Method for chondrocyte expansion with phenotype retention
View Patent ↗The present invention provides a method for expanding a population of chondrocytes that maintains chondrocyte phenotype during the expansion by culturing the population in a defined serum-free expansion medium containing one or more cytokines and under low attachment conditions. The method further solves diffusion problems during the subsequent stage of extracellular matrix production by use of a perforated polycarbonate substrate that results in a randomly organized cultured neocartilage tissue. Chondrocytes expanded and cultured in this manner can be used in various medical applications to repair cartilaginous tissues that have been injured by trauma or disease.
1. An in vitro method for producing cartilage tissue characterized by a random organization of cells, the method comprising:
(a) isolating a population of chondrocytes from donor cartilage tissue; then
(b) expanding the population of cartilage chondrocytes in an expansion medium on a substrate comprising polystyrene and hyaluronic acid covalently bound to each other to yield expanded chondrocytes, wherein the expansion conditions are effective to prevent attachment of a majority of the chondrocytes to the substrate; then
(c) seeding a plurality of expanded chondrocytes in a tissue production medium on a porous polycarbonate substrate having a plurality of pores therethrough, the pores having an inner diameter between about 1 micron and about 12 microns; and then
(d) culturing the expanded chondrocytes on the porous polycarbonate substrate for a period of time sufficient for the chondrocytes to produce extracellular matrix,
thereby producing in vitro a cultured cartilage tissue characterized by a random organization of cells.
2. A method according to claim 1 wherein expanding under conditions effective to prevent attachment of a majority of the chondrocytes to the substrate during expansion comprises use of a substrate having a coating that reduces cell attachment to the substrate relative to cell attachment to an uncoated substrate.
3. A method according to claim 1 wherein the expansion medium is a serum-free medium.
4. A method according to claim 1 wherein the tissue production medium comprises TGF-β.
5. A method according to claim 1 wherein the population of chondrocytes comprises synovial capsule chondrocytes or periosteum chondrocytes.
6. A method according to claim 1 wherein the population of chondrocytes comprises juvenile articular chondrocytes.
7. A method according to claim 1 wherein the population of chondrocytes comprises adult articular chondrocytes.
8. A method according to claim 1 comprising adding TGF-β to the tissue production medium in an amount of 10 ng/ml of the medium.
9. A method according to claim 1 , wherein expanding the population of chondrocytes comprises:
a first phase of cell expansion; and
a second phase of cell expansion following the first phase of cell expansion.
10. A method according to claim 9 further comprising maintaining the population of chondrocytes for a culture period of about forty-five (45) to about sixty-five (65) days in the tissue production medium.
11. A method according to claim 9 wherein maintaining the population of chondrocytes in a tissue production medium comprises maintaining a predetermined level of oxygen and carbon dioxide in the tissue production medium.
12. A method according to claim 1 wherein the polycarbonate substrate comprises a polycarbonate membrane, wherein the pores comprise perforations through the membrane.
13. A method according to claim 1 wherein the donor cartilage tissue comprises immature hyaline cartilage tissue and the chondrocytes comprise immature hyaline cartilage chondrocytes.
14. A method according to claim 13 wherein the conditions during expansion are further effective for the population of chondrocytes to undergo at least 3.8 doublings of the chondrocyte population while at least 50% of the chondrocytes retain rounded morphology and hyaline cartilage gene expression.
15. A method according to claim 1 wherein the step of expanding the population of chondrocytes under conditions that reduce chondrocyte attachment to the substrate during expansion comprises use of soluble hyaluronic acid in the expansion medium.