Method and apparatus for maintenance and expansion of hemopoietic stem cells and/or progenitor cells

• US 20090004738A1
• Filed: 09/02/2008
• Published: 01/01/2009
• Est. Priority Date: 02/04/1999
• Status: Abandoned Application

• Current Assignee: Pluristem Ltd. (Pluri Inc.)
• Original Assignee: Pluristem Life Systems Inc.
• Inventors: Meretski, Shai, Zipori, Dov, Merchav, Shoshana, Kadouri, Avinoam
• Application Number:
  US12/230,566
• Publication Number:
  US 20090004738A1
• Time in Patent Office: Days
• US Class Current: 435/396
• 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

1-17. -17. (canceled)

18. A three dimensional cell culture comprising cells derived from an adherent stromal cell culture on a substrate, said substrate forming a physiologically acceptable three-dimensional network of fibers, wherein said cells are capable of supporting expansion of undifferenciated hemopoietic stem cells, whereby said cells derived from said stromal cells culture are from a first individual and further whereby said undifferenciated hemopoietic stem cells are from a different individual.

19. The three dimensional cell culture of claim 18, wherein said undifferentiated hemopoietic stem cells are cells isolated from a tissue selected from the group consisting of cord blood, mobilized peripheral blood and bone-marrow.

20. The three dimensional cell culture of claim 18, wherein said first individual and said second individual are of different species.

21. The three dimensional cell culture of claim 18, wherein said cells derived from said adherent stromal cell culture are grown to a density of at least 5×

106 cells per a cubic centimeter of said substrate.

22. The three dimensional cell culture of claim 18, wherein said cells derived from said adherent stromal cell culture are grown to a density of at least 107 cells per a cubic centimeter of said substrate.

23. The three dimensional cell culture of claim 18, 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.

24. The three dimensional cell culture of claim 18, wherein said substrate is made of fibers 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.

25. The three dimensional cell culture of claim 18, wherein said substrate 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.

26. The three dimensional cell culture of claim 18, wherein said substrate has a pore volume as a percentage of total volume of from 60 to 95%.

27. The three dimensional cell culture of claim 18, wherein the substrate has a height of 50-1000 mm.

28. The three dimensional cell culture of claim 18, wherein the material of the substrate is selected from the group consisting of polyesters, polyalkylenes, polyfluorochloroethylenes, polyvinyl chloride, polystyrene, polysulfones, cellulose acetate, glass fibers, and inert metal fibers.

29. The three dimensional cell culture of claim 18, wherein the substrate is in a shape selected from the group consisting of squares, rings, discs, and cruciforms.

30. The three dimensional cell culture of claim 18, wherein the substrate is in the form of a disc.

31. The three dimensional cell culture of claim 18, wherein the substrate is coated with poly-D-lysine.