI (Sigma-Aldrich, St. Louis, MO), layering it on top of an equal volume of Ficoll-Hypaque (GE, Pittsburgh, PA) in a 50 mL conical tube, and centrifuging it at 400 x g for 35 minutes in a free-swinging rotor without the brake. After centrifugation, the top layer was discarded, and the interface PBMC fraction was transferred to a new 50 mL tube and brought to a total of 20 mL of RPMI. PBMCs were washed by centrifugation at 400 x g for 10 minutes, the supernatant discarded, and the pellet resuspended in 20 mL of RPMI and washed again at 200 x g for 10 minutes. The supernatant was removed, and the pellet was resuspended in complete medium containing RPMI-1640 + 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO), plus the antibiotics penicillin 100 units / mL and streptomycin 100 μg / mL (Thermo-Fisher, Waltham, MA).

[0182] Cell isolation, counting, and immunofluorescence staining. Before and after isolation using the above method, cell counts of the resulting product were determined using a blood cell analyzer (Beckman-Coulter AcT2 Diff2). Once during culture and after activation, cell counts were determined using a Scepter™ 2.0 manual cell counter (Millipore, Billerica, MA) and by absolute counting using flow cytometry. Cells from the input, product, and waste fractions were then plated onto polylysine-coated slides for 10 minutes and then fixed in 4% p-formaldehyde + 0.5% Triton X-100 in PBS for 15 minutes before washing three times in PBS by centrifugation. Slides were incubated with conjugated primary antibodies CD41-A647 and CD41-FITC (both BioLegend, San Diego, CA) for 60 minutes in the dark, washed three times with PBS, and then mounted in SlowFade mounting medium containing the DNA stain DAPI (Thermo-Fisher, Waltham, MA). Slides were viewed using an Etaluma™ Lumascope 620 inverted fluorescence microscope (Carlsbad, CA). The following fluorochrome-conjugated antibodies (mAbs) were obtained from BioLegend (San Diego, CA): CD25-PE, CD25-APC, CD95-FITC, CD45RA-BV605, CD45RO-PECy7, CD197 / CCR7 PE, CD279-PE, CD28 PE-Cy5, CD45-PerCP, CD3-FITC, CD3-BV421, CD4-AF700, CD8-APC-AF780, CD61-FITC, CD41-FITC, and CD45-Alexa647. Viability of DLD-obtained WBCs and Ficoll-Hypaque-purified PBMCs was determined by trypan blue exclusion.

[0183] Activation and magnetic separation. For T cell stimulation in the expansion group, DLD products, Ficoll products, and LRS products were added to 1 × 10 7The cells were diluted to 0.5 × 10 T cells / mL, then washed and activated by equilibration with anti-CD3 / CD28-conjugated magnetic beads (5.0 μm) (Thermo-Fisher, Waltham, MA) at a bead:cell ratio of 3.2:1 for 60 minutes, after which activated T cells were separated by magnetic depletion for 5 minutes. Unbound cells were removed, and bead-bound cells were further cultured in complete medium (described below). In the direct magnet protocol, 0.5 mL of LRS sample (same donor as processed with DLD or Ficoll) was incubated with immunomagnetic CD3 / CD28 beads for 1 hour. The mixture was then placed on a magnet for 5 minutes to capture T cells. The magnetic bead-bound cells (activated cells) were removed and then cultured in complete medium as described above at 0.5 × 10 6 Diluted to 1000 / mL.

[0184] After 3 days in culture, recombinant human IL-2 (BioLegend, San Diego, CA) was added to the wells at 200 IU / mL. After cell culture for up to 15 days, the beads were removed from the cells and the cells were counted at each time point. To remove the beads, the cells in the wells were resuspended by passing them through a 5 mL pipette 10 times. The cell suspension was then passed through a 1 mL pipette 40 times, followed by vigorous pipetting using a 200 μL tip for 1 minute. The cell suspension was then placed next to a magnet for 5 minutes, and the non-magnetic fraction was transferred to a fresh tube and counted. The number of cells in the culture wells was determined using a Scepter manual cell counter and by flow cytometry.

[0185] Cell culture and cell activation. For each T cell preparation put into cell culture, in addition to the stimulated cells described above, unstimulated cells (control) were added at 0.5 x 10 in complete medium (RPMI + 10% FBS + antibiotics). 6Cells were adjusted to cells / mL, placed in 6-well plates (Corning, NY), and cultured in a humidified incubator at 37°C and 5% CO. To eliminate any disruption during proliferation due to the de-beading behavior required for sampling and reliable counts, especially on day 3, individual wells for each condition, unstimulated, and stimulated with and without IL2, were dedicated to each donor at each time point.

[0186] Flow cytometry. Absolute, no-wash counting by flow cytometry was used for CD3+ cell counts at all time points. Initial counts on day 0 used TruCount tubes (BD Biosciences, San Jose, CA) to accurately determine the number of cells recovered and counted. Subsequent days used 25,000 123-beads (Affymetrix, Santa Clara, CA) indexed in TruCount tubes as an internal control. 100 μL of cell suspension was stained with CD3 FITC-, CD25 PE-, and CD45 PerCP-conjugated antibodies for 30 minutes in the dark, either in TruCount tubes or by adding 25,000 123-beads (Affymetrix, Santa Clara, CA). Cells were then diluted into 250 μL of PBS containing a final DRAQ5™ DNA dye (Thermo-Fisher, Waltham, MA) concentration of 1.0 mM. Stained cells were then fixed overnight with an additional 250 μL of 1.2% p-formaldehyde in PBS prior to acquisition. For absolute count cytometry, a minimum of 25,000 events or 2500 bead events were acquired on a BD FACSCalibur (BD Biosciences, San Jose, CA) using a fluorescence threshold (CD45 PerCP). Phenotypic analysis was also performed at all time points using a 7-color activation / anergy panel consisting of CD3, CD45RA, CD95, CD279, CD25, CD4, and CD8. On day 15, the panel was modified to create a 9-color panel focused on central memory T cells, adding CD45RO PE-Cy7, CD28 PE-Cy5, and substituting CD197 / CCR7 PE for CD279 / PD1 PE.For multicolor staining, 100 μl of cell suspension was stained as described above, resuspended in 750 μL PBS, washed by centrifugation at 400 × g, then resuspended in 250 μL 1.2% p-formaldehyde and fixed overnight, after which 20,000 events were acquired using a forward scatter threshold on a four-laser BD FACSAria II (BD Biosciences, San Jose, CA). All data analysis was performed using Flowlogic software (Inivai, Melbourne, Australia).

[0187] result DLD microchip and Ficoll processing of apheresis products Twelve LRS samples obtained from 12 different normal donors were processed using DLD and Ficoll separation methods. Of the 12 samples received and processed, 11 samples each yielded approximately 148.7 x 10 3 WBC count of 2.52 x 10 cells / µL 6 The mean platelet count was 313.3 x 10 cells / µL (Figures 2A and 2B). 3 WBC count of 4.87 x 10 cells / µL 6 The 12th sample, with a platelet count of 100 / µL, can be seen as a red triangle in the scatter plot (Figure 2A). This sample was well aggregated at the time of processing and rapidly clogged the 20 µm prefilter, thus not fully entering the DLD. Microscopic examination of the input sample showed that this sample was replete with platelet-WBC aggregates ranging in size from 25 to 50 µm, with numerous aggregates on the order of 250 µm in diameter observed (Figures 2C and 2D). Furthermore, both the WBC and platelet counts were 3 standard deviations higher than the mean WBC and platelet counts. Using the quartile method, this sample was classified as a minor outlier; using the Grubbs test for outliers and an alpha level of 0.05, this sample was also classified as an outlier. 20Consequently, this donor was excluded from the study based on extremely high WBC and platelet counts and excessively severe and impaired aggregation.

[0188] A representative image of the input material (LRS product diluted 0.2x) is shown in Figure 2A. Representative micrographs of DLD cell products (Figure 2E) and Ficoll cell products (Figure 2C) from the same input donor revealed significantly lower background platelet levels (green CD41-FITC) in DLD compared with Ficoll. The respective cell products as collected in tubes are also shown (Figure 2G, H). While DLD processing automates the process of removing WBCs from RBCs and platelets, resulting in one tube for product and one tube for waste, Ficoll samples still require additional manual processing by pipetting the PMB layer at the operationally defined interface between the upper plasma layer and the lower Ficoll layer (Figure 2H). In addition, a minimum of two additional centrifugal washes are required to remove the majority of contaminating platelets.

[0189] WBC recovery, as well as RBC and platelet depletion for the 11 samples, are summarized in Table 2. The mean cell recovery of PBMCs from DLD was approximately 80%, 17% higher than Ficoll (63%), and when counting CD3 cell numbers in both DLD and magnetic samples, the DLD product was 36% higher than direct magnet (44%). Mean platelet depletion with DLD (83%) was superior to both Ficoll (56.5%) and direct magnet (77%). Mean red blood cell depletion in these 24-hour samples was 97% for both DLD and Ficoll and 94% for the direct magnet approach. The mean viability of cells retrieved by DLD was 96% compared to 97% for Ficoll.

[0190] The average total time to process an equivalent aliquot of a single sample in a 50 mL conical tube using Ficoll technology took approximately 90 minutes, requiring approximately 30 minutes of hands-on time by skilled personnel. Timed runs using our single microchip layer breadboard system were much shorter, taking 50 minutes and requiring 25 minutes of hands-on time, with approximately 20 minutes solely due to assembly of the fluidics components, due to the prototype nature of the device, which would not otherwise require intervention.

[0191] Cell growth and characterization After DLD or Ficoll enrichment, cells were activated and separated using CD3 / CD28 magnetic beads for 60 minutes, using a target of 3.2 beads per CD3+ cell, and then counted before plating. Due to limited access to the flow cytometer and concerns about potential bead interference in the product cell count, we estimated T cell counts by counting both the input and non-magnetic fractions and obtaining the number of T cells bound to the magnet by subtraction, assuming a 90% efficiency of magnetic separation (based on the manufacturer's reported efficiency). Accurate T cell counts were determined after plating into culture by flow cytometry and by Coulter counter absolute counts × % CD3+ cells; these counts established that the initial magnetic CD3+ cell depletion process was only 44% efficient (Table 2). This means that the initial calculation relating to the target of 3.2 beads per CD3+ cell was in fact 2.3 on average for both the DLD and Ficoll fractions (fewer beads per T cell than the target number), with a 5:1 ratio in the direct magnet fraction (significantly more beads per T cell than the target number), potentially resulting in an even higher fold expansion in the direct magnet fraction compared to both the DLD and Ficoll arms.

[0192] Flow cytometric characterization of cultures was performed at each time point to assess the consistency of cell activation. Changes in CD25 expression of CD3+ cells, as measured on day 8, for Ficoll, DLD, and direct magnet (Figure 3). IL-2 receptor-positive (CD25) CD3 cells are shown in blue (CD4+ plot) and red (CD8+ plot). DLD-prepared cells show more consistent phenotypic expression of CD25 (an indicator of response to CD3 / CD28 stimulation) across four donors compared with both Ficoll-prepared and direct magnet preparations. DLD-prepared CD3+ cells produced an average 73% response to costimulation compared with Ficoll at 51% (both stimulated at 2.3 beads / cell), whereas the direct magnet fraction, stimulated at a higher 5:1 ratio, produced only a 54% response.

[0193] Unstimulated controls for Ficoll and DLD showed a striking difference, with DLD-prepared cells remaining CD25 negative compared to Ficoll (Figure 9). Interestingly, donor 37 in the direct magnetic fraction did not respond by day 8, but proliferated at later time points (also shown in Figure 5A), indicating a potential delay in the response of some samples to the direct magnetic approach.

[0194] In addition to assessing CD25 expression, conversion to a memory cell phenotype was tracked using the percentage of CD3+ cells that were CD45RA- and CD25+. On average, 58% of CD3+ cells were CD45RA- CD25+ in the DLD arm compared to 36% in the Ficoll arm and 439% in the direct magnet arm (Figure 4). These results indicate that a higher percentage of cultured cells were responsive to costimulation when generated by DLD compared to cells treated with Ficoll and direct magnet. Furthermore, the percent of CD3+ cells that were CD25- CD45RA- was lowest in the DLD fraction at 12% compared to 33% and 29% for Ficoll and direct magnet, respectively, indicating a more complete conversion to a CD25+ CD45RA- population for DLD CD3+ cells. The standard deviation of the CD45RA- CD25+ population at day 8 for DLD was 10.1% compared to 24.8% for Ficoll and 53.4% ​​for direct magnet.

[0195] The fold growth of individual cultures was determined at days 3, 8, and 15; the data are shown in Figure 5A. The plot shows the growth of each donor sample across methods. While the direct magnet approach appears to show higher growth, the counts were likely significantly affected by the different bead-to-cell ratios (and corresponding differences in plating density). Regardless, four donors show significant viability in fold growth. In addition, the day 15 culture for donor #21 in the direct magnet arm was contaminated and had to be discarded, despite the presence of antibiotics. It is not possible to know whether the day 8 growth data for donor #21 was affected by the contamination.

[0196] The comparison between Ficoll and DLD was both relevant and much more straightforward: the cells were plated at the same density and stimulated with the same bead:cell ratio. While the average fold expansion of DLD cells was not significantly higher than that of Ficoll cells, the consistency of expansion across the set of four donors and across all days examined is remarkable. Furthermore, the percentage of cells in culture with a central memory phenotype averaged 74% for the DLD arm, compared with 47% and 48% for the Ficoll and direct magnet arms, respectively. Multiplying the fold expansion in Figure 5A by the percent yield (Table 1) and percent memory (Figure 5B) shows that, despite the suboptimal comparison in bead:cell ratio, on average, twice as many memory cells were produced from the DLD arm compared with either the Ficoll or direct magnet arm.

[0197] Figure 6 shows the phenotypic approach to identifying memory cells used in this study, which was designed to eliminate any issues with shedding antigens such as CD62L (Mahnke, et al., Eur. J. of Immunol. 43:2797-2809 (2013)). Central memory cells were sequentially gated and then backgated to demonstrate that CD3+ T cells were positive for CD45R0+, CD95+, CD28+, and CD197 / CCR7+ relative to all other CD3+ cells in the culture. Using the conversion metric of any >50% of the culture being of the central memory phenotype, the DLD arm showed that 100% (4 / 4) donors achieved central memory conversion, with an average of 74% of cells being of the memory phenotype, with a coefficient of variation across donors of 13%. In contrast, the Ficoll arm demonstrated 50% (2 / 4) conversion with an average of 47% memory cells and a coefficient of variation of 29%. The direct magnet arm achieved 33% (1 / 3) conversion with an average of 48% memory cells and an associated coefficient of variation of 79%.

[0198] [Table 2]

[0199] Example 2: Platelet-added Back experiment principle Previously, WBCs derived from DLD isolation and purification were found to be healthy and differentiate into their Tcm (T central memory) phenotype in response to activation with CD3 / CD28 antibodies (Campos-Gonzalez, et al., SLAS, January 23, 2018, published online at doi.org / 10.1177 / 2472630317751214). Furthermore, in the presence of IL-2, Tcm cells expand and proliferate in a manner similar to cells derived from other methods, such as Ficoll.

[0200] A key feature of DLD cell purification is the thorough removal of red blood cells and platelets to provide a highly purified white blood cell (WBC) product. In comparison, Ficoll-derived white blood cells (PBMCs) exhibit higher red blood cell and platelet contamination, depending on the quality of the sample. On average, platelet "contamination" in Ficoll-derived cells is 44% with a variability range of approximately 22%, while DLD cells exhibit only 17% platelet contamination with a variability of + / - 12%.

[0201] Because of the marked difference in platelet depletion in DLD-treated apheresis blood compared with Ficoll-separated apheresis blood, an investigation was conducted into whether the addition of autologous platelets to DLD-purified leukocytes affects proliferation and Tcm production over time.

[0202] Experiment details Two different leukocyte reduction system apheresis ("LRS-apheresis") samples were collected from a local blood bank either as a control or in 2.0 mM EDTA. All four samples were processed identically in parallel using two different methods: DLD processing and Ficoll gradient centrifugation. The original platelet:WBC ratio provided by the blood bank was annotated and confirmed by Coulter counter measurement.

[0203] 3.0 ml of each LRS blood was processed by DLD according to our previously described protocol by diluting the blood 0.2X with 1.0% BSA / 5.0 mM EDTA in PBS. Samples were run in 1.0% BSA / PBS buffer under standard pressure and conditions using individual DLD-14 lane tips for each sample. Product and waste were collected, and cellularity was measured using a Coulter Counter.

[0204] 3.0 ml of each LRS blood product from two different donors and conditions (collected in 2.0 mM EDTA or control) was diluted 1:1 with 3.0 ml of phosphate-buffered saline (minus calcium and magnesium) and layered onto 6.0 ml of Ficoll-Paque in a 50 ml conical tube. PBMC peripheral mononuclear cells were obtained by centrifugation at 400 x g for 35 minutes without braking. The upper layer, or plasma-rich fraction, was removed, transferred to another tube, and diluted 1:1 with PBS / 1.0% BSA. PBMCs were washed with excess PBS by centrifugation once at 400 x g for 10 minutes and again at 200 x g for 10 minutes. Both supernatants were transferred to new 50 ml conical tubes and diluted 1:1 with PBS / 1.0% BSA. The diluted plasma-rich fraction and the two supernatants were centrifuged at 1,200 x g for 15 minutes. The supernatant was discarded, and the pellet was resuspended in PBS / 1.0% BSA, mixed, and centrifuged again at 1,200 x g for 15 minutes. The supernatant was discarded, and the pellet or platelet fraction was resuspended in 1.0 ml of PBS / 1.0% BSA by gentle pipetting. Platelets were counted using a Coulter counter. The corresponding platelets were added back to DLD-derived WBCs at the desired ratio (0, 0.1, 0.5, and 1.0x the original platelet count) and incubated for 1 hour before activation with CD3 / CD28 magnetic beads (Thermo Fisher).

[0205] After activation, cells were placed in complete RPMI medium + 10% FBS + antibiotics and cultured for a set period in a humidified incubator at 37°C and 5% CO2. Cell aliquots were analyzed by multicolor flow cytometry on days 3, 7, and 14 using the antibody combinations shown in the figure. Cell culture aliquots were obtained on different days, and the cells were de-beaded as described above before preparation for flow cytometry. Cell proliferation was also measured using a Scepter manual cell counter.

[0206] We next compared the differences between Ficoll-derived cells and those obtained from DLD treatment under control conditions and when platelets were back-added to DLD cells at different ratios. The parameters we used were cell counts at different time points and Tcm cell counts according to phenotype by flow cytometry.

[0207] The following scheme describes the overall experimental design followed during this experiment in steps proceeding from top to bottom. [ka]

[0208] Results and Conclusions Leukocytes obtained using DLD consistently displayed fewer platelets than leukocytes obtained using Ficoll (see Figures 19-21). Results also demonstrate clearly superior proliferation of DLD-derived T cells compared to their Ficoll-derived counterparts (Figure 22). Furthermore, back-addition of platelets to DLD-isolated cells reduced their ability to expand to the same level as platelet-free DLD cells (Figure 22). These results support the hypothesis that more efficient platelet reduction during DLD processing of blood products produces leukocytes that are more responsive to CD3 / CD28 activation and IL-2 expansion.

[0209] References

[0210] [Table 3]

[0211] [Table 4]

[0212] [Table 5]

[0213] All references cited herein are incorporated by reference in their entirety. Now that the invention has been fully described, it will be understood by those skilled in the art that the invention can be practiced within a wide and equivalent range of conditions, parameters, and the like, without affecting the spirit or scope of the invention or any embodiment thereof.

Claims

1. A method for producing chimeric antigen receptor (CAR) T cells, comprising the steps of: a) i) at least one channel extending from a sample inlet to one or more fluid outlets, said channel being bounded by a first wall and a second wall opposite said first wall; ii) an array of obstacles arranged in rows in the channel, with each successive row of obstacles being laterally shifted relative to the previous row, the obstacles being arranged in such a manner that when a crude fluid composition comprising T cells is applied to an inlet of the device and fluidly passes through the channel, the T cells in the crude fluid composition flow to one or more collection outlets where an enriched product is collected, and cells or particles in the crude fluid composition that are a different size than the T cells flow to another waste outlet that is separate from the collection outlets. performing deterministic lateral displacement (DLD) on the crude fluid composition in the absence of centrifugation or elutriation using a microfluidic device comprising: the crude fluid composition comprising T cells is an apheresis sample; a reduced ratio of platelets to white blood cells in the apheresis sample; a product is obtained in which the total number of platelets in the product is at least 90% lower than in the apheresis sample; Process; b) either before or after performing the deterministic lateral displacement (DLD), binding the T cells to one or more carriers to facilitate DLD separation; for the apheresis sample, not more than four hours have elapsed from the time apheresis is completed until the T cells are allowed to bind to the carrier; c) genetically engineering T cells in the enriched product obtained at the collection outlet of the microfluidic device in step a) to produce chimeric antigen receptors (CARs) on their surface.

2. A method for producing the chimeric antigen receptor (CAR) T cell of claim 1, further comprising the steps of: d) optionally, culturing the chimeric antigen receptor (CAR) T cells obtained in step c) to expand their numbers; and e) transferring the chimeric antigen receptor (CAR) T cells obtained in step c) or d) into a pharmaceutical composition for administration to a patient.

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