Methods for enhanced propagation of cells

• US 9,029,144 B2
• Filed: 06/16/2009
• Issued: 05/12/2015
• Est. Priority Date: 06/18/2008
• Status: Active Grant

• Current Assignee: Seastar Medical, Inc. (LMF Acquisition Opportunities, Inc.)
• Original Assignee: Cytopherx, Inc., Innovative Biotherapies, Inc.
• Inventors: Szczypka, Mark, Tziampazis, Evangelos, Westover, Angela J.
• Primary Examiner(s): LI, QIAN JANICE
• Application Number:
  US12/485,575
• Publication Number:
  US 20100136687A1
• Time in Patent Office: 2,156 Days
• Field of Search: 435/375, 435/395, 435/377
• US Class Current: 435/375
• CPC Class Codes: A61P 13/00 Drugs for disorders of the urinary system
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 5/0687 Renal stem cells; Renal progenitors

Abstract

The present invention relates generally to methods for the isolation and propagation of cells. For example, embodiments of the present invention relate to isolation and propagation methods for the manufacture of a large number of cells for use, for example, in biotherapeutic devices, such as devices for renal replacement therapy for the treatment of acute renal failure (ARF), acute tubular necrosis (ATN), multi-organ failure (MOF), sepsis, cardiorenal syndrome (CRS) and end-stage renal disease (ESRD).

Claims

1. A method for enhanced propagation of cells from a kidney sample, the method comprising culturing renal precursor cells from the sample while retaining non-adherent cells when culture or subculture growth medium is changed, thereby to enhance propagation of a cell population from the sample.

2. The method of claim 1, wherein the method maintains the precursor cells in an undifferentiated state.

3. The method of claim 1, wherein the method increases the number of precursor cells relative to the number present in the sample.

4. The method of claim 1, wherein the method comprises dilution of cells in the sample.

5. The method of claim 1, wherein the enhanced propagation cell population comprises renal cells.

6. The method of claim 1, further comprising administering retinoic acid.

7. The method of claim 1, the method comprising applying conditions that stimulate precursor cells.

8. The method of claim 1, the method comprising applying conditions that amplify precursor cells.

9. A method for propagating renal cells from a kidney sample, the method comprising:

(a) treating the kidney sample to produce tissue fragments having a dimension of about 212 μ

m or less in size;

(b) harvesting at least a portion of the tissue fragments by centrifugation at greater than 200×

(g) to produce a pelleted slurry;

(c) applying a portion of the pelleted slurry produced in step (b) to a solid support and culturing the cells from the pelleted slurry in the absence of retinoic acid and under conditions to permit the renal cells to attach to the solid support and divide;

(d) harvesting the cells produced in step (c) prior to reaching confluence on the solid support;

(e) applying a portion of the cells harvested in step (d) to a solid support and subculturing the cells in the absence of retinoic acid and under conditions to permit the renal cells to attach to the solid support and divide; and

(f) harvesting the cells produced in step (e) to produce a propagated population of renal cells.

10. The method of claim 9, wherein non-adherent cells are retained when culture or subculture growth medium is changed.

16. The method of claim 9, wherein step (e) further comprises permitting the cells to divide until confluence.

17. The method of claim 16, wherein step (f) further comprises seeding harvested cells onto a solid support.

18. The method of claim 17, further comprising adding retinoic acid to the cells seeded onto the solid support and permitting the cells to differentiate.

19. The method of claim 9, wherein after step (f), the cell yield is at least 3.22×

10¹⁰ renal epithelial cells per gram of kidney cortex tissue.

20. The method of claim 9, wherein the kidney sample is an adult kidney sample.

11. A method for enhanced propagation of cells from a kidney sample, the method comprising:

separating the kidney sample into fragments of about 212 μ

m or less in size to produce a slurry;

centrifuging the slurry at greater than 200×

(g) to produce a pelleted slurry;

creating a culture by applying to a growth surface about 10 μ

l or less of the pelleted slurry per square centimeter of surface area on which the culture is to be grown, wherein said 10 μ

l or less of the pelleted slurry is resuspended in a sufficient amount of a culture medium for plating; and

subculturing about 8.7×

10⁴ or fewer cells from the culture per square centimeter of surface area on which the subculture is to be grown.

12. The method of claim 11, further comprising adding retinoic acid after the subculturing step.

13. The method of claim 11, wherein non-adherent cells are retained when culture or subculture growth medium is changed.

14. The method of claim 11, wherein the subculturing step occurs prior to confluence.

15. The method of claim 11, further comprising subculturing about 8.7×

10⁴ or fewer cells from the subculture per square centimeter of surface area on which the further subculture is to be grown.

21. The method of claim 11, wherein the kidney sample is an adult kidney sample.