Methods and systems for conserving highly expanded cells
AssigneeThe Regents of the University of California
InventorsKyriacos A. Athanasiou, Jerry C. Hu, Heenam Kwon
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Abstract
The methods described herein are for conserving highly expanded cells that have functional properties such as potential for use in neotissue constructs. For example, highly expanded chondrocytes that can be used to construct neocartilage exhibiting functional properties similar to native articular cartilage. The methods and systems feature processes that form functional, human cartilage using cells that have been expanded to at least 1.5×105 times or P3 or greater. This enables a large quantity of engineered cartilage implants to be produced from few cells.
Claims20
A method of preparing human cartilage cells, expanded to passages N=5 (P5) or beyond, that are functional for neocartilage production, said method comprising:
- (a) culturing a population of human cartilage cells at passage 0 (P0) in monolayer in a medium comprising two or more of:
- a transforming growth factor (TGF)-β superfamily protein, a fibroblast growth factor, and a mitogen;
- and expanding said cells to passage 5 (P5) or beyond in said medium;
- (b) subjecting the expanded human cartilage cells from (a) to dissociation and subsequent culture in a three-dimensional environment in a medium comprising two or more of:
- a transforming growth factor (TGF)-β superfamily protein, a growth differentiation factor, and a bone morphogenetic protein;
- and
- (c) subjecting the human cartilage cells from (b) to a medium comprising:
- a transforming growth factor (TGF)-β superfamily protein, a proteoglycan and/or glycosaminoglycan degrading agent, and a cross-linking agent;
- wherein the human cartilage cells from (c) retain a phenotype that is similar to or better than the phenotype exhibited by the human cartilage cells formed by cartilage cells at P0 (native state) or at passage N-X, wherein N is the passage number, X is any integer between 1 and N.
A method of preparing human cartilage cells having a phenotype that is similar or better than a phenotype exhibited by cartilage cells at P0 (native state) or at passage N-X, wherein N is the passage number, X is any integer between 1 and N, said method comprising:
- (a) culturing a population of human cartilage cells at passage 0 (P0) in monolayer in a medium comprising two or more of:
- a transforming growth factor (TGF)-β superfamily protein, a fibroblast growth factor, and a mitogen;
- and expanding said cells to passage 5 (P5) or beyond in said medium;
- (b) subjecting the expanded human cartilage cells from (a) to dissociation and subsequent culture in a three-dimensional environment in a medium comprising two or more of:
- a transforming growth factor (TGF)-β superfamily protein, a growth differentiation factor, and a bone morphogenetic protein;
- (c) subjecting the human cartilage cells from (b) to a medium comprising a transforming growth factor (TGF)-β superfamily protein, a proteoglycan and/or glycosaminoglycan degrading agent, and a cross-linking agent;
- and
- (d) comparing a phenotype of the human cartilage cells in (c) to a phenotype of human cartilage cells at an earlier passage or P0 from (a), wherein the phenotype of the human cartilage cells from (c) is similar to or better than the phenotype exhibited by cartilage cells at P0 (native state) or at passage N-X, wherein N is the passage number, X is any integer between 1 and N;
- wherein the cells are functional for neocartilage production.
A method comprising:
- (a) culturing a population of human cartilage cells at passage 0 (P0) in monolayer in a medium comprising two or more of:
- a transforming growth factor (TGF)-β superfamily protein, a fibroblast growth factor, and a mitogen;
- and expanding said cells to passage 5 (P5) or beyond in said medium;
- (b) subjecting the expanded human cartilage cells from (a) to dissociation and subsequent culture in a three-dimensional environment in a medium comprising two or more of:
- a transforming growth factor (TGF)-β superfamily protein, a growth differentiation factor, and a bone morphogenetic protein;
- and
- (c) subjecting the cells from (b) to a medium comprising:
- a transforming growth factor (TGF)-β superfamily protein, a proteoglycan and/or glycosaminoglycan degrading agent, and a cross-linking agent;
- wherein the cells in (c) are functional for neocartilage production.
Dependencies are inferred from the claim text.