TANGENTIAL FLOW FILTRATION DEVICES AND METHODS FOR LEUKOCYTE ENRICHMENT

• US 20110189150A1
• Filed: 04/13/2010
• Published: 08/04/2011
• Est. Priority Date: 06/19/2002
• Status: Active Grant

• Current Assignee: Northwest Biotherapeutics Incorporated
• Original Assignee: Northwest Biotherapeutics Incorporated
• Inventors: Turner, Allen, Bosch, Marnix L., Boynton, Alton L., Monahan, Steven J., Lodge, Patricia A., Harris, Paul C.
• Granted Patent: US 8,518,636 B2
• Time in Patent Office: Days
• US Class Current: 424/93.71
• CPC Class Codes: A61M 1/3496 Plasmapheresis; Leucopheresis; Lymphopheresis A61M1/3472 takes precedence
• CPC Class Codes: A61M 1/3603 in the same direction
• CPC Class Codes: A61M 1/3633 Blood component filters, e.g. leukocyte filters
• CPC Class Codes: A61M 1/38 Removing constituents from donor blood and storing or returning remainder to body , e.g. for transfusion
• CPC Class Codes: A61M 2202/0439 White blood cells; Leucocytes lymphocytes A61M2202/0407
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: B01D 2315/10 Cross-flow filtration
• CPC Class Codes: B01D 61/147 Microfiltration
• CPC Class Codes: B01D 61/16 Feed pretreatment
• CPC Class Codes: B01D 61/18 Apparatus therefor
• CPC Class Codes: B01D 61/22 Controlling or regulating
• CPC Class Codes: Y10T 436/255 including use of a solid sorbent, semipermeable membrane, or liquid extraction

Abstract

The present invention provides tangential flow filtration devices and methods for enriching a heterogenous mixture of blood constituents for leukocytes by removal of non-leukocyte blood constituents. In one particular embodiment the device can provide a composition enriched in monocytes.

Claims

1. A method for preparing monocytic dendritic cell precursors from a sample of blood constituents from a subject wherein the sample comprises leukocytes, the method comprising:

(1) introducing the sample into a remover unit through an inlet in the remover unit;

(2) subjecting the sample to cross-flow substantially parallel to a filter having a pore size of about 1 to about 10 microns;

(3) subjecting the fluid to filtration through the filter;

(4) selectively removing non-leukocyte blood constituents from the sample to form a cell population enriched for leukocytes; and

(5) preparing monocytic dendritic cell precursors from the cell population enriched for leukocytes.

2. The method according to claim 1, wherein the isolation of monocytic dendritic cell precursors comprises:

contacting a monocytic dendritic cell precursor adhering substrate with the cell population enriched for leukocytes;

allowing monocytic dendritic cell precursors in the cell population to reversibly adhere to the substrate to form complexes comprising monocytic dendritic cell precursors and substrate;

separating the complexes from the non-adhering leukocytes to obtain complexes comprising monocytic dendritic cell precursors; and

culturing the monocytic dendritic cell precursors to differentiate the precursors to form immature or mature dendritic cells.

3. The method according to claim 2, wherein the monocytic dendritic cell precursors are eluted from the substrate prior to culturing.

4. The method according to claim 2, wherein the monocytic dendritic cell precursors are cultured on the substrate.

5. The method according to claim 2, wherein the substrate comprises glass, polystyrene, plastic or glass-coated polystyrene microbeads.

6. A method for enriching a sample of blood constituents for dendritic cells, comprising:

(1) introducing the sample into a tangential flow filtration (TFF) unit, the TFF unit comprising a cross-flow chamber, a filtrate chamber, and a filter in fluid communication with the cross-flow chamber and the filtrate chamber, the filter having a pore size of about 1 to about 10 microns;

(2) recirculating the sample through the TFF unit at a predetermined input rate and a predetermined filtration rate, the predetermined input rate at least five times the predetermined filtration rate;

wherein the predetermined filtration rate is less than the unopposed filtration rate for the filter;

(3) isolating a cell population enriched for leukocytes; and

(4) preparing dendritic cells from the enriched cell population.

7. The method according to claim 6, wherein the dendritic cells are prepared by:

contacting a monocytic dendritic cell precursor adhering substrate with the enriched cell population;

allowing monocytic dendritic cell precursors in the enriched cell population to reversibly adhere to the substrate to form complexes comprising monocytic dendritic cell precursors and substrate;

separating the complexes from the non-adhering leukocytes to obtain complexes comprising monocytic dendritic cell precursors; and

culturing the monocytic dendritic cell precursors to differentiate the precursors to form immature or mature dendritic cells.

8. The method according to claim 7, wherein the substrate comprises glass, polystyrene, plastic or glass-coated polystyrene microbeads.

9. The method according to claim 7, further comprising isolating immature or mature dendritic cells.

10. The method according to claim 7, wherein the monocytic dendritic cell precursors are cultured with cytokines that promote the differentiation of monocytic dendritic cell precursors into immature or mature dendritic cells.

11. The method according to claim 10, wherein the cytokines are GM-CSF, GM-CSF and IL-4.

12. The method according to claim 10, wherein the immature dendritic cells are matured into mature dendritic cells.

13. The method according to claim 10, wherein the immature dendritic cells are cultured with an antigen under conditions conducive for processing the antigen to form antigen loaded dendritic cells.

14. The method according to claim 13, further comprising the step of administering the antigen loaded dendritic cells to an individual.

15. The method according to claim 13, wherein the antigen loaded dendritic cells are cultured with a maturation agent to mature the cells into mature antigen presenting dendritic cells.

16. The method according to claim 6, wherein the leukocytes comprise CD34⁺ cells.

17. The method according to claim 16, wherein the sample of blood constituents is from a donor that has been treated with at least one stem cell mobilizing agent.

18. The method according to claim 17, wherein the stem cell mobilizing agent is G-CSF or cyclophosphamide.

19. The method according to claim 16, further comprising enriching the leukocytes for the CD34⁺ cells.

20. The method according to claim 19, wherein the enrichment of leukocytes for the CD34⁺ cells comprises using an anti-CD34 antibody conjugated to magnetic beads.

21. The method according to claim 16, further comprising expanding the CD34⁺ cells ex vivo.

22. The method according to claim 6, further comprising preparing monocyte-derived pluripotent stem cells from the cell population enriched for leukocytes.

23. The method according to claim 6, further comprising inducing differentiation of a progenitor or stem cell.

24. The method according to claim 6, further comprising inducing transdifferentiation of a differentiated cell.