Microfluidic cartridges for handling particles and cells

Abstract

The present invention describes a microfluidic box for purifying target particles or target cells of a predetermined size from contaminants in a sample, the box comprising a first planar support and a second planar support, the first planar support and the second planar support each having a top surface and a bottom surface, wherein the top surface of the first planar support and / or the second planar support comprises at least one embedded channel extending from one or more inlets to one or more outlets; the at least one embedded channel comprises a plurality of obstacles, wherein the microfluidic box comprises at least one void space, the void space being configured to deform when the first planar support and the second planar support are assembled into the microfluidic box.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 954,478, filed December 28, 2019, which is incorporated herein by reference in its entirety. Background Art

[0003] Preparation of cells for personalized therapies typically requires the collection of biological material from the patient, purification of specific cell types from the collected material, and engineering or culturing the purified cells. In the case of CAR T-cell therapy, large volumes of blood or blood-derived apheresis or leukapheresis preparations often need to be processed to obtain T-cell preparations suitable for genetic engineering and expansion. Microfluidic size-based procedures offer a rapid, gentle, and versatile processing option. However, there are several factors, including the deposition of biological debris during operation of microfluidic devices, that can slow processing and result in poor purification. Therefore, the development of better performing devices and methods to increase the speed of purification of biological materials is of great interest. Summary of the Invention

[0004] Certain separation cartridges for use with microfluidic devices are described herein that have been modified to allow for the fabrication of cartridges with fine features, such as columns or barriers for size-based separations, holding pens for cells, and other microfluidic features. Certain separation cartridges for use with microfluidic devices are also described that have been modified to allow for fluid flow in cartridges having multiple lanes or channels, such as dividing walls that extend a certain length to prevent unwanted mixing, turbulence caused by unwanted mixing, and the pulsatile nature of delivery due to some positive displacement pumps.

[0005] In one aspect, the present invention describes a microfluidic box for purifying target particles or target cells of a predetermined size from contaminants in a sample, the box comprising a first planar support and a second planar support, each of the first planar support and the second planar support having a top surface and a bottom surface, wherein the top surface of the first planar support and / or the second planar support comprises at least one embedded channel extending from one or more inlets to one or more outlets; at least one embedded channel comprises a plurality of obstacles.

[0006] In certain embodiments, the microfluidic cartridge includes at least one void space configured to deform when the first and second planar supports are assembled into the microfluidic cartridge. In certain embodiments, the bottom surfaces of the first and second planar supports include at least one void space configured to deform when the bottom of the first planar support is pressed against the bottom of the second planar support. In certain embodiments, the at least one void space is configured to prevent damage, displacement, or deformation of at least one embedded channel, one or more inlets, one or more outlets, multiple obstacles, or a combination thereof. In certain embodiments, the at least one void space is configured to prevent damage, displacement, or deformation of multiple obstacles. In certain embodiments, the microfluidic cartridge includes a 1:1 ratio of void space to channel. In certain embodiments, the at least one void space comprises a total surface area that is at least about 90% of the total surface area of ​​the at least one embedded channel. In certain embodiments, the at least one void space comprises a total surface area that is at least about 100% of the total surface area of ​​the at least one embedded channel. In certain embodiments, the at least one void space comprises a total surface area that is at least about 110% of the total surface area of ​​the at least one embedded channel. In certain embodiments, at least one void space is divided into two or more void spaces, and the two or more void spaces are positioned on the bottom surface of the first planar support and / or the second planar support opposite the obstacle array. In certain embodiments, the planar support is made of two layers of material bonded together. In certain embodiments, the microfluidic box also includes an obstacle adhesive layer that is bonded to the surface of the planar support and to the top surface of the multiple obstacles in at least one embedded channel to prevent the fluid or sample from flowing over the multiple obstacles during operation of the box. In certain embodiments, the obstacle adhesive layer includes one or more passages connected to the one or more inlet fluids of at least one embedded channel that allow the sample to flow into the at least one embedded channel and one or more passages connected to the one or more outlet fluids of at least one embedded channel that allow the fluid to flow out of the one or more outlets. In certain embodiments, the obstacles are positioned to define the critical size of the box so that when the sample is applied to the inlet of the box and flows to the outlet, particles or cells in the sample that are larger than the critical size are separated from particles or cells in the sample that are smaller than the critical size. In some embodiments, the one or more outlets include at least one product outlet, wherein target particles or target cells having a size greater than a critical size of the cartridge are directed to the at least one product outlet. In some embodiments, the one or more outlets include at least one waste outlet, and contaminants having a size less than a critical size of the cartridge flow to the at least one waste outlet. In some embodiments, the plurality of obstacles have a diamond or elongated diamond shape. In some embodiments, the plurality of obstacles have a circular or elliptical shape. In some embodiments, the plurality of obstacles have a hexagonal shape.In certain embodiments, a plurality of obstacles are elongated perpendicular to the direction of fluid flow so that they have a horizontal length (P1) that is different from their vertical length (P2). In certain embodiments, P1 is about 10 μm to about 160 μm and P2 is about 5 μm to about 80 μm. In certain embodiments, P1 is about 10 μm to about 80 μm and P2 is about 15 μm to about 60 μm. In certain embodiments, P1 is about 15 μm to about 30 μm and P2 is about 25 μm to about 45 μm. In certain embodiments, P1 is about 40 μm and P2 is about 20 μm. In certain embodiments, P1 is 50% to 150% longer than P2. In certain embodiments, a plurality of obstacles have vertices extending into parallel gaps so that the gaps are flanked on either side by one or more vertices pointing to each other but not directly opposite each other. In certain embodiments, a plurality of obstacles have vertices extending into vertical gaps so that the gaps are flanked on either side by vertices pointing to each other and directly opposite each other. In certain embodiments, a plurality of obstacles are arranged in at least 1 column. In certain embodiments, the plurality of obstacles are arranged in at least 10 columns. In certain embodiments, the plurality of obstacles are arranged in at least 30 columns. In certain embodiments, the plurality of obstacles are arranged in at least 50 columns. In certain embodiments, the plurality of obstacles are arranged in at least about 60 columns. In certain embodiments, the plurality of obstacles are arranged in at least about 50 rows. In certain embodiments, the plurality of obstacles are arranged in at least about 100 rows. In certain embodiments, the plurality of obstacles are arranged in at least about 300 rows. In certain embodiments, the plurality of obstacles are arranged in at least about 600 rows. In certain embodiments, the first planar support or the second planar support comprises at least 10 embedded channels. In certain embodiments, the first planar support and / or the second planar support comprises at least 20 embedded channels. In certain embodiments, the first planar support and / or the second planar support comprises about 28 embedded channels. In certain embodiments, the first planar support and / or the second planar support comprises about 30 embedded channels. In certain embodiments, the first planar support and / or the second planar support comprises at least about 50 embedded channels. In certain embodiments, the one or more inlets of the microfluidic cartridge include at least one or more sample inlets and at least one or more fluid inlets; wherein the at least one or more sample inlets are separated from the at least one or more fluid inlets by a partition wall, the partition wall extending from the one or more sample inlets toward the outlet into at least one array of obstacles embedded in the channel, and in a direction parallel to the direction of fluid flow. In certain embodiments, the partition wall extends at least 10% of the length of the plurality of obstacles. In certain embodiments, the partition wall extends at least 20% of the length of the plurality of obstacles. In certain embodiments, the partition wall extends at least 60% of the length of the plurality of obstacles.In certain embodiments, one or more inlets, one or more outlets, or both are fluidically connected to the first peristaltic pump, the second peristaltic pump, or both. In certain embodiments, the first peristaltic pump and the second peristaltic pump are fluidically connected in series. In certain embodiments, the first peristaltic pump and the second peristaltic pump are fluidically connected in parallel. In certain embodiments, the box is made of a polymer.

[0007] In certain embodiments, the polymer is a thermoplastic polymer. In certain embodiments, the thermoplastic polymer is selected from high density polyethylene, polypropylene, polyethylene terephthalate, polycarbonate or cycloolefin copolymer. In certain embodiments, the thermoplastic polymer is a cycloolefin copolymer.

[0008] In one aspect, the present invention describes a microfluidic cartridge for purifying target particles or cells of a predetermined size from contaminants in a sample, the cartridge comprising a first planar support and a second planar support, each having a top surface and a bottom surface, wherein the top surface of the first planar support and / or the second planar support comprises at least one embedded channel extending from one or more inlets to one or more outlets; at least one embedded channel comprises a plurality of obstacles, wherein the microfluidic cartridge comprises at least one void space configured to deform when the first and second planar supports are assembled into the microfluidic cartridge. In certain embodiments, the bottom surfaces of the first and second planar supports comprise at least one void space configured to deform when the bottom of the first planar support is pressed against the bottom of the second planar support. In certain embodiments, the at least one void space is configured to prevent damage, displacement, or deformation of at least one embedded channel, one or more inlets, one or more outlets, multiple obstacles, or a combination thereof. In certain embodiments, the at least one void space is configured to prevent damage, displacement, or deformation of the multiple obstacles. In certain embodiments, the microfluidic cartridge comprises a 1:1 ratio of void space to channel. In certain embodiments, the total surface area of ​​at least one void space is at least about 90% of the total surface area of ​​at least one embedded channel. In certain embodiments, the total surface area of ​​at least one void space is at least about 100% of the total surface area of ​​at least one embedded channel. In certain embodiments, the total surface area of ​​at least one void space is at least about 110% of the total surface area of ​​at least one embedded channel. In certain embodiments, at least one void space is divided into two or more void spaces, and the two or more void spaces are positioned on the bottom surface of the first planar support and / or the second planar support relative to the obstacle array. In certain embodiments, the planar support is made of two layers of material bonded together. In certain embodiments, the microfluidic box also includes an obstacle adhesive layer that is bonded to the surface of the planar support and to the top surface of the multiple obstacles in at least one embedded channel to prevent the fluid or sample from flowing over the multiple obstacles during the operation of the box. In certain embodiments, the obstacle adhesive layer includes one or more passages connected to the one or more inlet fluids of at least one embedded channel that allow the sample to flow into the at least one embedded channel and one or more passages connected to the one or more outlet fluids of at least one embedded channel that allow the fluid to flow out from the one or more outlets. In certain embodiments, the obstacles are positioned to define a critical size of the cartridge such that when a sample is applied to the inlet of the cartridge and flows toward the outlet, particles or cells in the sample that are larger than the critical size are separated from particles or cells in the sample that are smaller than the critical size.In certain embodiments, one or more outlets include at least one product outlet, wherein target particles or target cells having a size greater than the critical size of the box are directed to at least one product outlet. In certain embodiments, one or more outlets include at least one waste outlet, and pollutants having a size less than the critical size of the box flow to at least one waste outlet. In certain embodiments, a plurality of obstacles have a rhombus or elongated rhombus shape. In certain embodiments, a plurality of obstacles have a circular or elliptical shape. In certain embodiments, a plurality of obstacles have a hexagonal shape. In certain embodiments, a plurality of obstacles are elongated perpendicular to the direction of fluid flow so that they have a horizontal length (P1) that is different from their vertical length (P2). In certain embodiments, P1 is about 10 μm to about 160 μm and P2 is about 5 μm to about 80 μm. In certain embodiments, P1 is about 10 μm to about 80 μm and P2 is about 15 μm to about 60 μm. In certain embodiments, P1 is about 15 μm to about 30 μm and P2 is about 25 μm to about 45 μm. In certain embodiments, P1 is approximately 40 μm and P2 is approximately 20 μm. In certain embodiments, P1 is 50% to 150% longer than P2. In certain embodiments, the plurality of obstacles have vertices extending into the parallel gaps, such that the gaps are flanked by one or more vertices pointing toward each other but not directly opposite each other. In certain embodiments, the plurality of obstacles have vertices extending into the perpendicular gaps, such that the gaps are flanked by vertices pointing toward each other and directly opposite each other. In certain embodiments, the plurality of obstacles are arranged in at least one column. In certain embodiments, the plurality of obstacles are arranged in at least 10 columns. In certain embodiments, the plurality of obstacles are arranged in at least 30 columns. In certain embodiments, the plurality of obstacles are arranged in at least 50 columns. In certain embodiments, the plurality of obstacles are arranged in at least approximately 60 columns. In certain embodiments, the plurality of obstacles are arranged in at least approximately 50 rows. In certain embodiments, the plurality of obstacles are arranged in at least approximately 100 rows. In certain embodiments, the plurality of obstacles are arranged in at least approximately 300 rows. In certain embodiments, the plurality of obstacles are arranged in at least approximately 600 rows. In certain embodiments, the first planar support member or the second planar support member comprises at least 10 embedded channels. In certain embodiments, the first planar support member and / or the second planar support member comprises at least 20 embedded channels. In certain embodiments, the first planar support member and / or the second planar support member comprises approximately 28 embedded channels. In certain embodiments, the first planar support member and / or the second planar support member comprises approximately 30 embedded channels. In certain embodiments, the first and / or second planar support member comprises at least about 50 embedded channels.In certain embodiments, the one or more inlets of the microfluidic box include at least one or more sample inlets and at least one or more fluid inlets; wherein at least one or more sample inlets are separated from at least one or more fluid inlets by a partition wall, and the partition wall extends from the one or more sample inlets toward the outlet to the obstacle array embedded in at least one channel, and the direction is parallel to the direction of fluid flow. In certain embodiments, the partition wall extends at least 10% of the length of multiple obstacles. In certain embodiments, the partition wall extends at least 20% of the length of multiple obstacles. In certain embodiments, the partition wall extends at least 60% of the length of multiple obstacles. In certain embodiments, one or more inlets, one or more outlets or both are fluidically connected to the first peristaltic pump, the second peristaltic pump or both. In certain embodiments, the first peristaltic pump and the second peristaltic pump are fluidically connected in series. In certain embodiments, the first peristaltic pump and the second peristaltic pump are fluidically connected in parallel. In certain embodiments, the box is made of polymer.

[0009] In certain embodiments, the polymer is a thermoplastic polymer. In certain embodiments, the thermoplastic polymer is selected from high density polyethylene, polypropylene, polyethylene terephthalate, polycarbonate or cycloolefin copolymer. In certain embodiments, the thermoplastic polymer is a cycloolefin copolymer.

[0010] Also described is a microfluidic assembly comprising a plurality of microfluidic cartridges in fluidic connection. In certain embodiments, the microfluidic cartridges are stacked. In certain embodiments, the plurality of microfluidic cartridges is two. In certain embodiments, the microfluidic cartridges are in parallel fluidic connection. In certain embodiments, the microfluidic cartridges are in serial fluidic connection.

[0011] Also described herein is a method for making a microfluidic cartridge, wherein the cartridge is made by pressing the bottoms of a first planar support and a second planar support together so that the array of obstacles is not deformed. In some embodiments, at least one of the embedded channels, the obstacles, or both are made by molding, hot molding, roll-to-roll molding, or injection molding. In some embodiments, the microfluidic cartridge is UV cured during the manufacturing process. Also described herein is a method for enriching target particles or target cells of a predetermined size from contaminants in a sample, the method comprising: (a) obtaining a sample comprising target particles or target cells and contaminants; (b) separating the target particles or target cells from the contaminants by: (i) applying the sample to one or more sample inlets on the microfluidic cartridge; (ii) flowing the sample to an outlet on the cartridge; and (iii) obtaining a product enriched with the target particles or target cells from the one or more outlets while removing the contaminants. In certain embodiments, the size of the target particles or target cells is larger than the critical size of the obstacle array, and the size of at least some of the contaminants is smaller than the critical size of the obstacle array, and wherein the target cells or target particles flow to one or more product outlets, where a product enriched with the target cells or target particles is obtained, while contaminants smaller than the critical size of the obstacle array flow to one or more waste outlets. In certain embodiments, the flow rate of the box is about 400 mL per hour. In certain embodiments, the flow rate of the box is at least about 100 mL per hour or greater. In certain embodiments, the flow rate of the box is at least about 300 mL per hour or greater. In certain embodiments, the flow rate of the box is about 1000 mL per hour. In certain embodiments, the internal pressure of the box is at least about 1.5 pounds per square inch or greater. In certain embodiments, the internal pressure of the box is about 15 pounds per square inch. In certain embodiments, the internal pressure of the box is about 50 pounds per square inch or less. In certain embodiments, the internal pressure of the box is about 10 pounds per square inch to about 20 pounds per square inch. In certain embodiments, the sample is blood or a blood-related product. In certain embodiments, the sample is a single collection or leukocyte single collection sample. In certain embodiments, the sample includes platelets as contaminants. In certain embodiments, the method results in the removal of at least 80% of platelets from the sample. In certain embodiments, the method results in the removal of at least 90% of platelets from the sample. In certain embodiments, the method results in the removal of at least 95% of platelets from the sample. In certain embodiments, the enriched target cells include leukocytes. In certain embodiments, the enriched target cells include stem cells. In certain embodiments, the enriched target cells include peripheral blood mononuclear cells. In certain embodiments, the peripheral blood mononuclear cells include CD3+ cells. In certain embodiments, the method further includes genetically engineering the enriched target cells to obtain genetically engineered target cells. In certain embodiments, the genetic engineering includes transfecting or transducing the target cells with a recombinant nucleic acid.In certain embodiments, the enriched target cells or genetically engineered target cells are expanded by culturing them in vitro.

[0012] Another aspect described herein is a method for producing chimeric antigen receptor (CAR) T cells, the method comprising: (a) obtaining a sample including T cells; (b) separating T cells from contaminants by the following steps: (i) applying the sample to one or more sample inlets on a microfluidic box; (ii) causing the sample to flow to the outlet of the box; and (iii) obtaining a product enriched with T cells from the product outlet; (c) genetically engineering the T cells in the enriched product obtained in step b) to produce chimeric antigen receptors (CAR) on their surface. In certain embodiments, the sample is blood, a single-collection product, or a single-collection product of leukocytes. In certain embodiments, the genetic engineering of T cells includes transfection or transduction of target cells, and further amplifying genetically engineered target cells by culturing cells in vitro.

[0013] In another aspect described herein is a method for producing chimeric antigen receptor (CAR) natural killer cells, the method comprising: (a) obtaining a sample comprising natural killer cells; (b) separating the natural killer cells from contaminants by the following steps: (i) applying the sample to one or more sample inlets on a microfluidic cartridge; (ii) causing the sample to flow to an outlet of the cartridge; and (iii) obtaining a product enriched with natural killer cells from a product outlet; (c) genetically engineering the natural killer cells in the enriched product obtained in step b) to produce a chimeric antigen receptor (CAR) on its surface. In certain embodiments, the sample is a blood sample, a single collection product, or a leukocyte single collection product. In certain embodiments, the genetic engineering of the natural killer cells comprises transfecting or transducing target cells, and the genetically engineered target cells are further expanded by culturing cells in vitro.

[0014] Other aspects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be appreciated, the present disclosure is capable of other and different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Therefore, the drawings and descriptions should be considered to be exemplary rather than restrictive.

[0015] Incorporation by reference

[0016] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In addition, US 5,427,663; US 5,837,115; US 6,685,841; US ​​6,913,697; US 7,150,812; US 7,276,170; US 7,318,902; 7,472,794; US 7,735,652; US 7,988,840; US 8,021,614; US 8,282,799; US 8,304,230; US 8,579,117; US 10 / 324,011; US ​​2005 / 0282293; US 2006 / 0134599; US 2007 / 0160503; US 2006 / 0121624;US 2005 / 0266433; US2007 / 0026381; US2007 / 0026413; US 2007 / 0026414; US2007 / 0026415; US 2007 / 0026417; 2007 / 0059680; US2007 / 0059718; US 2007 / 0059781; US ​​2007 / 0059774; US 2007 / 0099207; US2007 / 0196820; US 2006 / 0223178; US 2008 / 0124721; US2008 / 0090239; US 2008 / 0113358; US Each of US2014 / 0342375; US2016 / 0139012; US2019 / 0071639; and WO2012094642 is incorporated herein by reference in its entirety. To the extent that publications and patents or patent applications incorporated by reference conflict with the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The novel features of the present invention are set forth with particularity in the appended claims. The features and advantages of the present invention may be better understood by reference to the following detailed description of illustrative embodiments in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as “Figures” and “FIG.”):

[0018] Figures 1A-1G Different operating modes of the DLD are shown.

[0019] Figure 2 shows various uses of the channel with respect to Figures 1A-1CAn alternative obstacle array to the obstacle array shown in .

[0020] Figures 3A-3D An embodiment of a device is shown that includes an arrangement of 14 parallel channels that can be used in a microfluidic device.

[0021] Figures 4A-4D 2 channels are shown. Figures 4B-4D An enlarged view of a section of the channel is shown.

[0022] Figure 5 is a cross-sectional view of a “bump array” device with equilateral triangle-shaped obstacles positioned in a microfluidic channel.

[0023] Figures 6A-6B An array of diamond-shaped pillars is shown.

[0024] Figures 7A-7C Depicted is a stacked separation assembly where two microfluidic devices are combined into a single unit.

[0025] Figures 8A-8B Two channels that might be found in the device depicted in FIG. 7 are depicted. Figure 8B A magnified view of a section of a channel is shown in . In this example, the channel has an array of asymmetrically spaced diamond-shaped obstacles, where G1 is larger than G2. The diamonds are offset so that each successive row is laterally displaced relative to the previous row.

[0026] Figure 9 Shown is a stacked assembly of microfluidic devices within a housing, which together with the housing may be referred to as a "cassette."

[0027] Figure 10A and 10B A channel defined by two walls is shown, having a sample inlet and a fluid inlet.

[0028] Figure 11 Comparison of the normalized velocity flow between two equilateral triangular cylinders (left panel) and the normalized velocity flow between two circular cylinders (right panel).

[0029] Figure 12 is a plot of the predicted critical diameter versus the array tilt angle (ε) for triangular (lower line) and circular (upper line) obstacle arrays.

[0030] Figure 13 is a graph showing the effect of the tilt angle ("array tilt" in the figure) on the gap length G.

[0031] Figure 14 is a graph showing the effect of obstacle edge roundness (expressed as r / S) on the critical dimension exhibited on one side of the gap defined by the edge.

[0032] Figure 15 is a graph showing the effect of applied pressure on particle velocity in a bump array with triangular pillars (data shown as triangles) and a bump array with circular pillars (data shown as circles).

[0033] Figure 16A and 16B : shows a cross-sectional view of a single-box DLD element comprising 6 layers: 2 layers of DLD micropillars, 2 layers of void space corrugated regions for the fluidic injector channels, and 2 layers of end layers. Figure 16B A top view of a non-limiting example DLD layer consisting of an array of elongated diamond-shaped or hexagonal pillars is shown.

[0034] Figure 17A-C Figure 17B shows a top view of a photograph of two DLD component cassettes loaded into a device cassette. Figure 17C shows a top view of the right side of a DLD cassette loaded into a device cassette.

[0035] Figure 18A Specific embodiments of the arrangement of void spaces are shown in FIG and B, which show a bottom view (18A) and a cross-sectional view (18B) of a planar support.

[0036] Figure 19A A and B show alternative embodiments of void spaces when planar supports are stacked to form a microfluidic cartridge (cross-sectional views shown). DETAILED DESCRIPTION

[0037] The present invention relates generally to size-based microfluidic separations, and more particularly to the use of DLD for the preparation of cells of therapeutic value. Guidance is provided herein for making and using microfluidic devices and for using DLD for separations involving biological materials.

[0038] While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, modifications, and substitutions will be apparent to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0039] Whenever the term "at least," "greater than," or "greater than or equal to" precedes the first value in a series of two or more values, the term "at least," "greater than," or "greater than or equal to" applies to every value in the series. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0040] When the term "not greater than," "less than," or "less than or equal to" precedes the first value in a series of two or more values, the term "not greater than," "less than," or "less than or equal to" applies to every value in the series. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0041] definition

[0042] Single sampling: As used herein, the term refers to the process of separating blood from a patient or donor into its components (e.g., white blood cells, platelets, and red blood cells). An "apheresis product" is the end result of this procedure. More specific terms are "platelet apheresis" (referring to the separation of platelets) and "leukocyte apheresis" (referring to the separation of white blood cells). As used herein, the term "separation" refers to obtaining a product that is enriched in a particular component compared to whole blood or other starting material, and does not imply that absolute purity has been achieved.

[0043] CAR T cells : The term "CAR" is an acronym for "chimeric antigen receptor." Therefore, a "CAR T cell" is a T cell that has been genetically engineered to express a chimeric receptor.

[0044] CAR T-cell therapy: The term refers to any procedure that uses CAR T cells to treat a disease or condition. Diseases that can be treated include blood and solid tumors, autoimmune diseases, and infectious diseases.

[0045] Carrier: As used herein, the term "carrier" refers to a medium, such as a bead or particle, made of biological or synthetic materials, that is added to a formulation to bind 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 are typically 1-1000 μm in size. They can be coated or uncoated and have affinity media that can be modified to include antigens or other molecules on the cell surface (e.g., antibodies, activators, haptens, aptamers, particles, or other compounds). Carriers can also be magnetized and can include particles that impart non-size-related secondary properties to cells or cell complexes (e.g., Janus or strawberry-like particles). For example, particles can produce chemical, electrochemical, or magnetic properties that can be used in downstream processes such as magnetic separation, electroporation, gene transfer, and / or specific analytical chemistry processes. Particles may also induce changes in cellular metabolism, activate cells, or promote cell division.

[0046] Supports that bind "in a manner that promotes DLD separation":This term refers to both the carrier and the method of binding the carrier, which, as the case may be, influences how cells, proteins, or particles behave during DLD. Specifically, "binding in a manner that facilitates DLD separation" means: a) binding must exhibit specificity for a particular target cell type, protein, or particle; and b) binding must result in a complex that increases in size relative to unbound cells, proteins, or particles. In the case of binding to target cells, the increase must be at least 2 μm (or, when expressed as a percentage, at least 20, 50, 100, 200, 500, or 1000%). In cases where therapeutic or other uses require release of the target cell, protein, or other particle from the complex to achieve its intended use, the term "in a manner that facilitates DLD separation" also requires that the complex permits such release, for example, by chemical or enzymatic cleavage, chemical dissolution, digestion, due to competition with other binding agents, or by physical shear (e.g., using a pipette to generate shear stress), and that the released target cell, protein, or other particle remains viable; for example, the therapeutic cell must retain the biological activity that enables its therapeutic use after release from the complex.

[0047] The vector can also be combined in a manner that complements the DLD separation: The 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 cells, whether or not they increase in size sufficient to facilitate DLD separation. Carriers that supplement DLD separation do not necessarily bind specifically to the target cells, i.e. they may have to be combined with some other mediator that makes them specific, or they may simply be added to the cell preparation and allowed to bind non-specifically. The terms "in a manner that supplements DLD separation" and "in a manner that facilitates DLD separation" are not mutually exclusive. Binding can either supplement DLD separation or facilitate DLD separation. For example, a polysaccharide carrier can have an activator on its surface that increases the rate of cell growth, and the binding of one or more of these carriers can also facilitate DLD separation. Alternatively, binding may simply facilitate DLD separation or simply supplement DLD separation.

[0048] sample:As used herein, the term "sample" generally refers to any sample containing or suspected of containing nucleic acid molecules or cells. For example, a sample can be a biological sample containing one or more nucleic acid molecules or cells. A biological sample can be obtained from (e.g., extracted or separated) or include: blood (e.g., whole blood), plasma, serum, urine, saliva, mucosal excretions, sputum, feces, and tears. A sample can include blood, blood products (such as leukocyte apheresis or apheresis products), and the blood products also include anticoagulants (e.g., EDTA, EGTA, heparin, citrate, ACD-A, or thrombin inhibitors). A biological sample can be a fluid or tissue sample (e.g., a skin sample). In some examples, a sample is obtained from acellular body fluids, such as whole blood. In some examples, a sample can include circulating tumor cells. In some examples, a sample is an environmental sample (e.g., soil, waste, ambient air, etc.), an industrial sample (e.g., a sample from any industrial process), and a food sample (e.g., dairy products, vegetable products, and meat products). The sample can be processed before being loaded into the microfluidic device. The sample may suitably be an apheresis product or a leukapheresis product (eg, leukopak).

[0049] target cells As used herein, a "target cell" is a cell that is required or designed to be purified, collected, engineered, etc., for the various procedures described herein. What the specific cell is will depend on the context in which the term is used. For example, if the goal of the procedure is to isolate a particular type of stem cell, then that cell would be the target cell for the procedure.

[0050] Separation or purification : Unless otherwise indicated, these terms are used herein as synonyms and refer to the enrichment of a desired product relative to unwanted material. These terms do not necessarily imply that a product is completely isolated or completely pure. For example, if the target cells of the starting sample comprise 2% of the cells in the sample, and a procedure is performed that results in a composition in which the target cells comprise 60% of the cells present, then the procedure has successfully isolated or purified the target cells.

[0051] The term "obstacle array" is used synonymously herein and describes an ordered array of obstacles positioned in a flow channel through which cells or a fluid containing particles can pass. An obstacle array comprises a plurality of obstacles arranged in a column (along the path of fluid flow). Gaps are formed between the obstacles (along the path of fluid flow) that allow cells or other particles to pass through. Such an array or column can be arranged in one or more repeating rows (perpendicular to the path of fluid flow).

[0052] As used herein, a "channel" or "lane" refers to a plurality of obstacles arranged into discrete, separated units, such that a channel can be bounded on either side by walls, thereby separating the discrete lanes. Channels can extend in parallel from one or more common inputs to one or more common outputs. Channels can be fluidically connected in series.

[0053] 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 of microfluidic obstacles based on their size. This process can be used to separate cells, which is typically the case discussed herein. However, it is important to recognize that DLD can also be used to concentrate cells and for buffer exchange (see Figure 1). The process is typically described herein in terms of continuous flow (DC conditions; i.e., the bulk fluid flows in only one direction). However, DLD can also work under oscillatory flow (AC conditions; i.e., the bulk fluid flows alternately between two directions).

[0054] critical size The "critical size," "critical diameter," or "predetermined size" of a particle passing through an array of obstacles describes the size limit of particles that can follow the laminar flow of a fluid. Particles larger than the critical size may be "dislodged" from the flow path of the fluid, while particles smaller than the critical size (or predetermined size) will not be displaced.

[0055] Fluid Flow: As used herein in conjunction with DLD, the terms "fluid flow" and "bulk fluid flow" refer to the macroscopic movement of a fluid in a general direction through an array of obstacles. These terms do not take into account temporary displacements of the fluid flow that cause the fluid to bypass obstacles so that the fluid continues to move in the general direction.

[0056] 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 arrangement of successive rows of obstacles in the array (see Figure 5 ).

[0057] Array direction In an obstacle array device, the "array direction" is the direction defined by the arrangement of consecutive rows of obstacles in the array. If, after traversing the gap and encountering a downstream obstacle, a particle is "deflected" within the obstacle array, the particle's overall trajectory follows the array direction of the obstacle array (i.e., travels at an inclination angle ε relative to the bulk fluid flow). If the particle's overall trajectory follows the direction of the bulk fluid flow under these circumstances, the particle will not be dislodged.

[0058] about : As used herein, the term "about" refers to an amount that is within 10% of the stated amount.

[0059] General Overview

[0060] The present invention relates to microfluidic devices in which size-based purification is performed by passing a biological sample through an array of obstacles in a microfluidic channel. It is based in part on the concept that cells of a given size can be processed more quickly by lengthening the obstacle gap perpendicular to the direction of fluid flow and reducing the gap length parallel to the fluid flow.

[0061] The device characteristics discussed above can be achieved by a series of elongated shaped obstacles, with the most preferred obstacles being diamond or hexagonal. Hexagonal obstacles are most preferred because they offer the same processing advantages as diamonds, but result in a device that is easier to manufacture and more resistant to biofouling.

[0062] While using asymmetric gaps improves throughput and allows the device to operate longer, narrowing the parallel gaps can present practical problems in large-scale production of the device, particularly in molding, forming, or demolding. This problem can be reduced and the need for narrow gaps offset to some extent by using elongated polygonal obstacles that preferably have vertices pointing toward each other in parallel gaps, but with vertices offset from each other (rather than directly opposite each other, see Figures 6A-6B ). This design reduces the flow through the parallel gaps (also known as secondary flux) by making the gaps longer rather than narrower. In contrast, the vertices in the vertical gaps are preferably directly opposite each other. Thus, a key feature of the device disclosed herein is the presence of an array of obstacles in which the vertical and parallel gaps are asymmetric, i.e., they are of different sizes. By varying the spacing, flow resistance can be reduced compared to a device that separates particles and cells within the same size range but has vertical and parallel gaps of the same length.

[0063] For some samples, biofouling and fluid mixing may persist and affect separation as the sample is fed into the device. For example, biofouling at the array inlet may force blood or aliquots of sample to diffuse prematurely into the secondary fluid stream, leading to platelet and red blood cell contamination of the leukocyte target cell product. A partition wall positioned to separate the sample inlet from the inlets of other fluids and terminating midway through the channel can be used to isolate the biofouled area and temporarily prevent contact between the flow streams. As a result, the co-flowing fluids have limited diffusional mixing time, and purification of target cells or particles can be improved. Typically, the partition wall will extend anywhere from 10% to 50% of the length of the microfluidic channel from the sample inlet, but the wall can be shorter or longer depending on the circumstances relevant to the separation.

[0064] Another advantage of dividing walls is that they reduce unwanted mixing that can occur when a fluctuating pressure source is used to propel samples and other fluids through the device. For example, a peristaltic pump can be used to drive fluids through the device and has the advantage of maintaining a closed system environment—that is, the sample does not contact the interior of the pump but travels only through tubing squeezed by the pump head. However, peristalsis can produce regular pressure fluctuations, which tend to cause the flow streams to mix. When a dividing wall is present, it can act as a baffle against these fluctuations, limiting the unwanted mixing that would otherwise occur. As a result, improved separation should be achieved.

[0065] Another feature of this microfluidic device is that they can be used as a part for assembly, wherein two or more devices are stacked together and sample introduction is passed through a common manifold.Each stacked microfluidic device comprises a planar support with one or more embedded channels, and each channel comprises an independent obstruction array.Support member generally has a plurality of channels, and in some cases, these channels can embed the top surface and bottom surface of support member.On one or more devices in assembly, use a plurality of channels can carry out microfluidic processing to a large amount of samples.For example, the assembly of microfluidic device as herein described can be designed to process the sample (for example, undiluted single sample) greater than 100mL per hour, and this depends on specific treatment target, and larger volume (greater than 200,300,400 or 500mL per hour) is preferred.

[0066] In general, the devices of the present invention are characterized by some or all of the following: 1) asymmetrically arranged obstacles, wherein the gap perpendicular to the flow of the bulk fluid is of different length than the gap parallel to the flow of the bulk fluid; 2) elongated polygonal obstacles with vertices extending into the gap; 3) vertices on either side of the parallel gap that are offset relative to each other; 4) vertices on either side of the perpendicular gap that are preferably directly opposite each other; 5) one or more partition walls that separate the sample inlet from the inlet of other fluids and extend downward along the channel portion; 6) the optional use of peristaltic or other fluctuating pressure sources to propel samples and other fluids through the device having the partition walls; and 7) the assembly of multiple individual microfluidic devices into a stacked assembly, each device having multiple channels.

[0067] Overview of specific implementation plans

[0068] In a first aspect, the present invention relates to a microfluidic device for purifying target particles or target cells of a predetermined size from contaminants in a sample. The device has a planar support that is generally rectangular and can be made of any material compatible with the separation method, including silicon, glass, hybrid materials or (preferably) polymers. The support will have a top surface and a bottom surface, one or both of which have at least one embedded channel extending from one or more sample inlets and one or more different fluid inlets to one or more product outlets and one or more different waste outlets. The fluid inlet (as opposed to the sample inlet) may sometimes be referred to as a "buffer" or "wash" inlet and, depending on the goal of the separation, may be used to deliver various fluids into the channel. Unless otherwise indicated by usage or context, it should be understood that a "fluid" can be a buffer, contain reagents, constitute a growth medium for cells or generally any liquid and contain any components compatible with the operation of the device and the user's goals.

[0069] When a fluid is applied to the device through a sample or fluid inlet, it flows through the channel toward the outlet, thereby defining the direction of the bulk fluid flow. To separate cells or particles of different sizes, the channel includes an array of obstacles arranged in columns extending longitudinally along the channel (from the inlet to the outlet) and rows extending transversely across the channel. Each subsequent row of obstacles is shifted laterally relative to the previous row, thereby defining an array direction that deviates from the direction of bulk fluid flow by an inclination angle (ε). The obstacles are positioned to define a critical size so that when a sample is applied to the inlet of the device and flows toward the outlet, particles or cells in the sample that are larger than the critical size follow the array direction, and particles that are smaller than the critical size flow in the direction of bulk fluid flow, resulting in separation.

[0070] Adjacent obstacles in a row of the array are separated by a gap G1 that is perpendicular to the direction of bulk fluid flow, while adjacent obstacles in a column are separated by a gap G2 that is parallel to the direction of bulk fluid flow (see Figure 6A and 6B ). A feature of the present device is that the ratio of the size of gap G2 to the size of gap G1 is not equal to 1, and G1 is typically wider than G2 (e.g., 10-100% wider). Each obstacle in the array has at least two vertices and is positioned so that there is at least one vertex on either side of each gap. In preferred embodiments, the vertices extend into parallel gaps such that either side of the gap is flanked by one or more vertices that point toward each other but are not directly opposite, and / or the obstacles have vertices that extend into perpendicular gaps such that either side of the gap is flanked by vertices that point toward each other and are directly opposite (see Figure 6A and 6B ).

[0071] Microfluidic devices also typically have a barrier adhesive layer that is bonded to the surface of the planar support and to the barriers in the channels to prevent fluid or sample from flowing over the barriers during device operation. The barrier adhesive layer can include one or more passages that are fluidically connected to the inlet and outlet of the channel, allowing fluid flow.

[0072] In general, microfluidic devices are used to separate target particles or target cells that are larger than the critical size of the device from contaminants that are smaller than the critical size. When a sample containing target cells or particles is applied to the device through a sample inlet and fluidly passes through a channel, the target cells or target particles will flow to one or more product outlets, where a product enriched with target cells or target particles is obtained. The term "enrichment" as used in this context means that the ratio of target cells or particles to contaminants in the product is higher than the ratio in the sample. Contaminants that are smaller than the critical size will primarily flow to one or more waste outlets, where they can be collected or discarded.

[0073] Although the goal of separation is typically to separate target cells or particles from smaller contaminants, sometimes the user may wish to separate target cells or particles from larger contaminants. In these cases, a microfluidic device can be used that has a critical size larger than the target cells or particles but smaller than the contaminants. A combination of two or more obstacle arrays with different critical sizes, whether on a single device or on multiple devices, can also be used for separation. For example, a device can have a channel that has a first array of obstacles with a critical size larger than T cells but smaller than granulocytes and monocytes and a second array with a critical size smaller than T cells but larger than platelets and red blood cells. Processing a blood sample on such a device allows collection of a product in which T cells have been separated from granulocytes, monocytes, platelets, and red blood cells. The order of the obstacle arrays should not be important to the results, i.e., the array with the smaller critical size may be located before or after the array with the larger critical size. Arrays with different critical sizes can also be located on separate devices through which cells pass.

[0074] Wide arrays and multiple outlets can be used to collect multiple products, for example, monocytes can be obtained at one outlet, while T cells can be obtained at a different outlet. Thus, the use of multiple arrays and multiple outlets can allow for the simultaneous collection of several products in a purer form than if a single array were used. As discussed further below, high throughput can be maintained by using many devices stacked together.

[0075] Preferably, the obstacles used in the microfluidic device have a polygonal shape, preferably diamond or hexagonal obstacles. The obstacles are also typically elongated so that their length perpendicular to the bulk fluid flow (P1) is different from their width parallel to the bulk fluid flow (P2) (P1 is typically longer), for example, by 10-100% (see Figure 6B Typically, P1 will be at least 15%, 30%, 50%, 100%, or 150% longer than P2. Expressed as a range, P1 may be 10-150% (15-100%; or 20-70%) longer than P2.

[0076] The microfluidic device may also include a dividing wall in an array of obstacles extending from a sample inlet of the device into the channel where it separates the sample inlet from the fluid inlet and prevents mixing (see Figure 10A and 10B ). The dividing wall is oriented parallel to the direction of bulk fluid flow and extends toward the sample and fluid outlet. The wall terminates before reaching the end of the channel, allowing the sample and fluid streams to come into contact with each other thereafter. It should typically extend for at least 10% of the length of the obstacle array, but may extend for at least 20%, 40%, 60%, or 70% of the array. Expressed as a range, the wall will typically extend 10-70% of the length of the obstacle array. More than one dividing wall may also be present in the device and may be positioned in different ways depending on the purpose of the separation.

[0077] In order to increase the rate at which the volume can be processed, a stacked separation assembly can be made by covering the first microfluidic device with one or more stacked devices, wherein the bottom surface of each stacked device is in contact with the top surface of the first microfluidic device or the obstacle adhesive layer on the top surface, or with the top surface of another stacked device or the obstacle adhesive layer on the top surface. The sample is supplied to the sample inlet of all devices through a first common manifold, and the fluid is supplied to the fluid inlet through a second manifold that may be the same as or different from the first manifold. The product is removed from the product outlet through one or more product conduits, and the waste is removed from the waste outlet through one or more waste conduits that are different from the product conduits. Typically, the stacked separation assembly will have 2 to 9 stacked devices and the first microfluidic device. However, a larger number of devices can also be used. In addition, the top surface and / or bottom surface of the support can have multiple (e.g., 2-40 or 2-30) embedded channels and be used to purify target particles or target cells.

[0078] The stacked separation assembly can have a reservoir adhesive layer attached to the bottom surface of the first microfluidic device and / or the top surface of the stacked microfluidic device. The reservoir adhesive layer should include a first end having one or more passages having inlets that allow fluid to flow into the channels, and optionally, one or more passages at a second end opposite the first end that allow fluid to flow into or out of the channels, product outlets and waste outlets, the passages being separated by a fluid-impermeable material.

[0079] like Figure 9As shown, the stacked device assembly can be supported in a cassette, characterized in that there is a housing with a port that allows samples and fluids to be transported into the cassette and products and waste to be transported out of the cassette. The figure shows a cassette with two inlet ports and two outlet ports. However, multiple ports for entering and exiting the cassette can be used, and several products can be collected substantially at the same time. It will also be appreciated that the cassette can be part of a system in which there are components known and commonly used in the art. Such common components include pumps, valves, and processors for controlling fluid flow; sensors for monitoring system parameters (such as flow and pressure); sensors for monitoring fluid properties (such as pH or salinity); sensors for determining cell or particle concentrations; and analyzers for determining the type of cells or particles present in the cassette or in the material collected from the cassette. More generally, any device known in the art and compatible with the cassette, the material being processed, and the processing target can be used.

[0080] In another aspect, the present invention relates to a method for purifying target particles or cells of a predetermined size from contaminants, the method comprising obtaining a sample comprising target particles or cells and contaminants and performing the purification using any of the microfluidic devices or stacked separation assemblies discussed herein. Purification is accomplished by applying the sample to one or more sample inlets on any of the microfluidic devices discussed above, or to a sample inlet on a first microfluidic device or stacked device in a device assembly. A manifold can be used to apply the sample to the inlets, particularly when using a stacked device. The sample then flows through channels to the device outlets. Typically, the size of the target particles or cells will be larger than the critical size of the array of obstacles on the device, and at least some of the contaminants will be smaller than the critical size. As a result, the target cells or particles will flow to one or more product outlets, where a product enriched with the target cells or particles is obtained, while contaminants smaller than the critical size will flow to one or more waste outlets. However, as previously discussed, there may be situations where the target cells or particles are smaller than the contaminants, and a device with a critical size larger than the target cells or particles and smaller than the contaminants is selected. In these cases, the general operation of the device will remain essentially the same, but the contaminants will flow in the direction of the array, and the target cells or particles will proceed in the direction of the bulk fluid flow.

[0081] Sample can be obtained from individual or patient, especially the patient with cancer, autoimmune disease or infectious disease.In certain embodiment, sample is blood or derives from blood (for example, single sampling or leukocyte single sampling sample), and target cell is dendritic cell, leukocyte (especially T cell), stem cell, B-cell, NK-cell, monocyte or progenitor cell.In these cases, contaminant generally includes red blood cell and / or platelet.Purification should produce the product of enrichment target cell, and wherein in sample, at least 80% (preferably 90%, more preferably 95%) platelet and / or red blood cell have been removed.

[0082] Once purified target cells are obtained, they can be genetically engineered by transfecting or transducing them with recombinant nucleic acids. They can then optionally be expanded in culture and ultimately used to treat the patient from whom the sample was obtained.

[0083] Of particular interest is that the present invention includes a method for producing chimeric antigen receptor (CAR) T cells by: a) obtaining a sample including T cells; b) separating T cells from contaminants by applying the sample to one or more sample inlets on any microfluidic device or stacked device discussed herein; c) causing the sample to flow to the outlet of the device; and d) obtaining a product enriched with T cells from the product outlet. Once the T cells are recovered, they are genetically engineered, preferably by transfecting or transducing them with recombinant nucleic acids so that they express chimeric antigen receptors on their surfaces. The genetically engineered target cells are expanded by culturing cells in vitro and can be therapeutically administered to patients providing samples.

[0084] The sample containing T cells is preferably blood, apheresis products or leukocyte apheresis products from a patient with cancer, autoimmune disease or infectious disease or from an HLA-matched (matched to the patient to be treated) donor. The cells can be combined with one or more carriers in a manner that promotes or supplements DLD separation, and the cells or complexes can then be purified by DLD. The present invention includes manufactured CAR T cells and CAR T cell therapies using CAR T cells therein.

[0085] I. Design of Microfluidic Cassette

[0086] The present disclosure provides a microfluidic cartridge (i.e., a device, a chip, a cassette, a plate, a microfluidic device, a cartridge, a DLD device, etc.) for purifying particles or cells. The microfluidic cartridge of the present disclosure can be operated using a DLD method. The microfluidic cartridge of the present disclosure can be formed of a polymeric material (e.g., a thermoplastic) and can include one or more of a first planar support having a top surface and a bottom surface and a second planar support having a top surface and a bottom surface, wherein the top surface of the first and second planar supports includes at least one embedded channel extending from one or more inlets to one or more outlets; at least one embedded channel includes an array of obstacles, wherein the bottom surfaces of the first planar support and the second planar support include a void space, the void space being configured to deform when the bottom of the first planar support is pressed toward the bottom of the second planar support. The microfluidic cartridge of the present disclosure can be a single use or disposable device. Alternatively, the microfluidic cartridge can be a multiple use device. Using polymers (e.g., thermoplastics) to form microfluidic structures can allow for the use of inexpensive and highly scalable soft molding processes, while void spaces can provide improved rapid manufacturing capabilities and avoid damage to obstacles (i.e., pillars, DLD arrays, etc.) during the fabrication process.

[0087] The cartridges described herein can be operated by deterministic lateral displacement or DLD operation. Referring Figures 1A-1G , DLD can include three different operating modes. The operating modes include: i) separation ( Figure 1A ), ii) buffer exchange ( Figure 1B ) and iii) concentration ( Figure 1C ). In each mode, particles above the critical diameter are deflected from the entry point along the array direction, enabling size selection, buffer exchange or concentration according to the geometry of the device. In all cases, particles below the critical diameter pass directly through the device under laminar flow conditions and subsequently exit the device. Figure 1D Shows a 14-lane DLD design for the separation mode. The full length of the separation zone of the microfluidic cartridge can be approximately 75 mm and the width can be approximately 40 mm, with the width of each individual channel being approximately 1.8 mm. Figures 1E-1F Is an enlarged view of the merged collection port of the plastic diamond pillar array and the outlet. Figure 1G Depicts a leukapheresis product processed using the device at 10 PSI.

[0088] The cartridges described herein can be arranged in multiple orientations to achieve different DLD modes or product outcomes ( Figure 2 ). Figure 2 Shows four channels, with sidewalls (1) and an array of obstacles (2). A sample containing blood, cells or particles enters the channels through the sample inlet (3) at the top, and buffer, reagent or medium enters the channels at a separate fluid inlet (4). As they flow towards the bottom of the channels, cells or particles larger than the array critical diameter (>Dc) flow at an angle determined by the array direction of the obstacles and are separated from cells and particles smaller than the array critical diameter (<Dc).

[0089] Referring Figures 3A-3D , embodiments of the cartridge can include an arrangement of 14 parallel channels, which can be used in a microfluidic device or cartridge. Figures 3B-3D Shows an enlarged view of a section of the cartridge. In this figure, the channels have three zones (sections) with gradually decreasing gaps. The cartridge has a common sample inlet, e.g., for blood, which feeds the sample into the inlets on each channel. The buffer channels have separate inlets, but depending on the processing objective, they can be used to introduce fluids with reagents, growth medium or other fluids into the channels. At the bottom of each channel there is a product outlet, which is typically used to recover target cells or particles larger than the critical diameter of the obstacle array in the channel. The outlets of the individual channels feed into a common product outlet from which the target cells or particles can be recovered. A waste outlet is also shown, where cells and particles smaller than the critical diameter of the obstacle array in the channel are discharged.

[0090] Referring Figures 4A-4D , embodiments of the box may include 2 channels. Figures 4B-4D An enlarged view of a section of a channel is shown. The channel has three sections designed with obstacles and gaps of gradually decreasing diameter.

[0091] Some cartridges may have a "bump array" with equilateral triangle obstacles positioned in the microfluidic channels, e.g. Figure 5 As shown in a cross-sectional view. In the figure, the fluid flows from left to right, as shown by the arrow marked "fluid". In this array, equilateral triangular columns are arranged in a parallelogram lattice that is tilted relative to the direction of fluid flow. Other lattice arrangements (e.g., square, rectangular, trapezoidal, hexagonal, etc. lattices) can also be used. The tilt angle ∈ (epsilon) is selected so that the device is periodic. In this embodiment, the tilt angle of 18.4 degrees (1 / 3 radian) makes the device periodic after three rows. The tilt angle ε also represents the angle at which the array direction deviates from the direction of fluid flow. The gap between the columns is represented by G using the length of the equilateral triangle side S. Streamlines extending between the columns are shown, dividing the fluid flow between the columns into three regions ("flow tubes") of equal volume flow. When the fluid flows in the direction shown, relatively large particles (larger than the critical size of the array) follow the array tilt angle. Relatively small particles (smaller than the critical size of the array) follow the direction of fluid flow.

[0092] The cartridges provided herein may include arrays of diamond-shaped pillars, such as Figures 6A-6B As shown in . Figure 6A A symmetrical array of obstacles is shown, in which the gaps perpendicular to the direction of fluid flow (e.g., Gap 1 (G1)) and the gaps parallel to the direction of fluid flow (e.g., Gap 2 (G2)) are approximately the same length. Diamond-shaped obstacles may have two diameters, one perpendicular to the direction of fluid flow (P1) and the other parallel to the direction of fluid flow (P2). The right side of the figure shows an asymmetric array, in which the parallel gaps are shorter than the perpendicular gaps. Although G1 has been widened in the asymmetric array compared to the symmetric array, the reduction in gap G2 results in the critical diameter of the array being the same as the symmetric array. Therefore, these two arrays should be roughly equally effective in separating particles or cells of a given diameter in a sample. However, the widening of G1 allows for higher sample throughput and reduces channel clogging. Figure 6B On the left is shown an array of diamond-shaped obstacles that have been elongated so that their vertical diameter is longer than their horizontal diameter. The middle section of Figure 6 shows diamond-shaped columns that have been elongated so that their horizontal diameter is greater than their vertical diameter, and the rightmost section of the figure shows hexagonal obstacles that have been elongated horizontally.

[0093] refer to Figures 7A-7C, the cartridges described herein may comprise stacked discrete assemblies in which two microfluidic devices or cartridges are combined into a single unit. The topmost device (5) comprises a planar support (6) which may be made from a variety of materials but is most preferably a polymeric material and has a top surface (7) and a bottom surface (12). The top surface (7) of the support contains reservoirs which provide a sample inlet (9) and a buffer or other fluid inlet (10) at one end of the support and a product outlet (14) and a waste outlet (13) at the other end. Each reservoir is fluidically connected through the support using a small through hole (inside (9), (10), (13), (14)) which connects the top surface (7) to a channel on the bottom surface (12). The bottom surface (12) of the support has a number of embedded microfluidic channels (8) each having an array of obstacles connected by the channel (see Figures 1A-1C , 2, 3B-3D, 4B-4D, 5, 6A and 6B and 8B). The embedded microfluidic layer is bonded to an obstacle adhesive layer (15) that seals the first device and prevents fluid from flowing over the obstacle during operation. The second microfluidic device in the stack is shown as (16), which contains an embedded microfluidic channel on the topmost surface and is sealed by the same obstacle adhesive layer (15) as the topmost device. The reservoir adhesive layer (18) is also shown as having an elongated opening (19) that allows liquid to pass through the channel inlet and liquid to pass through the channel outlet. The reservoir adhesive layer is similar to the obstacle adhesive layer, except that it is attached to the surface of the device rather than the obstacle and can be connected to one or more reservoirs that feed the device stack or to a manifold. A hole (11) is shown for aligning the stacked devices. As described above, the two embedded microfluidic surfaces face the same obstacle adhesive layer. An alternative configuration is to have embedded channels on the top surface of both devices, with an intermediate layer between the devices that acts both as a barrier adhesion layer for the embedded channels below and as a distribution layer for the reservoirs above. Figure 7B A stack of multiple microfluidic devices forming a single assembled unit is shown. At the top of this stack (optionally at the top and bottom) is a manifold (22) having an inlet tube (23) for a manifold inlet distributor (24) and a conduit (28) leading from a manifold product outlet (27). An inlet tube leading to a fluid inlet (25) and a conduit for discharging fluid from a waste outlet (26) will also be present, but not shown. Figure 7C The stacked separation assembly (20) is shown installed in a housing (21).

[0094] The two channels that may exist in the device depicted in Figure 7 are also Figures 8A-8B Shown in. Figure 8BA magnified view of a section of a channel is shown in . In this example, the channel has an array of asymmetrically spaced diamond-shaped obstacles, where G1 is larger than G2. The diamonds are offset so that each successive row is laterally displaced relative to the previous row.

[0095] The present disclosure provides herein a stacked assembly of microfluidic devices (20) within a housing (21), which together may be referred to as a "cassette" ( Figure 9 ). Port (29) serves as an inlet for the sample, which is fed through the shell into the manifold (22). Port (29) is connected to the manifold inlet (23), which distributes the sample to the channel sample inlet via the manifold sample inlet (24). After application, the sample flows through a channel containing an array of obstacles (see Figures 3-6), and products with particles or cells larger than a critical size leave the stack at the manifold product outlet (27). The product then flows from the manifold outlet through the product conduit (28) and is transported out of the cassette via the product outlet port (31). The fluid flows into the cassette and flows to the manifold via port (51), which is connected to the manifold fluid inlet (49). It is distributed to the channel fluid inlet via the manifold fluid inlet (25). The fluid flows through the channel, and particles or cells smaller than the critical size leave the stack primarily via the manifold waste outlet (26). These particles or cells then flow through a waste conduit (50) which transports waste out of the cartridge through an outlet port (30).

[0096] One embodiment of a cartridge or device provided herein may include a channel defined by two walls (32) having a sample inlet (33) and a fluid inlet (34) ( Figure 10A -B). A partition wall (35) is present to prevent the sample flow stream from mixing with the fluid flow stream. The partition wall extends into the obstacle array (36) and ends approximately halfway. The arrows in the array show the direction of travel of target cells whose size is larger than the critical size of the array. Initially, after entering the obstacle array, the target cells turn away from the direction of fluid flow until they reach the partition wall. They then travel along the wall until the wall ends. Thereafter, they continue to turn away until they exit the channel at the product outlet (37). Particles whose size is smaller than the critical size of the obstacle array do not turn away and exit the channel at the waste outlet (38). Figure 10BAlso shown is a channel defined by a wall (43) having a sample inlet (39), a reagent inlet (40), and an inlet (42) for a buffer or other fluid. The sample enters at the inlet and flows onto the obstacle array (44). There, particles or cells larger than the array's critical diameter are diverted into the reagent stream, where they react. A partition wall (41) extends downward from the reagent inlet along the obstacle array (44) portion and separates the reagent stream from the buffer stream or other fluid stream. The wall holds the cells or particles in the reagent stream for a longer time, thereby providing more time for the reaction. At the end of the partition wall, the particles or cells continue to divert to the product outlet (48), where they can be collected. In this process, the cells or particles are separated from the unreacted reagent. A second partition wall (45) extends from the end of the first partition wall (41) to a waste outlet (47), where the buffer or other fluid, reagent, and small particles or cells can leave the device and can be collected or discarded. A second waste outlet (46) is used to remove reagents, fluids in which particles or cells are suspended in the sample, and particles or cells smaller than the obstacle array's critical diameter. These materials can be recycled or discarded.

[0097] The normalized velocity flow between two equilateral triangular cylinders (left panel) and the normalized velocity flow between two circular cylinders (right panel) can be compared ( Figure 11 ), demonstrating the influence of the obstacle or column shape. Figure 11 The shaded portion of represents an equal proportion of the area under the curve, demonstrating that the critical radius of particles flowing through the points of the triangle (<15% gap width) is significantly smaller than that of particles flowing through the cylinder (>20% gap width).

[0098] Figure 12 is a plot of the predicted critical diameter versus the array tilt angle (ε) for triangular (lower line) and circular (upper line) obstacle arrays. Figure 12 The analysis further proves that the column shape plays an important role in Figure 11 The effects on particle or cell displacement are shown in .

[0099] refer to Figure 13 , we can show the effect of the tilt angle ("array tilt" in the figure) on the gap length G. T Refers to the gap length between triangular columns, G C Refers to the gap length between the circular pillars. As the array inclination increases, the array specific critical dimension (D C )The required gap length difference is reduced.

[0100] Figure 14The effect of obstacle edge roundness (expressed as r / S) on the critical dimension exhibited on one side of the gap defined by the edge is shown. For a given gap length, increasing the roundness of the pillars increases the critical dimension value of the pillars.

[0101] In addition to the critical size, different shapes of pillars may also affect the particle velocity under constant applied pressure. Figure 15 The effect of applied pressure on particle velocity in a bump array with triangular pillars (data shown as triangles) and a bump array with circular pillars (data shown as circles) is shown. Given an applied pressure, the array with triangular pillars results in a greater particle velocity than the array with circular pillars. Furthermore, the rate at which particle velocity increases with increasing pressure is greater in the triangular pillar array than in the circular pillar array.

[0102] refer to Figure 16A , the cartridge described herein includes a seal / lid 1600 at the top and / or bottom and a separation layer 1605 including a plurality of barriers 1620 to facilitate separation, a fluidic layer 1610, and a void space or crumple zone that allows the cartridge to be manufactured without deforming the plurality of barriers. Figure 16B , multiple obstacles 1620 can be arranged in rows 1625 and columns 1630 such that gaps 1635 are configured to allow passage of fluids and cells. The obstacles can be arranged so that they are stacked with no or minimal offset between repeated rows. Referring to Figures 17A to C, two or more cartridges can be stacked or connected in series or in parallel to achieve greater separation or higher throughput.

[0103] Because similar devices or microfluidic cartridges operate on the submillimeter scale and process microliter, nanoliter, or smaller amounts of fluid, a major hurdle in manufacturing is avoiding damage or deformation of the obstacles during the molding or assembly process. For example, the handling of the chip may cause stress on the planar supports, especially when the planar supports are pressed together, which may result in deformation or damage to one or more of the planar supports, the obstacles (i.e., the obstacle array), and the various separation lanes. Such deformation or damage may result in a significant loss of performance in purifying particles or cells, or may completely impair the function of the microfluidic cartridge. To avoid potential deformation and defects during manufacturing and assembly, other microfluidic systems require slower manufacturing runs or accept reduced performance.

[0104] In one aspect, the present disclosure provides a microfluidic cartridge for purifying cells or particles. The microfluidic cartridge may include a first planar support member. The first planar support member may include a top surface and a bottom surface. The device may include a second planar support member. The second planar support member may include a top surface and a bottom surface. The top surface may include at least one embedded channel extending from one or more inlets to one or more outlets. At least one embedded channel may include an array of obstacles. The bottom surfaces of the first planar support member and the second planar support member may include a void space. The void space may be configured to deform when the bottom of the first planar support member is pressed against the bottom of the second planar support member.

[0105] Separation according to the present description occurs along a channel embedded in a planar support that includes a plurality of obstacles. For the cartridges of the present description, a first and a second planar surface can be used. The first and second planar surfaces can be stacked (e.g., bottom to bottom or top to bottom, with spacers that double the flux and separation capability while maintaining a small footprint. The top surface of the first and / or second planar surface can include at least 1 embedded channel to about 500 embedded channels. The top surface can include at least 1 embedded channel to about 2 embedded channels, 1 embedded channel to about 5 embedded channels, 1 embedded channel to about 20 embedded channels, 1 embedded channel to about 50 embedded channels, 1 embedded channel to about 10 ... The number of embedded channels is from about 2 to about 500 embedded channels, from about 2 to about 5 embedded channels, from about 2 to about 20 embedded channels, from about 2 to about 50 embedded channels, from about 2 to about 100 embedded channels, from about 2 to about 500 embedded channels, from about 5 to about 20 embedded channels, from about 5 to about 50 embedded channels, from about 5 to about 100 embedded channels, from about 5 to about 500 embedded channels, from about 20 to about 50 embedded channels, The top surface may include at least 1 embedded channel, about 2 embedded channels, about 5 embedded channels, about 20 embedded channels to about 100 embedded channels, about 20 embedded channels to about 500 embedded channels, about 50 embedded channels to about 100 embedded channels, about 50 embedded channels to about 500 embedded channels, or about 100 embedded channels to about 500 embedded channels. The top surface may include at least 1 embedded channel, about 2 embedded channels, about 5 embedded channels, about 20 embedded channels, about 50 embedded channels, about 100 embedded channels, or about 500 embedded channels. The top surface may include at least 1 embedded channel, about 2 embedded channels, about 5 embedded channels, about 20 embedded channels, about 50 embedded channels, about 100 embedded channels, or about 500 embedded channels. In some embodiments, the top surface of the present invention can include at least about 2 embedded channels, about 5 embedded channels, about 20 embedded channels, about 50 embedded channels or about 100 embedded channels. The top surface can include at least about 2 embedded channels, about 5 embedded channels, about 20 embedded channels, about 50 embedded channels, about 100 embedded channels or about 500 embedded channels. The top surface or the first or second planar surface can include about 28 channels (56 when stacked). The other third, fourth, fifth or sixth planar surface can also include the embedded channels of similar number to the first or second planar surface.

[0106] The microfluidic box can include at least 1 inlet to about 50 inlets. The microfluidic box can include at least 1 inlet to about 2 inlets, 1 inlet to about 5 inlets, 1 inlet to about 10 inlets, 1 inlet to about 20 inlets, 1 inlet to about 50 inlets, about 2 inlets to about 5 inlets, about 2 inlets to about 10 inlets, about 2 inlets to about 20 inlets, about 2 inlets to about 50 inlets, about 5 inlets to about 10 inlets, about 5 inlets to about 20 inlets, about 5 inlets to about 50 inlets, about 10 inlets to about 20 inlets, about 10 inlets to about 50 inlets, or about 20 inlets to about 50 inlets. The microfluidic box can include at least 1 inlet, about 2 inlets, about 5 inlets, about 10 inlets, about 20 inlets, or about 50 inlets. The microfluidic box can include at least 1 inlet, about 2 inlets, about 5 inlets, about 10 inlets, about 20 inlets, or about 50 inlets. The microfluidic box can include at least 1 inlet, about 2 inlets, about 5 inlets, about 10 inlets, or about 20 inlets. The microfluidic cartridge can include at least up to about 2 inlets, about 5 inlets, about 10 inlets, about 20 inlets, or about 50 inlets. The inlets can be fed by a common fluid system or a dual fluid system (one for buffer / diluent and one for sample).

[0107] The microfluidic cartridge may include at least 1 outlet to about 50 outlets. The microfluidic cartridge may include at least 1 outlet to about 2 outlets, 1 outlet to about 5 outlets, 1 outlet to about 10 outlets, 1 outlet to about 20 outlets, 1 outlet to about 50 outlets, about 2 outlets to about 5 outlets, about 2 outlets to about 10 outlets, about 2 outlets to about 20 outlets, about 2 outlets to about 50 outlets, about 5 outlets to about 10 outlets, about 5 outlets to about 20 outlets, about 5 outlets to about 50 outlets, about 10 outlets to about 20 outlets, about 10 outlets to about 50 outlets, or about 20 outlets to about 50 outlets. The microfluidic cartridge may include at least 1 outlet, about 2 outlets, about 5 outlets, about 10 outlets, about 20 outlets, or about 50 outlets. The microfluidic cartridge may include at least 1 outlet, about 2 outlets, about 5 outlets, about 10 outlets, or about 20 outlets. The microfluidic cartridge can include at least up to about 2 outlets, about 5 outlets, about 10 outlets, about 20 outlets, or about 50 outlets. The outlets can feed a common fluid system or a dual fluid system (one for waste and one for enriched target cells or particles).

[0108] A cartridge comprising two or more planar surfaces may include void spaces to protect arrays of obstacles in the lanes, as their small size may cause them to be susceptible to deformation, leading to malfunction.

[0109] The void space of the microfluidic cartridge can be configured to deform, bend, expand, collapse, or wrinkle. The void space can be configured to protect an obstacle, channel, inlet, outlet, planar surface, or any combination thereof from damage, displacement, deformation, or failure. The void space can include a wrinkle region configured to protect an obstacle, channel, inlet, outlet, planar surface, or any combination thereof from damage, displacement, deformation, or failure. The void space can have a volume of about 1 cubic micron to about 10,000 cubic microns. The void spaces may have a volume of about 1 cubic micron to about 5 cubic microns, about 1 cubic micron to about 10 cubic microns, about 1 cubic micron to about 30 cubic microns, about 1 cubic micron to about 50 cubic microns, about 1 cubic micron to about 100 cubic microns, about 1 cubic micron to about 300 cubic microns, about 1 cubic micron to about 1,000 cubic microns, about 1 cubic micron to about 3,000 cubic microns, about 1 cubic micron to about 10,000 cubic microns, about 5 cubic microns to about 10 cubic microns, about 5 cubic microns to about 30 cubic microns, about 5 cubic microns to about 50 cubic microns, about 5 cubic microns to about 100 cubic microns, about 5 cubic microns to about 300 cubic microns, about 5 cubic microns to about 1,000 cubic microns, about 5 cubic microns to about 3,000 cubic microns, about 5 cubic microns to about 10,000 cubic microns, about 10 cubic microns to about 30 cubic microns, about 10 cubic microns to about 50 cubic microns, about 10 cubic microns to about 100 cubic microns, about 10 cubic microns to about 300 cubic microns, about 10 cubic microns to about 1,000 cubic microns, about 10 cubic microns to about 3,000 cubic microns, about 10 cubic microns to about 10,000 cubic microns, About 30 cubic microns to about 50 cubic microns, about 30 cubic microns to about 100 cubic microns, about 30 cubic microns to about 300 cubic microns, about 30 cubic microns to about 1,000 cubic microns, about 30 cubic microns to about 3,000 cubic microns, about 30 cubic microns to about 10,000 cubic microns, about 50 cubic microns to about 100 cubic microns, about 50 cubic microns to about 300 cubic microns, about 50 cubic microns to about 1,000 cubic microns, about 50 cubic microns to about 3,000 cubic microns, about 50 cubic microns to about 10,000 cubic microns, about 100 cubic microns The volume of the present invention is from about 100 cubic microns to about 300 cubic microns, from about 100 cubic microns to about 1,000 cubic microns, from about 100 cubic microns to about 3,000 cubic microns, from about 100 cubic microns to about 10,000 cubic microns, from about 300 cubic microns to about 1,000 cubic microns, from about 300 cubic microns to about 3,000 cubic microns, from about 300 cubic microns to about 10,000 cubic microns, from about 1,000 cubic microns to about 3,000 cubic microns, from about 1,000 cubic microns to about 10,000 cubic microns, or from about 3,000 cubic microns to about 10,000 cubic microns.The void space may have a volume of about 1 cubic micron, about 5 cubic microns, about 10 cubic microns, about 30 cubic microns, about 50 cubic microns, about 100 cubic microns, about 300 cubic microns, about 1,000 cubic microns, about 3,000 cubic microns, or about 10,000 cubic microns. The void space may have a volume of at least about 1 cubic micron, about 5 cubic microns, about 10 cubic microns, about 30 cubic microns, about 50 cubic microns, about 100 cubic microns, about 300 cubic microns, about 1,000 cubic microns, or about 3,000 cubic microns. The void space may have a volume of at most about 5 cubic microns, about 10 cubic microns, about 30 cubic microns, about 50 cubic microns, about 100 cubic microns, about 300 cubic microns, about 1,000 cubic microns, about 3,000 cubic microns, or about 10,000 cubic microns. The void space may be about X cubic microns.

[0110] refer to Figure 18A , which shows a non-limiting view of the bottom surface 1812 of the planar support 1806 of the present disclosure. The bottom surface can include a plurality of void spaces 1815, which are shown here arranged in strips extending parallel to the length of the planar support. The void spaces extend below the swim lanes formed by the array or columns of obstacles (not shown) or by the columns of obstacles (not shown) fabricated on the top surface of the planar support. Figure 18B , showing a cross-sectional view of a planar support 1806. The top surface 1807 of the planar support includes a plurality of individual obstacles 1820 formed into an array or column, forming gaps 1835 to allow the flow of fluids, cells and / or particles. Below the obstacles embedded in the bottom surface 1812 of the planar support is a void space 1815. The area (length x width) of the void space opposite the swimming lane can be at least about 80% of the area (length x width) of the swimming lane. In certain embodiments, the area (length x width) of the void space opposite the swimming lane can be at least about 90%, 100%, 110% or 120%, up to and including about 150%, of the area (length x width) of the swimming lane.

[0111] In one configuration, the void spaces between the two planar supports are symmetrical or nearly symmetrical. Figure 16A However, an alternative arrangement is shown in FIG19 . In such a case, the supports are not compacted back to back but are stacked, with the void space being above (as shown in FIG19A ) or below (as shown in FIG19B ) the obstacle layer.

[0112] The void space can be divided into two or more void spaces. The void space can be divided into at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 void spaces. The void space can be divided into exactly two void spaces. For each planar support including an obstacle, the ratio between the channel or lane and the void space can be 1:1.

[0113] The planar support member can be made of two layers of material bonded together. The layers can be bonded together using an adhesive, a polymer, or a thermoplastic. The layers can include a polymer or thermoplastic. The polymer or thermoplastic layer or the bonding material can include high-density polyethylene (HDPE), polypropylene (PP), polyethylene terephthalate (PT), polycarbonate (PC), or cyclic olefin copolymer (COC).

[0114] The top layer of the box can include an array of obstacles in at least one embedded channel, void space, at least one inlet, at least one outlet, or a combination thereof. The bottom layer of the box can include an array of obstacles in at least one embedded channel, void space, at least one inlet, at least one outlet, or a combination thereof. These layers can be positioned so that the planar supports are bonded together on their side surfaces, bottom surfaces, or top surfaces. The void space can be within the interface of the bonded planar supports, or outside the interface.

[0115] The microfluidic box can also include an obstacle adhesive layer that is bonded to the surface of the planar support and the top surface of the obstacle array embedded in the channel to prevent fluid or sample from flowing over the obstacle array during box operation. The obstacle adhesive layer can be metal, polymer or thermoplastic. The obstacle adhesive layer can be a covering or a film. The polymer or thermoplastic layer or adhesive material can include high-density polyethylene (HDPE), polypropylene (PP), polyethylene terephthalate (PT), polycarbonate (PC) or cyclic olefin copolymer (COC). The microfluidic box can include two obstacle adhesive layers located outside the top planar support. The microfluidic box can include a single obstacle adhesive layer located in the middle of the box as an adhesive medium for the planar support. The obstacle adhesive layer can include one or more passages connected to one or more inlet fluids embedded in the channel that allow the sample to flow into the channel and one or more passages connected to one or more outlet fluids of the channel that allow fluid to flow out from one or more outlets. Such an obstacle layer may include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 50, or at least about 100 passages fluidly connected to one or more inlets or one or more outlets of the embedded channel.

[0116] A microfluidic cartridge may have obstacles positioned to define a critical size for the cartridge, such that when a sample is applied to an inlet of the cartridge and flows toward an outlet, particles or cells in the sample larger than the critical size are separated from particles or cells in the sample smaller than the critical size. Each obstacle may have its own individual subcritical size, with the sum of the obstacles defining the critical size of the cartridge. The one or more outlets of the cartridge may include at least one product outlet, into which target particles or cells larger than the critical size of the cartridge are directed. The one or more outlets of the cartridge may include at least one product outlet, into which target particles or cells smaller than the critical size of the cartridge are directed. The cartridge may have at least about 1, at least about 2, at least about 3, at least about 5, at least about 10, or at least about 50 product outlets. The one or more outlets may include at least one waste outlet. Contaminants, particles, or cells smaller than the critical size may flow toward the at least one waste outlet. Contaminants, particles, or cells larger than the critical size may flow toward the at least one waste outlet. The cartridge may have at least about 1, at least about 2, at least about 3, at least about 5, at least about 10, or at least about 50 waste outlets.

[0117] The obstacles used in the box can be cylindrical or triangular, square, rectangular, diamond, trapezoidal, hexagonal, teardrop-shaped, circular, semicircular, triangular with horizontal top side and triangular with horizontal bottom side. In addition, adjacent obstacles can have such a geometric shape that the obstacle portion defining the gap is symmetrical or asymmetrical about the gap axis extending in the direction of the main fluid flow. The obstacles can have vertices extending into parallel gaps, so that the gap is flanked by one or more vertices pointing to each other but not directly opposite each other. The obstacles can have vertices extending into perpendicular gaps, so that the gap is flanked by vertices pointing to each other and directly opposite each other on either side. The position and shape of the obstacles can vary within a single chip. Additional obstacles can be added to any position of the device according to any specific requirements. In addition, the shape of the obstacles in the device can be different. Any combination of column shapes, sizes and positions can be used to meet specific requirements. The box can include only diamond or hexagonal obstacles.

[0118] The obstacle shapes may be elongated perpendicular to the direction of fluid flow such that they have a horizontal length (P1) that is different from their vertical length (P2).P1 may have a length of about 1 μm to about 160 μm. P1 can have a diameter of about 1 μm to about 10 μm, about 1 μm to about 15 μm, about 1 μm to about 30 μm, about 1 μm to about 40 μm, about 1 μm to about 80 μm, about 1 μm to about 160 μm, about 10 μm to about 15 μm, about 10 μm to about 30 μm, about 10 μm to about 40 μm, about 10 μm to about 80 μm, about 10 μm to about 160 μm, about 15 μm to about 30 μm, about 15 μm to about 40 μm, about 15 μm to about 80 μm, about 15 μm to about 160 μm, about 30 μm to about 40 μm, about 30 μm to about 80 μm, about 30 μm to about 160 μm, about 40 μm to about 80 μm, about 40 μm to about 160 μm, or about 80 μm to about 160 μm. P1 may have a length of about 1 μm, about 10 μm, about 15 μm, about 30 μm, about 40 μm, about 80 μm, or about 160 μm. P1 may have a length of at least about 1 μm, about 10 μm, about 15 μm, about 30 μm, about 40 μm, or about 80 μm. P1 may have a length of at most about 10 μm, about 15 μm, about 30 μm, about 40 μm, about 80 μm, or about 160 μm. P2 may have a length of about 1 μm to about 160 μm. P2 may have a length of about 1 μm to about 10 μm, about 1 μm to about 15 μm, about 1 μm to about 30 μm, about 1 μm to about 40 μm, about 1 μm to about 80 μm, about 1 μm to about 160 μm, about 10 μm to about 15 μm, about 10 μm to about 30 μm, about 10 μm to about 40 μm, about 10 μm to about 80 μm, about 10 μm to about 160 μm, about 15 μm to about 30 μm, about 15 μm to about 40 μm, about 15 μm to about 80 μm, about 15 μm to about 160 μm, about 30 μm to about 40 μm, about 30 μm to about 80 μm, about 30 μm to about 160 μm, about 40 μm to about 80 μm, about 40 μm to about 160 μm, or about 80 μm to about 160 μm. P2 may have a length of about 1 μm, about 10 μm, about 15 μm, about 30 μm, about 40 μm, about 80 μm, or about 160 μm. P2 may have a length of at least about 1 μm, about 10 μm, about 15 μm, about 30 μm, about 40 μm, or about 80 μm. P2 may have a length of at most about 10 μm, about 15 μm, about 30 μm, about 40 μm, about 80 μm, or about 160 μm. P1 may be about 25% to about 200% longer than P2.P1 can be about 25% to about 50%, about 25% to about 75%, about 25% to about 100%, about 25% to about 150%, about 25% to about 200%, about 50% to about 75%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 75% to about 100%, about 75% to about 150%, about 75% to about 200%, about 100% to about 150%, about 100% to about 200%, or about 150% to about 200% longer than P2. P1 can be about 25%, about 50%, about 75%, about 100%, about 150%, or about 200% longer than P2. P1 can be at least about 25%, about 50%, about 75%, about 100%, or about 150% longer than P2. PI can be up to about 50%, about 75%, about 100%, about 150%, or about 200% longer than P2.

[0119] The microfluidic cartridge can include obstacles as obstacle arrays. The obstacles can be arranged in columns and rows to form a discrete array. The obstacle array can include at least about 5 columns to about 50 columns. The obstacle array can include at least about 5 columns to about 10 columns, about 5 columns to about 28 columns, about 5 columns to about 29 columns, about 5 columns to about 30 columns, about 5 columns to about 50 columns, about 10 columns to about 28 columns, about 10 columns to about 29 columns, about 10 columns to about 30 columns, about 10 columns to about 50 columns, about 28 columns to about 29 columns, about 28 columns to about 30 columns, about 28 columns to about 50 columns, about 29 columns to about 30 columns, about 29 columns to about 50 columns, or about 30 columns to about 50 columns. The obstacle array can include at least about 5 columns, about 10 columns, about 28 columns, about 29 columns, about 30 columns, or about 50 columns. The obstacle array can include at least about 5 columns, about 10 columns, about 28 columns, about 29 columns, or about 30 columns. The obstacle array may include at least up to about 10 rows, about 28 rows, about 29 rows, about 30 rows, or about 50 rows. The obstacle array may include at least about 20 rows to about 500 rows. The obstacle array may include at least about 20 rows to about 30 rows, about 20 rows to about 60 rows, about 20 rows to about 100 rows, about 20 rows to about 200 rows, about 20 rows to about 500 rows, about 30 rows to about 60 rows, about 30 rows to about 100 rows, about 30 rows to about 200 rows, about 30 rows to about 500 rows, about 60 rows to about 100 rows, about 60 rows to about 200 rows, about 60 rows to about 500 rows, about 100 rows to about 200 rows, about 100 rows to about 500 rows, or about 200 rows to about 500 rows. The obstacle array may include at least about 20 rows, about 30 rows, about 60 rows, about 100 rows, about 200 rows, or about 500 rows. The obstacle array can include at least about 20 rows, about 30 rows, about 60 rows, about 100 rows, or about 200 rows. The obstacle array can include at least about 30 rows, about 60 rows, about 100 rows, about 200 rows, or about 500 rows. Multiple obstacle arrays can be arranged in discrete lanes. The obstacle arrays of the first planar support or the second planar support can form about 10 to about 50 lanes. The array of obstacles on the first planar support or the second planar support forms about 10 swimming lanes to about 20 swimming lanes, about 10 swimming lanes to about 28 swimming lanes, about 10 swimming lanes to about 30 swimming lanes, about 10 swimming lanes to about 50 swimming lanes, about 20 swimming lanes to about 28 swimming lanes, about 20 swimming lanes to about 30 swimming lanes, about 20 swimming lanes to about 50 swimming lanes, about 28 swimming lanes to about 30 swimming lanes, about 28 swimming lanes to about 50 swimming lanes, or about 30 swimming lanes to about 50 swimming lanes. The array of obstacles on the first planar support or the second planar support forms about 10 swimming lanes, about 20 swimming lanes, about 28 swimming lanes, about 30 swimming lanes, or about 50 swimming lanes. The array of obstacles on the first planar support or the second planar support forms at least about 10 swimming lanes, about 20 swimming lanes, about 28 swimming lanes, or about 30 swimming lanes.The array of obstacles of the first planar support or the second planar support forms up to about 20 lanes, about 28 lanes, about 30 lanes, or about 50 lanes.

[0120] In one embodiment, the present invention provides a plurality of barrier surfaces, and each barrier surface comprises a plurality of barrier surfaces. Each box can comprise at least one group, at least two groups, at least three groups or at least four groups of barrier arrays. Each flat top surface can comprise at least one or at least two arrays. The box can comprise approximately 20 swimming lanes to approximately 100 swimming lanes altogether. The box can comprise approximately 20 swimming lanes to approximately 40 swimming lanes, approximately 20 swimming lanes to approximately 56 swimming lanes, approximately 20 swimming lanes to approximately 60 swimming lanes, approximately 20 swimming lanes to approximately 100 swimming lanes, approximately 40 swimming lanes to approximately 56 swimming lanes, approximately 40 swimming lanes to approximately 60 swimming lanes, approximately 40 swimming lanes to approximately 100 swimming lanes, approximately 56 swimming lanes to approximately 60 swimming lanes, approximately 56 swimming lanes to approximately 100 swimming lanes or approximately 60 swimming lanes to approximately 100 swimming lanes. The box can comprise approximately 20 swimming lanes, approximately 40 swimming lanes, approximately 56 swimming lanes, approximately 60 swimming lanes or approximately 100 swimming lanes altogether. The cassette may comprise a total of at least about 20 lanes, about 40 lanes, about 56 lanes, or about 60 lanes. The cassette may comprise a total of at most about 40 lanes, about 56 lanes, about 60 lanes, or about 100 lanes.

[0121] The inlet, outlet, or both of the microfluidic cartridge can be fluidically connected to a pump or motor to drive fluid flow inside and outside the cartridge. The inlet, outlet, or both can be fluidically connected to at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 pumps. The pumps can be peristaltic pumps. The pumps can be fluidically connected or isolated from each other. The inlet and outlet of the cartridge can be fluidically connected to two peristaltic pumps connected in parallel. The inlet and outlet of the cartridge can be fluidically connected to two peristaltic pumps connected in series.

[0122] The microfluidic cartridge can be made of metal, polymer, or thermoplastic. The polymer or thermoplastic can include high-density polyethylene (HDPE), polypropylene (PP), polyethylene terephthalate (PT), polycarbonate (PC), or cyclic olefin copolymer (COC). In one example, the microfluidic cartridge includes cyclic olefin copolymer.

[0123] The present disclosure also provides a microfluidic assembly comprising a plurality of fluidically connected microfluidic cartridges. The cartridges in the assembly can be stacked or layered. The plurality of microfluidic cartridges can include at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 30 cartridges. The plurality of cartridges can be fluidically connected in series or in parallel.

[0124] Cells, for example, in a composition prepared by apheresis or leukapheresis, can be separated by DLD using a microfluidic cartridge having a channel through which a fluid flows from an inlet at one end to an outlet at the other 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 hereby incorporated herein in their entirety) and are also summarized in the following sections.

[0125] During DLD, a fluid sample containing cells is introduced into the device at the inlet and carried to the outlet along with the fluid flowing through the device. As cells in the sample traverse the device, they encounter posts or other obstacles positioned to create gaps or pores through which the cells must pass. Each successive row of obstacles is displaced relative to the previous row to create an array orientation that differs from the direction of fluid flow in the flow channel. The "tilt angle" defined by these two orientations, along with the width of the gaps between obstacles, the shape of the obstacles, and the orientation of the obstacles forming the gaps, are the primary factors determining the array's "critical size." Cells larger than the critical size travel in the array direction rather than in the direction of the bulk fluid flow, while particles smaller than the critical size travel in the direction of the bulk fluid flow. In devices used to produce leukocyte apheresis-derived compositions, array features can be selected to cause leukocytes to be diverted in the array direction while red blood cells and platelets continue in the direction of bulk fluid flow. To separate selected leukocyte types from other leukocytes of similar size, a carrier can then be used that binds to the cells in a manner that facilitates DLD separation, resulting in complexes larger than uncomplexed leukocytes. Separation can then be performed on a device with a critical size that is smaller than the complexes but larger than the uncomplexed cells.

[0126] II. Fabrication and Operation of Microfluidic Devices

[0127] General procedures for making and using microfluidic devices capable of separating cells based on size are well known in the art. Such devices include 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 may be helpful in making and using the devices of 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; US 2007 / 0026415; US 2008 / 0113358; and WO 2012094642, each of which is also incorporated herein by reference in its entirety. Of the various references describing the manufacture and use of devices, US 7,150,812 provides particularly good guidance, while 7,735,652 is particularly concerned with microfluidic devices for separating samples having cells found in blood (see also US 2007 / 0160503 in this regard).

[0128] The device can be fabricated using any material commonly used to manufacture micro- and nanoscale fluid handling devices, including silicon, glass, plastic, and hybrid materials. 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 customized for specific applications. In one aspect, the microfluidic cartridge can be fabricated using soft molding and UV light curing.

[0129] Microfluidic boxes (or devices, cassettes, chips, etc.) can be manufactured by the following techniques, including replica molding, soft lithography using PDMS, thermosetting polyesters, molding, soft molding, hot molding, roll-to-roll molding, injection molding, laser ablation, UV light curing, and combinations thereof. More detailed information 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 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 manufacturing methods, which is incorporated herein by reference in its entirety.

[0130] High-throughput molding methods, such as reel-to-reel processing of thermoplastics, are an attractive approach for industrial microfluidic chip production. Using single-chip hot-molding technology can be a cost-effective technique for achieving high-quality microfluidic devices in the prototyping stage. Yang et al., "Microfluidic device fabrication by thermoplastic hot-embossing," Methods Mol. Biol. 949: 115-23 (2013), describe a method for replicating microscale features in two thermoplastics, polymethyl methacrylate (PMMA) and / or polycarbonate (PC), which is incorporated herein by reference in its entirety.

[0131] The flow channel can be constructed using two or more components that, when assembled, form a closed cavity (preferably with an orifice for adding or removing fluid) in which the obstruction is positioned. The obstruction can be fabricated on one or more components that assemble into the flow channel, or it can be fabricated as an insert sandwiched between two or more components that define the boundaries of the flow channel.

[0132] The obstacles can be solid objects that extend transversely across the flow channel in an array and longitudinally along the channel from the inlet to the outlet. When an obstacle is integral with (or extends from) one face of the flow channel at one end of the obstacle, the other end of the obstacle can seal against or press against the opposite face of the flow channel. A small space (preferably too small to accommodate any particles of interest for the intended use) can be tolerated between one end of the obstacle and the face of the flow channel, as long as the space does not adversely affect the structural stability of the obstacle or the relevant flow characteristics of the device.

[0133] Surfaces can be coated to alter their properties, and the polymer materials used to make devices can be modified in a variety of ways. In some cases, functional groups such as amines or carboxylic acids, present in natural polymers 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. By using surfactants Addition to PDMS formulations renders the surface hydrophilic and protein repellent. 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 alter its contact angle.

[0134] In order to reduce the non-specific adsorption of cells or compounds (for example, released by lysed cells or found in biological samples) on the channel wall, one or more walls can be chemically modified to be non-adhesive or repulsive. The wall can be coated with a thin film coating (for example, a monolayer) of a commercial non-stick reagent (such as those used to form a hydrogel). Other examples of chemicals that can be used to modify the channel wall include oligoethylene glycols, fluorinated polymers, organosilanes, thiols, polyethylene glycol, hyaluronic acid, bovine serum albumin, polyvinyl alcohol, mucin, poly-HEMA, methacrylate PEG and agarose. Charged polymers can also be used to repel substances with opposite charges. The type of chemical used to repel and the method for attaching to the channel wall can depend on the properties of the substance being repelled and the properties of the wall and the substance being attached. Such surface modification techniques are well known in the art. The wall can be functionalized before or after assembling the device.

[0135] III. CAR T and NK cells

[0136] Methods for preparing and using CAR T and natural killer (NK) cells are well known in the art. Procedures have been described, for example, in 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.

[0137] The present disclosure provides microfluidic cartridges (i.e., devices, chips, cassettes, plates, microfluidic devices, cartridges, DLD devices, etc.) and methods for purifying particles or cells that may include chimeric antigen receptor (CAR) T and NK cells. The microfluidic cartridge (i.e., device, chip, cassette, plate, microfluidic device, cartridge, DLD device, etc.) may be any of those described herein. The use of the described cartridges may allow for the production of more efficient CAR T or NK cells by providing purer T or NK cell products for downstream genetic engineering and CAR T or NK cell production. More efficient CAR T or NK cells may be produced by removing platelets that are not removable by other methods for producing CAR T or NK cells.

[0138] The method for producing chimeric antigen receptor (CAR) T or NK cells may include obtaining a sample including T or NK cells and separating T or NK cells from pollutants. Pollutants may include platelets or other pollutants as described herein. Separating pollutants may include applying the sample to one or more sample inlets of any box or device described herein, causing the sample to flow to the outlet of the box, obtaining a product enriched with T or NK cells from the product outlet, and genetically engineering the T cells in the enriched product to produce chimeric antigen receptors on the surface of T or NK cells. The sample of the method may include a single sampling product or a single leukocyte sampling product. The genetic engineering of the method may include a genetic engineering method as described herein. The method may further include amplifying CAR T or NK cells by culturing cells in vitro.

[0139] Some commercial examples of CAR T cell therapies that can be designed according to the devices and methods herein include axicabtagene ciloleucel, tisagenlecleucel, and brexucabtagene autoleucel.

[0140] IV. Separation process using DLD

[0141] The DLD devices described herein can be used to purify cells, cell fragments, cell adducts, or nucleic acids. Separation and purification of blood components using the device can be found, for example, in U.S. Publication No. US 2016 / 0139012, the teachings of which are incorporated herein by reference in their entirety.

[0142] The purity, yield, and viability of cells produced by the DLD method will vary depending on many factors, including the nature of the starting material, the exact procedure employed, and the characteristics of the DLD apparatus. Preferably, a purity, yield, and viability of at least 60% should be achieved, with higher percentages, such as at least 70, 80, or 90%, being more preferred.

[0143] In one aspect, the present disclosure provides a method for enriching a target particle or target cell of a predetermined size from a pollutant in a sample. The method for enriching a target particle or target cell uses any box, microfluidic box, cassette, chip, device, fluid device or microfluidic device as described elsewhere herein. A method may include obtaining a sample comprising a target particle or target cell and a pollutant. The method may also include separating the target particle or target cell from the pollutant by applying the sample to one or more sample inlets on any box, cassette or device as described herein. The method may further include causing the sample to flow to an outlet on any box, cassette or device as described herein. The method may further include obtaining a product enriched with a target particle or target cell from one or more outlets while removing pollutants. The method may result in an excellent ability to purify or separate cells or particles from pollutants, produce higher cell yields, improve the ability of the product to amplify in vitro, and the enriched cell product is more suitable for transduction or other genetic engineering.

[0144] The method may entail using a deterministic lateral displacement, whereby the device has a critical size as described herein and contaminants and target particles or target cells are separated based on having different critical sizes. The method may include flowing a sample containing target particles or target cells and contaminants to any box, cassette, or device described herein, wherein the target particles or target cells have a size greater than the critical size of the obstacle array and at least some of the contaminants have a size less than the critical size of the obstacle array, and wherein the target cells or target particles flow to one or more product outlets where a product enriched in the target cells or target particles is obtained, while contaminants having a size less than the critical size of the obstacle array are flowed to one or more waste outlets. The method may include flowing a sample containing target particles or target cells and contaminants to any box, cassette, or device described herein, wherein the target particles or target cells have a size less than the critical size of the obstacle array and at least some of the contaminants have a size greater than the critical size of the obstacle array, and wherein the target cells or target particles flow to one or more product outlets where a product enriched in the target cells or target particles is obtained, while contaminants having a size greater than the critical size of the obstacle array are flowed to one or more waste outlets.

[0145] The method can include making the sample containing target particles or target cells and pollutants flow to any box, cassette or device described herein with a constant flow rate or a variable flow rate. The box flow rate of the method can be about 400mL per hour. The box flow rate of the method can be about 100mL per hour to about 1,000mL per hour. The box flow rate of the method can be about 100mL per hour to about 200mL per hour, about 100mL per hour to about 400mL per hour, about 100mL per hour to about 800mL per hour, about 100mL per hour to about 1,000mL per hour, about 200mL per hour to about 400mL per hour, about 200mL per hour to about 800mL per hour, about 200mL per hour to about 1,000mL per hour, about 400mL per hour to about 800mL per hour, about 400mL per hour to about 1,000mL per hour or about 800mL per hour to about 1,000mL per hour. The cassette flow rate of the method can be about 100 mL per hour, about 200 mL per hour, about 400 mL per hour, about 800 mL per hour, or about 1,000 mL per hour. The cassette flow rate of the method can be at least about 100 mL per hour, about 200 mL per hour, about 400 mL per hour, or about 800 mL per hour. The cassette flow rate of the method can be at most about 200 mL per hour, about 400 mL per hour, about 800 mL per hour, or about 1,000 mL per hour.

[0146] The method can include an internal pressure within the box. The internal pressure of the box can be at least about 15 pounds per square inch. The internal pressure of the box can be at least about 1.5 pounds per square inch to about 50 pounds per square inch. The internal pressure of the box can be at least about 1.5 pounds per square inch to about 5 pounds per square inch, about 1.5 pounds per square inch to about 10 pounds per square inch, about 1.5 pounds per square inch to about 15 pounds per square inch, about 1.5 pounds per square inch to about 20 pounds per square inch, about 1.5 pounds per square inch to about 50 pounds per square inch, about 5 pounds per square inch to about 10 pounds per square inch, about 5 pounds per square inch to about 15 pounds per square inch, about 5 pounds per square inch to about 20 pounds per square inch, about 5 pounds per square inch to about 50 pounds per square inch, about 10 pounds per square inch to about 15 pounds per square inch, about 10 pounds per square inch to about 20 pounds per square inch, about 10 pounds per square inch to about 50 pounds per square inch, about 15 pounds per square inch to about 20 pounds per square inch, about 15 pounds per square inch to about 50 pounds per square inch, or about 20 pounds per square inch to about 50 pounds per square inch. The internal pressure of box can be at least about 1.5 pounds per square inches, about 5 pounds per square inches, about 10 pounds per square inches, about 15 pounds per square inches, about 20 pounds per square inches or about 50 pounds per square inches. The internal pressure of box can be at least about 1.5 pounds per square inches, about 5 pounds per square inches, about 10 pounds per square inches, about 15 pounds per square inches or about 20 pounds per square inches. The internal pressure of box can be at least about 5 pounds per square inches, about 10 pounds per square inches, about 15 pounds per square inches, about 20 pounds per square inches or about 50 pounds per square inches at the most.

[0147] The target particles or target cells of the method can include stem cells, platelets, synoviocytes, fibroblasts, β cells, hepatocytes, megakaryocytes, pancreatic cells, DE3 lysogens, yeast cells, plant cells, algae cells, monocytes, T cells, B cells, regulatory T cells, macrophages, dendritic cells, granulocytes, innate lymphocytes, natural killer cells, leukocytes, peripheral blood mononuclear cells, CD3+ cells, neurons, platelets, cancer cells, muscle cells or epithelial cells. The method can include enriching target particles or target cells to produce enriched target cells, wherein the enriched target cells include stem cells, platelets, synoviocytes, fibroblasts, beta cells, hepatocytes, megakaryocytes, pancreatic cells, DE3 lysogens, yeast cells, plant cells, algae cells, monocytes, T cells, B cells, regulatory T cells, macrophages, dendritic cells, granulocytes, innate lymphocytes, natural killer cells, leukocytes, peripheral blood mononuclear cells, CD3+ cells, neurons, platelets, cancer cells, muscle cells or epithelial cells. The pollutant of the method can include stem cells, platelets, synoviocytes, fibroblasts, beta cells, hepatocytes, megakaryocytes, pancreatic cells, DE3 lysogens, yeast cells, plant cells, algae cells, monocytes, T cells, B cells, regulatory T cells, macrophages, dendritic cells, granulocytes, innate lymphocytes, natural killer cells, leukocytes, peripheral blood mononuclear cells, CD3+ cells, neurons, platelets, cancer cells, muscle cells or epithelial cells. For example, the target cell can be a peripheral blood mononuclear cell, and the pollutant can be a platelet. For example, the target cell can be a CD3+ cell, and the pollutant can be a platelet. The method can result in removing more than 90% platelets. The method can result in removing about 50% platelets to about 99% platelets. The method can result in the removal of about 50% of the platelets to about 75% of the platelets, about 50% of the platelets to about 80% of the platelets, about 50% of the platelets to about 90% of the platelets, about 50% of the platelets to about 95% of the platelets, about 50% of the platelets to about 99% of the platelets, about 75% of the platelets to about 80% of the platelets, about 75% of the platelets to about 90% of the platelets, about 75% of the platelets to about 90% of the platelets, about 75% of the platelets to about 99% of the platelets. Platelets to about 95% platelets, about 75% platelets to about 99% platelets, about 80% platelets to about 90% platelets, about 80% platelets to about 95% platelets, about 80% platelets to about 99% platelets, about 90% platelets to about 95% platelets, about 90% platelets to about 99% platelets, or about 95% platelets to about 99% platelets. The method can result in the removal of about 50% platelets, about 75% platelets, about 80% platelets, about 90% platelets, about 95% platelets, or about 99% platelets.The method can result in the removal of at least about 50% of the platelets, about 75% of the platelets, about 80% of the platelets, about 90% of the platelets, or about 95% of the platelets. The method can result in the removal of up to about 75% of the platelets, about 80% of the platelets, about 90% of the platelets, about 95% of the platelets, or about 99% of the platelets.

[0148] The method may include modifying the enriched target cells. The method may include genetically engineering the enriched target cells to obtain genetically engineered target cells. Genetic engineering includes transfecting or transducing the target cells with recombinant nucleic acids. Genetic engineering methods may include using TALEN, zinc finger nucleases, CRISPR-Cas related proteins, homologous recombination, viral vectors or heterologous plasmids. The method may also include amplifying the enriched target cells or genetically engineered cells by in vitro culture.

[0149] V. Technical Background

[0150] "Obstacle array" devices have been described, and their basic operation is explained, for example, in U.S. Patent No. 7,150,812 (which is incorporated herein by reference in its entirety). Bump arrays operate primarily by separating particles that pass through an array of obstacles (usually a periodically arranged array), with the separation occurring between particles that follow the direction of the bulk fluid flow and particles that follow an "array direction" that deviates from the direction of the bulk fluid flow.

[0151] A. Fractionation range

[0152] Objects separated by size in microfluidic devices include cells, biomolecules, inorganic beads, and other objects. The typical size range for fractionation is 100 nanometers to 50 micrometers. However, larger and smaller particles can also be fractionated.

[0153] B. Capacity

[0154] The speed at which a sample can be processed will vary widely depending on the design of the device or device combination. Preferably, the device and assembly should be able to process more than 500 ml of sample in one hour.

[0155] C. Channel

[0156] Device can comprise one or more channels, and one or more channels has one or more entrances and one or more outlets.The entrance can be used for sample or thick (that is, not purified) fluid composition, buffer or introduce reagent.The outlet can be used for collecting product or can be used as the outlet of waste.The channel can be about 0.5 to 100mm wide and about 2-200mm long, but different widths and lengths are also possible.The depth can be 1-1000 μm, and 1 to 500 or more channels can be present on the device.

[0157] Specific embodiments of the various aspects described herein may be illustrated by the following numbered embodiments.

[0158] 1. A microfluidic device for purifying target particles or target cells of a predetermined size from contaminants in a sample, the device comprising a planar support having a top surface and a bottom surface, wherein the top surface and / or the bottom surface comprises at least one embedded channel, the at least one embedded channel extending from one or more sample inlets and one or more different fluid inlets to one or more product outlets and one or more different waste outlets; wherein: (a) when a fluid is applied to the channel through the sample and / or fluid inlets, it flows through the channel toward the outlet, thereby defining a direction of bulk fluid flow; (b) the channel comprises an array of obstacles arranged in columns extending longitudinally along the channel and rows extending transversely across the channel, wherein the obstacles are positioned to define a critical size such that When a sample is applied to an inlet of the device and flows toward an outlet, particles or cells in the sample that are larger than a critical size are separated from particles or cells in the sample that are smaller than the critical size; and wherein: (i) adjacent obstacles in a row are separated by a gap G1 that is perpendicular to the direction of bulk fluid flow; (ii) adjacent obstacles in a column are separated by a gap G2 that is parallel to the direction of bulk fluid flow; (iii) the ratio of the size of gap G2 to the size of gap G1 is not equal to 1; (iv) each subsequent row of obstacles is laterally shifted relative to the previous row, thereby defining an array direction that is tilted away from the direction of bulk fluid flow by an angle (ε); and (v) the obstacles have at least two vertices such that each gap is flanked on either side by at least one vertex. 2. The device of embodiment 1, further comprising an obstacle adhesive layer, the obstacle adhesive layer being adhered to the surface of the planar support and to the obstacles in the channels embedded in the surface to prevent fluid or sample from flowing over the obstacles during operation of the device. 3. The microfluidic device of embodiment 2, wherein the barrier adhesive layer comprises one or more passages connected to the sample inlet fluid of the channel to allow sample to flow into the channel and one or more passages connected to the outlet fluid of the channel to allow fluid to flow out of the outlet. 4. The microfluidic device of any one of embodiments 1-3, wherein the size of the target particles or target cells is larger than the critical size of the device and the size of at least some contaminants is smaller than the critical size, and wherein the obstacles are arranged in a manner such that when the sample is applied to the inlet of the device and the fluid flows through the channel, the target cells or target particles flow to one or more product outlets, where an enriched product including the target cells or target particles is obtained, while contaminants smaller than the critical size flow to one or more waste outlets. 5. The microfluidic device of any one of embodiments 1-4, wherein the obstacles have a polygonal shape. 6. The microfluidic device of embodiment 5, wherein the obstacles have a diamond or hexagonal shape. 7. The microfluidic device of embodiment 5 or embodiment 6, wherein the obstacles are elongated perpendicular to the direction of the bulk flow such that they have a horizontal length (P1) that is different from their vertical length (P2). 8. The microfluidic device of embodiment 6, wherein P1 is at least 15% longer than P2.9. The microfluidic device of embodiment 6, wherein P1 is 10%-150% longer than P2. 10. The microfluidic device of embodiment 6, wherein P1 is 15%-100% longer than P2. 11. The microfluidic device of embodiment 6, wherein P1 is 20%-70% longer than P2. 12. The microfluidic device of any of embodiments 1-11, wherein the obstacles have vertices extending into the parallel gaps, such that the gaps are flanked on either side by one or more vertices pointing toward each other but not directly opposite each other. 13. The microfluidic device of any of embodiments 1-12, wherein the obstacles have vertices extending into the perpendicular gaps, such that the gaps are flanked on either side by vertices pointing toward each other and directly opposite each other. 14. The microfluidic device of any of embodiments 1-13, wherein one or more sample inlets are separated from one or more fluid inlets by a partition wall, the partition wall extending from the one or more sample inlets toward the outlet into the array of obstacles in the channel and in a direction parallel to the direction of bulk fluid flow. 15. The microfluidic device of embodiment 14, wherein the partition wall extends at least 10% of the length of the array of obstacles. 16. The microfluidic device of embodiment 14, wherein the dividing wall extends at least 20% of the length of the obstacle array. 17. The microfluidic device of embodiment 14, wherein the dividing wall extends at least 40% of the length of the obstacle array. 18. The microfluidic device of embodiment 14, wherein the dividing wall extends at least 60% of the length of the obstacle array. 19. The microfluidic device of any one of embodiments 1-18, wherein the inlet and / or outlet of the device is connected to a peristaltic pump. 20. A stacked separation assembly comprising at least two microfluidic devices of any one of embodiments 1-19. 21. A stacked separation assembly comprising a first microfluidic device selected from the microfluidic devices of any one of embodiments 1-19, and one or more stacked microfluidic devices also selected from the microfluidic devices of any one of embodiments 1-19, wherein: (a) the bottom surface of each stacked device contacts the top surface or the barrier adhesive layer on the top surface of the first microfluidic device, or contacts the top surface or the barrier adhesive layer on the top surface of another stacked device; (b) a sample is provided to the sample inlet through a first common manifold; (c) a fluid is supplied to the fluid inlet through a second manifold, which may or may not be the same as the first manifold; (d) a product is removed from the product outlet through one or more conduits; (e) waste is removed from the waste outlet through one or more conduits different from the one or more conduits of (d); and (f) the first microfluidic device and the stacked microfluidic devices are optionally mounted in a common housing. 22. A stacked separation assembly as described in embodiment 22, wherein the assembly comprises at least two stacked microfluidic devices.23. The stacked separation assembly of embodiment 22, further comprising at least one reservoir adhesive layer attached to the bottom surface of the first microfluidic device and / or the top surface of the stacked microfluidic device, and comprising one or more passages at a first end that allow fluid to flow into an inlet on a channel and comprising one or more passages at a second end opposite the first end that allow fluid to flow out of a product outlet and a waste outlet of the channel, wherein the passages at the first and second ends of the reservoir layer are separated by a fluid-impermeable material. 24. The stacked separation assembly of any one of embodiments 22 or 23, wherein both the top and bottom surfaces of the planar support of one or more microfluidic devices comprise one or more channels having obstacles for separating target particles or target cells. 25. A method for purifying target particles or target cells of a predetermined size from contaminants in a sample, the method comprising: (a) obtaining a sample comprising the target particles or target cells and the contaminants; (b) separating the target particles or target cells from the contaminants by: (i) applying the sample to one or more sample inlets on a microfluidic device as described in any of embodiments 1-21 or a first microfluidic device or a stacked device as described in any of embodiments 22-24; (ii) flowing the sample to an outlet on a device as described in any of embodiments 1-21 or a first microfluidic device or a stacked device as described in any of embodiments 22-24; and (iii) obtaining a product enriched in the target particles or target cells from the one or more outlets. 26. The method of embodiment 25, wherein the target particles or target cells are larger than a critical size of the obstacle array and at least some of the contaminants are smaller than the critical size, and wherein the target particles or target cells flow to one or more product outlets, where a product enriched in the target cells or target particles is obtained, while contaminants smaller than the critical size flow to one or more waste outlets. 27. The method of embodiment 26, wherein the sample is blood or derived from blood. 28. The method of embodiment 26, wherein the sample is an apheresis sample or a leukocyte apheresis sample. 29. The method of embodiment 27 or 28, wherein the sample includes platelets as a contaminant. 30. The method of embodiment 29, wherein the method results in the removal of at least 80% of platelets from the sample. 31. The method of embodiment 29, wherein the method results in the removal of at least 90% of platelets from the sample. 32. The method of embodiment 29, wherein the method results in the removal of at least 95% of platelets from the sample. 33. The method of any one of embodiments 27-31, wherein the target cell is a leukocyte. 34. The method of any one of embodiments 27-31, wherein the target cell is a stem cell. 35. The method of any one of embodiments 27-31, wherein the target cell is a B cell, a T cell, a NK cell, a monocyte, or a progenitor cell.36. The method of any one of embodiments 27-31, wherein the target cells are dendritic cells. 37. The method of any one of embodiments 25-36, wherein the sample is obtained from a patient. 38. The method of embodiment 37, wherein the patient has cancer, an autoimmune disease, or an infectious disease. 39. The method of any one of embodiments 25-38, further comprising genetically engineering the purified target cells. 40. The method of embodiment 39, wherein the genetic engineering comprises transfecting or transducing the target cells with a recombinant nucleic acid. 41. The method of embodiment 39 or 40, wherein the genetically engineered target cells are expanded by culturing them in vitro. 42. A method for producing chimeric antigen receptor (CAR) T cells, the method comprising: (a) obtaining a sample comprising T cells; (b) separating T cells from contaminants by the following steps: (i) applying the sample to one or more sample inlets on a microfluidic device as described in any one of embodiments 1-21 or a first microfluidic device or a stacked device as described in any one of embodiments 22-24; (ii) causing the sample to flow to the outlet of the device; and (iii) obtaining a product enriched with T cells from the product outlet; (c) genetically engineering the T cells in the enriched product obtained in step b) to produce a chimeric antigen receptor (CAR) on its surface. 43. The method of embodiment 42, wherein the sample is blood, apheresis product, or leukocyte apheresis product from a patient. 44. The method of embodiment 42 or 43, wherein the genetic engineering comprises transfecting or transducing target cells, and the genetically engineered target cells are further expanded by culturing cells in vitro. 45. The method of any one of embodiments 42-44, wherein separation is achieved by performing a deterministic lateral displacement on a microfluidic device. 46. ​​The method of any one of embodiments 42-44, wherein the sample is obtained from a patient suffering from cancer, an autoimmune disease, or an infectious disease. 47. The method of embodiment 46, wherein after obtaining the sample, the T cells are bound to one or more carriers in a manner that promotes DLD separation. 48. CART cells prepared by the method of any one of embodiments 42-47.

[0159] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited to the specific embodiments provided in the specification. Although the present invention has been described with reference to the foregoing description, the description and illustration of the embodiments herein are not intended to be interpreted in a restrictive sense. Without departing from the present invention, those skilled in the art will now appreciate many variations, changes, and substitutions. In addition, it should be understood that all aspects of the present invention are not limited to the specific descriptions, configurations, or relative proportions described herein that depend on a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein can be adopted in practicing the present invention. Therefore, it is contemplated that the present invention also encompasses any such alternatives, modifications, variations, or equivalents. The appended claims are intended to define the scope of the present invention and thus cover methods and structures within the scope of these claims and their equivalents.

[0160] 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 performed within a wide and equivalent range of conditions, parameters, etc. without affecting the spirit or scope of the invention or any embodiment thereof.

Claims

1. A microfluidic cartridge for purifying target particles or target cells of a predetermined size from contaminants in a sample, The microfluidic cartridge comprises a first planar support and a second planar support as planar supports, the first planar support and the second planar support each having a top surface and a bottom surface, the top surface of the first planar support and / or the second planar support comprising at least one embedded channel extending from one or more inlets to one or more outlets; The at least one embedded channel includes an obstacle array formed of a plurality of obstacles arranged in columns extending longitudinally along the channel and rows extending transversely across the channel, the obstacles being positioned to define a critical dimension of the microfluidic cartridge, wherein Adjacent obstacles in the row are separated by a gap G1 perpendicular to the direction of the main fluid flow, and adjacent obstacles in the column are separated by a gap G2 parallel to the direction of the main fluid flow, and the size of the gap G1 is larger than the size of the gap G2. Each subsequent row of obstacles is laterally shifted relative to the previous row, thereby defining an array direction that is offset from the direction of the bulk fluid flow by an oblique angle, When a sample is applied to the inlet of the microfluidic cartridge and flows toward the outlet, particles or cells in the sample that are larger than the critical size are separated from particles or cells in the sample that are smaller than the critical size.

2. The microfluidic cartridge of claim 1 , wherein the microfluidic cartridge comprises at least one void space configured to deform when the first planar support and the second planar support are assembled into the microfluidic cartridge, Wherein the bottom surfaces of the first and second planar support members include at least one void space, the void space being configured to deform when the bottom of the first planar support member is pressed toward the bottom of the second planar support member.

3. The microfluidic cartridge of claim 2, wherein the at least one void space is configured to prevent damage, displacement, or deformation of the at least one embedded channel, the one or more inlets, the one or more outlets, the plurality of obstacles, or a combination thereof.

4. The microfluidic cartridge of claim 2, wherein the at least one void space comprises a total surface area that is at least 90% of the total surface area of ​​the at least one embedded channel.

5. The microfluidic cartridge of claim 2, wherein the at least one void space comprises a total surface area that is at least 100% of the total surface area of ​​the at least one embedded channel.

6. The microfluidic cartridge of claim 2, wherein the at least one void space comprises a total surface area that is at least 110% of the total surface area of ​​the at least one embedded channel.

7. A microfluidic box as described in claim 2, wherein the at least one void space is divided into two or more void spaces, and the two or more void spaces are positioned on the bottom surface of the first planar support and / or the second planar support opposite to the obstacle array.

8. The microfluidic cartridge of claim 2, wherein the planar support is made of two layers of material bonded together.

9. The microfluidic cartridge of claim 2 , further comprising an obstacle adhesive layer bonded to a surface of the planar support and to a top surface of the plurality of obstacles in the at least one embedded channel to prevent fluid or sample from flowing over the plurality of obstacles during operation of the microfluidic cartridge.

10. The microfluidic cartridge of claim 9 , wherein the barrier adhesive layer comprises one or more passages fluidically connected to the one or more inlets of the at least one embedded channel for allowing sample to flow into the at least one embedded channel and one or more passages fluidically connected to the one or more outlets of the at least one embedded channel for allowing fluid to flow out of the one or more outlets.

11. The microfluidic cartridge of claim 1 , wherein the one or more outlets include at least one product outlet, wherein the target particles or target cells having a size larger than the critical size of the microfluidic cartridge are directed to the at least one product outlet.

12. The microfluidic cartridge of claim 1, wherein the one or more outlets include at least one waste outlet, and contaminants having a size smaller than the critical size of the microfluidic cartridge flow to the at least one waste outlet.

13. The microfluidic cartridge of claim 1, wherein the plurality of obstacles have a diamond shape.

14. The microfluidic cartridge of claim 1, wherein the plurality of obstacles have a circular shape or an elliptical shape.

15. The microfluidic cartridge of claim 1, wherein the plurality of obstacles have a hexagonal shape.

16. The microfluidic cartridge of claim 13, wherein the plurality of obstacles are elongated perpendicular to the direction of fluid flow such that they have a horizontal length P1 that is different from their vertical length P2. The microfluidic cartridge of claim 16 , wherein P1 is 10 μm to 160 μm and P2 is 5 μm to 80 μm. The microfluidic cartridge of claim 16 , wherein P1 is 10 μm to 80 μm and P2 is 15 μm to 60 μm. The microfluidic cartridge of claim 16 , wherein P1 is 15 μm to 30 μm and P2 is 25 μm to 45 μm.

20. The microfluidic cartridge of claim 16, wherein P1 is 40 μm and P2 is 20 μm.

21. The microfluidic cartridge of claim 16, wherein P1 is 50% to 150% longer than P2.

22. The microfluidic cartridge of claim 1 , wherein the plurality of obstacles have vertices extending into parallel gaps such that the gaps are flanked on either side by one or more vertices pointing toward but not directly opposite each other.

23. The microfluidic cartridge of claim 1 , wherein the plurality of obstacles have vertices extending into a vertical gap such that the gap is flanked on either side by vertices pointing toward and directly opposite each other.

24. The microfluidic cartridge of claim 1, wherein the plurality of obstacles are arranged in at least 10 columns.

25. The microfluidic cartridge of claim 1, wherein the plurality of obstacles are arranged in at least 30 columns.

26. The microfluidic cartridge of claim 1, wherein the plurality of obstacles are arranged in at least 50 columns.

27. The microfluidic cartridge of claim 1, wherein the plurality of obstacles are arranged in at least 60 columns.

28. The microfluidic cartridge of claim 1, wherein the plurality of obstacles are arranged in at least 50 rows.

29. The microfluidic cartridge of claim 1, wherein the plurality of obstacles are arranged in at least 100 rows.

30. The microfluidic cartridge of claim 1, wherein the plurality of obstacles are arranged in at least 300 rows.

31. The microfluidic cartridge of claim 1 , wherein the plurality of obstacles are arranged in at least 600 rows.

32. The microfluidic cartridge of claim 1, wherein the first planar support or the second planar support comprises at least 10 embedded channels.

33. The microfluidic cartridge of claim 1, wherein the first planar support and / or the second planar support comprises at least 20 embedded channels.

34. The microfluidic cartridge of claim 1, wherein the first planar support and / or the second planar support comprises 28 embedded channels.

35. The microfluidic cartridge of claim 1, wherein the first planar support and / or the second planar support comprises 30 embedded channels.

36. The microfluidic cartridge of claim 1, wherein the first planar support and / or the second planar support comprises at least 50 embedded channels.

37. A microfluidic box as described in claim 1, wherein the one or more inlets include at least one or more sample inlets and at least one or more fluid inlets; wherein the at least one or more sample inlets are separated from the at least one or more fluid inlets by a partition wall, and the partition wall extends from the one or more sample inlets toward the outlet into the obstacle array in the at least one embedded channel, and the direction is parallel to the fluid flow direction.

38. The microfluidic cartridge of claim 37, wherein the partition wall extends at least 10% of the length of the plurality of obstacles.

39. The microfluidic cartridge of claim 37, wherein the partition wall extends at least 20% of the length of the plurality of obstacles.

40. The microfluidic cartridge of claim 37, wherein the dividing wall extends at least 60% of the length of the plurality of obstacles.

41. The microfluidic cartridge of claim 1, wherein the one or more inlets, the one or more outlets, or both are fluidly connected to a first peristaltic pump, a second peristaltic pump, or both.

42. The microfluidic cartridge of claim 41 , wherein the first peristaltic pump and the second peristaltic pump are in serial fluidic connection.

43. The microfluidic cartridge of claim 41 , wherein the first peristaltic pump and the second peristaltic pump are fluidically connected in parallel.

44. The microfluidic cartridge of claim 1, wherein the microfluidic cartridge is made of a polymer.

45. The microfluidic cartridge of claim 44, wherein the polymer is a thermoplastic polymer.

46. ​​The microfluidic cartridge of claim 45, wherein the thermoplastic polymer is selected from high density polyethylene, polypropylene, polyethylene terephthalate, polycarbonate, or cyclic olefin copolymer.

47. A microfluidic assembly comprising a plurality of microfluidic cartridges as claimed in any one of claims 1 to 46, wherein the plurality of microfluidic cartridges are in fluid connection.

48. The microfluidic assembly of claim 47, wherein the microfluidic cartridges are stacked.

49. The microfluidic assembly of claim 47, wherein the plurality of microfluidic cartridges is two.

50. The microfluidic assembly of claim 47, wherein the microfluidic cartridges are in parallel fluidic connection.

51. The microfluidic assembly of claim 47, wherein the microfluidic cartridges are in serial fluidic connection.

52. A method of manufacturing a microfluidic cartridge as claimed in any one of claims 1 to 46, wherein the microfluidic cartridge is manufactured by pressing the bottoms of the first planar support and the second planar support together so that the obstacle array is not deformed.

53. The method of manufacturing of claim 52, wherein the at least one embedded channel, obstacle, or both are manufactured by compression molding or injection molding.

54. The manufacturing method of claim 52, wherein the microfluidic cartridge is UV light cured during manufacturing.

55. A method for enriching target particles or target cells of a predetermined size from contaminants in a sample, comprising: a) obtaining a sample comprising the target particles or target cells and the contaminants; b) separating the target particles or target cells from the contaminants by the following steps: i) applying the sample to one or more sample inlets on the microfluidic cartridge according to any one of claims 1 to 46; ii) allowing the sample to flow toward the outlet on the microfluidic cartridge according to any one of claims 1 to 46; and iii) obtaining the product enriched with the target particles or target cells from one or more outlets while removing the contaminants.

56. A method as claimed in claim 55, wherein the size of the target particles or target cells is larger than the critical size of the obstacle array, and the size of at least some contaminants is smaller than the critical size of the obstacle array, and wherein the target cells or target particles flow to one or more product outlets, where a product enriched with target cells or target particles is obtained, while the contaminants having a size smaller than the critical size of the obstacle array flow to one or more waste outlets.

57. The method of claim 55, wherein the flow rate of the microfluidic cartridge is 400 mL per hour.

58. The method of claim 55, wherein the flow rate of the microfluidic cartridge is at least 100 mL per hour.

59. The method of claim 55, wherein the flow rate of the microfluidic cartridge is at least 300 mL per hour.

60. The method of claim 55, wherein the flow rate of the microfluidic cartridge is 1000 mL per hour.

61. The method of claim 55, wherein the internal pressure of the microfluidic cartridge is at least 1.5 pounds per square inch.

62. The method of claim 55, wherein the internal pressure of the microfluidic cartridge is 15 pounds per square inch.

63. The method of claim 55, wherein the internal pressure of the microfluidic cartridge is 50 pounds per square inch or less.

64. The method of claim 55, wherein the internal pressure of the microfluidic cartridge is between 10 and 20 psi.

65. The method of claim 55, wherein the sample is blood or a blood-related product.

66. The method of claim 55, wherein the sample is a single sample.

67. The method of claim 55, wherein the sample is a leukocyte apheresis sample.

68. The method of claim 55, wherein the sample comprises platelets as contaminants.

69. The method of claim 68, wherein said method results in the removal of at least 80% of said platelets from said sample.

70. The method of claim 68, wherein said method results in the removal of at least 90% of said platelets from said sample.

71. The method of claim 68, wherein said method results in the removal of at least 95% of said platelets from said sample.

72. The method of claim 55, wherein the enriched target cells comprise leukocytes.

73. The method of claim 55, wherein the enriched target cells comprise stem cells.

74. The method of claim 55, wherein the enriched target cells comprise peripheral blood mononuclear cells.

75. The method of claim 74, wherein the peripheral blood mononuclear cells comprise CD3 + cell.

76. The method of claim 55, further comprising genetically engineering the enriched target cells to obtain genetically engineered target cells.

77. The method of claim 76, wherein the genetic engineering comprises transfecting or transducing the target cell with a recombinant nucleic acid.

78. The method of claim 76, wherein the enriched target cells or genetically engineered target cells are expanded by culturing them in vitro.

79. A method for producing chimeric antigen receptor T cells, comprising: a) obtaining a sample comprising T cells; b) separating the T cells from contaminants by: i) applying the sample to one or more sample inlets on the microfluidic cartridge according to any one of claims 1 to 46; ii) allowing the sample to flow toward the outlet of the microfluidic cartridge; and iii) obtaining a product enriched with T cells from a product outlet; c) genetically engineering the T cells in the enriched product obtained in step b) to produce chimeric antigen receptors on their surface.

80. The method of claim 79, wherein the sample is blood or an apheresis product.

81. The method of claim 79, wherein the sample is a leukapheresis product.

82. The method of claim 79, wherein genetically engineering the T cells comprises transfecting or transducing the target cells, and further expanding the genetically engineered target cells by culturing the cells in vitro.

83. A method for producing chimeric antigen receptor natural killer cells, comprising: a) obtaining a sample comprising natural killer cells; b) separating the natural killer cells from contaminants by the following steps: i) applying the sample to one or more sample inlets on the microfluidic cartridge according to any one of claims 1 to 46; ii) allowing the sample to flow toward the outlet of the microfluidic cartridge; and iii) obtaining a product enriched with natural killer cells from a product outlet; c) genetically engineering the natural killer cells in the enriched product obtained in step b) to produce chimeric antigen receptors on their surface.

84. The method of claim 83, wherein the sample is a blood sample or an apheresis product.

85. The method of claim 83, wherein the sample is a leukapheresis product.

86. The method of claim 83, wherein genetically engineering the natural killer cells comprises transfecting or transducing the target cells, and further expanding the genetically engineered target cells by culturing the cells in vitro.

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