High efficiency microfluidic purification of stem cells to improve transplants
Described herein is a novel, highly efficient system to remove erythrocytes and purify leukocytes would raise the quality of UCB and other transplant grafts, thereby significantly improving patient outcomes.
1. A method for purifying leukocytes for therapeutic use from a sample comprising leukocytes and erythrocytes, the method comprising:
(a) providing a sample comprising erythrocytes and leukocytes, wherein the sample volume is at least 100 ml and less than 300 mL;
(b) separating said erythrocytes from said leukocytes by deterministic lateral displacement on a microfluidic device, wherein said microfluidic device comprises posts with one or more non-rounded vertices, wherein said posts are organized into an array of rows and columns such that adjacent posts form a gap through which fluid may pass and adjacent columns are offset from one another to form an array direction that differs from the flow direction of the device by a tilt angle (c) and wherein the array has a critical diameter such that, when said sample is flowed through the device, leukocytes travel in the array direction and erythrocytes travel in the direction of fluid flow;
(c) collecting a product from said microfluidic device, wherein: i) the product comprises leukocytes; ii) relative to the sample applied to the device, the product is depleted in erythrocytes; and iii) the product comprises less than 10% erythrocytes;
wherein the device is operated at a flow rate of at least 100 ml per hour and a linear flow rate of 5 mm/sec to 150 mm/sec, and wherein greater than 90% of the leukocytes collected in the product are viable.
2. The method of claim 1 wherein adjacent posts define a gap through which fluid flows and at least one vertex of a post points into the gap and wherein the erythrocytes in the sample are reduced by greater than 99% in the product.
3. The method of claim 1 , wherein said posts have at least one vertex in which a ratio of the radius of curvature to characteristic dimension is not greater than 0.25.
4. The method of claim 1 , wherein said posts are triangular.
5. The method of claim 1 , wherein said posts are square, rectangular, trapezoidal or hexagonal.
6. The method of claim 1 , wherein said device is operated at a flow rate of 100-300 ml/hr and wherein the cells isolated are suitable for transplantation.
7. The method of claim 1 , wherein said device is operated at a fluid speed of at least 150/mm/sec.
8. The method of claim 1 , wherein the product comprising leukocytes includes hematopoietic stem cells.
9. The method of claim 1 , wherein the product has a yield of leukocytes of greater than 90%.
10. The method of claim 1 , wherein the method does not use centrifugation or differential sedimentation.
11. The method of claim 1 , wherein the sample is umbilical cord blood.
12. The method of claim 11 , wherein the umbilical cord blood is not cryopreserved.
13. The method of claim 11 , wherein the method does not use centrifugation or differential sedimentation.
14. The method of claim 11 , wherein the posts in the microfluidic device are triangular.
15. The method of claim 11 , wherein the posts in the microfluidic device are square, rectangular, trapezoidal or hexagonal.
16. The method of claim 1 , wherein the microfluidic device is operated at a flow rate of 100-300 ml/hr and the erythrocytes in the sample are reduced by greater than 99% in the product.
17. A method for purifying leukocytes for therapeutic use from a sample comprising leukocytes and erythrocytes, the method comprising:
(a) providing a sample comprising erythrocytes and leukocytes, wherein the sample volume is at least 100 ml;
(b) separating said erythrocytes from said leukocytes by deterministic lateral displacement on a microfluidic device, wherein said microfluidic device comprises posts with one or more non-rounded vertices, wherein said posts are organized into an array of rows and columns such that adjacent posts form a gap through which fluid may pass and adjacent columns are offset from one another to form an array direction that differs from the flow direction of the device by a tilt angle (c) and wherein the array has a critical diameter such that, when said sample is flowed through the device, leukocytes travel in the array direction and erythrocytes travel in the direction of fluid flow;
(c) collecting a product comprising leukocytes from said microfluidic device;
wherein:
i) relative to the sample applied to the device, the product is depleted in erythrocytes;
ii) the product comprises less than 0.01 M/μl of erythrocytes;
iii) the device is operated at a linear flow rate of 5 mm/sec to 150 mm/sec,
iv) greater than 90% of the leukocytes collected in the product are viable.
18. The method of claim 17 , wherein the erythrocytes in the sample are reduced by greater than 99% in the product.
19. The method of claim 18 , wherein the product has a yield of leukocytes of greater than 90%.
20. The method of claim 18 , wherein the method is performed in less than one hour and does not use centrifugation.
21. The method of claim 17 , wherein the product has a yield of leukocytes of greater than 90%.
22. The method of claim 21 , wherein the product comprising leukocytes includes hematopoietic stem cells.
23. The method of claim 17 , wherein the product comprising leukocytes includes hematopoietic stem cells.