Method of expanding undifferentiated hemopoietic stem cells

• US 7,534,609 B2
• Filed: 04/11/2005
• Issued: 05/19/2009
• Est. Priority Date: 02/04/1999
• Status: Expired due to Term

• Current Assignee: Pluristem Ltd. (Pluri Inc.)
• Original Assignee: Pluristem Life Systems Inc.
• Inventors: Meretski, Shai, Merchav, Shoshana
• Primary Examiner(s): Naff; David M
• Application Number:
  US11/102,625
• Publication Number:
  US 20050176143A1
• Time in Patent Office: 1,499 Days
• Field of Search: None
• US Class Current: 435/395
• CPC Class Codes: A61F 2/00 Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents as cosmetic articles, see the relevant subclasses, e.g. wigs, hair pieces, A41G3/00, A41G5/00; artificial nails A45D31/00; dental prostheses A61C13/00; materials for prostheses A61L27/00; artificial hearts A61M60/00; artificial kidneys A61M1/14
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: C12M 25/14 Scaffolds; Matrices in general C12N5/0068
• CPC Class Codes: C12N 11/096 Polyesters; Polyamides
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/145 Thrombopoietin [TPO]
• CPC Class Codes: C12N 2501/26 Flt-3 ligand (CD135L, flk-2 ligand)
• CPC Class Codes: C12N 2502/1394 Bone marrow stromal cells; whole marrow
• CPC Class Codes: C12N 2533/30 Synthetic polymers
• CPC Class Codes: C12N 5/06 Animal cells or tissues; Human cells or tissues preservation of living cells or tissues A01N1/02
• CPC Class Codes: C12N 5/0647 Haematopoietic stem cells; Uncommitted or multipotent progenitors

Abstract

A method of expanding undifferentiated hemopoietic stem cells to increase the number of the hemopoietic stem cells is provided. The method comprising: (a) obtaining the undifferentiated hemopoietic stem cells; and (b) culturing the undifferentiated hemopoietic stem cells in a medium containing a stromal cell conditioned medium, the stromal cell conditioned medium being derived from a stationary phase plug-flow bioreactor in which a three dimensional stromal cell culture has been established under continuous flow of a culture medium on a substrate in the form of a sheet, the substrate including a non-woven fibrous matrix forming a physiologically acceptable three-dimensional network of fibers, thereby expanding the undifferentiated hemopoietic stem cells.

Claims

1. A method of expanding an undifferentiated hemopoietic stem cell population to increase the number of the hemopoietic stem cells without the undifferentiated hemopoietic stem cells undergoing differentiation, the method comprising:

(a) culturing stromal cells to confluence in a stationary phase plug-flow bioreactor under continuous flow of medium on a substrate in the form of a sheet, said substrate including a non-woven fibrous matrix forming a physiologically acceptable three-dimensional network of fibers to thereby generate a stationary phase three-dimensional confluent stromal cell culture;

(b) separating a stromal cell conditioned medium from said stationary phase three-dimensional confluent stromal cell culture;

(c) collecting said stromal cell conditioned medium;

(d) obtaining an undifferentiated hemopoietic stem cell population;

(e) adding said undifferentiated hemopoietic stem cell population to a medium containing said stromal cell conditioned medium; and

(f) culturing said undifferentiated hemopoietic stem cell populations in a medium containing said stromal cell conditioned medium, thereby expanding the undifferentiated hemopoietic stem cell population to increase the number of undifferentiated hemopoietic stem cells without the undifferentiated hemopoietic stem cells undergoing differentiation to obtain an expanded undifferentiated hematopoietic stem cell population.

2. The method of claim 1, wherein a rate of said continuous flow is in a range of 0.1 to 25 ml/minute.

3. The method of claim 1, wherein a rate of said continuous flow is in a range of 1 to 10 ml/minute.

4. The method of claim 1, wherein the undifferentiated hemopoietic stem cell population comprises cells isolated from a tissue selected from the group consisting of cord blood, mobilized peripheral blood and bone-marrow.

5. The method of claim 1, wherein the undifferentiated hemopoietic stem cell population and stromal cells of said stromal cell culture share common HLA antigens.

6. The method of claim 1, wherein the undifferentiated hemopoietic stem cells population and stromal cells of said stromal cell culture are from a single individual.

7. The method of claim 1, wherein the undifferentiated hemopoietic stem cell population and stromal cells of said stromal cell culture are from different individuals.

8. The method of claim 1, wherein the undifferentiated hemopoietic stem cell population and stromal cells of said stromal cell culture are from the same species.

9. The method of claim 1, wherein the undifferentiated hemopoietic stem cell population and stromal cells of said stromal cell culture are from different species.

10. The method of claim 1, wherein said three dimensional confluent stromal cell culture has a density of at least 5×

10⁶ cells per a cubic centimeter of said substrate.

11. The method of claim 1, wherein said three-dimensional confluent stromal cell culture has a density of at least 10⁷ cells per a cubic centimeter of said substrate.

12. The method of claim 1, wherein said fibers form a pore volume as a percentage of total volume of from 40 to 95% and a pore size of from 10 microns to 100 microns.

13. The method of claim 1, wherein said matrix is made of a fiber selected from the group consisting of flat, non-round, and hollow fibers and mixtures thereof, said fibers being of from 0.5 microns to 50 microns in diameter or width.

14. The method of claim 1, wherein said matrix is composed of ribbon formed fibers having a width of from 2 microns to 20 microns, and wherein the ratio of width to thickness of the fibers is at least 2:

1.

15. The method of claim 1, wherein said matrix has a pore volume as a percentage of total volume of from 60 to 95%.

16. The method of claim 1, wherein said matrix has a height of 50-1000 μ

m.

17. The method of claim 1, wherein a material of said matrix is selected from the group consisting of polyesters, polyalkylenes, polyfluorochloroethylenes, polyvinyl chloride, polystyrene, polysulfones, cellulose acetate, glass fibers, and inert metal fibers.

18. The method of claim 1, wherein said matrix is in a shape selected from the group consisting of squares, rings, discs, and cruciforms.

19. The method of claim 1, wherein said matrix is in the form of a disc.

20. The method of claim 1, wherein said matrix is coated with poly-D-lysine.

21. The method of claim 1, wherein said culturing of said stromal cells to confluence a primary culturing of said stromal cells.

22. The method of claim 1, wherein said stromal cells are of a cell line when culturing stromal cells to confluence.

23. The method of claim 1, wherein said stromal cell conditioned medium is devoid of added cytokines.

24. The method of claim 1, wherein said medium is serum free medium when culturing stromal cells to confluence.

25. The method of claim 1, wherein said continuous flow is stopped for 16 hours when culturing stromal cells to confluence.