Three-dimensional stromal cell and tissue culture system
AssigneeAdvanced Tissue Sciences, Inc.
InventorsSandra R. Slivka, Lee Landeen
Life of this patent
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Abstract
The present invention relates to an improved three-dimensional cell culture system in which cells are grown on a three-dimensional matrix while cycling the cultures between metabolically favorable and metabolically unfavorable (but noncytotoxic) conditions. The invention is based, at least in part, on the discovery that cycling the cultures in this manner optimizes the formation of extracellular matrix and produces an overall structure that more closely resembles naturally occurring tissue.
Claims14
An improved method of preparing a living stromal tissue in vitro that comprises stromal cells and connective tissue proteins naturally secreted by the stromal cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells, wherein the improvement comprises:
- (a) culturing the stromal cells inoculated onto the three-dimensional framework in a nutrient medium under a temperature, CO.sub.2 concentration and humidity that promotes cell division, so that the inoculated stromal cells attach to the three-dimensional framework and cell division is promoted to form a three-dimensional stromal cell culture;
- (b) incubating the three-dimensional stromal cell culture of step (a) in a buffer under a temperature, CO.sub.2 concentration and humidity that inhibits cell division, so that cell division is inhibited yet the cells remain viable;
- (c) culturing the three-dimensional stromal cell culture of step (b) in a nutrient medium under a temperature, CO.sub.2 concentration and humidity that promotes cell division, so that cell division is promoted;
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- d) repeating steps (b) and (c) as necessary to produce extracellular matrix proteins in the three dimensional stromal culture.
An improved method of preparing a living stromal tissue in vitro that comprises stromal cells and connective tissue proteins naturally secreted by the stromal cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells, wherein the improvement comprises:
- (a) culturing the stromal cells inoculated onto the three-dimensional framework in a nutrient medium under a temperature, CO.sub.2 concentration and humidity that promotes cell division for 3-6 cell cycles, so that the inoculated stromal cells attach to the three-dimensional framework and cell division is promoted to form a three-dimensional stromal cell culture;
- (b) incubating the three-dimensional stromal cell culture of step (a) in a buffer under a temperature, CO.sub.2 concentration and humidity that inhibits cell division for 1-2 cell cycles, so that cell division is inhibited, yet the cells remain viable;
- (c) culturing the three-dimensional stromal cell culture of step (b) in a nutrient medium under a temperature, CO.sub.2 concentration and humidity that promotes cell division for 3-4 cell cycles, so that cell division is promoted;
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- (d) repeating steps (b) and (c) as necessary to produce extracellular matrix proteins in the three dimensional stromal culture.
An improved method of producing a dermal equivalent which is a living stromal tissue prepared and cultured in vitro that comprises fibroblasts that have been seeded onto a framework and connective tissue proteins naturally secreted by the fibroblasts attached to and substantially enveloping the framework in which the framework is composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the fibroblasts, wherein the improvement comprises:
- (a) culturing the living stromal tissue immediately after fibroblast seeding in nutrient medium at 37.degree. C. for 12 to 13 days;
- (b) culturing the living stromal tissue produced in step (a) in phosphate buffered saline at 4.degree. to 8.degree. C. for 3 to 4 days;
- (c) culturing the living stromal tissue produced in step (b) in nutrient medium at 37.degree. C. for 3 to 5 days;
- (d) culturing the living stromal tissue produced in step (c) in phosphate buffered saline at 4.degree. to 8.degree. C. for 3 to 4 days;
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- (e) culturing the living stromal tissue produced in step (d) in nutrient medium at 37.degree. C. for 3 to 5 days.
An improved method of culturing skin tissue, in which keratinocytes are grown on a dermal equivalent which is a living stromal tissue prepared and cultured in vitro that comprises fibroblasts that have been seeded onto a framework and connective tissue proteins naturally secreted by the fibroblasts attached to and substantially enveloping the framework, in which the framework is composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the fibroblasts, wherein the improvement comprises:
- (a) culturing the living stromal tissue immediately after fibroblast seeding in nutrient medium at 37.degree. C. for 12 to 13 days;
- (b) culturing the living stromal tissue produced in step (a) in phosphate buffered saline at 4.degree. to 8.degree. C. for 3 to 4 days;
- (c) culturing the living stromal tissue produced in step (b) in nutrient medium at 37.degree. C. for 3 to 5 days,
- (d) culturing the living stromal tissue produced in step (c) in phosphate buffered saline at 4.degree. to 8.degree. C. for 3 to 4 days;
- (e) culturing the living stromal tissue produced in step (d) in nutrient medium at 37.degree. C. for 3 to 5 days;
- (f) equilibrating the living stromal tissue produced in step (e) in stratification medium which is Dulbecco's Modified Eagle's Medium with 5 percent fetal bovine serum, 100 .mu.g/ml ascorbate, and 0.5 .mu.g/ml hydrocortisone, for 24 to 48 hours;
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- (g) seeding keratinocytes onto the equilibrated living stromal tissue produced in step (f) and;
- (h) culturing the product of step (g) in stratification medium.
Dependencies are inferred from the claim text.