Method of preparing a conditioned medium from a confluent stromal cell culture

• US 7,678,573 B2
• Filed: 04/11/2005
• Issued: 03/16/2010
• Est. Priority Date: 02/04/1999
• Status: Expired due to Fees

• Current Assignee: Pluristem Ltd. (Pluri Inc.)
• Original Assignee: Pluristem Ltd. (Pluri Inc.)
• Inventors: Meretski, Shai, Zipori, Dov, Merchav, Shoshana, Kadouri, Avinoam
• Primary Examiner(s): Naff; David M
• Application Number:
  US11/102,623
• Publication Number:
  US 20050181504A1
• Time in Patent Office: 1,800 Days
• Field of Search: None
• US Class Current: 435/404
• 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 preparing a stromal cell conditioned medium useful in expanding undifferentiated hemopoietic stem cells to increase the number of the hemopoietic stem cells is provided. The method comprising: (a) establishing a stromal cell culture in a stationary phase plug-flow bioreactor under continuous flow 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 undifferentiated hemopoietic stem cells; and (b) when a desired stromal cell density has been achieved, collecting medium from the stationary phase plug-flow bioreactor, thereby obtaining the stromal cell conditioned medium useful in expanding the undifferentiated hemopoietic stem cells.

Claims

1. A method of preparing a conditioned medium from a confluent stromal cell culture, 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 to obtain a stationary phase three-dimensional confluent stromal cell culture, wherein said stromal cell culture is grown to a density of at least 5×

10⁶ cells per a cubic centimeter of said substrate; and

(b) separating and collecting a stromal cell conditioned medium from said stationary phase confluent stromal cell culture;

thereby obtaining the conditioned medium, said medium useful in expanding an undifferentiated hemopoietic stem cell culture to increase the number of the undifferentiated hemopoietic stem cells.

2. The method of claim 1, wherein said stromal cell culture is grown to a density of at least 10⁷ cells per a cubic centimeter of said substrate.

3. The method of claim 1, wherein said fibrous matrix forms 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.

4. The method of claim 1, wherein said matrix is made of 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.

5. 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.

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

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

m.

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

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

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

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

12. The method of claim 1, wherein said stromal cell culture comprises a primary culture.

13. The method of claim 1, wherein said stromal cell culture comprises a culture of a stromal cell line.

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

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

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