Microfluidic cartridges for processing particles and cells

Abstract

Described herein is a microfluidic cartridge for purifying target particles or cells of a predetermined size from contaminants in a sample, the cartridge comprising first and second planar supports, each having a top surface and a bottom surface, the top surface of the first and / or second planar supports comprising at least one embedded channel extending from one or more inlets to one or more outlets, the at least one embedded channel comprising a plurality of obstacles, and the microfluidic cartridge comprising at least one void configured to be deformed upon assembling the microfluidic cartridge from the first and second planar supports.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of U.S. Provisional Application No. 62 / 954,478, filed Dec. 28, 2019, the entire disclosure of which is incorporated herein by reference.

Background Art

[0002] For the preparation of cells for individualized therapy, in many cases, it is necessary to collect biological material from a patient, purify a specific cell type from the collected material, and manipulate or grow the purified cells. In the case of CAR T cell therapy, typically, it is necessary to process a large volume of blood or a preparation of blood - derived apheresis or leukapheresis to obtain a T cell preparation suitable for genetic manipulation and proliferation. Procedures based on microfluidic size are rapid, gentle, and provide versatile processing options. However, there are factors, including the deposition of biological debris during the operation of microfluidic devices, that can slow down processing and lead to poor purification. Therefore, the development of better devices and better methods for increasing the rate at which biological materials can be purified is of great interest.

Summary of the Invention

Means for Solving the Problems

[0003] A fixed separation cartridge for use with a microfluidic device, improved to enable the manufacture of cartridges with precise features such as posts or obstacles for size - based separation, storage sections for cells, and other microfluidic features, is described herein. A fixed separation cartridge for use with a microfluidic device having a plurality of lanes or channels, such as a separation wall extending over a certain length, to allow fluid flow in the cartridge, is also described to prevent unwanted mixing, turbulent flow due to unwanted mixing, and the pulsating nature of delivery caused by some positive displacement pumps.

[0004] A microfluidic cartridge for purifying target particles or target cells of a predetermined size from contaminants in a sample is described herein in one embodiment, wherein the cartridge comprises first and second planar supports, each having a top and a bottom surface, and the top surface of the first and / or second planar supports comprises at least one embedding channel extending from one or more inlets to one or more outlets, and the at least one embedding channel comprises a plurality of obstacles.

[0005] In some embodiments, the microfluidic cartridge includes at least one void configured to deform when the microfluidic cartridge is assembled from first and second planar supports. In some embodiments, the bottom surfaces of the first and second planar supports include at least one void configured to deform when the bottom of the first planar support is pressed against the bottom of the second planar support. In some embodiments, the at least one void 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 some embodiments, the at least one void is configured to prevent damage, displacement, or deformation of multiple obstacles. In some embodiments, the microfluidic cartridge has a void-to-channel ratio of 1:1. In some embodiments, the total surface area of ​​the at least one void is at least about 90% of the total surface area of ​​at least one embedded channel. In some embodiments, the total surface area of ​​the at least one void is at least about 100% of the total surface area of ​​at least one embedded channel. In some embodiments, at least one void has a total surface area of ​​at least about 110% of the total surface area of ​​at least one embedding channel. In some embodiments, at least one void is separated into two or more voids located on the bottom surface of a first and / or second planar support on the opposite side of the array of obstacles. In some embodiments, the planar support is fabricated from two layers of material bonded to each other. In some embodiments, the microfluidic cartridge further includes an obstacle bonding layer bonded to the surface of the planar support and to the top surface of the multiple obstacles of at least one embedding channel to prevent the fluid or sample from flowing over the multiple obstacles during the operation of the cartridge.In some embodiments, the obstruction bonding layer comprises one or more passages fluid-connected to one or more inlets of at least one embedding channel, allowing sample flow into at least one embedding channel, and one or more passages fluid-connected to one or more outlets of at least one embedding channel, allowing fluid flow out of one or more outlets. In some embodiments, the obstruction is positioned to define the critical size of the cartridge, so that as the sample is poured into the cartridge inlet and flows to the 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. In some embodiments, one or more outlets comprises at least one product outlet, and target particles or target cells having a size larger than the critical size of the cartridge are directed to at least one product outlet. In some embodiments, one or more outlets comprises at least one waste outlet, and contaminants having a size smaller than the critical size of the cartridge flow to at least one waste outlet. In some embodiments, the multiple obstructions are diamond-shaped or elongated diamond-shaped. In some embodiments, the multiple obstructions are circular or ellipsoidal. In some embodiments, the multiple obstructions are hexagonal. In some embodiments, the obstacles are elongated in a direction perpendicular to the direction of fluid flow, and therefore have a horizontal length (P1) that is different from their vertical length (P2). In some embodiments, P1 is about 10 μm to about 160 μm and P2 is about 5 μm to about 80 μm. In some embodiments, P1 is about 10 μm to about 80 μm and P2 is about 15 μm to about 60 μm. In some embodiments, P1 is about 15 μm to about 30 μm and P2 is about 25 μm to about 45 μm. In some embodiments, P1 is about 40 μm and P2 is about 20 μm. In some embodiments, P1 is 50 to 150% longer than P2.In some embodiments, the obstacles have vertices that extend through parallel gaps such that one or more vertices facing each other but not directly opposite each other are adjacent (flank) on each side of the gap. In some embodiments, the obstacles have vertices that extend through the gaps such that vertices facing each other are adjacent on each side of a vertical gap and are directly opposite each other. In some embodiments, the obstacles are arranged in at least one vertical column. In some embodiments, the obstacles are arranged in at least ten vertical columns. In some embodiments, the obstacles are arranged in at least thirty vertical columns. In some embodiments, the obstacles are arranged in at least fifty vertical columns. In some embodiments, the obstacles are arranged in at least about sixty vertical columns. In some embodiments, the obstacles are arranged in at least about fifty horizontal rows. In some embodiments, the obstacles are arranged in at least about fifty horizontal rows. In some embodiments, the obstacles are arranged in at least about 100 horizontal rows. In some embodiments, the obstacles are arranged in at least about 300 horizontal rows. In some embodiments, the obstacles are arranged in at least about 600 horizontal rows. In some embodiments, the first or second planar support comprises at least 10 embedding channels. In some embodiments, the first and / or second planar support comprises at least 20 embedding channels. In some embodiments, the first and / or second planar support comprises about 28 embedding channels. In some embodiments, the first and / or second planar support comprises about 30 embedding channels. In some embodiments, the first and / or second planar support comprises at least about 50 embedding channels.In some embodiments, one or more inlets of a microfluidic cartridge consist of at least one or more sample inlets and at least one or more fluid inlets, the at least one or more sample inlets being separated from at least one or more fluid inlets by a separation wall, the separation wall extending from one or more sample inlets through an array of at least one embedded channel obstruction toward an outlet and oriented parallel to the direction of fluid flow. In some embodiments, the separation wall extends over at least 10% of the length of the multiple obstructions. In some embodiments, the separation wall extends over at least 20% of the length of the multiple obstructions. In some embodiments, the separation wall extends over at least 60% of the length of the multiple obstructions. In some embodiments, one or more inlets, one or more outlets, or both are fluid-coupled to a first peristaltic pump, a second peristaltic pump, or both. In some embodiments, the first and second peristaltic pumps are fluid-coupled in series. In some embodiments, the first and second peristaltic pumps are fluid-coupled in parallel. In some embodiments, the cartridge is made from a polymer. In some embodiments, the polymer is a thermoplastic polymer. In some embodiments, the thermoplastic polymer is selected from the group including high-density polyethylene, polypropylene, polyethylene terephthalate, polycarbonate, or cyclic olefin copolymers. In some embodiments, the thermoplastic polymer is a cyclic olefin copolymer.

[0006] A microfluidic cartridge for purifying target particles or target cells of a predetermined size from contaminants in a sample is described herein in one embodiment, wherein the cartridge comprises first and second planar supports, each having a top and a bottom surface, and the top surface of the first and / or second planar supports comprises at least one embedding channel extending from one or more inlets to one or more outlets, the at least one embedding channel comprising a plurality of obstructions, and the microfluidic cartridge comprises at least one cavity configured to deform when the microfluidic cartridge is assembled from the first and second planar supports. In some embodiments, the bottom surfaces of the first and second planar supports comprises at least one cavity configured to deform when the bottom of the first planar support is pressed against the bottom of the second planar support. In some embodiments, the at least one cavity is configured to prevent damage, displacement, or deformation of at least one embedding channel, one or more inlets, one or more outlets, a plurality of obstructions, or a combination thereof. In some embodiments, at least one void is configured to prevent damage, displacement, or deformation of multiple obstacles. In some embodiments, the microfluidic cartridge has a void-to-channel ratio of 1:1. In some embodiments, the total surface area of ​​at least one void is at least about 90% of the total surface area of ​​at least one embedded channel. In some embodiments, the total surface area of ​​at least one void is at least about 100% of the total surface area of ​​at least one embedded channel. In some embodiments, the total surface area of ​​at least one void is at least about 110% of the total surface area of ​​at least one embedded channel. In some embodiments, at least one void is separated into two or more voids positioned on the bottom surface of a first and / or second planar support on the opposite side of the array of obstacles. In some embodiments, the planar support is made from two layers of material joined together.In some embodiments, the microfluidic cartridge further comprises an obstacle bonding layer bonded to the surface of a planar support and to the upper surfaces of multiple obstacles in at least one embedding channel to prevent the fluid or sample from flowing over multiple obstacles during the operation of the cartridge. In some embodiments, the obstacle bonding layer comprises one or more passages fluid-connected to one or more inlets of at least one embedding channel, allowing the flow of sample into at least one embedding channel, and one or more passages fluid-connected to one or more outlets of at least one embedding channel, allowing the flow of fluid out of one or more outlets. In some embodiments, the obstacles are positioned to define the critical size of the cartridge, so that when the sample is poured into the cartridge inlet and flows to the 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. In some embodiments, one or more outlets comprises at least one product outlet, and target particles or target cells having a size larger than the critical size of the cartridge are directed to at least one product outlet. In some embodiments, one or more outlets are provided with at least one waste outlet, and contaminants having a size smaller than the critical size of the cartridge flow to at least one waste outlet. In some embodiments, the multiple obstacles are diamond-shaped or elongated diamond-shaped. In some embodiments, the multiple obstacles are circular or ellipsoidal. In some embodiments, the multiple obstacles are hexagonal. In some embodiments, the multiple obstacles are elongated in the direction perpendicular to the direction of fluid flow, and therefore have a left-right length (P1) different from their up-down length (P2). In some embodiments, P1 is about 10 μm to about 160 μm and P2 is about 5 μm to about 80 μm. In some embodiments, P1 is about 10 μm to about 80 μm and P2 is about 15 μm to about 60 μm. In some embodiments, P1 is about 15 μm to about 30 μm and P2 is about 25 μm to about 45 μm. In some embodiments, P1 is about 40 μm and P2 is about 20 μm.In some embodiments, P1 is 50 to 150% longer than P2. In some embodiments, the obstacles have vertices that extend through the gaps such that one or more vertices facing each other but not directly opposite each other are adjacent on each side of the parallel gaps. In some embodiments, the obstacles have vertices that extend through the gaps such that vertices facing each other are adjacent on each side of the vertical gaps and are directly opposite each other. In some embodiments, the obstacles are arranged in at least one column. In some embodiments, the obstacles are arranged in at least ten columns. In some embodiments, the obstacles are arranged in at least 30 columns. In some embodiments, the obstacles are arranged in at least 50 columns. In some embodiments, the obstacles are arranged in at least about 60 columns. In some embodiments, the obstacles are arranged in at least about 50 rows. In some embodiments, the obstacles are arranged in at least about 100 rows. In some embodiments, the obstacles are arranged in at least about 300 rows. In some embodiments, the obstacles are arranged in at least approximately 600 horizontal rows. In some embodiments, the first or second planar support comprises at least 10 embedding channels. In some embodiments, the first and / or second planar support comprises at least 20 embedding channels. In some embodiments, the first and / or second planar support comprises approximately 28 embedding channels. In some embodiments, the first and / or second planar support comprises approximately 30 embedding channels. In some embodiments, the first and / or second planar support comprises at least approximately 50 embedding channels.In some embodiments, one or more inlets of a microfluidic cartridge consist of at least one or more sample inlets and at least one or more fluid inlets, the at least one or more sample inlets being separated from at least one or more fluid inlets by a separation wall, the separation wall extending from one or more sample inlets through an array of at least one embedded channel obstruction toward an outlet and oriented parallel to the direction of fluid flow. In some embodiments, the separation wall extends over at least 10% of the length of the multiple obstructions. In some embodiments, the separation wall extends over at least 20% of the length of the multiple obstructions. In some embodiments, the separation wall extends over at least 60% of the length of the multiple obstructions. In some embodiments, one or more inlets, one or more outlets, or both are fluid-coupled to a first peristaltic pump, a second peristaltic pump, or both. In some embodiments, the first and second peristaltic pumps are fluid-coupled in series. In some embodiments, the first and second peristaltic pumps are fluid-coupled in parallel. In some embodiments, the cartridge is made from a polymer. In some embodiments, the polymer is a thermoplastic polymer. In some embodiments, the thermoplastic polymer is selected from the group including high-density polyethylene, polypropylene, polyethylene terephthalate, polycarbonate, or cyclic olefin copolymers. In some embodiments, the thermoplastic polymer is a cyclic olefin copolymer.

[0007] Also described are microfluidic assemblies comprising multiple microfluidic cartridges, which are fluid-connected. In some embodiments, the microfluidic cartridges are stacked. In some embodiments, there are two microfluidic cartridges. In some embodiments, the microfluidic cartridges are fluid-connected in parallel. In some embodiments, the microfluidic cartridges are fluid-connected in series.

[0008] A method for manufacturing a microfluidic cartridge is also described herein, wherein the cartridge is manufactured by pressing the bottoms of first and second planar supports together so that an array of obstructions is not deformed. In some embodiments, at least one embedded channel, obstruction, or both are manufactured by embossing, hot embossing, roll-to-roll embossing, or injection molding. In some embodiments, the microfluidic cartridge is UV cured during manufacturing. A method for enriching a target particle or target cell of a predetermined size from contaminants in a sample is also described herein, comprising (a) obtaining a sample containing the target particle or target cell and the contaminants, and (b) separating the target particle or target cell from the contaminants by (i) pouring the sample into one or more sample inlets of a microfluidic cartridge, (ii) allowing the sample to flow to the outlets of the cartridge, and (iii) obtaining a product enriched with the target particle or target cell from one or more outlets while removing the contaminants. In some embodiments, target particles or target cells have a size greater than the critical size of the obstacle array, and at least some contaminants have a size smaller than the critical size of the obstacle array, and the target cells or target particles flow to one or more product outlets where the product enriched with the target cells or target particles is obtained, and contaminants having a size smaller than the critical size of the obstacle array flow to another waste outlet. In some embodiments, the cartridge flow rate is about 400 mL per hour. In some embodiments, the cartridge flow rate is at least about 100 mL per hour or more. In some embodiments, the cartridge flow rate is at least about 300 mL per hour or more. In some embodiments, the cartridge flow rate is about 1000 mL per hour. In some embodiments, the cartridge internal pressure is at least about 1.5 pounds / square inch or more. In some embodiments, the cartridge internal pressure is about 15 pounds / square inch. In some embodiments, the cartridge internal pressure is about 50 pounds / square inch or less. In some embodiments, the cartridge internal pressure is about 10 pounds / square inch to about 20 pounds / square inch.In some embodiments, the sample is blood or blood-related products. In some embodiments, the sample is an apheresis sample or a leukocyte apheresis sample. In some embodiments, the sample contains platelets as contaminants. In some embodiments, the method removes at least 80% of platelets from the sample. In some embodiments, the method removes at least 90% of platelets from the sample. In some embodiments, the method removes at least 95% of platelets from the sample. In some embodiments, the enriched target cells include leukocytes. In some embodiments, the enriched target cells include stem cells. In some embodiments, the enriched target cells include peripheral blood mononuclear cells. In some embodiments, the peripheral blood mononuclear cells include CD3+ cells. In some embodiments, the method further includes genetically engineering the enriched target cells to obtain genetically engineered target cells. In some embodiments, the genetic engineering includes transfecting or transducing the target cells with recombinant nucleic acids. In some embodiments, the enriched target cells or genetically engineered target cells are grown by culturing them in vitro.

[0009] In another embodiment, a method for producing chimeric antigen receptor (CAR) T cells is described herein, comprising (a) obtaining a sample containing T cells; (b) separating T cells from contaminants by (i) pouring the sample into one or more sample inlets of a microfluidic cartridge, (ii) allowing the sample to flow to the outlet of the cartridge, and (iii) obtaining a T cell-enriched product from the product outlet; and (c) genetically engineering the T cells in the enriched product obtained in step b) to produce chimeric antigen receptors (CARs) on their surfaces. In some embodiments, the sample is blood, an apheresis product, or a leukocyte apheresis product. In some embodiments, the genetic engineering of T cells comprises transfecting or transducing target cells, the genetically engineered target cells being further proliferated by cell growth in vitro.

[0010] In another embodiment, a method for producing chimeric antigen receptor (CAR) natural killer cells is described herein, comprising: (a) obtaining a sample containing natural killer cells; (b) separating natural killer cells from contaminants by (i) pouring the sample into one or more sample inlets of a microfluidic cartridge, (ii) allowing the sample to flow to the outlet of the cartridge, and (iii) obtaining a natural killer cell-enriched product from the product outlet; and (c) genetically engineering the natural killer cells in the enriched product obtained in step b) to produce chimeric antigen receptors (CARs) on their surfaces. In some embodiments, the sample is a blood sample, an apheresis product, or a leukocyte apheresis product. In some embodiments, the genetic engineering of natural killer cells comprises transfecting or transducing target cells, the genetically engineered target cells being further grown by cell growth in vitro.

[0011] Additional aspects and advantages of the present disclosure will be readily apparent to those skilled in the art from the following detailed description, which shows and describes only exemplary embodiments of the present disclosure. As will be understood, various other embodiments are possible of the present disclosure, and some of its details can be modified in various obvious ways, all of which do not depart from the present disclosure. Accordingly, the drawings and description should be considered illustrative and not restrictive.

[0012] Embedding by reference Any publication, patent, or patent application referred to herein is incorporated by reference to the same extent as any individual publication, patent, or patent application is incorporated by reference specifically and individually. Furthermore, U.S. Patents 5,427,663, 5,837,115, 6,685,841, 6,913,697, 7,150,812, 7,276,170, 7,318,902, 7,472,794, 7,735,652, 7,988,840, 8,021,614, 8,282,799, and 8,3 U.S. Patent No. 04,230, U.S. Patent No. 8,579,117, U.S. Patent Publication No. 10 / 324,011, U.S. Patent Publication No. 2005 / 0282293, U.S. Patent Publication No. 2006 / 0134599, U.S. Patent Publication No. 2007 / 0160503, U.S. Patent Publication No. 2006 / 0121624, U.S. Patent Publication No. 2005 / 0266433, U.S. Patent Publication No. 2007 / 0026381, U.S. Patent Publication No. 2007 / 0026413 , U.S. Patent Application Publication No. 2007 / 0026414, U.S. Patent Application Publication No. 2007 / 0026415, U.S. Patent Application Publication No. 2007 / 0026417, U.S. Patent Application Publication No. 2007 / 0059680, U.S. Patent Application Publication No. 2007 / 0059718, U.S. Patent Application Publication No. 2007 / 0059781, U.S. Patent Application Publication No. 2007 / 0059774, U.S. Patent Application Publication No. 2007 / 0099207, U.S. Patent Application Publication No. 2007 / 01968 Applications 20, U.S. Patent Publication No. 2006 / 0223178, U.S. Patent Publication No. 2008 / 0124721, U.S. Patent Publication No. 2008 / 0090239, U.S. Patent Publication No. 2008 / 0113358, U.S. Patent Publication No. 2014 / 0342375, U.S. Patent Publication No. 2016 / 0139012, U.S. Patent Publication No. 2019 / 0071639, and WO2012094642 are each incorporated in their entirety by reference.To the extent that any publications and patents or patent applications incorporated by reference conflict with the disclosures contained herein, this Specified Specified supersedes and / or replaces any such conflicting material.

[0013] Novel features of the present invention are described in particular in the appended claims. A better understanding of the features and advantages of the present invention is obtained by referring to the following detailed description and the appended drawings (also "figure" and "FIG." herein) which describe exemplary embodiments in which the principles of the present invention are utilized. [Brief explanation of the drawing]

[0014] [Figure 1] Figures 1A to 1G show various operating modes of the DLD.

[0015] [Figure 2] Figure 2 shows various uses of a channel with an array of obstacles different from those shown in Figures 1A to 1C.

[0016] [Figure 3-1] Figures 3A to 3D show an embodiment of the device with a configuration of 14 parallel channels that can be used in a microfluidic device. [Figure 3-2] Same as above.

[0017] [Figure 4-1] Figures 4A to 4D show two channels. Figures 4B to 4D show magnified views of parts of the channels. [Figure 4-2] Same as above.

[0018] [Figure 5] Figure 5 is a cross-sectional view of a "bump array" device in which equilateral triangular obstacles are arranged in a microfluidic channel.

[0019] [Figure 6] Figures 6A and 6B show an array of diamond-shaped posts.

[0020] [Figure 7] Figures 7A - 7C show a stacked separation assembly in which two microfluidic devices are combined to form a single unit.

[0021] [Figure 8-1] Figures 8A - 8B show two channels that may be found in the device shown in FIG. 7. A partial enlarged view of the channel is shown in FIG. 8B. In this example, the channel has an array of asymmetrically spaced diamond - shaped obstacles, and G1 is larger than G2. The diamonds are offset, and thus successive rows are shifted transversely with respect to the previous row. [Figure 8-2] The same as above.

[0022] [Figure 9] FIG. 9 shows a stacked assembly of microfluidic devices inside a casing, which may be collectively referred to as a "cassette".

[0023] [Figure 10] Figures 10A and 10B show a channel enclosed by two walls having a sample inlet and a fluid inlet.

[0024] [Figure 11] FIG. 11 is a comparison of the normalized velocity flow between two equilateral - triangle posts (left panel) and the normalized velocity flow between two circular posts (right panel).

[0025] [Figure 12] FIG. 12 is a graph of the predicted critical diameter versus the array tilt angle (ε) for an array of triangular obstacles (lower line) and an array of circular obstacles (upper line).

[0026] [Figure 13] FIG. 13 is a graph showing the effect of the tilt angle ( "array tilt" in the figure) on the gap length G.

[0027] [Figure 14] Figure 14 is a graph showing the effect (expressed as r / S) of the curvature of the obstacle's edge on the critical size of the side of the gap enclosed by the edge.

[0028] [Figure 15] Figure 15 is a graph showing the effect of applied pressure on particle velocity in bump arrays with triangular posts (data shown as triangles) and bump arrays with circular posts (data shown as circles).

[0029] [Figure 16-1] Figures 16A and 16B show cross-sectional views of a single cartridge DLD element comprising six layers: two DLD micropost layers, two layers with crumple zones for fluid feeder channels, and two end layers. Figure 16B shows a top view of a non-limiting example of a DLD layer consisting of an array of elongated diamond or hexagonal posts. [Figure 16-2] Same as above.

[0030] [Figure 17] Figures 17A to 17C show top views of two DLD element cartridges mounted in a device cassette. Figure 17B shows a view from the upper left to the lower part of the DLD cartridge mounted in the device cassette. Figure 17C shows a view from the upper right to the lower part of the DLD cartridge mounted in the device cassette.

[0031] [Figure 18] Figures 18A and 18B show a bottom view (18A) and a cross-sectional view (18B) of a planar support, illustrating a specific embodiment of the configuration of the cavity.

[0032] [Figure 19]Figures 19A and 19B show alternative embodiments of voids when planar supports are stacked to form a microfluidic cartridge (cross-sectional views are shown). [Modes for carrying out the invention]

[0033] This invention primarily relates to size-based microfluidic separation, and more particularly to the use of DLD in the preparation of therapeutically useful cells. The language of this specification provides guidance on the fabrication and use of microfluidic devices and the use of DLD for performing separations involving biological materials.

[0034] While various embodiments of the present invention are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided merely as examples. Many variations, modifications, and substitutions can be conceived by 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 adopted.

[0035] Whenever the terms “at least,” “greater than,” or “greater than or equal to” precede the first number in a set of two or more numbers, the terms “at least,” “greater than,” or “greater than or equal to” apply to each number in that set. For example, 1, 2, or 3 or more is equivalent to 1 or more, 2 or more, or 3 or more.

[0036] Whenever the terms “no more than,” “less than,” or “less than or equal to” precede the first number in a set of two or more numbers, the terms “no more than,” “less than,” or “less than or equal to” apply to each number in that set. For example, 3, 2, or 1 or less is equivalent to 3 or less, 2 or less, or 1 or less.

[0037] definition Apheresis: As used herein, this term refers to the procedure by which blood from a patient or donor is separated into its components, such as leukocytes, platelets, and erythrocytes. “Apheresis sample” is the final product of this procedure. More specific terms are “platelet apheresis” (referring to the separation of platelets) and “leukocyte apheresis” (referring to the separation of leukocytes). In this context, the term “separation” refers to the acquisition of a product enriched with specific components compared to whole blood or other starting material, and does not imply that absolute purity is achieved.

[0038] CAR T cells: The term "CAR" is an acronym for "chimeric antigen receptor." Therefore, "CAR T cells" are T cells that have been genetically modified to express chimeric receptors.

[0039] CAR T-cell therapy: This term refers to any procedure in which a disease or medical condition is treated using CAR T cells. Diseases that can be treated include hematological malignancies and solid tumors, autoimmune diseases, and infections.

[0040] Carrier: As used herein, the term “carrier” refers to an active substance, e.g., beads or particles, made of biomaterials or synthetic materials, added to a preparation to bind directly or indirectly (i.e., through one or more intermediate cells, particles, or compounds) to some or all of the existing compounds or cells. Carriers may be made from a variety of different materials, including DEAE-dextran, glass, polystyrene plastic, acrylamide, collagen, and arginate, and typically have a size of 1 to 1000 μm. They may be coated or uncoated and may have a surface that can be modified to contain affinity substances (e.g., antibodies, activators, haptens, aptamers, particles, or other compounds) that identify antigens or other molecules on the cell surface. Carriers may be magnetized and may contain particles (e.g., Janus-like or strawberry-like particles) that impart size-independent secondary properties to cells or cell complexes. As a result, for example, the particles may have chemical, electrochemical, or magnetic properties that can be used in downstream processes such as magnetic separation, electroporation, gene transfer, and / or certain analytical chemistry processes. The particles may also induce metabolic changes in cells, activate cells, or promote cell division.

[0041] Carriers that bind "to facilitate DLD separation": This term refers, depending on the context, to carriers and methods of binding that influence how cells, proteins, or particles behave in DLDs. More specifically, "binding to facilitate DLD separation" means that a) the binding must be specific to a particular target cell type, protein, or particle, and b) the size of the complex resulting from the binding must be increased compared to unbound cells, proteins, or particles. If binding to target cells, the increase must be at least 2 μm (alternatively, expressed as a percentage, at least 20, 50, 100, 200, 500, or 1000%). If a treatment or other use requires target cells, proteins, or other particles to be released from the complex in order to fulfill its intended use, the term "to facilitate DLD separation" means that the complex must enable such release, for example by chemical or enzymatic cleavage, chemical lysis, digestion, competition with other binders, or physical shear (e.g., creating shear stress using a pipette), and the released target cells, proteins, or other particles must maintain their activity, and for example, therapeutic cells must also maintain biological activity that makes them therapeutically useful after release from the complex.

[0042] Carriers may be bound “to complement DLD separation”: This term refers to carriers and methods of binding that alter the chemical, electrochemical, or magnetic properties of cells or cell complexes, or alter the biological activity of one or more cells, regardless of whether they increase in size sufficiently to promote DLD separation. Carriers that complement DLD separation do not necessarily bind with specificity to target cells; that is, they may need to be combined with some other active ingredient to make them specific, or they may simply be added to cell preparations to enable non-specific binding. The terms “to complement DLD separation” and “to promote DLD separation” are not mutually exclusive. Binding can both complement and promote DLD separation. For example, polysaccharide carriers may have activators on their surface that increase cell growth rate, and binding of one or more of these carriers can also promote DLD separation. Alternatively, binding may only promote DLD separation or only complement DLD separation.

[0043] Sample: The term “sample” as used herein generally refers to any sample that contains or is suspected of containing nucleic acid molecules or nucleic acid cells. For example, a sample may be a biological sample containing one or more nucleic acid molecules or nucleic acid cells. A biological sample may be obtained (e.g., extracted or isolated) from or contain blood (e.g., whole blood), plasma, serum, urine, saliva, mucosal discharge, sputum, feces, and tears. A sample may also contain blood, blood products (such as leukocyte apheresis products or apheresis products), and anticoagulants (e.g., EDTA, EGTA, heparin, citrate, ACD-A, or thrombin inhibitors). A biological sample may be a fluid sample or a tissue sample (e.g., a skin sample). In some examples, a sample may be obtained from a cell-free fluid such as whole blood. In some examples, a sample may contain circulating tumor cells. In some examples, the samples are environmental samples (e.g., soil, waste, outside air), industrial samples (e.g., samples from any industrial process), and food samples (e.g., dairy products, vegetable products, and meat products). The samples may be processed before being placed in the microfluidic device. The samples may, appropriately, be apheresis products or leukocyte apheresis products [e.g., leukopak].

[0044] Target Cells: As used herein, “target cells” refers to cells required by the various procedures described herein, or cells designed to be purified, harvested, manipulated, etc. What constitutes a specific cell varies depending on the context in which the term is used. For example, if the objective of a procedure is to isolate a particular type of stem cell, then that cell is the target cell for that procedure.

[0045] Isolate or purify: Unless otherwise specified, these terms are synonymous as used herein and refer to the enrichment of an unwanted material with a desired product. This term does not necessarily mean that the product is completely isolated or completely pure. For example, if a starting sample has target cells that make up 2% of the cells in the sample, and the procedure is performed results in a composition in which 60% of the present cells are target cells, then the procedure has succeeded in isolating or purifying the target cells.

[0046] The term “obstacle array” is used herein as synonymous with and refers to an aligned array of obstacles arranged in a flow channel through which a fluid carrying cells or particles can pass. An obstacle array comprises a number of obstacles arranged in a column (along the fluid flow path). Gaps are formed between the obstacles (along the fluid flow path) to allow the passage of cells or other particles. Such arrays or columns are arranged in a repeating manner of one or more transverse rows (perpendicular to the fluid flow path).

[0047] As described herein, “channel” or “lane” refers to a group of obstacles arranged to form separate isolation units, and such channels may be enclosed on each side by walls so that separate lanes are isolated. Channels may extend in parallel from one or more common inputs to one or more common outputs. Channels may be fluidly connected in series.

[0048] Deterministic Lateral Displacement: As used herein, the term “Deterministic Lateral Displacement,” or “DLD,” refers to a process in which particles are deterministically deflected based on their size in a channel through a microfluidic obstacle array. This process can be used to separate cells, which is generally the context in which it is discussed herein. However, it is important to recognize that DLD can also be used to enrich cells and for buffer exchange (see Figure 1). The process is described herein in terms of continuous flow (DC state, i.e., bulk fluid flow in only one direction). However, DLD can also function under oscillatory flow (AC state, i.e., bulk fluid flow alternating between two directions).

[0049] Critical Size: The "critical size," "critical diameter," or "predetermined size" of particles passing through an obstacle array represents the limit of particle size that can follow the laminar flow of a fluid. Particles larger than the critical size may be "pushed" out of the fluid flow path, while particles smaller than the critical size (or predetermined size) are not displaced.

[0050] As used herein in relation to fluid flow: DLD, the terms “fluid flow” and “bulk fluid flow” refer to the macroscopic movement of fluid in the overall direction across an obstacle array. These terms do not consider the transient displacement of the fluid stream as the fluid moves around the obstacles in order for the fluid to continue moving in the overall direction.

[0051] In a bump array device, the inclination angle ε is the angle between the direction of the bulk fluid flow and the direction determined by the alignment of a series of obstacles in the array (see Figure 5).

[0052] Array Direction: In an obstacle array device, the "array direction" is the direction determined by the alignment of a series of obstacles in the array. When a particle passes through a gap and encounters a downstream obstacle, it is "deflected" in the obstacle array (i.e., moves at an angle ε relative to the bulk fluid flow) if the overall trajectory of the particle follows the array direction of the obstacle array. The particle is not displaced if its overall trajectory follows the direction of the bulk fluid flow under such circumstances.

[0053] As used herein, the term "approximately" refers to an amount within 10% of the stated amount.

[0054] Overall Overview The present invention relates to a microfluidic device in which size-based purification is performed by passing a biological sample through an array of obstacles in a microfluidic channel. This is based in part on the concept that cells of a given size can be processed more rapidly by increasing the length of the gaps of obstacles perpendicular to the direction of fluid flow and decreasing the length of the gaps parallel to the fluid flow.

[0055] The device characteristics discussed above can be realized by the shape of the obstacle, which is elongated, with the most preferred shapes being diamond-shaped or hexagonal. The hexagonal obstacle is most preferred because, while it offers the same processing advantages as the diamond shape, it is easier to manufacture and has better resistance to biofouling.

[0056] Using asymmetric gaps improves processing capacity and allows devices to operate for longer periods, but narrowing parallel gaps can create practical challenges in large-scale production of devices, particularly with respect to embossing, molding, or demolding. These challenges can be mitigated by using polygonal and elongated obstacles that are offset to some extent, resulting in narrower gaps where the obstacles preferably have vertices facing each other in the parallel gaps, but where the vertices are offset from each other (not directly opposite each other; see Figures 6A-6B). Such a design reduces flow through the parallel gaps (also known as minor flux) by lengthening rather than narrowing the gaps. In contrast, the vertices of the vertical gaps preferably face each other directly. Therefore, a key feature of the devices disclosed herein is the presence of obstacle arrays where the vertical and parallel gaps are asymmetric, i.e., not the same size. By varying the spacing, it is possible to reduce resistance to flow compared to devices that separate particles and cells of the same size range but have vertical and parallel gaps of the same length.

[0057] For some samples, biofouling and mixing of the fluid as the sample is fed into the device can continue to affect separation. For example, biofouling at the array inlet can prematurely spread blood or apheresis samples into a second fluid stream, resulting in contamination of leukocyte target cell products with platelets and erythrocytes. By using a separation wall, positioned to separate the sample inlet from the inlets for other fluids and terminating midway down the channel, biofouling areas can be isolated, temporarily preventing contact between flow streams. As a result, the parallel-flowing fluids have limited time for diffusion mixing, which can improve the purification of target cells or target particles. Typically, the separation wall extends from the sample inlet for a distance of about 10 to 50 percent of the microfluidic channel length, but the wall may be shorter or longer depending on the circumstances related to separation.

[0058] Another advantage of separation walls is that they reduce undesirable mixing that can occur when using a fluctuating pressure source to propel a sample and other fluids through a device. For example, a peristaltic pump may be used to push fluid through a device, and a peristaltic pump has the advantage of maintaining a closed system environment, i.e., the sample does not touch the inside of the pump but only moves through a tube constricted by the pump head. However, peristalsis can generate normal pressure surges that tend to mix the flowstream. In the presence of a separation wall, it acts as a baffle for those surges, reducing the undesirable mixing that would occur if the wall were not present. As a result, improved separation can be achieved.

[0059] Another feature of this microfluidic device is that two or more devices can be used as part of an assembly stacked on top of each other and supplied through a common manifold. Each stacked microfluidic device comprises a planar support having one or more embedded channels, each channel containing a separate obstacle array. The support typically has multiple channels, and in some examples, the channels may be embedded on both the top and bottom surfaces of the support. By using multiple channels in the device and multiple devices in the assembly, large volumes of samples can be microfluidically processed. For example, an assembly of microfluidic devices described herein may be designed to process more than 100 mL of sample per hour (e.g., undiluted apheresis sample), and higher volumes (more than 200, 300, 400, or 500 mL per hour) are preferred depending on the specific processing purpose.

[0060] Overall, the devices of the present invention are characterized in some or all of the following: 1) asymmetrically arranged obstacles in which the gap perpendicular to the bulk fluid flow is of a different length from the gap parallel to the bulk fluid flow; 2) elongated and polygonal obstacles whose vertices extend through the gaps; 3) vertices on each side of the parallel gaps that are offset from each other; 4) vertices on each side of the perpendicular gaps, preferably directly opposite each other; 5) one or more segregating walls that extend partway through the channel and separate the sample inlet from the inlet for other fluids; 6) the appropriate use of a peristaltic or fluctuating pressure source to propel the sample and other fluids through the device having the segregating walls; and 7) assembling a stacked assembly from a plurality of individual microfluidic devices, each having a plurality of channels.

[0061] Overview of a Specific Embodiment 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, which is typically rectangular and can be made of any material suitable for the separation method, including silicon, glass, hybrid materials, or (preferably) polymers. The support has a top and a bottom, one or both of which have at least one embedded channel extending from one or more sample inlets and one or more separate fluid inlets to one or more product outlets and one or more separate waste outlets. The fluid inlets (unlike the sample inlets) may, in some cases, be referred to as "buffer" inlets or "wash" inlets and may be used to transport various fluids into the channel, depending on the purpose of separation. Unless otherwise specified by use or context, "fluid" may be a buffer, may contain reagents, may constitute a cell growth medium, and may generally be any liquid and may contain any components suitable for the operation of the device and the user's purpose.

[0062] When a fluid is poured into the device through the sample inlet or fluid inlet, it flows through the channel toward the outlet, thereby determining the direction of the bulk fluid flow. To separate cells or particles of varying sizes, the channel includes an array of obstacles aligned in longitudinal rows extending along the channel (from inlet to outlet) and transverse rows extending across the channel. Each subsequent transverse row of obstacles is transversely offset from the preceding transverse row, thus determining an array direction that is offset by an angle (ε) from the direction of the bulk fluid flow. The obstacles are positioned to define a critical size, so that when a sample is poured into the device inlet and flows to the outlet, particles or cells in the sample larger than the critical size follow the array direction, while particles smaller than the critical size flow in the direction of the bulk fluid flow, resulting in separation.

[0063] Adjacent obstacles in a row of the array are separated by a gap G1 perpendicular to the direction of bulk fluid flow, and adjacent obstacles in a column are separated by a gap G2 parallel to the direction of bulk fluid flow (see Figures 6A and 6B). A feature of this 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%). Each obstacle in the array has at least two vertices, and at least one vertex is positioned adjacent to each gap on each side. In a preferred embodiment, the vertices have one or more vertices that face each other but are not directly opposite each other, extending through the gaps so that they are adjacent to the parallel gaps on each side, and / or the obstacles have vertices that face each other and are directly opposite each other, extending through the gaps so that they are adjacent to the perpendicular gaps on each side (see Figures 6A and 6B).

[0064] Microfluidic devices typically also have an obstacle bonding layer bonded to the surface of a planar support and to the obstacle in the channel, in order to prevent the fluid or sample from flowing over the obstacle during the operation of the device. This obstacle bonding layer may have one or more passages that are fluid-connected to the inlet and outlet of the channel, thereby enabling fluid flow.

[0065] Generally, microfluidic devices are used to separate target particles or target cells that are larger than the device's critical size from contaminants that are smaller than the critical size. As a sample containing target cells or target particles is poured into the device through a sample inlet and fluidly passes through the channels, the target cells or target particles flow to one or more product outlets where enriched products are obtained. The term "enriched," as used in this context, means that the ratio of target cells or target particles to contaminants is higher in the product than in the sample. Contaminants that are smaller than the critical size mostly flow to another waste outlet where they can be collected or discarded.

[0066] The purpose of separation is generally to separate target cells or target particles from smaller contaminants, but sometimes users may want to separate target cells or target particles from larger contaminants. In those cases, a microfluidic device with a critical size larger than the target cells or target particles but smaller than the contaminants can be used. A combination of two or more obstacle arrays with different critical sizes may be used for separation in a single device or multiple devices. For example, a device may have a channel with a first obstacle array having a critical size larger than T cells but smaller than granulocytes and monocytes, and a second array having a critical size smaller than T cells but larger than platelets and erythrocytes. Processing a blood sample with such a device would allow for the collection of products separated from granulocytes, monocytes, platelets, and erythrocytes by T cells. The order of the obstacle arrays should not be particularly important to the outcome; that is, arrays with smaller critical sizes can come before or after arrays with larger critical sizes. Also, arrays with different critical sizes can be present in separate devices through which the cells pass.

[0067] A wide array and multiple outlets may be used to collect multiple products; for example, monocytes may be obtained at one outlet and T cells at a different outlet. Therefore, using multiple arrays and multiple outlets allows for the simultaneous collection of several products, which are purified more effectively than when a single array is used. High throughput can be maintained by using multiple devices stacked on top of each other, as will be discussed further below.

[0068] Preferably, the obstacles used in microfluidic devices are polygonal, with diamond-shaped or hexagonal obstacles being preferred. These obstacles are also generally elongated such that their length (P1) perpendicular to the bulk fluid flow is, for example, 10–100% different (generally longer) from their width (P2) parallel to the bulk fluid flow (see Figure 6B). Typically, P1 is at least 15%, 30%, 50%, 100%, or 150% longer than P2. Expressed as a range, P1 is 10–150% (15–100%, or 20–70%) longer than P2.

[0069] Microfluidic devices may include a separation wall, which extends from the sample inlet of the device into an array of obstacles in the channel, separating the sample inlet from the fluid inlet in the device and preventing mixing (see Figures 10A and 10B). The separation wall is oriented parallel to the direction of the bulk fluid flow and extends toward the sample outlet and fluid outlet. The wall terminates before reaching the ends of the channel, after which the sample stream and fluid stream can come into contact with each other. The wall should generally extend over a distance of at least 10% of the length of the array of obstacles, but may extend over at least 20%, 40%, 60%, or 70% of the array. Expressed as a range, the wall typically extends over 10–70% of the length of the array of obstacles. Two or more separation walls may be present in the device and may be positioned differently depending on the purpose of separation.

[0070] To increase the rate at which the volume can be processed, a stacked separation assembly can be fabricated by overlaying one or more stacked devices onto a first microfluidic device, wherein the bottom surface of each stacked device is in contact with the top surface of the first microfluidic device or its obstruction bonding layer, or with the top surface of another stacked device or its obstruction bonding layer. Samples are supplied to the sample inlets of all devices through a first common manifold, and fluid is supplied to the fluid inlets through a second manifold, which may or may not be the same as the first manifold. Products are removed from the product outlet through one or more product conduits, and waste is removed from the waste outlet through one or more waste conduits separate from the product conduits. Generally, a stacked separation assembly has two to nine stacked devices together with the first microfluidic device; however, a larger number of devices may be used. Furthermore, the top and / or bottom surfaces of the support may have multiple (e.g., 2 to 40 or 2 to 30) embedding channels which may be used for purifying target particles or target cells.

[0071] The stacked separation assembly may have a reservoir junction layer attached to the bottom surface of a first microfluidic device and / or the top surface of the stacked microfluidic devices. The reservoir junction layer should include a first end through which one or more passages allow fluid flow to the channel inlet, and a second end which may include one or more passages allowing fluid flow to and from the channel's product outlet and waste outlet. The second end is on the opposite side of the first end and is separated by a fluid-impermeable material.

[0072] As shown in Figure 9, the stacked assembly of the device may be supported in a cassette characterized by the presence of an outer casing having ports that allow for the transport of samples and fluids into the cassette and the transport of products and wastes out of the cassette. The figure shows a cassette having two inlet ports and two outlet ports. However, several products may be collected essentially simultaneously using multiple ports to the inside and outside of the cassette. It is also recognized that the cassette may be part of a system, of which there are components that are well 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 rate and pressure, sensors for monitoring fluid characteristics such as pH or salinity, sensors for determining the concentration of cells or particles, and analyzers for determining the type of cells or particles present in the cassette or the material collected from the cassette. More conceptually, any equipment, material to be processed, and processing purpose that are well known in the art and compatible with the cassette may be used.

[0073] In another aspect, the present invention relates to a method for purifying target particles or target cells of a predetermined size from contaminants by obtaining a sample containing target particles or target cells and contaminants, and performing purification using either a microfluidic device or a stacked separation assembly discussed herein. Purification is achieved by pouring the sample into one or more sample inlets of any of the microfluidic devices discussed above, or into the sample inlet of a stacked device of the first microfluidic device or device assembly. In particular, when using a stacked device, a manifold may be used to pour the sample into the inlet. The sample is then flowed through the channels of the device to the outlet. Generally, the target particles or target cells are larger than the critical size of the device's obstacle array, and at least some of the contaminants are smaller than the critical size. As a result, the target cells or target particles flow to one or more product outlets from which the enriched product of the target cells or target particles is obtained, and the contaminants having a size smaller than the critical size flow to another waste outlet. However, as mentioned earlier, there are also cases where the target cells or target particles are smaller than the contaminants, and the device is selected to have a critical size that is larger than the target cells or target particles but smaller than the contaminants. In those cases, the overall operation of the device is essentially the same, but the contaminants flow in the direction of the array, and the target cells or target particles move in the direction of the bulk fluid flow.

[0074] The sample may be obtained from an individual or patient, particularly from a cancer patient, a patient with an autoimmune disease, or a patient with an infectious disease. In some embodiments, the sample is blood or derived from blood (e.g., an apheresis sample or a leukocyte apheresis sample), and the target cells are dendritic cells, leukocytes (especially T cells), stem cells, B cells, NK cells, monocytes, or progenitor cells. Contaminants in these examples typically include erythrocytes and / or platelets. The result of the purification should be a product enriched with target cells and with at least 80% (preferably 90%, more preferably 95%) of platelets and / or erythrocytes removed from the sample.

[0075] Once purified target cells are obtained, these cells may be genetically engineered by transfecting them with recombinant nucleic acids or by transduction. These cells may then be grown in culture medium and finally used to treat the patient from whom the sample was obtained.

[0076] The present invention, in particular, includes a method for producing chimeric antigen receptor (CAR) T cells by a) obtaining a sample containing T cells, b) separating the T cells from contaminants by pouring the sample into one or more sample inlets of either a microfluidic device or a stacked device as discussed herein, c) flowing the sample to the outlet of the device, and d) obtaining a T cell-enriched product from the product outlet. Once the T cells are recovered, they are genetically engineered, preferably by transfecting or transducing them with recombinant nucleic acids, to express the chimeric antigen receptor on the cell surface. The genetically engineered target cells are grown in vitro by cell growth and may be administered as therapy to the patient who provided the sample.

[0077] The sample containing T cells is preferably blood, apheresis product, or leukocyte apheresis product from a cancer patient, a patient with an autoimmune disease or an infectious disease, or from an HLA-matched (to the patient being treated) donor. The cells may be surrounded by one or more carriers to facilitate or complement DLD isolation, and the cells or complex may then be purified by DLD. The present invention comprises the CAR T cells produced and CAR T cell therapy in which the CAR T cells are used.

[0078] I. Design of Microfluidic Cartridges This disclosure provides microfluidic cartridges (i.e., devices, chips, cassettes, plates, microfluidic devices, cartridges, DLD devices, etc.) for purifying particles or cells. The microfluidic cartridges of this disclosure may operate using DLD methods. The microfluidic cartridges of this disclosure may be formed from a polymer material (e.g., thermoplastic) and may include one or more first planar supports having a top and bottom surface and second planar supports having a top and bottom surface, wherein the top surfaces of the first and second planar supports include at least one embedding channel extending from one or more inlets to one or more outlets, the at least one embedding channel includes an array of obstacles, and the bottom surfaces of the first and second planar supports include at least one cavity configured to deform when the bottom of the first planar support is pressed against the bottom of the second planar support. The microfluidic cartridges of this disclosure may be single-use or disposable devices. Alternatively, the microfluidic cartridges may be multi-use devices. Using polymers (e.g., thermoplastics) to form microfluidic structures allows for the use of inexpensive and highly scalable soft embossing processes, improving the ability to rapidly manufacture voids and avoiding damage to obstacles during the manufacturing process (i.e., posts, DLD arrays, etc.).

[0079] The cartridges described herein can operate by a deterministic lateral displacement method, i.e., DLD. Referring to FIGS. 1A-1G, DLD may include three different operating modes. The operating modes include i) separation (FIG. 1A), ii) buffer exchange (FIG. 1B), and iii) concentration (FIG. 1C). In each mode, particles above the critical diameter are deflected in the direction of the array from the point of entry, and as a result, size selection, buffer exchange, or concentration is performed as a function of the shape of the device. In all cases, particles below the critical diameter pass directly through the device under laminar flow conditions and then exit the device. FIG. 1D shows a 14-lane DLD design used in the separation mode. The total length of the separation zone of the microfluidic cartridge may be about 75 mm, the width may be about 40 mm, and the individual channels may each have a width of about 1.8 mm. FIGS. 1E-1F are enlarged views of a plastic diamond-shaped post array and a converging collection port for the outlet. FIG. 1G shows where leukocyte apheresis products are being processed at 10 PSI using the device.

[0080] The cartridges described herein may be arranged in various orientations to achieve different DLD modes or product results (FIG. 2). FIG. 2 shows four channels having sidewalls (1) and an array of obstacles (2). A sample containing blood, cells, or particles enters the channel through an upper sample inlet (3), and buffer, reagent, or media enters the channel at a separate fluid inlet (4). As they flow towards the bottom of the channel, cells or particles having a size greater than (>Dc) the critical diameter of the array flow at an angle defined by the direction of the array of obstacles and are separated from cells and particles having a size less than (<Dc) the critical diameter of the array.

[0081] Referring to Figures 3A–3D, an embodiment of the cartridge may have a configuration of 14 parallel channels that can be used in a microfluidic device or cartridge. Figures 3B–3D show a partially enlarged view of the cartridge. In this figure, the channels have three zones (sections) where the gaps gradually become smaller. The cartridge has a common sample inlet, for example, for blood, which supplies a sample to the inlet of each channel. There are separate inlets to channels for buffers, but these can also be used to introduce a fluid containing reagents, growth medium or other fluids into the channels, depending on the processing purpose. At the bottom of each channel is a product outlet, which will typically be used to collect target cells or target particles that are larger in size than the critical diameter of the channel's obstacle array. The outlets from the individual channels can feed into the common product outlet, from which target cells or target particles can be collected. A waste outlet is also shown from which cells and particles smaller than the critical diameter of the channel's obstacle array exit.

[0082] Referring to Figures 4A to 4D, the cartridge embodiment may have two channels. Figures 4B to 4D show magnified views of the channels. The channels have three parts, designed to have obstacles and gaps of gradually decreasing diameter.

[0083] Some cartridges may have a “bump array” in which equilateral triangular obstacles are arranged in the microfluidic channel, as shown in the cross-sectional view of Figure 5. In the figure, the fluid flows from left to right, as indicated by the arrow labeled “fluid”. In this array, the equilateral triangular posts are arranged in a parallelogram grid configuration, inclined with respect to the direction of fluid flow. Other grid configurations (e.g., square, rectangular, trapezoidal, hexagonal grids) can also be used. The inclination angle ε (epsilon) is selected so that the device is periodic. In this embodiment, an inclination angle of 18.4 degrees (1 / 3 radian) results in the device being periodic every three rows. The inclination angle ε also represents the angle at which the array direction is offset from the fluid flow direction. The gap between posts is represented by G, and the side length of the equilateral triangle is S. Streamlines extending between posts are shown, dividing the fluid flow between posts into three regions ("flow tubes") of equal volume flow. Relatively large particles (those exceeding the critical size of the array) follow the inclination angle of the array when the fluid flow is in the direction shown in the diagram. Relatively small particles (those smaller than the critical size of the array) follow the direction of the fluid flow.

[0084] The cartridges provided herein may comprise an array of diamond-shaped posts, as shown in Figures 6A and 6B. Figure 6A shows an array of symmetrical obstructions, where gaps perpendicular to the direction of fluid flow, e.g., gap 1 (G1), and gaps parallel to the direction of fluid flow, e.g., gap 2 (G2), are all of approximately the same length. The diamond-shaped obstructions may have two diameters, one perpendicular to the direction of fluid flow (P1) and the other parallel to the direction of fluid flow (P2). On the right side of the figure is an asymmetrical array in which the parallel gap is shorter than the perpendicular gap. G1 of the asymmetrical array is wider than that of the symmetrical array, but by reducing gap G2, the critical diameter of the array becomes the same as that of the symmetrical array. As a result, the two arrays should be virtually equal in separating particles or cells of a given diameter in a sample. However, widening G1 allows for increased sample handling and reduces channel clogging. Figure 6B shows an array of diamond-shaped obstacles on the left, where the obstacles are elongated such that their vertical diameter is longer than their horizontal diameter. The middle section of Figure 6 shows diamond-shaped posts, where the posts are elongated such that their horizontal diameter is longer than their vertical diameter, and the far right section of the figure shows elongated hexagonal obstacles in the horizontal direction.

[0085] Referring to Figures 7A to 7C, the cartridges described herein may constitute a stacked separation assembly in which two microfluidic devices or cartridges are combined to form a single unit. The uppermost device (5) comprises a planar support (6), which may be made from a variety of materials, most preferably a polymer, and has a top surface (7) and a bottom surface (12). The top surface (7) of the support houses a reservoir, which provides a sample inlet (9) and an inlet (10) for a buffer or other fluid at one end of the support, and a product outlet (14) and a waste outlet (13) at the other end. Each reservoir is fluid-connected through the support using a small via [(9), (10), (13), (14)] that connects the top surface (7) to a channel in the bottom surface (12). The bottom surface (12) of the support has a number of embedded microfluidic channels (8), each of which has 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 bonding layer (15), which seals the first device and prevents the fluid from flowing over the obstacles during operation. A second microfluidic device (16) of the laminate is shown, housing the embedded microfluidic channels on its uppermost surface, and the device (16) is sealed by the same obstacle bonding layer (15) as the uppermost device. A reservoir bonding layer (18) having an elongated opening (19) is also shown, which allows liquid to pass through the channel inlet and outlet. The reservoir bonding layer is similar to the obstruction bonding layer, except that it is attached to the surface of the device rather than the obstruction, and may be connected to one or more reservoirs that supply the device stack or manifold. Holes (11) used for aligning the stacked devices are shown. As described above, the two embedded microfluidic surfaces face the same obstruction bonding layer.An alternative configuration has embedded channels on the top surface of both devices, with an intermediate layer between the devices serving both functions: as an obstacle bonding layer for the lower embedded channel and as a distribution layer for the upper reservoir. Figure 7B shows a stack of multiple microfluidic devices that together form a single assembly unit. At the top of this stack (or possibly both top and bottom) is a manifold (22) which has a supply section (23) for the manifold inlet distribution section (24) and a conduit (28) connected from the manifold's product outlet (27). Although not shown in the figure, there are also conduits for extracting fluid from the supply section connected to the fluid inlet (25) and the waste outlet (26). Figure 7C shows a stacked separation assembly (20) mounted on a casing (21).

[0086] Two channels, which may be found in the device shown in Figure 7, are shown in Figures 8A and 8B. A magnified view of a portion of the channel is shown in Figure 8B. In this example, the channel has an array of asymmetrically spaced diamond-shaped obstacles, where G1 is larger than G2. The diamonds are offset, and therefore each successive row is shifted transversely relative to the previous row.

[0087] This disclosure provides a stacked assembly of microfluidic devices (20) inside a casing (21), which may collectively be referred to herein as “multiple cassettes” or “cassette” (Figure 9). A port (29) acts as a feeder for the sample supplied to a manifold (22) through the casing. The port (29) is connected to a manifold feeder (23), which distributes the sample to the sample inlet of the channel through a manifold sample inlet (24). Once poured, the sample flows through the channel containing an obstacle array (see Figures 3–6), and products with particles or cells larger than critical size exit the stacked assembly of the device at the manifold product outlet (27). The products then flow from the manifold outlet through a product conduit (28) and are transported out of the cassette through a product outlet port (31). The fluid flows through the cassette and reaches a manifold through port (51) connected to a manifold fluid supply unit (49). The fluid is distributed to the channel fluid inlet by a manifold fluid inlet (25). The fluid flows through the channel, and particles or cells smaller than the critical size mostly exit the device stack through the manifold waste outlet (26). These particles or cells then flow through a waste conduit (50) that transports the waste out of the cassette through an outlet port (30).

[0088] Embodiments of the cartridge or device provided herein may comprise a channel enclosed by two walls (32) having a sample inlet (33) and a fluid inlet (34) (Figures 10A–10B). A separation wall (35) is present to prevent the sample flow stream from mixing with the fluid flow stream. The separation wall extends into an obstacle array (36) and terminates at approximately the halfway point. Arrows on the array indicate the direction in which target cells having a size larger than the critical size of the array move. Upon entering the obstacle array, target cells are first deflected away from the direction of the fluid flow until they reach the separation wall. They then move along the wall until it terminates. They are then deflected again until they exit the channel at the product outlet (37). Particles having a size smaller than the critical size of the obstacle array are not deflected and exit the channel at the waste outlet (38). Figure 10B also shows a walled channel (43) with 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 over an obstacle array (44), where particles or cells larger than the critical diameter of the array are deflected into the reagent stream that will be reacted. A separation wall (41) extends from the reagent inlet to partway across the obstacle array (44), separating the reagent stream from the buffer or other fluid stream. This wall keeps cells or particles in the reagent stream for a longer time, thereby providing a longer time for the reaction. At the end of the separation wall, the particles or cells are again deflected to a product outlet (48) where they can be collected. During this process, the cells or particles are separated from the unreacted reagent. A second separation wall (45) extends from the end of the first separation wall (41) to a waste outlet (47), where buffers or other fluids, reagents, and small particles or cells may exit the device and 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 critical diameter of the obstacle array. These materials may be collected or discarded.

[0089] A comparison can be made between the normalized velocity flow between two equilateral triangular posts (left panel) and the normalized velocity flow between two circular posts (right panel) (Figure 11), illustrating the effect of obstacles or post shape. The shaded areas in Figure 11 represent equal proportions of under-curve areas and show that the critical radius (<15% gap width) of particles flowing over the triangular points is significantly smaller than the critical radius (>20% gap width) of particles flowing over the circular posts.

[0090] Figure 12 is a graph of the predicted critical diameter against the array tilt angle (ε) for arrays of triangular (lower line) and circular (upper line) obstacles. The analysis in Figure 12 further shows the effect of the post shape on displacing the particles or cells shown in Figure 11.

[0091] Referring to Figure 13, the effect of the inclination angle (referred to as "array inclination" in the figure) on the gap length G is clearly shown. T This refers to the length of the gap between the triangular posts, G C This refers to the length of the gap between the round posts. As the array inclination increases, there is a specific critical size (D) of the array between the triangular and round posts. C The difference in gap length becomes smaller.

[0092] Figure 14 shows the effect of the curvature of the obstacle's edge (expressed as r / S) on the critical size, which is represented for the side of a gap enclosed by the edge. Increasing the curvature of the post increases the critical size of that post with respect to a given gap length.

[0093] In addition to the critical size, posts of different shapes may affect particle velocity when a certain pressure is applied. Figure 15 shows the effect of applied pressure on particle velocity for bump arrays with triangular posts (data shown as triangles) and bump arrays with circular posts (data shown as circles). When pressure is applied, the array with triangular posts increases particle velocity more than the array with circular posts. Furthermore, the rate of increase in particle velocity when the pressure is increased is also greater for the triangular post array than for the circular post array.

[0094] Referring to Figure 16A, the cartridge described herein comprises a top and / or bottom seal / lid 1600 and a separation layer 1605. The separation layer 1605 comprises a plurality of obstacles 1620 to facilitate separation, a fluid layer 1610, and a void or crumple zone that allows the cartridge to be fabricated without deforming the plurality of obstacles. Referring to Figure 16B, the plurality of obstacles 1620 may be arranged in transverse rows 1625 and longitudinal rows 1630 such that gaps 1635 configured to allow fluid and cells to pass through are formed. The obstacles may be arranged so as not to be offset or with minimal offset between repeating transverse rows. Referring to Figures 17A to 17C, two or more cartridges may be stacked in series or in parallel or linked together to achieve greater separation or higher processing capacity.

[0095] Since similar devices or microfluidic cartridges operate on a sub-millimeter scale and handle volumes of fluid below microliters or nanoliters, the main obstacles during manufacturing are those that are damaged or deformed during embossing or assembly. For example, handling tips can put pressure on planar supports, especially when planar supports are pressed against each other, which can then cause deformation or destruction of the planar supports, obstacles (i.e., arrays of obstacles), and various separation lanes. Such deformation or destruction can significantly reduce performance when purifying particles or cells, or completely impair the function of the microfluidic cartridge. To avoid possible deformations and malfunctions during manufacturing and assembly, other microfluidic systems require slower manufacturing processes or accept reduced performance.

[0096] In one embodiment, the disclosure provides a microfluidic cartridge for purifying cells or particles. The microfluidic cartridge may include a first planar support. The first planar support may have a top and a bottom surface. The device may include a second planar support. The second planar support may have a top 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. The at least one embedded channel may include an array of obstacles. The bottom surfaces of the first and second planar supports may include a cavity. The cavity may be configured to deform when the bottom of the first planar support is pressed against the bottom of the second planar support.

[0097] The separation described herein occurs along channels embedded in a planar support, the channels comprising multiple obstacles. The cartridge described herein may utilize first and second planar surfaces. The first and second planar surfaces may be stacked (e.g., bottom and bottom or top and bottom, using spacers that double the processing and separation capacity while maintaining a small footprint). The top surface of the first and / or second planar surface may comprise at least one embedded channel and approximately 500 embedded channels. The top surface has approximately 2 embedding channels from at least 1 embedding channel, approximately 5 embedding channels from 1 embedding channel, approximately 20 embedding channels from 1 embedding channel, approximately 50 embedding channels from 1 embedding channel, approximately 100 embedding channels from 1 embedding channel, approximately 500 embedding channels from 1 embedding channel, approximately 5 embedding channels from approximately 2 embedding channels, approximately 20 embedding channels from approximately 2 embedding channels, approximately 50 embedding channels from approximately 2 embedding channels, approximately 100 embedding channels from approximately 2 embedding channels, and approximately 500 embedding channels from approximately 2 embedding channels. The top surface may have at least one embedded channel, approximately 2 embedded channels, approximately 5 embedded channels, approximately 5 embedded channels, approximately 100 embedded channels, approximately 5 embedded channels, approximately 500 embedded channels, approximately 20 embedded channels, approximately 100 embedded channels, approximately 20 embedded channels, approximately 500 embedded channels, approximately 50 embedded channels, or approximately 100 to 500 embedded channels.The top surface may have at least one embedded channel, about two embedded channels, about five embedded channels, about 20 embedded channels, about 50 embedded channels, or about 100 embedded channels. The top surface may have at least up to about two embedded channels, about five 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 may have about 28 channels (56 if stacked). An additional third, fourth, fifth, or sixth planar surface may have a similar number of embedded channels as the first or second planar surface.

[0098] The microfluidic cartridge may have at least one inlet and approximately 50 inlets. The microfluidic cartridge may have at least one inlet leading to about two inlets, one inlet leading to about five inlets, one inlet leading to about ten inlets, one inlet leading to about twenty inlets, one inlet leading to about fifty inlets, about two inlets leading to about five inlets, about two inlets leading to about ten inlets, about two inlets leading to about twenty inlets, about two inlets leading to about fifty inlets, about five inlets leading to about ten inlets, about five inlets leading to about twenty inlets, about five inlets leading to about fifty inlets, about ten inlets leading to about twenty inlets, about ten inlets leading to about fifty inlets, or about twenty inlets leading to about fifty inlets. A microfluidic cartridge may have at least one inlet, approximately two inlets, approximately five inlets, approximately ten inlets, approximately twenty inlets, or approximately fifty inlets. A microfluidic cartridge may have at least one inlet, approximately two inlets, approximately five inlets, approximately ten inlets, or approximately twenty inlets. A microfluidic cartridge may have at least up to approximately two inlets, approximately five inlets, approximately ten inlets, approximately twenty inlets, or approximately fifty inlets. The inlets may be supplied by a common fluid system or a dual fluid system (one for buffer / diluent and one for sample).

[0099] The microfluidic cartridge may have at least one outlet and approximately 50 outlets. The microfluidic cartridge may have at least one outlet leading to about two outlets, one outlet leading to about five outlets, one outlet leading to about ten outlets, one outlet leading to about twenty outlets, one outlet leading to about fifty outlets, about two outlets leading to about five outlets, about two outlets leading to about ten outlets, about two outlets leading to about twenty outlets, about two outlets leading to about fifty outlets, about five outlets leading to about ten outlets, about five outlets leading to about twenty outlets, about five outlets leading to about fifty outlets, about ten outlets leading to about twenty outlets, about ten outlets leading to about fifty outlets, or about twenty outlets leading to about fifty outlets. The microfluidic cartridge may have at least one outlet, approximately two outlets, approximately five outlets, approximately ten outlets, approximately twenty outlets, or approximately fifty outlets. The microfluidic cartridge may have at least one outlet, approximately two outlets, approximately five outlets, approximately ten outlets, or approximately twenty outlets. The microfluidic cartridge may have at least up to approximately two outlets, approximately five outlets, approximately ten outlets, approximately twenty outlets, or approximately fifty outlets. The outlets may supply a common fluid system or a dual fluid system (one for waste and one for enriched target cells or target particles).

[0100] Cartridges with two or more planar surfaces are prone to deformation and malfunction due to the small array of obstacles in the lane; therefore, they may have voids to protect them.

[0101] The void in the microfluidic cartridge may be configured to deform, bend, expand, fold, or crumple. The void may be configured to protect obstacles, channels, inlets, outlets, planar surfaces, or any combination thereof from damage, displacement, deformation, or failure. The void may include a crumple zone configured to protect obstacles, channels, inlets, outlets, planar surfaces, or any combination thereof from damage, displacement, deformation, or failure. The volume of the void may range from approximately 1 cubic micrometer to approximately 10,000 cubic micrometers.The volume of the void is approximately 1 cubic μm to 5 cubic μm, approximately 1 cubic μm to 10 cubic μm, approximately 1 cubic μm to 30 cubic μm, approximately 1 cubic μm to 50 cubic μm, approximately 1 cubic μm to 100 cubic μm, approximately 1 cubic μm to 300 cubic μm, approximately 1 cubic μm to 1,000 cubic μm, approximately 1 cubic μm to 3,000 cubic μm, approximately 1 cubic μm to 10,000 cubic μm, approximately 5 cubic μm to 10 cubic μm, approximately 5 cubic μm to 30 cubic μm, approximately 5 cubic μm to 50 cubic μm, and approximately 5 cubic μm. From approximately 100 cubic μm, from approximately 5 cubic μm to approximately 300 cubic μm, from approximately 5 cubic μm to approximately 1,000 cubic μm, from approximately 5 cubic μm to approximately 3,000 cubic μm, from approximately 5 cubic μm to approximately 10,000 cubic μm, from approximately 10 cubic μm to approximately 30 cubic μm, from approximately 10 cubic μm to approximately 50 cubic μm, from approximately 10 cubic μm to approximately 100 cubic μm, from approximately 10 cubic μm to approximately 300 cubic μm, from approximately 10 cubic μm to approximately 1,000 cubic μm, from approximately 10 cubic μm to approximately 3,000 cubic μm, from approximately 10 cubic μm to approximately 10,000 cubic μm, 30 cubic μm to approximately 50 cubic μm, approximately 30 cubic μm to approximately 100 cubic μm, approximately 30 cubic μm to approximately 300 cubic μm, approximately 30 cubic μm to approximately 1,000 cubic μm, approximately 30 cubic μm to approximately 3,000 cubic μm, approximately 30 cubic μm to approximately 10,000 cubic μm, approximately 50 cubic μm to approximately 100 cubic μm, approximately 50 cubic μm to approximately 300 cubic μm, approximately 50 cubic μm to approximately 1,000 cubic μm, approximately 50 cubic μm to approximately 3,000 cubic μm, approximately 50 cubic μm to approximately 10,000 cubic μm, approximately 100 cubic μm It is acceptable to use approximately 300 cubic μm from m, approximately 1,000 cubic μm from approximately 100 cubic μm, approximately 3,000 cubic μm from approximately 100 cubic μm, approximately 10,000 cubic μm from approximately 100 cubic μm, approximately 1,000 cubic μm from approximately 300 cubic μm, approximately 3,000 cubic μm from approximately 300 cubic μm, approximately 10,000 cubic μm from approximately 1,000 cubic μm, approximately 10,000 cubic μm from approximately 1,000 cubic μm, or approximately 3,000 cubic μm to approximately 10,000 cubic μm. The volume of the void may be approximately 1 cubic μm, 5 cubic μm, 10 cubic μm, 30 cubic μm, 50 cubic μm, 100 cubic μm, 300 cubic μm, 1,000 cubic μm, 3,000 cubic μm, or 10,000 cubic μm.The void may have a volume of at least approximately 1 cubic μm, approximately 5 cubic μm, approximately 10 cubic μm, approximately 30 cubic μm, approximately 50 cubic μm, approximately 100 cubic μm, approximately 300 cubic μm, approximately 1,000 cubic μm, or approximately 3,000 cubic μm. The void may have a maximum volume of approximately 5 cubic μm, approximately 10 cubic μm, approximately 30 cubic μm, approximately 50 cubic μm, approximately 100 cubic μm, approximately 300 cubic μm, approximately 1,000 cubic μm, approximately 3,000 cubic μm, or approximately 10,000 cubic μm. The void may be approximately X cubic μm.

[0102] Figure 18A shows a non-limiting diagram of the bottom surface 1812 of a planar support 1806 of the present disclosure. The bottom surface may comprise a plurality of cavities 1815, which are arranged to form strips extending parallel to the length of the planar support. The cavities extend below an array or column (not shown) of obstacles or a lane (not shown) formed by a column of obstacles, which is fabricated on the top surface of the planar support. Referring to Figure 18B, a cross-sectional view of the planar support 1806 is shown. The top surface 1807 of the planar support comprises a plurality of individual obstacles 1820 formed to form an array or column, creating gaps 1835 that allow for the flow of fluids, cells, and / or particles. The cavities 1815 are located below the obstacles embedded in the bottom surface of the planar support 1812. The area (length × width) of the cavities on the opposite side of the lane can be at least about 80% of the area (length × width) of the lane. In some embodiments, the area (length × width) of the space on the opposite side of the lane may be less than or equal to at least about 90%, 100%, 110%, 120%, or about 150% of the area (length × width) of the lane.

[0103] In one configuration, the voids between the two planar supports are symmetrical or nearly symmetrical, with their backs pressed together as shown in Figure 16A. However, an alternative configuration is shown as shown in Figure 19. In such cases, the supports are stacked rather than pressed together back to back, and the voids are either above the obstacle layer as in 19A or below as in 19B.

[0104] A void may be divided into two or more voids. A void may 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 voids. A void may be divided into exactly two voids. For each planar support with an obstacle, the ratio of channels or lanes to voids may be 1:1.

[0105] A planar support may be made from two layers of material joined together. The layers may be joined together by an adhesive, polymer, or thermoplastic. The layers may consist of a polymer or thermoplastic. The polymer layer or thermoplastic layer or joining material may consist of high-density polyethylene (HDPE), polypropylene (PP), polyethylene terephthalate (PT), polycarbonate (PC), or cyclic olefin copolymer (COC).

[0106] The upper layer of the cartridge may comprise an array of obstacles for at least one embedded channel, a void, at least one inlet, at least one outlet, or a combination thereof. The bottom layer of the cartridge may comprise an array of obstacles for at least one embedded channel, a void, at least one inlet, at least one outlet, or a combination thereof. The layers may be positioned where planar supports are joined to each other on the sides, bottom, or top. The void may be inside or outside the interface of the joined planar supports.

[0107] The microfluidic cartridge may further include an obstacle bonding layer, which is bonded to the surface of a planar support and the top surface of the array of obstacles in the embedded channel to prevent fluid or sample from flowing over the array of obstacles during the operation of the cartridge. The obstacle bonding layer may be metal, polymer, or thermoplastic. The obstacle bonding layer may be a cover or film. The polymer layer or thermoplastic layer or bonding material may consist of high-density polyethylene (HDPE), polypropylene (PP), polyethylene terephthalate (PT), polycarbonate (PC), or cyclic olefin copolymer (COC). The microfluidic cartridge may have two obstacle bonding layers on the outside of the upper planar support. The microfluidic cartridge may have an obstacle bonding layer in the middle of the cartridge as a bonding material for the planar support. The obstacle bonding layer may include one or more passages fluid-connected to one or more inlets of the embedded channel, allowing sample flow into the channel, and one or more passages fluid-connected to one or more outlets of the channel, allowing fluid flow out of one or more outlets. Such an obstruction layer may have 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.

[0108] A microfluidic cartridge may have obstacles positioned to define the cartridge's critical size, so that as the sample is poured into the cartridge's inlet and flows to the 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, and the sum of the individual obstacles defines the cartridge's critical size. One or more outlets of the cartridge may have at least one product outlet, and target particles or cells larger than the cartridge's critical size are directed to at least one product outlet. One or more outlets of the cartridge may have at least one product outlet, and target particles or cells smaller than the cartridge's critical size are directed to at least one product 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 product outlets. One or more outlets may have at least one waste outlet. Contaminants, particles, or cells smaller than the critical size may flow to at least one waste outlet. Contaminants, particles, or cells larger than the critical size may flow to 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.

[0109] The obstacles used in the cartridge may take the form of a vertical column, or they may be triangular, square, rectangular, diamond-shaped, trapezoidal, hexagonal, teardrop-shaped, circular, semicircular, a triangle with a horizontal top, or a triangle with a horizontal base. Furthermore, adjacent obstacles may have a shape such that the portion of the obstacle defining the gap is symmetrical or asymmetrical about the axis of the gap extending in the direction of the bulk fluid flow. Obstacles may have vertices that extend through the gaps such that one or more vertices facing each other but not directly opposite each other are adjacent to parallel gaps on each side. Obstacles may have vertices that extend through the gaps such that vertices facing each other are adjacent to vertical gaps on each side and are directly opposite each other. The position and shape of the obstacles can be varied within a single chip. Additional obstacles can be added to any position in the device for any specific requirements. Also, the shape of the obstacles can vary within the device. Any combination of post shape, size, and position can be used for any specific requirements. The cartridge may consist only of diamond-shaped or hexagonal obstacles.

[0110] The shape of the obstacles may be elongated in the direction perpendicular to the fluid flow direction, and therefore have a left-right length (P1) that is different from their vertical length (P2). P1 may be approximately 1 μm to 160 μm. P1 may be approximately 1 μm to 10 μm, approximately 1 μm to 15 μm, approximately 1 μm to 30 μm, approximately 1 μm to 40 μm, approximately 1 μm to 80 μm, approximately 1 μm to 160 μm, approximately 10 μm to 15 μm, approximately 10 μm to 30 μm, approximately 10 μm to 40 μm, approximately 10 μm to 80 μm, or approximately 10 μm to 160 μm. The lengths of the particles may be approximately 15 μm to 30 μm, 15 μm to 40 μm, 15 μm to 80 μm, 15 μm to 160 μm, 30 μm to 40 μm, 30 μm to 80 μm, 30 μm to 160 μm, 40 μm to 80 μm, 40 μm to 160 μm, or 80 μm to 160 μm. P1 may have lengths of approximately 1 μm, 10 μm, 15 μm, 30 μm, 40 μm, 80 μm, or 160 μm. P1 may have lengths of at least approximately 1 μm, 10 μm, 15 μm, 30 μm, 40 μm, or 80 μm. P1 can have a maximum length of approximately 10 μm, 15 μm, 30 μm, 40 μm, 80 μm, or 160 μm. P2 can have a length from approximately 1 μm to approximately 160 μm. P2 can have a length of approximately 1 μm to 10 μm, 1 μm to 15 μm, 1 μm to 30 μm, 1 μm to 40 μm, 1 μm to 80 μm, 1 μm to 160 μm, 10 μm to 15 μm, 10 μm to 30 μm, 10 μm to 40 μm, 10 μm to 80 μm, or 10 μm to 160 μm. The length of P2 may be approximately μm, approximately 15 μm to approximately 30 μm, approximately 15 μm to approximately 40 μm, approximately 15 μm to approximately 80 μm, approximately 15 μm to approximately 160 μm, approximately 30 μm to approximately 40 μm, approximately 30 μm to approximately 80 μm, approximately 30 μm to approximately 160 μm, approximately 40 μm to approximately 80 μm, approximately 40 μm to approximately 160 μm, or approximately 80 μm to approximately 160 μm. The length of P2 may be approximately 1 μm, approximately 10 μm, approximately 15 μm, approximately 30 μm, approximately 40 μm, approximately 80 μm, or approximately 160 μm. The length of P2 may be at least approximately 1 μm, approximately 10 μm, approximately 15 μm, approximately 30 μm, approximately 40 μm, or approximately 80 μm. P2 can have a maximum length of approximately 10 μm, 15 μm, 30 μm, 40 μm, 80 μm, or 160 μm.P1 may be approximately 25% to 200% longer than P2. P1 may be approximately 25% to 50%, 25% to 75%, 25% to 100%, 25% to 150%, 25% to 200%, 50% to 75%, 50% to 100%, 50% to 150%, 50% to 200%, 75% to 100%, 75% to 150%, 75% to 200%, 100% to 150%, 100% to 200%, or 150% to 200% longer than P2. P1 may be approximately 25%, 50%, 75%, 100%, 150%, or 200% longer than P2. P1 may be at least approximately 25%, 50%, 75%, 100%, or 150% longer than P2. P1 may be up to approximately 50%, 75%, 100%, 150%, or 200% longer than P2.

[0111] The microfluidic cartridge may include obstacles as an array of obstacles. The obstacles may be arranged in columns and rows, forming separate arrays. The array of obstacles may consist of at least about 5 columns and about 50 columns. The array of obstacles may consist of at least about 5 to about 10 columns, about 5 to about 28 columns, about 5 to about 29 columns, about 5 to about 30 columns, about 5 to about 50 columns, about 10 to about 28 columns, about 10 to about 29 columns, about 10 to about 30 columns, about 10 to about 50 columns, about 28 to about 29 columns, about 28 to about 30 columns, about 28 to about 50 columns, about 29 to about 30 columns, about 29 to about 50 columns, or about 30 to about 50 columns. An array of obstacles may have at least about 5 columns, about 10 columns, about 28 columns, about 29 columns, about 30 columns, or about 50 columns. An array of obstacles may have at least about 5 columns, about 10 columns, about 28 columns, about 29 columns, or about 30 columns. An array of obstacles may have at least up to about 10 columns, about 28 columns, about 29 columns, about 30 columns, or about 50 columns. An array of obstacles may have at least about 20 rows and about 500 rows. The array of obstacles may consist of at least approximately 20 to 30 rows, approximately 20 to 60 rows, approximately 20 to 100 rows, approximately 20 to 200 rows, approximately 20 to 500 rows, approximately 30 to 60 rows, approximately 30 to 100 rows, approximately 30 to 200 rows, approximately 30 to 500 rows, approximately 60 to 100 rows, approximately 60 to 200 rows, approximately 60 to 500 rows, approximately 100 to 200 rows, approximately 100 to 500 rows, or approximately 200 to 500 rows. The array of obstacles may consist of at least approximately 20 rows, approximately 30 rows, approximately 60 rows, approximately 100 rows, approximately 200 rows, or approximately 500 rows.An array of obstacles may have at least about 20 rows, about 30 rows, about 60 rows, about 100 rows, or about 200 rows. An array of obstacles may have at least up to about 30 rows, about 60 rows, about 100 rows, about 200 rows, or about 500 rows. Multiple arrays of obstacles can be arranged in separate lanes. An array of obstacles on a first or second planar support forms about 10 to about 50 lanes. The array of obstacles of the first or second planar support forms approximately 10 lanes to approximately 20 lanes, approximately 10 lanes to approximately 28 lanes, approximately 10 lanes to approximately 30 lanes, approximately 10 lanes to approximately 50 lanes, approximately 20 lanes to approximately 28 lanes, approximately 20 lanes to approximately 30 lanes, approximately 20 lanes to approximately 50 lanes, approximately 28 lanes to approximately 30 lanes, approximately 28 lanes to approximately 50 lanes, or approximately 30 lanes to approximately 50 lanes. The array of obstacles of the first or second planar support forms approximately 10 lanes, approximately 20 lanes, approximately 28 lanes, approximately 30 lanes, or approximately 50 lanes. The array of obstacles of the first or second planar support forms at least approximately 10 lanes, approximately 20 lanes, approximately 28 lanes, or approximately 30 lanes. The array of obstacles of the first or second planar support forms up to approximately 20 lanes, approximately 28 lanes, approximately 30 lanes, or approximately 50 lanes.

[0112] Each cartridge may have at least one, at least two, at least three, or at least four arrays of obstacles. Each planar top surface may have at least one or at least two arrays. A cartridge may have a total of approximately 20 to approximately 100 lanes. A cartridge may have a total of approximately 20 to approximately 40 lanes, approximately 20 to approximately 56 lanes, approximately 20 to approximately 60 lanes, approximately 20 to approximately 100 lanes, approximately 40 to approximately 56 lanes, approximately 40 to approximately 60 lanes, approximately 40 to approximately 100 lanes, approximately 56 to approximately 60 lanes, approximately 56 to approximately 100 lanes, or approximately 60 to approximately 100 lanes. A cartridge may have a total of approximately 20, approximately 40, approximately 56, approximately 60, or approximately 100 lanes. The cartridge may have a total of at least approximately 20 lanes, approximately 40 lanes, approximately 56 lanes, or approximately 60 lanes. The cartridge may have a total of up to approximately 40 lanes, approximately 56 lanes, approximately 60 lanes, or approximately 100 lanes.

[0113] The inlet, outlet, or both of the microfluidic cartridge may be fluid-connected to a pump or motor to facilitate the flow of fluid in and out of the cartridge. The inlet, outlet, or both may be fluid-connected to at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 pumps. The pumps may be peristaltic pumps. The pumps may be fluid-connected to each other or disconnected. The cartridge inlet and outlet may be fluid-connected to two peristaltic pumps connected in parallel to each other. The cartridge inlet and outlet may be fluid-connected to two peristaltic pumps connected in series to each other.

[0114] Microfluidic cartridges may be made from metal, polymer, or thermoplastic. The polymer or thermoplastic may consist of high-density polyethylene (HDPE), polypropylene (PP), polyethylene terephthalate (PT), polycarbonate (PC), or cyclic olefin copolymer (COC). In one example, the microfluidic cartridge is made from a cyclic olefin copolymer.

[0115] The disclosure also provides a microfluidic assembly comprising a plurality of fluid-connected microfluidic cartridges. The cartridges of the assembly may be stacked or arranged in layers. The plurality of microfluidic cartridges may comprise at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 30 cartridges. The plurality of cartridges may be fluid-connected in series or in parallel.

[0116] Cells, for example, cells in a composition prepared by apheresis or leukocyte apheresis, may be isolated by performing DLD using a microfluidic cartridge having a channel through which fluid flows from an inlet at one end to an outlet at the other. The basic principles of size-based microfluidic separation and the design of obstacle arrays for separating cells are provided in other literature (see U.S. Patent Application Publication No. 2014 / 0342375, U.S. Patent Application Publication No. 2016 / 0139012, U.S. Patent No. 7,318,902 and U.S. Patent No. 7,150,812, which are incorporated herein in their entirety) and summarized in the following paragraphs.

[0117] During DLD, a fluid sample containing cells is introduced into the device at the inlet and carried along with the fluid flowing through the device to the outlet. As the cells in the sample traverse the device, they encounter posts or other obstacles positioned to form gaps or pores through which they must pass. Each successive row of obstacles is displaced relative to the preceding row so as to form an array direction different from the direction of fluid flow in the flow channel. The “angle of inclination” determined by the two directions, 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 main factors in determining the “critical size” of the array. Cells larger than the critical size move in the array direction rather than in the direction of the bulk fluid flow, while particles smaller than the critical size move in the direction of the bulk fluid flow. In devices used for compositions derived from leukocyte apheresis, the array features may be selected so that leukocytes are deflected in the array direction, while erythrocytes and platelets remain in the direction of the bulk fluid flow. Next, a carrier may be used to separate the selected type of leukocytes from others of similar size, the carrier binding to the cells in a manner that facilitates DLD separation, thereby causing them to form a complex larger than that of uncomplexed leukocytes. Separation can then be performed with a device having a critical size smaller than that of the complexes but larger than that of the uncomplexed cells.

[0118] II. Fabrication and Operation of Microfluidic Devices General procedures for fabricating and using microfluidic devices capable of separating cells based on size are well known in the art. Such devices include U.S. Patent Nos. 5,837,115, 7,150,812, 6,685,841, 7,318,902, 7,472,794, and 7,735,652, all of which are incorporated herein by reference in their entirety. Other references that may provide guidance useful for the fabrication and use of the devices of the present invention include U.S. Patent Nos. 5,427,663, 7,276,170, 6,913,697, 7,988,840, 8,021,614, 8,282,799, 8,304,230, 8,579,117, and U.S. Patent Application Publication No. U.S. Patent Application Publication No. 2006 / 0134599, U.S. Patent Application Publication No. 2007 / 0160503, U.S. Patent Application Publication No. 20050282293, U.S. Patent Application Publication No. 2006 / 0121624, U.S. Patent Application Publication No. 2005 / 0266433, U.S. Patent Application Publication No. 2007 / 0026381, U.S. Patent Application Publication No. 2007 / 0026414, U.S. Patent Application Publication No. 2007 / 0026417, U.S. Patent Application Publication No. U.S. Patent Application Publication No. 2007 / 0026415, U.S. Patent Application Publication No. 2007 / 0026413, U.S. Patent Application Publication No. 2007 / 0099207, U.S. Patent Application Publication No. 2007 / 0196820, U.S. Patent Application Publication No. 2007 / 0059680, U.S. Patent Application Publication No. 2007 / 0059718, U.S. Patent Application Publication No. 2007 / 005916, U.S. Patent Application Publication No. 2007 / 0059774, U.S. Patent This includes U.S. Patent Application Publication No. 2007 / 0059781, U.S. Patent Application Publication No. 2007 / 0059719, U.S. Patent Application Publication No. 2006 / 0223178, U.S. Patent Application Publication No. 2008 / 0124721, U.S. Patent Application Publication No. 2008 / 0090239, U.S. Patent Application Publication No. 2008 / 0113358, and WO2012094642, all of which are incorporated into this application by reference.Among the various references describing the fabrication and use of devices, U.S. Patent No. 7,150,812 provides particularly good guidance regarding microfluidic devices for separation of cells found in blood samples, and Patent No. 7,735,652 is particularly interesting (see also U.S. Patent Application Publication 2007 / 0160503 in this regard).

[0119] The devices can be fabricated using any of the materials typically used to create devices that handle microscale and nanoscale fluids, including silicon, glass, plastics, and hybrid materials. A wide range of thermoplastic materials suitable for microfluidic fabrication are available, offering a broad selection of mechanical and chemical properties that can be utilized and further prepared for specific applications. In one embodiment, the microfluidic cartridge may be fabricated by soft embossing and UV curing.

[0120] Microfluidic cartridges (or devices, cassettes, chips, etc.) can be fabricated by techniques including replica molding, soft lithography using PDMS, thermosetting polyester, embossing, soft embossing, hot embossing, roll-to-roll embossing, injection molding, laser ablation, UV curing, and combinations thereof. Further details can be found in "Disposable microfluidic devices: fabrication, function and application" by Fiorini, et al., [BioTechniques 38:429-446 (March 2005)], which is incorporated herein by reference 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 on fabrication methods, which is also incorporated herein by reference in its entirety.

[0121] High-volume embossing methods, such as reel-to-reel processing of thermoplastics, are attractive for the production of industrial microfluidic chips. The use of single-chip hot embossing can be a cost-effective technique for achieving high-quality microfluidic devices during the prototyping phase. A method for reproducing microscale features in two thermoplastics, polymethyl methacrylate (PMMA) and / or polycarbonate (PC), is described in "Microfluidic device fabrication by thermoplastic hot-embossing" by Yang, et al., [Methods Mol. Biol. 949: 115-23 (2013)], which is incorporated herein by reference in its entirety.

[0122] A flow channel can be constructed using two or more components (preferably one of which has an orifice for adding or withdrawing fluid) that, when assembled, form a closed void with an obstacle positioned inside. The obstacle can be fabricated on one or more components assembled to form the flow channel, or in the form of an insert sandwiched between two or more components that define the boundary of the flow channel.

[0123] The obstruction may be a solid object in the array that extends transversely across the flow channel and longitudinally along the channel from inlet to outlet. If the obstruction is integrated with (or an extension thereof of) one of the faces of the flow channel at one end of the obstruction, the other end of the obstruction may be sealed to or pressed against the opposite face of the flow channel. Small spaces (preferably too small to accommodate any of the particles for the intended use) are permitted between one end of the obstruction and the face of the flow channel, provided that the space does not adversely affect the structural stability of the obstruction in the device or the nature of the flow involved.

[0124] Surfaces can be coated to alter their properties, and the polymer materials used to fabricate devices can be modified in various ways. In some cases, functional groups such as amines or carboxylic acids in the original or additional polymers are used to crosslink proteins or other molecules through wet chemical treatment or plasma treatment. Surface amine groups can be used to attach DNA to COC and PMMA substrates. Surfactants such as Pluronic® can be used to make the surface hydrophilic and protein-repulsive by adding Pluronic® to PDMS formulations. In some cases, a layer of PMMA is spin-coated onto the device, and for example, the microfluidic chip and PMMA are “doped” with hydroxypropyl cellulose to alter their contact angle.

[0125] For example, one or more walls may be chemically modified to be non-adherent or repulsive in order to reduce nonspecific adsorption to the channel walls of cells or compounds released by lysed cells or found in biological samples. The walls may be coated with a thin film coating (e.g., a monolayer) of commercially available nonstick reagents, such as those used to form hydrogels. Additional examples of chemical species that may be used to modify the channel walls include oligoethylene glycol, fluorinated polymers, organosilanes, thiols, polyethylene glycol, hyaluronic acid, bovine serum albumin, polyvinyl alcohol, mucin, poly-HEMA, methacrylated PEG, and agarose. Charged polymers may be employed to repel reverse-charged species. The type of chemical species used for repulsion and the method of attachment to the channel walls may vary depending on the properties of the repelled species as well as the properties of the wall and the attached species. Such surface modification techniques are well known in the art. The walls may be functionally assigned before or after the device is assembled.

[0126] III. CAR T cells and NK cells Methods for producing and using CAR T cells and natural killer (NK) cells are well known in the art. Procedures are described, for example, in U.S. Patent Nos. 9,629,877, 9,328,156, 8,906,682, U.S. Patent Publication Nos. 2017 / 0224789, 2017 / 0166866, 2017 / 0137515, 2016 / 0361360, 2016 / 0081314, 2015 / 0299317, and 2015 / 0024482, all of which are incorporated by reference in their entirety in this application.

[0127] This 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 contain chimeric antigen receptor (CAR) T cells and NK cells. The microfluidic cartridges (i.e., devices, chips, cassettes, plates, microfluidic devices, cartridges, DLD devices, etc.) may be any of those described herein. Using the described cartridges, it is possible to enable more effective CAR T cell or NK cell production by providing a purer T cell or NK cell product for downstream genetic manipulation and CAR T cell or NK cell production. More effective CAR T cells or NK cells that cannot be achieved by other methods of producing CAR T cells or NK cells may be produced by removing platelets.

[0128] A method for producing chimeric antigen receptor (CAR) T cells or NK cells may include obtaining a sample containing T cells or NK cells and separating the T cells or NK cells from a contaminant. The contaminant may include platelets or other contaminants as described herein. Separating the contaminant may include pouring the sample into one or more sample inlets of any cartridge or device as described herein, flowing the sample to the outlet of the cartridge, obtaining a product enriched with T cells 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 the T cells or NK cells. The sample of this method may include apheresis products or leukocyte apheresis products. Genetic engineering in this method may include genetic engineering methods as described herein. This method may further include growing CAR T cells or NK cells in vitro.

[0129] Some commercially available examples of CAR T-cell therapies that can be operated according to the devices and methods described herein include axicaptagen silolucel, tisagenlecleucel, and brexcaptagen oatlucel.

[0130] IV. Isolation process using DLD 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 devices can be found, for example, in U.S. Patent Application Publication No. 2016 / 0139012, the teachings of which are incorporated in their entirety by reference.

[0131] The purity, yield, and viability of cells produced by the DLD method vary based on several factors, including the properties of the starting material, the precise procedure employed, and the characteristics of the DLD device. Preferably, a purity, yield, and viability of at least 60% should be obtained, with higher percentages, at least 70%, 80%, or 90%, being more preferable.

[0132] In one embodiment, the Disclosure provides a method for enriching a sample with target particles or target cells of a predetermined size from contaminants. The method for enriching the target particles or target cells uses any cartridge, microfluidic cartridge, cassette, tip, device, fluid device, or microfluidic device as described elsewhere in this Spec. The method may include obtaining a sample containing the target particles or target cells and contaminants. The method may further include separating the target particles or target cells from the contaminants by pouring the sample into one or more sample inlets of any of the cartridges, cassettes, or devices described herein. The method may further include flowing the sample to one of the outlets of any of the cartridges, cassettes, or devices described herein. The method may further include obtaining a product enriched with target particles or target cells from one or more outlets while removing the contaminants. The method may provide a superior ability to purify or separate cells or particles from contaminants, increase cell yield, improve the ability to grow the product in vitro, and make the enriched cell product more compliant with transduction or other genetic manipulation.

[0133] The method may include the use of a deterministic transverse displacement method, the device having a critical size as described herein, and separation based on the fact that contaminants and target particles or target cells have different critical sizes. The method may include flowing a sample containing target particles or target cells and contaminants to one of the cartridges, cassettes, or devices described herein, wherein the target particles or target cells are larger than the critical size of the obstacle array, and at least some contaminants are smaller than the critical size of the obstacle array, the target cells or target particles flow to one or more product outlets from which a product enriched with target cells or target particles is obtained, and the contaminants having a size smaller than the critical size of the obstacle array flow to another waste outlet. The method may include flowing a sample containing target particles or target cells and contaminants to one of the cartridges, cassettes, or devices described herein, wherein the target particles or target cells are smaller than the critical size of the obstacle array, and at least some of the contaminants are larger than the critical size of the obstacle array, the target cells or target particles flow to one or more product outlets from which a product enriched with the target cells or target particles is obtained, and the contaminants larger than the critical size of the obstacle array flow to another waste outlet.

[0134] This method may involve flowing a sample containing target particles or target cells and contaminants through a cartridge, cassette, or device as described herein at a constant or variable flow rate. The cartridge flow rate in this method may be approximately 400 mL per hour. The cartridge flow rate in this method may range from approximately 100 mL per hour to approximately 1,000 mL per hour. The cartridge flow rate for this method may be approximately 100 mL to 200 mL per hour, approximately 100 mL to 400 mL per hour, approximately 100 mL to 800 mL per hour, approximately 100 mL to 1,000 mL per hour, approximately 200 mL to 400 mL per hour, approximately 200 mL to 800 mL per hour, approximately 200 mL to 1,000 mL per hour, approximately 400 mL to 800 mL per hour, approximately 400 mL to 1,000 mL per hour, or approximately 800 mL to 1,000 mL per hour. The cartridge flow rate for this method may be approximately 100 mL per hour, approximately 200 mL per hour, approximately 400 mL per hour, approximately 800 mL per hour, or approximately 1,000 mL per hour. The cartridge flow rate for this method may be at least approximately 100 mL per hour, approximately 200 mL per hour, approximately 400 mL per hour, or approximately 800 mL per hour. The cartridge flow rate for this method may be at most approximately 200 mL per hour, approximately 400 mL per hour, approximately 800 mL per hour, or approximately 1,000 mL per hour.

[0135] This method may include the internal pressure within the cartridge. The internal pressure of the cartridge may be at least approximately 15 pounds / square inch. The internal pressure of the cartridge may be at least approximately 1.5 pounds / square inch to approximately 50 pounds / square inch. The internal pressure of the cartridge may be at least approximately 1.5 pounds / square inch to approximately 5 pounds / square inch, approximately 1.5 pounds / square inch to approximately 10 pounds / square inch, approximately 1.5 pounds / square inch to approximately 15 pounds / square inch, approximately 1.5 pounds / square inch to approximately 20 pounds / square inch, approximately 1.5 pounds / square inch to approximately 50 pounds / square inch, approximately 5 pounds / square inch to approximately 10 pounds / square inch, approximately 5 pounds / square inch to approximately 15 pounds / square inch, approximately 5 pounds / The pressure may range from one square inch to approximately 20 pounds / square inch, from approximately 5 pounds / square inch to approximately 50 pounds / square inch, from approximately 10 pounds / square inch to approximately 15 pounds / square inch, from approximately 10 pounds / square inch to approximately 20 pounds / square inch, from approximately 10 pounds / square inch to approximately 50 pounds / square inch, from approximately 15 pounds / square inch to approximately 20 pounds / square inch, from approximately 15 pounds / square inch to approximately 50 pounds / square inch, or from approximately 20 pounds / square inch to approximately 50 pounds / square inch. The internal pressure of the cartridge may be at least approximately 1.5 pounds / square inch, approximately 5 pounds / square inch, approximately 10 pounds / square inch, approximately 15 pounds / square inch, approximately 20 pounds / square inch, or approximately 50 pounds / square inch. The internal pressure of the cartridge may be at least approximately 1.5 lbs / square inch, 5 lbs / square inch, 10 lbs / square inch, 15 lbs / square inch, or 20 lbs / square inch. The internal pressure of the cartridge may be at least and at most approximately 5 lbs / square inch, 10 lbs / square inch, 15 lbs / square inch, 20 lbs / square inch, or 50 lbs / square inch.

[0136] The target particles or target cells of this method may include stem cells, platelets, synovial cells, fibroblasts, beta cells, hepatocytes, megakaryocytes, pancreatic cells, DE3 lysogenized cells, yeast cells, plant cells, algal 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 may include enriching target particles or target cells to produce enriched target cells, the target cells including stem cells, platelets, synovial cells, fibroblasts, beta cells, hepatocytes, megakaryocytes, pancreatic cells, DE3 lysogenized cells, yeast cells, plant cells, algal 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 contaminants in this method may include stem cells, platelets, synovial cells, fibroblasts, beta cells, hepatocytes, megakaryocytes, pancreatic cells, DE3 lysogenized cells, yeast cells, plant cells, algal 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 cells may be peripheral blood mononuclear cells and the contaminants may be platelets. For example, the target cells may be CD3+ cells and the contaminants may be platelets. More than 90% of platelets can be removed by this method. Approximately 50% to 99% of platelets can be removed by this method.This method can remove approximately 50% to 75% of platelets, approximately 50% to 80%, approximately 50% to 90%, approximately 50% to 95%, approximately 50% to 99%, approximately 75% to 80%, approximately 75% to 90%, approximately 75% to 95%, approximately 75% to 99%, approximately 80% to 90%, approximately 80% to 95%, approximately 80% to 99%, approximately 90% to 95%, approximately 90% to 99%, approximately 90% to 99%, or approximately 95% to 99% of platelets. This method can remove approximately 50%, 75%, 80%, 90%, 95%, or 99% of platelets. This method can remove at least approximately 50%, 75%, 80%, 90%, or 95% of platelets. This method can remove up to approximately 75%, 80%, 90%, 95%, or 99% of platelets.

[0137] This method may include modifying enriched target cells. This method may include genetically engineering enriched target cells to obtain genetically engineered target cells. Genetic engineering includes transfecting or transducing target cells with recombinant nucleic acids. Genetic engineering methods may include the use of TALENs, zinc finger nucleases, CRISPR-Cas-related proteins, homologous recombination, viral vectors, or non-homologous plasmids. This method may also include growing enriched target cells or genetically engineered cells in vitro by culturing them.

[0138] V. Technical background This invention describes “obstacle array” devices, the basic operation of which is described, for example, in U.S. Patent No. 7,150,812, which is incorporated in its entirety by reference. Bump arrays essentially operate by separating particles passing through an array of obstacles (generally, a periodically aligned array), the separation occurring between particles following the direction of the bulk fluid flow and particles following an “array direction” offset from the direction of the bulk fluid flow.

[0139] A. Scope of sorting Objects separated by size in microfluidic devices include cells, biomolecules, inorganic beads, and other objects. Typical sizes separated range from 100 nanometers to 50 micrometers. However, larger and smaller particles may also be separated in some cases.

[0140] B. Capacity The rate at which samples can be processed varies greatly depending on the design of the device or combination of devices. Preferably, the device and assembly should be able to process more than 500 ml of sample per hour.

[0141] C channel The device may comprise one or more channels, each having one or more inlets and one or more outlets. Inlets may be used for samples or raw (i.e., unpurified) fluid compositions, or for buffers, or for introducing reagents. Outlets may be used for collecting products or as waste outlets. Channels may be approximately 0.5 to 100 mm wide and approximately 2 to 200 mm long, but different widths and lengths are possible. Depths may be 1 to 1000 μm, and there may be 1 to 500 or more channels in a single device.

[0142] Specific embodiments of the various aspects described herein can be illustrated by the following numbered embodiments.

[0143] 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 and a bottom surface, the top and / or bottom surface comprising at least one embedded channel extending from one or more sample inlets and one or more separate fluid inlets to one or more product outlets and one or more separate waste outlets, (a) when fluid is poured into the channel through the sample inlets and / or fluid inlets, it flows through the channel toward the outlets, thereby determining the direction of bulk fluid flow, and (b) the channel comprising an array of obstacles arranged in longitudinal rows extending along the channel and transverse rows extending across the channel, the obstacles being positioned to determine a critical size Therefore, as the sample is poured into the inlet of the device and flows to the 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, (i) adjacent obstacles in a transverse row are separated by a gap G1 perpendicular to the direction of bulk fluid flow, (ii) adjacent obstacles in a longitudinal row are separated by a gap G2 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 transverse row of obstacles is shifted transversely with respect to the preceding transverse row, so that an array direction is set that is off by an angle of inclination (ε) from the direction of bulk fluid flow, and (v) each obstacle has at least two vertices such that at least one vertex is adjacent to each gap on each side, the microfluidic device. 2. The device according to Embodiment 1, further comprising an obstacle bonding layer bonded to obstacles in channels bonded to the plane of a planar support and embedded in the surface, to prevent fluid or sample from flowing over obstacles during the operation of the device. 3. The microfluidic device according to Embodiment 2, wherein the obstruction bonding layer comprises one or more fluid-connected passages at the sample inlet of the channel, which allow the flow of a sample into the channel, and one or more fluid-connected passages at the outlet of the channel, which allow the flow of fluid out of the outlet.4. A microfluidic device according to any one of Embodiments 1 to 3, wherein the target particles or target cells are larger than the critical size of the device, and at least some contaminants are smaller than the critical size, and the obstacles are arranged such that when the sample is poured into the inlet of the device and fluidly passes through the channels, the target cells or target particles flow to one or more product outlets from which an enriched product containing the target cells or target particles is obtained, and contaminants having a size smaller than the critical size flow to another waste outlet. 5. A microfluidic device according to any one of Embodiments 1 to 4, wherein the obstacles are polygonal. 6. A microfluidic device according to Embodiment 5, wherein the obstacles are diamond-shaped or hexagonal. 7. A microfluidic device according to Embodiment 5 or Embodiment 6, wherein the obstacles are elongated perpendicular to the direction of the bulk fluid, and therefore have a left-right length (P1) different from their up-down length (P2). 8. A microfluidic device according to Embodiment 6, wherein P1 is at least 15% longer than P2. 9. The microfluidic device according to Embodiment 6, wherein P1 is 10-150% longer than P2. 10. The microfluidic device according to Embodiment 6, wherein P1 is 15-100% longer than P2. 11. The microfluidic device according to Embodiment 6, wherein P1 is 20-70% longer than P2. 12. The microfluidic device according to any one of Embodiments 1-11, wherein the obstacles have vertices that extend through the gaps such that one or more vertices facing each other but not directly opposite each other are adjacent on each side of the parallel gaps. 13. The microfluidic device according to any one of Embodiments 1-12, wherein the obstacles have vertices that extend through the gaps such that one or more vertices facing each other are adjacent on each side of the vertical gaps and are directly opposite each other. 14. The microfluidic device according to any one of Embodiments 1-13, wherein the sample inlet or entrance is separated from the fluid inlet or entrance by a separation wall, the separation wall extending from the sample inlet or entrance through an array of obstacles in the channel toward the exit and oriented parallel to the direction of bulk fluid flow. 15. The microfluidic device according to Embodiment 14, wherein the separation wall extends over at least 10% of the length of the array of obstacles.16. The microfluidic device according to Embodiment 14, wherein the separation wall extends over at least 20% of the length of the array of obstacles. 17. The microfluidic device according to Embodiment 14, wherein the separation wall extends over at least 40% of the length of the array of obstacles. 18. The microfluidic device according to Embodiment 14, wherein the separation wall extends over at least 60% of the length of the array of obstacles. 19. The microfluidic device according to any one of Embodiments 1 to 18, wherein the inlet and / or outlet of the device are connected to a peristaltic pump. 20. A stacked separation assembly comprising at least two of the microfluidic devices according to any one of Embodiments 1 to 19. 21. A stacked separation assembly comprising a first microfluidic device selected from any one of the microfluidic devices described in any one of Embodiments 1 to 19, and one or more stacked microfluidic devices also selected from any one of the microfluidic devices described in any one of Embodiments 1 to 19, wherein (a) the bottom surface of each stacked device is in contact with the top surface or an obstacle bonding layer on the top surface of the first microfluidic device, or the top surface or an obstacle bonding layer on the top surface of another stacked device, (b) a sample is supplied 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 in (d), and (f) the first microfluidic device and the stacked microfluidic devices may be mounted in a common outer casing. 22. The stacked separation assembly according to Embodiment 22, comprising at least two stacked microfluidic devices.23. A stacked separation assembly according to Embodiment 22, further comprising at least one reservoir bonding layer, the reservoir bonding layer being attached to the bottom surface of a first microfluidic device and / or the top surface of a stacked microfluidic device, the first end comprising one or more passages enabling fluid flow to a channel inlet, the second end opposite the first end comprising one or more passages enabling fluid flow from a product outlet and waste outlet of the channel, and the passages at the first and second ends of the reservoir layer being separated by a fluid-impermeable material. 24. A stacked separation assembly according to any one of Embodiments 22 or 23, wherein both the top and bottom surfaces of a 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, comprising: (a) obtaining a sample containing the target particles or target cells and the contaminants; (b) (i) pouring the sample into one or more sample inlets of a microfluidic device according to any one of Embodiments 1 to 21, or a first microfluidic device or stacked device according to any one of Embodiments 22 to 24; (ii) flowing the sample to an outlet of the device according to any one of Embodiments 1 to 21, or a first microfluidic device or stacked device according to any one of Embodiments 22 to 24; and (iii) separating target particles or target cells from contaminants by obtaining a product enriched with target particles or target cells from one or more outlets. 26. The method according to Embodiment 25, wherein the target particles or target cells are larger than the critical size of the obstacle array, and at least some contaminants are smaller than the critical size, and the target cells or target particles flow to one or more product outlets where the product enriched with the target cells or target particles is obtained, and the contaminants having a size smaller than the critical size flow to another waste outlet. 27. The method according to Embodiment 26, wherein the sample is blood or derived from blood.28. The method according to Embodiment 26, wherein the sample is an apheresis sample or a leukocyte apheresis sample. 29. The method according to Embodiment 27 or 28, wherein the sample contains platelets as a contaminant. 30. The method according to Embodiment 29, wherein at least 80% of platelets are removed from the sample by this method. 31. The method according to Embodiment 29, wherein at least 90% of platelets are removed from the sample by this method. 32. The method according to Embodiment 29, wherein at least 95% of platelets are removed from the sample by this method. 33. The method according to any one of Embodiments 27-31, wherein the target cells are leukocytes. 34. The method according to any one of Embodiments 27-31, wherein the target cells are stem cells. 35. The method according to any one of Embodiments 27-31, wherein the target cells are B-cells, T-cells, NK-cells, monocytes, or progenitor cells. 36. The method according to any one of Embodiments 27-31, wherein the target cells are dendritic cells. 37. The method according to any one of Embodiments 25-36, wherein the sample is obtained from a patient. 38. The method according to Embodiment 37, wherein the patient has cancer, an autoimmune disease, or an infection. 39. The method according to any one of Embodiments 25 to 38, further comprising genetically engineering purified target cells. 40. The method according to Embodiment 39, wherein the genetic engineering includes transfecting or transducing target cells with recombinant nucleic acid. 41. The method according to Embodiment 39 or 40, wherein the genetically engineered target cells are grown by culturing them in vitro. 42. A method for producing chimeric antigen receptor (CAR) T cells, comprising: (a) obtaining a sample containing T cells; (b) separating T cells from contaminants by (i) pouring the sample into one or more sample inlets of a microfluidic device according to any one of Embodiments 1 to 21, or a first microfluidic device or stacked device according to any one of Embodiments 22 to 24; (ii) allowing the sample to flow to the outlet of the device; and (iii) obtaining a T cell-enriched product from the product outlet; and (c) genetically engineering the T cells in the enriched product obtained in step b) to produce chimeric antigen receptors (CARs) on their surfaces.43. The method according to Embodiment 42, wherein the sample is blood, apheresis product, or leukocyte apheresis product from a patient. 44. The method according to either Embodiment 42 or 43, wherein the genetic engineering comprises transfecting or transducing target cells, and the genetically engineered target cells are further proliferated by cell growth in vitro. 45. The method according to any one of Embodiments 42-44, wherein separation is achieved by performing a deterministic transverse displacement method in a microfluidic device. 46. The method according to any one of Embodiments 42-44, wherein the sample is obtained from a cancer patient, an autoimmune disease patient, or an infectious disease patient. 47. The method according to Embodiment 46, wherein, after obtaining the sample, the T cells are surrounded by one or more carriers to facilitate DLD separation. 48. CAR T cells produced by the method according to any one of Embodiments 42-47.

[0144] Preferred embodiments of the present invention have been illustrated and described herein, but it will be apparent to those skilled in the art that such embodiments are provided merely as examples. The present invention is not intended to be limited by any particular example provided herein. The present invention has been described with reference to the preceding specification, but the descriptions and examples of embodiments herein are not intended to be restrictive. Hereinafter, numerous variations, modifications, and substitutions will come to mind for those skilled in the art without departing from the present invention. Furthermore, it will be understood that all aspects of the present invention are not limited to any particular description, configuration, or relative proportion described herein, which will vary depending on various conditions and variables. It will be understood that various alternatives to the embodiments of the present invention described herein can be adopted in the practice of the present invention. Accordingly, the present invention is intended to encompass any such alternatives, modifications, variations, or equivalents. The following claims define the scope of the present invention, and the methods and structures within the scope of these claims and their equivalents are intended to be encompassed thereby.

[0145] Any references listed herein are incorporated entirely by reference. Having described the present invention in detail, it will be understood by those skilled in the art that the present invention can be practiced within a broad and equivalent range of conditions, parameters, etc., without affecting the spirit or scope of the invention or any embodiment thereof.

Claims

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 extending from one or more sample inlets and one or more separate fluid inlets to one or more product outlets and one or more separate waste outlets, (a) When fluid is poured into the channel through the sample inlet and / or fluid inlet, the fluid flows through the channel toward the outlet, thereby determining the direction of the bulk fluid flow; (b) The channel comprises an array of obstacles arranged in longitudinal rows along the channel and transverse rows across the channel, wherein the obstacles are positioned to define a critical size, so that as the sample is poured into the inlet and flows out of the device, particles or cells larger than the critical size in the sample are separated from particles or cells smaller than the critical size in the sample; where (i) Adjacent obstacles in a row are separated by a gap G1 perpendicular to the direction of bulk fluid flow; (ii) Adjacent obstacles in a column are separated by a gap G2 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, and the size of gap G1 is greater than the size of gap G2; (iv) Each subsequent row of obstacles is offset transversely from the preceding row, thus determining an array direction that is at least 1 angle of inclination from the direction of the bulk fluid flow. (v) The obstacle has at least two vertices such that at least one vertex is adjacent to each gap on each side. (vi) The obstruction is elongated perpendicular to the direction of bulk fluid flow and therefore has a left-right length (P1) that is different from its up-down length (P2). (vii) P1 is 10-150% longer than P2 (viiii) P1 is between 1 μm and 160 μm; and (ix) One or more sample inlets are separated from one or more fluid inlets by a separation wall, the separation wall extending from one or more sample inlets through an array of channel obstructions toward the outlet, the separation wall extending for at least 10% of the length of the array of obstructions in a direction parallel to the direction of bulk fluid flow, To prevent fluid or sample from flowing over the upper surface of the obstacle during the operation of the device, the device further comprises an obstacle bonding layer bonded to the surface of the planar support and bonded to the obstacle in the channel embedded in the surface, The obstruction bonding layer comprises one or more fluid-connected passages at the sample inlet of the channel, which enable the flow of a sample into the channel, and one or more fluid-connected passages at the outlet of the channel, which enable the flow of fluid out of the outlet. Microfluidic devices.

2. The microfluidic device according to claim 1, wherein the obstacle is polygonal.

3. The microfluidic device according to claim 2, wherein the obstacle is rhomboid or hexagonal.

4. The microfluidic device according to claim 1, wherein P1 is at least 15% longer than P2.

5. The microfluidic device according to claim 1, wherein P1 is 20 to 70% longer than P2.

6. The microfluidic device according to any one of claims 1 to 5, wherein the obstacle has vertices that extend through the gap such that one or more vertices facing each other but not directly opposite each other are adjacent on each side of the parallel gap.

7. The microfluidic device according to any one of claims 1 to 5, wherein the obstacles have vertices that extend through the gap such that the vertices facing each other and directly opposite each other are adjacent to a vertical gap on each side.

8. The microfluidic device according to any one of claims 1 to 7, wherein the separation wall extends over at least 20% of the length of the array of obstacles.

9. A stacked separation assembly comprising a first microfluidic device selected from the microfluidic devices described in any one of claims 1 to 8, and one or more stacked microfluidic devices also selected from the microfluidic devices described in any one of claims 1 to 8, wherein (a) The bottom surface of each stacked device is in contact with the top surface or an obstacle bonding layer on the top surface of the first microfluidic device, or with the top surface or an obstacle bonding layer on the top surface of another stacked device; (b) The sample is provided to the sample inlet through the first common manifold; (c) The fluid is supplied to the fluid inlet through a second manifold which may or may not be the same as the first manifold; (d) The product is removed from the product outlet through one or more conduits; (e) the waste is removed from the waste outlet through one or more conduits different from one or more conduits in (d); and (f) A stacked separation assembly configured such that the first microfluidic device and the stacked microfluidic devices are optionally mounted within a common outer casing.

10. A stacked separation assembly according to claim 9, further comprising at least one reservoir bonding layer, the reservoir bonding layer being attached to the bottom surface of a first microfluidic device and / or the top surface of a stacked microfluidic device, wherein the reservoir bonding layer has one or more passages at a first end that allow fluid to flow to a channel inlet, wherein the reservoir bonding layer has one or more passages at a second end opposite to the first end that allow fluid to flow from a product outlet and a waste outlet of the channel, and wherein the passages at the first and second ends of the reservoir bonding layer are separated by a fluid-impermeable material.

11. The stacked separation assembly according to claim 10, wherein both the top and bottom surfaces of a planar support of one or more microfluidic devices comprise one or more channels having obstacles for separating target particles or target cells, and one or more cavities configured to prevent damage, displacement, or deformation of the obstacles.

12. A method for purifying target particles or target cells of a predetermined size from contaminants in a sample, (a) obtaining a sample containing the target particles or target cells and the contaminants; and (b) Below: (i) Pouring the sample into one or more sample inlets of the microfluidic device according to any one of claims 1 to 8, or the first microfluidic device or stacked device according to any one of claims 9 to 11. (ii) Flowing a sample to the outlet of the microfluidic device according to any one of claims 1 to 8, or the first microfluidic device or stacked device according to any one of claims 9 to 11, and (iii) Separating the target particles or target cells from the contaminant by obtaining a product enriched with the target particles or target cells from one or more outlets; a method comprising:

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