[미국특허]
Method of expanding undifferentiated hemopoietic stem cells
원문보기
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C12N-005/06
C12N-005/08
C12N-011/08
C12N-011/00
C12M-003/00
A61F-002/00
출원번호
UP-0102625
(2005-04-11)
등록번호
US-7534609
(2009-07-01)
발명자
/ 주소
Merchav, Shoshana
Meretski, Shai
출원인 / 주소
Pluristem Life Systems Inc.
인용정보
피인용 횟수 :
12인용 특허 :
29
초록▼
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 st
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.
대표청구항▼
What is claimed is: 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
What is claimed is: 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 μm. 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.
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