Combined Purification and Concentration by Deterministic Lateral Displacement With Recirculation of Product

1 . A method of separating target cells or target particles of a predetermined size from a sample comprising cells or particles of less than the predetermined size, the method comprising:

a) applying both the sample and a wash fluid to a microfluidic device at separate inlets, wherein:

i) the wash fluid applied to the device is devoid of said target cells or target particles and devoid of said cells or particles of less than the predetermined size;

ii) the microfluidic device comprises an array of obstacles arranged in rows, with each subsequent row of obstacles shifted laterally with respect to a previous row, and wherein the obstacles are positioned so as to differentially deflect target cells or particles to a first outlet where they may be recovered as a target cell or target particle product, and to direct the cells or particles of less than the predetermined size to a second outlet where they may be collected or discarded as waste;

b) performing deterministic lateral displacement (DLD) by flowing the sample and wash fluid through the device, wherein during the performance of said DLD, at least a portion of the target cell or target particle product is recirculated one or more times so as to replace, all, or at least a portion, of the wash fluid being applied to the device;

c) during, or at the end of, step b), collecting a final product comprising target cells or particles from the first outlet.

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

3 . The method of claim 1 , wherein the wash fluid is water or an aqueous buffer, and/or a) comprises reagents that chemically react with cells, particles or other components in the wash fluid; or b) comprises antibodies, carriers or activators that interact specifically with target cells or target particles.

4 . The method of claim 1 , wherein the first outlet comprises, or is connected to, a valve that can be used to divert the target cell or target particle product to a conduit that recycles the product to an inlet on the microfluidic device.

5 . The method of claim 1 , wherein the microfluidic device comprises an inlet that comprises or is connected to a valve that can be used to switch the feed entering the device through the inlet from a conduit feeding wash buffer to a conduit feeding cell or target particle product.

6 . The method of claim 1 , wherein the target cell or target particle product being recirculated to said microfluidic device is reacted with, or bound to, a carrier, antibody, fluorescent tag, activator or compound prior to, during or after being reapplied to the microfluidic device.

7 . The method of claim 1 , wherein cell or particle counts are made of the target cell or target particle product.

8 . The method of claim 1 , wherein recirculation is continued until, relative to the concentration in the sample, cells or particles are concentrated by a factor of at least 3.

9 . The method of claim 1 , wherein recirculation is continued until, relative to the concentration in the sample, cells or particles are concentrated by a factor of at least 5.

10 . The method of claim 1 , wherein recirculation is continued until, relative to the concentration in the sample, cells or particles are concentrated by a factor of at least 10.

11 . The method of claim 1 , wherein recirculation together with microfluidic processing is the sole method used for concentrating cells or particles.

12 . The method of claim 1 , wherein the sample comprises target cells or stem cells of a predetermined size and cells less than the predetermined size.

13 . The method of claim 12 , wherein the target cells are leukocytes and cells less than the predetermined size are platelets or red blood cells.

14 . The method of claim 12 , wherein the sample is blood or a composition that has been obtained by performing apheresis or leukapheresis on blood.

15 . The method of claim 12 , wherein the leukocytes or stem cells being recirculated are bound to a carrier, antibody, or activator in a way that promotes or complements DLD separation.

16 . The method of claim 13 , wherein the leukocytes are T cells.

17 . The method of claim 16 , wherein said method is being used in a process for producing CAR-T cells.

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

19 . The method of claim 17 , wherein said method is used to concentrate cells sufficiently to allow for their administration to a patient.

20 . The method of claim 19 , wherein the sample is obtained from a patient and no more than four hours elapse from the time that the obtaining of the sample is complete until DLD is completed.

21 . (canceled)

22 . A method of making purified genetically engineered target cells, comprising:

a) obtaining a sample comprising target cells of a predetermined size and one or more contaminant cells or contaminant particles that are smaller than the predetermined size;

b) applying the sample to a microfluidic device at a first inlet and a wash fluid at a second inlet, wherein the microfluidic device comprises an array of obstacles positioned so as to differentially deflect a flow of target cells to a first outlet where they may be recovered as a target cell product, and to direct contaminant cells or contaminant particles that are smaller than the predetermined size to a second outlet;

c) flowing the sample and wash fluid through the device, wherein the concentration of target cells at the first outlet is determined and at least a portion of the target cells are recirculated from the outlet so as to replace, all, or at least a portion, of the wash fluid being applied to an inlet of the device, said recirculation being continued or repeated until a desired product cell concentration, PC, is reached;

d) once PC is reached, directing the flow of target cells from the first outlet to a site where the target cells are transformed or transfected to form genetically engineered target cells;

e) flowing the genetically engineered target cells to a device where they are separated from reagents, virus or other materials used in transforming or transfecting the target cells to form purified genetically engineered target cells;

f) either collecting the purified genetically engineered target cells or flowing the purified genetically engineered target cells to another site where they are further processed before collection.

23 - 44 . (canceled)