Combined purification and concentration by deterministic lateral displacement with recirculation product

Abstract

To provide a method of separating target cells or target particles of a predetermined size from a sample containing cells or particles of the predetermined size.SOLUTION: By recirculating product streams back onto microfluidic devices and applying them in the place of wash fluid, the final concentration of products is controlled. Initially, both sample and wash fluid are fed onto a device, and deterministic lateral displacement is performed on microfluidic devices while recirculating products, thereby simultaneously purifying and concentrating cells or other particles.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 670,839, filed March 13, 2018.

[0002] FIELD OF THEINVENTION The present invention relates to a method for simultaneously purifying and concentrating cells or other particles by performing deterministic lateral displacement on a microfluidic device while recirculating the product. [Background technology]

[0003] Deterministic lateral displacement (DLD) typically uses two co-flowing liquids: one containing the particles (sample) and one a wash / collection fluid (the "run" or "wash" fluid). During DLD, particles above a critical diameter for the array are deflected into the wash fluid, while particles below the critical diameter follow the flow direction of the sample stream. The concentration of particles in the final product (collected) stream depends on the input concentration of particles in the sample stream, the ratio of sample to wash fluid, and the percentage of the total amount collected as product. Summary of the Invention [Problem to be solved by the invention]

[0004] It is possible to obtain high concentration factors using modern DLD procedures, but this may increase the risk of contamination (particles smaller than the critical diameter being collected in the product stream) or may require very long DLD arrays with extra redundancy to efficiently hit the narrow collection width. It is also difficult to adjust the concentration of the product generated by DLD to achieve consistency from run to run or to standardize results obtained across multiple donor samples with different input cell numbers. Samples can always be diluted, but concentrating is much more difficult. Samples with different input concentrations cannot be standardized to a single output concentration without pre-diluting each sample differently.

[0005] Another problem with controlling product concentration using current procedures is that relatively large volumes of wash solutions are usually required. Limiting these volumes would result in higher product concentrations, but there always needs to be enough wash solution to process the entire sample volume. [Means for solving the problem]

[0006] General description The invention is based on the concept that it is possible to control the final concentration of DLD products by recirculating product streams to a microfluidic device and applying them instead of wash solutions. First, both sample and wash solutions (also referred to more specifically herein as wash buffers or reagents) are fed into the device. Then, at a selected point in the run, the inlet carrying the wash solution is sealed and the product from the outlet of the device is fed through the inlet at the wash solution location. This can be achieved using standard valves. The flow of recycled product is propelled using, for example, a pressurized vessel, a peristaltic pump, or a syringe pump.

[0007] Two processes occur while the collected product (P1) is recirculated back into the device. First, the sample flowing adjacent to the recirculated product is fractionated based on size such that larger particles are deflected into the "wash stream" (here P1) and directed to the product outlet. Second, the product concentration increases because the particle count in P1 is increasing. A typical set of ratios is 60% sample and 40% wash at the inlet, 20% product and 80% waste at the outlet. In this situation, if P1 is recirculated as wash, the particles in the wash stream will be concentrated two-fold (40% / 20%) since they are still deflected into the array and into the product collection stream. The collected product (P2) therefore contains both P1 cells, concentrated two-fold, in addition to the cells from the sample processed during that period.

[0008] One advantage of this method is that the final concentration of the output product depends on the number of recirculations and the initial wash volume used. It can therefore be easily adjusted. For example, for a very dilute donor sample, the operator can choose to run only a small amount of the sample through the wash solution and recirculate the product several times to process the remaining sample. A higher concentration factor can be obtained as a result. This allows the operator (or the instrumentation) to provide any output concentration, regardless of the input concentration. It also means that the instrument can be optimized to provide a fixed product concentration required for a downstream assay step, regardless of the input donor concentration. The user simply inputs the initial donor concentration and selects the desired output concentration. Using this information, the instrument adjusts the number of recirculations to achieve the selected value.

[0009] Another advantage of this method is that enrichment is achieved without the need for a separate enrichment device (performed in-line or as a second pass). Enrichment occurs in the same DLD used for cell isolation, and the time required to process the donor sample remains the same as if the sample had only been run on wash buffer.

[0010] The volume of wash solution required is also significantly reduced. Instead of needing enough wash solution to process the entire sample, the wash volume is reduced by an amount corresponding to the volume of recirculated product. For example, if recirculation is initiated after about 50% of the sample has been processed, the volume of wash solution is reduced by about half.

[0011] Embodiment In its first aspect, the present invention relates to a method for separating target cells or particles of a predetermined size from a sample containing cells or particles below the predetermined size. The method comprises applying a sample and a washing solution to a microfluidic device at separate inlets. The washing solution may be water or an aqueous buffer, and may optionally contain a reagent that chemically reacts with the sample components, or an antibody, carrier or activator that specifically interacts with the target cells or particles. As initially applied to the device, i.e., before product recirculation, the washing solution should be devoid of target cells or particles and free of cells or particles below the predetermined size.

[0012] The microfluidic device is preferably configured for deterministic lateral displacement (DLD), a process that involves flowing a sample through an array of specially designed microposts that are inclined at a small 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)). Thus, the microfluidic device has an array of obstacles arranged in rows, with each subsequent row of obstacles being shifted laterally relative to the previous row. The obstacles should be positioned to deflect target cells or target particles into a first outlet (where they can be collected as target cell or target particle product) and direct cells or particles below a predetermined size into a second outlet (where they can be collected or discarded as waste).

[0013] DLD is performed by flowing a sample and a wash solution through the microfluidic device. During this process, at least a portion of the target cell or target particle product is recirculated from a first outlet to replace all or at least a portion of the wash solution applied to the inlet of the device. During or at the end of the procedure, a final product containing the target cells or target particles is obtained from the first outlet.

[0014] To facilitate recirculation, a first outlet of the microfluidic device may include or be connected to a valve that can be used to divert the target cell or target particle product to a conduit that recirculates the target cell or target particle product to the inlet of the microfluidic device. Similarly, the microfluidic device may have an inlet that includes or is connected to a valve that can be used to switch the feed entering the device through the inlet from a conduit that provides a wash fluid to a conduit that provides the target cell or target particle product.

[0015] In some embodiments, the target cells or target particles recirculated to the microfluidic plate react or bind with a carrier, antibody, fluorescent tag, activator or compound before, during or after reapplication to the microfluidic device. In a preferred embodiment, target cells, e.g., white blood cells, or more specifically, T cells, are the target cells and are specifically bound by the carrier, antibody or activator. When used in this context, the word "specificity" means that for each non-target cell bound to the sample, at least 100 (preferably at least 1000) target cells are bound by the carrier. Such binding can be used to promote cell division (in the case of activators) or to facilitate assay or further purification of the cells (in the case of antibodies).

[0016] Carriers can be used to modify the behavior of cells during the DLD procedure. For example, cells that exit the device at one location can be bound to a carrier to form a complex that exits the same device at a different location. Thus, size-based separations that would not be possible otherwise can be achieved. In this case, the binding of the carrier must be done "to facilitate DLD separation". This term, as used in this context, means that the method must ultimately result in binding that exhibits specificity for a particular target cell type, providing an increase in the size of the complex compared to unbound cells of at least 2 μm (or, if expressed as a percentage, at least 20, 50, 100, 200, 500, or 1000%). If the therapeutic or other application requires free target cells, it allows the target cells to be released from the complex by chemical or enzymatic cleavage, chemical lysis, digestion, for example, by physical shear using a pipette, to generate shear stress, or by other means, for competition with other binding agents. Carriers can also be bound to complement DLD separation.

[0017] To determine the concentration, a cell or particle count can be made from the target cell or target particle product. Based on this, it can be decided whether to continue recycling the product. Depending on the objectives of the party performing the process, the cells or particles can be concentrated at least three-fold or at least ten-fold compared to their concentration in the sample. In the case of cells, it is preferred that recirculation in combination with microfluidic processing is the only method used for concentration during this process. This is especially true for cells that are genetically engineered and / or used therapeutically.

[0018] The methods described herein may be used to separate and enrich cells from preparations derived from bioreactors, flasks, culture plates, culture bags, biological fluids or extracts, and tissues. Particularly preferred are leukocytes (preferably T cells, most preferably CAR-T cells) or stem cells of a certain size. Samples containing these cells may be blood, apheresis, leukapheresis, or leukapheresis compositions obtained by leukopheresis, or leukoreduction, and typically contain platelets or red blood cells smaller than a certain size. Cells may also be genetically engineered by any method used in the art, including electrical, chemical, or nanoparticle transfection or viral transduction.

[0019] In some cases, the recirculated white blood cells or stem cells may be bound to carriers, antibodies, or activators in a manner that enhances or complements the DLD separation. Binding should occur before, during, or after the cells have passed through the microfluidic device at least once before being recirculated into the microfluidic device. If the cells are to be used therapeutically, the process of preparing the cells should not include a centrifugation step, and recirculation should occur until or after a sufficient concentration of cells is obtained to be administered therapeutically to the patient. Similarly, the addition of virus or genetic material to the concentration loop before release may be possible. Furthermore, if a sample is obtained from a patient for isolation of cells to be used therapeutically, it is preferred that no more than four hours elapse between the completion of obtaining the sample and the completion of processing of the cells by DLD.

[0020] It will be readily apparent to those skilled in the art that, with some modifications, the procedures described herein can be adapted to separate target cells or particles from larger sized contaminants. Apart from being directed to the above methods, the present invention includes cells (including white blood cells, stem cells and CAR-T cells) or particles produced by the methods.

[0021] Integrated method for producing purified genetically engineered target cells In a preferred embodiment, the above procedure may be used as part of a method for producing genetically engineered target cells. In particular, the present invention includes a method for producing purified genetically engineered target cells by: a) obtaining a sample containing target cells of a predetermined size and one or more contaminating cells or particles smaller than the predetermined size; b) applying the sample to a microfluidic device at a first inlet and a washing fluid at a second inlet, where the microfluidic device includes an array of obstacles arranged to differentially deflect a stream of target cells to a first outlet where they are collected as a target cell product and direct contaminating cells or particles smaller than the predetermined size to a second outlet where they can be collected or discarded as waste; c) flowing the sample and the washing fluid through the device, where the concentration of the target cells at the first outlet is determined, and at least a portion of the target cells are recirculated from the outlet to replace all or at least a portion of the washing fluid applied to the inlet of the device; The circulation is continued or repeated until the desired product cell concentration, PC, is reached; d) once the PC is reached, direct the flow of target cells to be collected from the first outlet or to a site where they are transformed or transfected to form genetically engineered target cells; e) in step d), if the target cells are transformed or transfected, form the genetically engineered target cells, preferably by flowing the genetically engineered target cells to a site or device where this purification takes place, and purify the genetically engineered target cells from viruses, reagents or other materials used during the genetic manipulation to form purified genetically engineered target cells; f) collect the purified genetically engineered target cells produced in step e) or flow the purified genetically engineered target cells produced in step e) to a site where they are further processed before collection. In a preferred embodiment, all steps from application of the cells to the microfluidic device in step b) to collection of the cells are carried out as a single continuous process. It is also preferred that no other means are used to concentrate the target cells during this process other than the microfluidic separation by product recirculation procedure described above.

[0022] The term "cells of a predetermined size" as used herein refers to the size of the target cells selected for separation. The size can be either experimentally determined or known from the art. For example, the size of human T cells is known in the art as are the sizes of platelets and red blood cells. Thus, a separation could be performed in which T cells are the "target cells of a predetermined size" and platelets and red blood cells are the contaminant cells to be separated. The term "predetermined size" can also be used in the context of designing a microfluidic device to perform a separation. For example, the term "critical size" or "predetermined size" of a particle passing through an array of obstacles can be used to describe the size limit of a particle that can be tracked through a laminar flow of a fluid. Larger particles can, for example, be "bumped" out of the flow channel of the fluid, while particles having a size smaller than the critical size (or predetermined size) are not necessarily displaced.

[0023] Many types of cells can be used as target cells, including therapeutically active cells such as lymphocytes and stem cells. T lymphocytes are particularly preferred, as they can be genetically engineered to produce chimeric antigen receptors (CARs) on their surface, forming therapeutically valuable chimeric antigen receptor T cells.

[0024] The most preferred process for separating target cells from cells or particles smaller than a given size, i.e., steps b) and c), is by DLD. DLD may also be used in step e) to separate the genetically engineered target cells from reagents, viruses or other materials used for transformation or transfection of the target cells. If the genetically engineered cells are grown in cell culture (usually performed in the production of CAR T cells), the materials used for transformation or transfection of the target cells may be removed in step e) while simultaneously transferring the cells to a growth medium. The transferred cells can be collected and cultured. Alternatively, they can be directly flowed to the place where the culture is performed.

[0025] The wash fluid used in the above methods may be water or an aqueous buffer and may contain reagents that chemically react with cells, particles or other components in the wash fluid, or may contain antibodies, carriers or activators that specifically interact with target cells.

[0026] Generally, the direction of target cells exiting the device is by an outlet that includes or is connected to a valve. Depending on whether the valve is open or closed, the valve directs the target cells: a) from the outlet to a conduit that recirculates the cells to an inlet of the microfluidic device; or b) from the outlet to a site where the target cells are transformed or transfected to form genetically engineered target cells. Preferably, the valve electronically responds to a concentration of target cells below a desired concentration, PC, by directing the target cells to a conduit that recirculates the cells to an inlet on the microfluidic device, and electronically responds to a concentration of target cells above PC by directing the target cells to a site where they are collected or where they are transformed or transfected to form genetically engineered target cells.

[0027] Target cells that are recirculated to the microfluidic device inlet may react or bind with carriers, antibodies, fluorescent tags, activators or compounds before, during, or after reapplication to the microfluidic device. For example, white blood cells or stem cells may bind to carriers, antibodies, or activators in a manner that enhances or complements DLD separation.

[0028] The recirculation of the target cells may continue until the target cells are concentrated at least 3-fold, preferably at least 5-fold, and even more preferably at least 10-fold relative to the concentration in the sample. It is also preferred that the target cells are not centrifuged or frozen between taking the sample in step a) and taking the cells. Furthermore, it is preferred that the sample is taken from a patient who will ultimately be treated with the generated cells, and that the process from taking the sample to step f) is completed within 10 hours (preferably within 5 hours).

[0029] The most preferred sample is blood or a composition obtained by processing blood, for example by apheresis or leukapheresis. This type of sample may be used, for example, to separate white blood cells from platelets and / or red blood cells.

[0030] The present invention encompasses not only the above methods, but also purified genetically engineered target cells produced by the methods, therapeutic compositions based on these cells, and methods in which the therapeutic compositions are used to treat patients. [Brief description of the drawings]

[0031] [Figure 1A-1C] Figures 1A-1C illustrate the various modes of operation of one type of DLD device. These include i) separation (Figure 1A), ii) buffer exchange (Figure 1B), and iii) concentration (Figure 1C). In each mode, essentially all particles above a critical diameter are deflected from the entry point towards the array, resulting in size selection, buffer exchange, or concentration as a function of the geometry of the device. In all cases, particles below the critical diameter pass directly through the device under laminar flow conditions and then exit the device at the exit. DLD devices have been described in the literature, along with methods for making and using the devices (see, e.g., U.S. Patent Application Publication No. 2016 / 0139012, U.S. Patent Application Publication No. 2017 / 0333900; U.S. Patent Application Publication No. 2016 / 0047735; U.S. Patent Application Publication No. 2017 / 0209864; U.S. Patent Application Publication No. 2017 / 0248508; and U.S. Patent Application Publication No. 2019 / 0071639, the entireties of which are incorporated by reference). [Figure 1D] Figure ID shows a 14-lane DLD design used in separation mode. The overall length of the illustrated array and microchannels is 75 mm, width is 40 mm, and individual lane width is 1.8 mm. [Figure 1E] FIG. 1E is a close-up view of the plastic diamond post array and the integrated collection port at the outlet. [Figure 1F]FIG. 1E is a close-up view of the plastic diamond post array and the integrated collection port at the outlet. [Figure 1G] FIG. 1G shows a photograph of a leukapheresis product being processed using the device. [Diagram 2] FIG. 2 is a schematic diagram showing how individual chips today are designed to be stackable in layers to deliver the throughput required for a particular application. [Diagram 3] The leftmost diagram in Figure 3 shows the movement of cells during DLD. Buffer and sample are applied to the device at separate inlets. As the sample progresses towards the outlet, cells with sizes larger than the critical size of the array move from the sample stream (outside hatched part of the channel) to the buffer stream (center of the channel, stippled part) and finally exit the product outlet. Panels 1-3 show the various steps of the DLD procedure where product recirculation takes place. In panel 1, sample (white in the upper sample reservoir) and buffer (stippled part in the upper buffer reservoir) are applied to the microfluidic device at separate inlets. The product, which contains cells larger than the critical size of the array, is collected from the product outlet (stippled part in the lower product reservoir) and waste is collected from the waste outlet (clear part in the lower waste reservoir). Note that the valve from the product reservoir to the buffer inlet is closed, whereas the valve from the buffer reservoir to the buffer inlet is open. In panel 2, the valve from the product reservoir to the buffer inlet is open and the valve from the buffer reservoir to the buffer inlet is closed. As a result, the product is recycled back to the microfluidic device. In panel 3, the process is nearly complete. The total amount of waste increases and the total amount of product decreases. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Illustrative Table The expected calculated results from the procedure are shown in the table below. The numbers are based on a selected set of input and output conditions for a 14-lane microfluidic device. It should be recognized that microfluidic devices with different layouts (and inlet / outlet ratios) will have numbers on a different scale.

[0033] [Table 1]

[0034] [Table 2]

[0035] [Table 3]

[0036] [Table 4]

[0037] [Table 5]

[0038] 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 "leukoapheresis" (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.

[0039] 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. Methods for making and using CAR T cells are well known in the art. Procedures are described, for example, in U.S. Pat. No. 9,629,877; U.S. Pat. No. 9,328,156; U.S. Pat. No. 8,906,682; U.S. Patent Application Publication No. 2017 / 0224789; U.S. Patent Application Publication No. 2017 / 0166866; U.S. Patent Application Publication No. 2017 / 0137515; U.S. Patent Application Publication No. 2016 / 0361360; U.S. Patent Application Publication No. 2016 / 0081314; U.S. Patent Application Publication No. 2015 / 0299317 and U.S. Patent Application Publication No. 2015 / 0024482, which are incorporated herein by reference.

[0040] 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 blood and solid tumor cancers, autoimmune diseases, and infectious diseases.

[0041] Carrier: As used herein, the term "carrier" refers to an agent, e.g., a bead or particle, made of either biological or synthetic materials, that is added to a preparation for the purpose of binding directly or indirectly (i.e., via one or more intermediary cells, particles or compounds). Carriers can be made of a variety of materials, such as DEAE-dextran, glass, polystyrene plastic, acrylamide, collagen, alginate, etc., and are typically 1-1000 μm in size. They may be coated or uncoated, and carriers with surfaces that are modified to include affinity agents (e.g., antibodies, activators, haptens, aptamers, particles or other compounds) that recognize antigens or other molecules on the surface of cells may be magnetized, which may provide an additional purification means to complement DLD, and they may include particles (e.g., Janus or strawberry-like particles) that impart secondary properties to cells or cell complexes that are not size-related.

[0042] For example, particles may provide 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. Particles may also cause metabolic changes in cells, activate cells, or promote cell division.

[0043] Carriers that bind "in a manner that promotes DLD separation": This term refers to carriers and methods of binding carriers that affect how cells, proteins, or particles behave during DLD, depending on the context. 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) it must produce a complex that increases the size of the complex compared to the unbound cell, protein, or particle. When binding to a target cell, an increase of at least 2 μm is required (or, when expressed as a percentage, at least 20, 50, 100, 200, 500, or 1000%). If a therapeutic or other application requires that the target cells, proteins, or other particles be released from the complex to fulfill their intended use, the term "in a manner that promotes DLD separation" also requires that the complex allows for such release. Chemical or enzymatic cleavage, chemical lysis, digestion, and the liberated target cells, proteins, or other particles must remain active, whether by competition with other binding agents or by physical shear (e.g., using a pipette to generate shear stress). For example, the therapeutic cells after release from the complex must still maintain the biological activity that makes them therapeutically useful.

[0044] Carriers may be bound "in a manner that complements DLD separation": this term refers to carriers and methods of binding carriers that change the chemical, electrochemical, or magnetic properties of a cell or cell complex, or change one or more biological activities of the cell, regardless of whether it increases the size sufficiently to promote DLD separation. Carriers that complement DLD separation also do not necessarily bind specifically to the target cells, i.e., they may need to be combined with other agents that make them specific, or they may simply be added to the cell preparation to allow non-specific binding. The terms "in a manner that complements DLD separation" and "in a manner that promotes DLD separation" are not mutually exclusive. Binding may complement DLD separation and promote DLD separation. For example, a polysaccharide carrier may have an activator on its surface that increases the rate of cell proliferation, and binding of one or more of these carriers may also promote DLD separation. Alternatively, binding may only promote DLD separation or complement DLD separation.

[0045] Target Cell: As used herein, a "target cell" is a cell that the various procedures described herein require or are designed to purify, collect, manipulate, etc. What a particular cell is will depend on the context in which the term is used. For example, if the purpose of the procedure is to isolate a particular type of stem cell, then that cell will be the target cell for the procedure.

[0046] Isolation, purification: Unless otherwise specified, these terms, as used herein, are synonymous and refer to the enrichment of a desired product relative to undesired materials. These terms do not necessarily mean that the product is completely separated or completely pure. For example, if a starting sample has target cells that make up 2% of the cells in the sample, and a procedure is performed that results in a composition in which the target cells are 60% of the cells present, the procedure will have been successful in isolating or purifying the target cells.

[0047] Deterministic Lateral Displacement: As used herein, the term "deterministic lateral displacement" or "DLD" refers to a process in which particles are deterministically deflected on a path through an array based on their size, which is related to some array parameters. The process is generally described herein in terms of continuous flow (DC conditions; i.e., bulk fluid flow in only one direction). However, DLD also works with oscillatory flow (AC conditions; i.e., bulk fluid flow that alternates between two directions).

[0048] Critical Size: The "critical size" or "predetermined size" of a particle passing through an obstacle array represents the size limit of a particle that can track through the laminar flow of a fluid. Particles larger than the critical size may be "dropped" out of the fluid flow channel, while particles smaller than the critical size (or predetermined size) are not necessarily so displaced. If the fluid flow profile through the gap is symmetrical with respect to a plane that bisects the gap in the direction of bulk fluid flow, the critical size may be the same on both sides of the gap; however, if the profile is asymmetrical, the critical size on both sides of the gap may be different.

[0049] Fluid Flow: As used herein in connection with DLDs, the terms "fluid flow" and "bulk fluid flow" refer to the macroscopic movement of fluid in a general direction across an array of obstacles. These terms do not take into account temporary displacements of the fluid flow as the fluid moves around the obstacles so that the fluid continues to move in all directions.

[0050] 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.

[0051] Array Orientation: In a bump array device, the "array orientation" is the direction defined by the arrangement of successive rows of obstacles in the array. A particle is "bumped" (dropped) in the bump array if, when passing through a gap and encountering a downstream obstacle, the particle's overall trajectory follows the array orientation of the bump array (i.e., it moves at an oblique angle ε relative to the bulk fluid flow). Under such circumstances, if the particle's overall trajectory follows the direction of the bulk fluid flow, the particle is not dropped.

[0052] Detailed Description of the Invention The present invention relates to the use of DLD in preparing cells of therapeutic value. The following text provides general guidance regarding the methods disclosed herein, as well as information that may assist in the manufacture and use of devices involved in carrying out those methods.

[0053] I. Microfluidic Plate Design Cells, particularly cells in compositions prepared by apheresis or leukapheresis, and particles can be separated by performing DLD using a microfluidic device that includes a channel through which fluid flows from one or more inlets at or near one end of the device to an outlet at or near or at the opposite end. The basic principles of size-based microfluidic separation and the design of obstacle arrays for separating cells are provided in other applications (see U.S. Patent Application Publication No. 2014 / 0342375; U.S. Patent Application Publication No. 2016 / 0139012; U.S. Patent No. 7,318,902 and U.S. Patent No. 7,150,812, which are incorporated herein by reference). These are also summarized in the following sections.

[0054] During DLD, a fluid sample containing particles or cells is introduced into the device at an inlet and carried along with the fluid flowing through the device to an outlet. As cells in the sample pass through the device, they encounter struts or other obstacles that are arranged in rows and form gaps or pores through which the cells must pass. Each successive row of obstacles is displaced relative to the previous row to form an array direction that is different from the direction of fluid flow in the flow channel. The "tilt angle" defined by these two directions, along with the width of the gap between the obstacles, the shape of the obstacles, and the direction of the obstacles that form the gap, are the primary factors that determine the "critical size" of the array. Cells with a size larger than the critical size will migrate in the direction of the array rather than in the direction of bulk fluid flow, and particles with a size smaller than the critical size will migrate in the direction of bulk fluid flow. In devices used for apheresis or leukoapheresis derived compositions, array characteristics can be selected that result in leukocytes being diverted in the direction of the array while red blood cells and platelets continue in the direction of bulk fluid flow. To separate a selected type of white blood cell from other white blood cells of similar size, a carrier can then be used that binds to the cells in a manner that promotes 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.

[0055] The obstacles used in the device may be in the form of a pillar, or in the form of a triangle, square, rectangle, rhombus, trapezoid, hexagon, or teardrop. Furthermore, adjacent obstacles may have shapes such that the portions of the obstacles that define a gap are either symmetrical or asymmetrical with respect to the axis of the gap that extends in the direction of bulk fluid flow.

[0056] II. Microfluidic Device Fabrication and Operation General procedures for making and using microfluidic devices capable of separating cells or particles based on size are well known in the art. Such devices include those described in U.S. Patent No. 5,837,115; U.S. Patent No. 7,150,812; U.S. Patent No. 6,685,841; U.S. Patent No. 7,318,902; U.S. Patent No. 7,472,794; and U.S. Patent No. 7,735,652, all of which are incorporated herein by reference. Other references that provide guidance that may be useful in making and using the devices of the present invention include: U.S. Patent No. 7,276,170; U.S. Patent No. 6,913,697; U.S. Patent No. 7,988,840; U.S. Patent No. 8,021,614; U.S. Patent No. 8,282,799; U.S. Patent No. 8,304,230; U.S. Patent No. 8,579,117; U.S. Patent Application Publication No. 2006 / 0134599; U.S. Patent Application Publication No. 2007 / 0160503; U.S. Patent Application Publication No. Publication No. 2005 / 0282293; U.S. Patent Application Publication No. 2006 / 0121624; U.S. Patent Application Publication No. 2005 / 0266433; U.S. Patent Application Publication No. 2007 / 0026381; U.S. Patent Application Publication No. 2007 / 0026414; U.S. Patent Application Publication No. 2007 / 0026417; U.S. Patent Application Publication No. 2007 / 0026415; U.S. Patent Application Publication No. 007 / 0026413;U.S. Patent Application Publication No. 2007 / 0099207;U.S. Patent Application Publication No. 2007 / 0196820;U.S. Patent Application Publication No. 2007 / 0059680;U.S. Patent Application Publication No. 2007 / 0059718;U.S. Patent Application Publication No. 2007 / 005916;U.S. Patent Application Publication No. 2007 / 0059774;U.S. Patent Application Publication No. 2007 / 0 No. 2008 / 0124721; U.S. Patent Application Publication No. 2008 / 0090239; U.S. Patent Application Publication No. 2008 / 0113358; and WO2012 / 094642, all of which are hereby incorporated by reference.Among the various references describing the manufacture and use of the devices, U.S. Pat. No. 7,150,812 provides particularly good guidance, and U.S. Pat. No. 7,735,652 is of particular interest with respect to microfluidic devices for separations performed on samples containing cells found in blood (see, in this regard, U.S. Patent Application Publication No. 2007 / 0160503).

[0057] Devices can be fabricated using any of the materials in which micro- and nanoscale fluid handling devices are typically fabricated, including silicon, glass, plastics, hybrid materials, etc. A wide variety of thermoplastic materials suitable for microfluidic fabrication are available, offering a wide selection of mechanical and chemical properties that can be exploited and further tailored to specific applications.

[0058] Techniques for fabricating the devices include replica molding, soft lithography using PDMS, thermosetting polyesters, embossing, injection molding, laser ablation, and combinations thereof. Further details can be found in "Disposable microfluidic devices: fabrication, function and application" by Fiorini et al., BioTechniques 38:429-446 (March 2005), which is incorporated herein by reference. "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.

[0059] High-throughput embossing methods, such as reel-to-reel processing of thermoplastics, are attractive methods for industrial microfluidic chip manufacturing. The use of single-chip hot embossing is a cost-effective approach to achieve high-quality microfluidic devices at the prototyping stage. Methods for replicating microscale features in two thermoplastics, polymethylmethacrylate (PMMA) and / or polycarbonate (PC), are described by Yangra in "Fabrication of Microfluidic Devices by Thermoplastic Hot Embossing," Methods Mol. Biol., 949:115-23 (2013), which is incorporated herein by reference.

[0060] A flow channel can be constructed using two or more parts that, when assembled, form a closed cavity (preferably one with an orifice for adding or withdrawing fluid) within which an obstacle is located. The obstacle can be fabricated on one or more of the parts that are assembled to form the flow channel, or can be fabricated in the form of an insert sandwiched between two or more parts that define the boundaries of the flow channel.

[0061] The obstacle may be a solid that crosses the flow channel, possibly extending from one side of the flow channel to the opposite side of the flow channel. If the obstacle is integral with (or an extension of) one of the faces of the flow channel at one end of the obstacle, the other end of the obstacle may be sealed or pressed against the opposite face of the flow channel. A small space (preferably too small to accommodate particles of interest for the intended application) between one end of the obstacle and the face of the flow channel is acceptable, as long as it does not adversely affect the structural stability of the obstacle or the proper flow characteristics of the device.

[0062] The number of obstacles present must be sufficient to achieve the particle separation properties of the array. The obstacles may typically be organized into rows and columns (Note: the use of the term "rows and columns" does not imply or imply that the rows and columns are perpendicular to one another). Obstacles that are generally aligned transverse to the flow of fluid in a flow channel may be referred to as obstacles in a column. Obstacles adjacent to one another in a column may define gaps through which fluid may flow.

[0063] Obstacles in adjacent columns may be offset from one another by a degree characterized by an inclination angle called ε (epsilon). Thus, for several columns adjacent to one another (i.e., several columns of obstacles passed successively by a generally unidirectional fluid flow across the columns), corresponding obstacles in the columns may be offset from one another. form a row of obstacles extending at an angle ε relative to the direction of fluid flow through the columns. The inclination angle may be selected and the columns spaced apart from one another such that 1 / ε (when expressed in radians) is an integer and the columns of obstacles are periodically repeated. Obstacles within a single column may also be offset from one another by the same or different inclination angles. By way of example, rows and columns may be positioned at 90 degree angles relative to one another, with both rows and columns inclined at the same angle of ε relative to the direction of bulk fluid flow through the flow channel.

[0064] Surfaces can be coated to modify their properties, and polymeric materials used to fabricate devices can be altered in a variety of ways. In some cases, functional groups such as amines or carboxylic acids, either present in the natural polymer or added by 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 the PDMS formulation. In some cases, a layer of PMMA is spin-coated onto a device, e.g., a microfluidic chip, and the PMMA is "doped" with hydroxypropyl cellulose to change its contact angle.

[0065] To reduce non-specific adsorption of cells or compounds, e.g., released by lysed cells or found in biological samples, to the channel walls, one or more walls may be chemically modified to be non-adhesive or repelling. The walls may be coated with a thin coating (e.g., a monolayer) of commercially available non-adhesive reagents, such as those used to form hydrogels. Further 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, polyHEMA, methacrylated PEG, and agarose. Charged polymers, such as heparin, may be used to repel oppositely charged species. The type of chemical species used for repulsion and the method of attachment to the channel walls may depend on the nature of the species to be repelled, and the nature of the walls and the species to be attached. Such surface modification techniques are well known in the art. The walls may be functionalized before or after the device is assembled.

[0066] III. Separation Process Using DLD The DLD devices described herein may be used to purify cells, cell fragments, cell adducts, or nucleic acids. As discussed herein, these devices may also be used to separate a cell population of interest from a number of other cells. Separation and purification of blood components using the devices can be found, for example, in U.S. Patent Application Publication No. US2016 / 0139012, the teachings of which are incorporated herein by reference. A brief description of some exemplary separations is provided below.

[0067] A. Viable cells In one embodiment, the device is used in a procedure designed to separate viable cells from non-viable cells. The term "viable cells" refers to cells that can grow, actively divide, replicate, etc. If the viable cells have a larger size than the non-viable cells, the DLD device may be designed to include a critical size that is larger than the predetermined size of the non-viable cells and smaller than the predetermined size of the viable cells. The critical size may be only 1.1 times larger (or smaller) than the predetermined size of the non-viable cells, but generally is an order of magnitude larger (or smaller), for example, about 1.2 times to 2 times, preferably 3 to 10 times.

[0068] B. Adherent cells In another embodiment, the DLD device may be used to separate adherent cells. As used herein, the term "adherent cells" refers to cells that are capable of adhering to a surface. Adherent cells include immortalized cells used in cell culture and may be derived from a mammalian host. In some instances, adherent cells may be trypsinized prior to purification. Examples of adherent cells include MRC-5 cells; HeLa cells; Vero cells; NIH3T3 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-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-WRC256 cells; McCoy cells; MCF7 cells; WI-38 cells; WISH cells; XC cells; Y-1 cells; CHO cells; Raw264.7; BHK-21 cells; HEK293 cells, including 293A, 293T, etc.; HEP G2 cells; BAE-1 cells; SH-SY5Y cells; and derivatives, engineered strains and recombinant strains thereof.

[0069] In some embodiments, the procedure may include separating the cells from a diluent, such as a growth medium, which may provide efficient maintenance of the culture of adherent cells. For example, a culture of adherent cells in a growth medium may be exchanged for a transfection medium containing a transfection reagent, a second growth medium designed to induce a change in the adherent cells, such as differentiation of stem cells, or a series of washing buffers designed to remove compounds from the culture medium.

[0070] In a preferred procedure, adherent cells are purified through association with one or more carriers to which they bind in a manner that facilitates DLD separation. The carriers may be of the type described herein, and the binding may stabilize and / or activate the cells. Carriers are typically in the range of 1-1000 μm, but may be outside this range.

[0071] The association between the carrier and the cells should produce a complex that is increased in size compared to other material not associated with the carrier. Depending on the particular size of the cells and carriers and the number of cells and carriers present, the complex may be anywhere 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.

[0072] C. Activated cells The DLD device may also be used in procedures to separate activated or activatable cells from a plurality of other cells. Although cells undergoing activation can be expanded 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 after collection. The terms "activated cells" or "activatable cells" refer to cells that have been activated or can be activated, respectively, through association, incubation, or contact with a cell activator. Examples of activatable cells may include T cells, B cells; cells that play a role in immune or inflammatory responses, such as regulatory T cells, macrophages, dendritic cells, granulocytes, innate lymphoid cells, megakaryocytes, natural killer cells, platelets, synoviocytes; cells that play a role in metabolism, such as beta cells, hepatocytes, pancreatic cells; and recombinant cells capable of inducible protein expression, such as DE3 lysogenized E. coli cells, yeast cells, plant cells, and the like.

[0073] Typically, an activator is present on the surface of one or more carriers. Examples of cell activators include proteins, antibodies, cytokines, CD3, CD28, antigens against specific proteins, helper T cells, receptors, and glycoproteins; hormones such as insulin, glucagon; IPTG, lactose, allolactose, lipids, glycosides, terpenes, steroids, and alkaloids. The activatable cells should be at least partially associated with the carrier through an interaction between the activatable cells and the cell activator on the surface of the carrier. The complex formed may be only a few percent larger than the uncomplexed cells or many times larger than the size of the uncomplexed cells. An increase of at least 20% is desirable to facilitate separation, with higher percentages (40, 50, 100, 1000 or more) being preferred.

[0074] D. Separation of cells from toxic substances DLDs may also be used in purification designed to remove compounds that may be toxic to cells or to protect cells from contamination with toxic compounds. Examples include antibiotics, cryopreservatives, antifungals, toxic metabolites, sodium azide, metal ions, metal ion chelators, endotoxins, plasticizers, pesticides, and any combination thereof. The device may be used to remove toxic compounds from cells and ensure consistent production of material from the cells. In some examples, the cells may be log phase cells. The term "log phase cells" refers to cells that are actively dividing in a growth phase characterized by exponential logarithmic growth. In log phase, a cell population may double at a constant rate such that plotting the natural log of cell number against time produces a straight line.

[0075] The ability to isolate toxic substances was demonstrated in bacterial strains such as BL21, Tuner, Origami, Origami B, Rosetta, C41, C43, DH5α, DH10β or XL1Blue; yeast strains such as Saccharomyces, Pichia, Kluyveromyces, Hansenula and Yarrowia; algae; MRC-5 cells; HeLa cells; Vero cells; NIH3T3 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; 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-6 0 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; It is of interest for a wide variety of cells, including L2 cells; LLC-WRC256 cells; McCoy cells; MCF7 cells; WI-38 cells; WISH cells; XC cells; Y-1 cells; CHO cells; Raw264.7; BHK-21 cells; HEK293 cells (including 293A, 293T, etc.); HEPG2 cells; BAE-1 cells; SH-SY5Y cells; stem cells and their derivatives (including engineered and recombinant lines).

[0076] V. Technical background Without being bound to any particular theory, a general discussion of some technical aspects of microfluidics may help to understand the factors that affect the separations carried out in this field. A variety of microfabricated sieving matrices for particle separation have been disclosed (Choura, Proc. Natl. Acad. Sci., 96:13762 (1999); Hanra, Science, 288:1026 (2000); Huangra, 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 described, for example, in U.S. Patent No. 7,150,812, which is incorporated herein by reference in its entirety. Bump arrays essentially operate by separating particles passing through an array of obstacles (usually a periodically ordered array), with separation occurring between particles that follow an "array direction" that is offset from the direction of bulk fluid flow or the direction of an applied field (U.S. Patent No. 7,150,812).

[0077] A. Bump Array In some arrays, the geometry of adjacent obstacles is such that the portions of the obstacles that define a gap are symmetrical about the axis of the gap that extends in the direction of bulk fluid flow. The velocity or volume profile of fluid flow through such a gap is approximately parabolic throughout the gap, with fluid velocity and flux being zero (assuming no-slip flow conditions) at the surface of each obstacle that defines the gap, and reaching a maximum value at the center point of the gap. Because the profile is parabolic, a fluid layer of a particular width adjacent one of the obstacles that define the gap contains the same proportion of fluid flux as a fluid layer of the same width adjacent the other obstacle that defines the gap, which means that the size of a particle that is "bumped" while passing through the gap will be equal regardless of which obstacle the particle moves near.

[0078] In some cases, the particle size separation performance of an obstacle array can be improved by shaping and arranging the obstacles such that the portion of the adjacent obstacles that deflects the fluid flow into the gap between the obstacles is not symmetrical with respect to the axis of the gap that extends in the direction of the bulk fluid flow into the obstacle. Such lack of flow symmetry into the gap can lead to an asymmetric fluid flow profile in the gap. Concentration of the fluid flow toward one side of the gap (i.e., the result of an asymmetric fluid flow profile through the gap) can reduce the critical size of a particle that is induced to move in the direction of the array rather than in the direction of the bulk fluid flow. This is because the asymmetry of the flow profile creates a difference between the width of the flow layer adjacent to one obstacle containing a selected percentage of the fluid flux through the gap and the width of the flow layer containing the same percentage of the fluid flux adjacent to the other obstacle that defines the gap. The different widths of the fluid layers adjacent to the obstacles define a gap that exhibits two different critical particle sizes. A particle passing through the gap can collide (i.e., move in the direction of the array rather than in the direction of the bulk fluid flow) if it exceeds the critical size of the fluid layer through which it is carried. Thus, particles passing through a gap with an asymmetric flow profile may collide if the particles travel through the fluid layer adjacent to one obstacle, but not if the particles travel through the fluid layer adjacent to the other obstacle that defines the gap.

[0079] In another aspect, the particle size separation performance of an obstacle array can be improved by reducing the roundness of the edges of the obstacles that define the gaps. As an example, an array of obstacles having a triangular cross section 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.

[0080] Thus, by sharpening the edges of the obstacles that define the gaps in the obstacle array, the critical size of particles that will be 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 will provide equivalent particle separation characteristics as obstacles of the same size that do not have sharper edges, but can still be spaced farther apart.

[0081] B. Fractionation Range Objects that can be separated by size on microfluidic devices include cells, biomolecules, inorganic beads, and other objects. Typical sizes separated range from 100 nanometers to 50 micrometers, although larger and smaller particles can also be fractionated in some cases.

[0082] C. Volume Depending on the design, a device or combination of devices may be used to process samples of about 10 μl to at least 500 μl, about 500 μl to about 40 mL, about 500 μl to about 20 mL, about 20 mL to about 200 mL, about 40 mL to about 200 mL, or at least 200 mL. The total amount of material processed may be 50 mL to 5000 mL, 100 mL to 4000 mL, or 500 mL to 2000 mL, as the case may be. Starting materials include blood, blood-derived preparations (e.g., apheresis or leukapheresis preparations), other body fluids or extracts, cells grown in culture, and the like.

[0083] D. Channel The device may contain one or more channels with one or more inlets and one or more outlets. The inlets can be used for the introduction of samples or crude (i.e., unpurified) fluid compositions, buffers, or reagents. The outlets can be used to collect the product or as waste outlets. The channels can be about 0.5 to 100 mm wide and about 2-200 mm long, but can be of different widths and lengths. They can be 1-1000 μm deep and there can be anywhere from 1 to 100 channels or more. The volume can vary over a very wide range, from a few μl to several ml, and the device can have multiple zones (stages or sections) with different configurations of obstacles.

[0084] E. Gap size (distance from edge to edge between posts or obstacles) The gap size of the array of obstacles (the end-to-end distance between posts or obstacles) can be on the order of a few micrometers (e.g., 1-500) micrometers to over 1 millimeter. The obstacles, in some embodiments, can have diameters of 1-3000 micrometers and can have a variety of shapes (circular, triangular, teardrop, diamond, square, rectangular, etc.). The first row of posts can be located close to the inlet (e.g., within 5 μm) or 1 mm or more away.

[0085] F. Stackable Chips The device can include multiple stackable chips. The device can include about 1 to 50 chips. In some examples, the device can have multiple chips arranged in series or parallel or both. EXAMPLES

[0086] Working Example The following examples are intended to illustrate, but not limit, the invention. A normal blood sample was diluted 0.2x and processed on the DLD in normal separation mode (running the sample against buffer). The first product fraction collected had the expected concentration (a further 0.28x dilution) based on the input numbers and inlet to outlet DLD device ratio. Midway through the run, the collected product was recirculated to the DLD device as a "wash stream". Various fractions were collected at different times while continuing to recirculate the DLD product. To obtain recovery values, the final volume and final concentration of the DLD product were compared to the input volume and input concentration of the sample. The net recovery of WBCs after multiple passes was 97.5%, with a concentration coefficient change from input material to product material of 2.9x (compared to the initial change from input to product of 0.28x when the sample was run against running buffer).

[0087] All references cited herein are incorporated by reference. Having fully described the invention, it will be understood by those skilled in the art that the invention can be practiced within a broadly equivalent range of conditions, parameters, and the like, without affecting the spirit or scope of the invention or any embodiment thereof.

Claims

1. 1. A method for isolating a target cell or target particle of a predetermined size from a sample containing cells or particles of the predetermined size, comprising: a) Both sample and wash solutions are applied to the microfluidic device at separate inlets, where: i) the wash solution applied to the device is devoid of said target cells or target particles and is devoid of said cells or particles below a predetermined size; ii) the microfluidic device comprises an array of obstacles arranged in rows, where each subsequent row of obstacles is laterally shifted relative to the previous row, and the obstacles are arranged to differentially deflect and direct target cells or particles into a first outlet where they may be collected as a target cell or target particle product, and direct cells or particles below a predetermined size to a second outlet where they are collected or discarded as waste; b) performing a deterministic lateral displacement (DLD) by flowing a sample and a wash solution through the device, wherein during said DLD, at least a portion of the target cell or target particle product is recirculated one or more times such that all or at least a portion of the wash solution is applied to the device; c) collecting the final product comprising the target cells or particles from the first outlet during or at the end of step b). The method includes:

2. 13. The method of claim 1, wherein after the target cell or target particle product has been recirculated, recirculation is stopped and a wash solution is again applied to the microfluidic device.

3. The method of claim 1 or 2, wherein the washing solution is water or an aqueous buffer and / or comprises a) a reagent that chemically reacts with cells, particles or other components in the washing solution; or b) an antibody, carrier or activator that specifically interacts with target cells or target particles.

4. 4. The method of any one of claims 1 to 3, wherein the first outlet comprises or is connected to a valve that can be used to divert the target cell or target particle product to a conduit that recirculates the target cell or target particle product to the inlet of the microfluidic device.

5. 5. The method of any one of claims 1 to 4, wherein the microfluidic device comprises an inlet that includes or is connected to a valve that can be used to switch the supply entering the device through the inlet from a conduit supplying a wash buffer to a conduit supplying a cell or target particle product.

6. 6. The method of any one of claims 1 to 5, wherein the target cells or target particle products that are recycled to the microfluidic device react or bind with a carrier, antibody, fluorescent tag, activator or compound before, during or after being reapplied to the microfluidic device.

7. The method of any one of claims 1 to 6, wherein the number of cells or particles consists of target cells or target particle products.

8. The method of any one of claims 1 to 7, wherein recirculation is continued until the cells or particles are concentrated at least three-fold relative to their concentration in the sample.

9. The method of any one of claims 1 to 7, wherein recirculation is continued until the cells or particles are concentrated at least 5-fold relative to their concentration in the sample.

10. The method of any one of claims 1 to 7, wherein recirculation is continued until the cells or particles are concentrated at least 10-fold relative to their concentration in the sample.

11. The method according to any one of claims 1 to 10, wherein recirculation with microfluidic processing is the only method used to concentrate the cells or particles.

12. The method of any one of claims 1 to 11, wherein the sample comprises target cells or stem cells of a predetermined size and cells below the predetermined size.

13. 13. The method of claim 12, wherein the target cells are white blood cells and the cells below a predetermined size are platelets or red blood cells.

14. 14. The method of claim 12 or 13, wherein the sample is blood or a composition obtained by performing apheresis or leukoapheresis on blood.

15. The method of any one of claims 12 to 14, wherein the recirculated white blood cells or stem cells are bound to a carrier, antibody or activator in a manner that promotes or complements DLD separation.

16. The method according to any one of claims 13 to 15, wherein the leukocytes are T cells.

17. 17. The method of claim 16, wherein the method is used in a process for generating CAR-T cells.

18. The method of claim 17 , wherein the process does not include a centrifugation step.

19. 19. The method of claim 17 or 18, wherein the method is used to concentrate the cells sufficiently to allow administration of the cells to a patient.

20. 20. The method of claim 19, wherein a sample is obtained from a patient and less than four hours elapse between the completion of sample acquisition and the completion of DLD.

21. 21. A CAR-T cell or other genetically engineered cell, NK cell, B cell, monocyte or stem cell produced by the method of any one of claims 12 to 20.

22. 1. A method of producing a purified genetically engineered target cell, comprising: a) obtaining a sample containing target cells of a predetermined size and one or more contaminating cells or particles smaller than the predetermined size; b) applying a sample at a first inlet and a wash fluid at a second inlet to a microfluidic device, where the microfluidic device includes an array of obstacles arranged to parallaxically deflect a stream of target cells to a first outlet and contaminating cells or particles smaller than a predetermined size to a second outlet, where the target cells can be collected as a target cell product; c) flowing sample and wash fluid through the device, where the concentration of target cells at a first outlet is determined and at least a portion of the target cells are recirculated from the outlet to replace all or at least a portion of the wash fluid applied to the inlet of the device, said recirculation being continued or repeated until a desired product cell concentration, PC, is achieved; d) upon reaching the PC, directing the stream of target cells out a first outlet to a site where the target cells are transformed or transfected to form genetically engineered target cells; e) flowing the genetically engineered target cells into the device where they are separated from reagents, viruses, or other materials used to transform or transfect the target cells to form purified genetically engineered target cells; f) Either collect the purified genetically engineered target cells or flow the purified genetically engineered target cells to another site where they are further processed prior to collection. The method includes:

23. 23. The method of claim 22, wherein all steps from application of the cells to the microfluidic device in step b) to collection of the cells are carried out as a single continuous process.

24. 24. The method of claim 22 or 23, wherein the target cell is a lymphocyte or a stem cell.

25. 24. The process of claim 22 or 23, wherein the target cell is a T lymphocyte.

26. 26. The method of claim 25, wherein the T lymphocytes are genetically engineered to produce a chimeric antigen receptor (CAR) on their surface.

27. The method according to any one of claims 22 to 26, wherein in steps b) and c) the target cells are separated from cells or particles larger or smaller than a predetermined size by DLD.

28. The method according to any one of claims 22 to 27, wherein in step e) the device is a microfluidic device and the genetically engineered target cells are separated from reagents, viruses or other materials used for transformation or transfection of the target cells by DLD.

29. 29. The method of any one of claims 22 to 28, wherein in step e) during separation of reagents, viruses or other materials used to transform or transfect the target cells, the purified genetically engineered target cells are transferred to a growth medium and harvested in step f) and either grown in cell culture or flowed to a place where the cells are cultivated and then harvested.

30. 30. The method according to any one of claims 22 to 29, wherein the washing solution is water or an aqueous buffer and / or: a) comprises a reagent that chemically reacts with cells, particles or other components in the washing solution; or b) comprises an antibody, carrier or activator that specifically interacts with the target cells.

31. 31. The method of any one of claims 22 to 30, wherein the first outlet comprises or is connected to a valve that can be used to direct the target cells to a conduit that recirculates the product to an inlet of a microfluidic device or that can direct the flow of target cells from the first outlet to a site where the target cells are transformed or transfected to form genetically engineered target cells.

32. 32. The method of claim 31 , wherein the valve is configured to electronically respond to concentrations of target cells below the PC by directing the target cells to a conduit that recirculates the product to an inlet on the microfluidic device, and to electronically respond to concentrations of target cells above the PC by directing the target cells to a site where the target cells are transformed or transfected to form a genetically engineered target cell.

33. 33. The method of any one of claims 22 to 32, wherein the target cells that are recirculated to the microfluidic device react or bind with a carrier, antibody, fluorescent tag, activator or compound before, during or after being reapplied to the microfluidic device.

34. The method of any one of claims 22 to 33, wherein recirculation is continued until the target cells are enriched at least three-fold relative to their concentration in the sample.

35. The method of any one of claims 22 to 33, wherein recirculation is continued until the target cells are enriched at least 5-fold relative to their concentration in the sample.

36. The method of any one of claims 22 to 33, wherein recirculation with microfluidic processing is the only method used to concentrate the cells or particles.

37. The method according to any one of claims 22 to 36, wherein the target cells are not centrifuged during the entire time between obtaining the sample in step a) and collecting the cells in step f).

38. The method according to any one of claims 22 to 37, wherein the target cells are not frozen during the entire time between obtaining the sample in step a) and collecting the cells in step f).

39. The method according to any one of claims 22 to 38, wherein the sample is blood or a composition obtained by performing apheresis or leukapheresis on blood.

40. The method of claim 39, wherein the target cells are white blood cells and in steps b) and c) DLD is performed to separate white blood cells from platelets and / or red blood cells.

41. The method of any one of claims 22 to 40, wherein the recirculated leukocytes or stem cells are bound to a carrier, antibody or activator in a manner that promotes or complements DLD separation.

42. 42. The method of any one of claims 22 to 41, wherein the sample is obtained from the patient and no more than 10 hours elapse between the completion of obtaining the sample and the completion of step f).

43. 43. The method of claim 42, wherein step f) is completed within 5 hours from the completion of sample acquisition.

44. A purified genetically engineered target cell produced by the method of any one of claims 22 to 43.

Images