Three-dimensional stromal cell and tissue culture system

Claim 2

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 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 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 2 concentration and humidity that promotes cell division, so that cell division is promoted; and d) repeating steps (b) and (c) as necessary to produce extracellular matrix proteins in the three dimensional stromal culture.

Claim 3

The method of claim 1 in which the temperature that promotes cell division is 37° C.

Claim 4

The method of claim 1 in which the temperature that does not promote cell division is 4° to 8° C. and the buffer is phosphate buffered saline.

Claim 5

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 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 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 2 concentration and humidity that promotes cell division for 3-4 cell cycles, so that cell division is promoted; and (d) repeating steps (b) and (c) as necessary to produce extracellular matrix proteins in the three dimensional stromal culture.

Claim 6

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 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 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 2 concentration and humidity that promotes cell division for 3-4 cell cycles, so that cell division is promoted; and (d) repeating steps (b) and (c) as necessary to produce extracellular matrix proteins in the three dimensional stromal culture.

Claim 7

The method of claim 4 in which the temperature that promotes cell division is 37° C.

Claim 8

The method of claim 4 in which the temperature that does not promote cell division is 4° to 8° C. and the buffer is phosphate buffered saline.

Claim 9

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° C. for 12 to 13 days; (b) culturing the living stromal tissue produced in step (a) in phosphate buffered saline at 4° to 8° C. for 3 to 4 days; (c) culturing the living stromal tissue produced in step (b) in nutrient medium at 37° C. for 3 to 5 days; (d) culturing the living stromal tissue produced in step (c) in phosphate buffered saline at 4° to 8° C. for 3 to 4 days; and (e) culturing the living stromal tissue produced in step (d) in nutrient medium at 37° C. for 3 to 5 days.

Claim 10

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° C. for 12 to 13 days; (b) culturing the living stromal tissue produced in step (a) in phosphate buffered saline at 4° to 8° C. for 3 to 4 days; (c) culturing the living stromal tissue produced in step (b) in nutrient medium at 37° C. for 3 to 5 days; (d) culturing the living stromal tissue produced in step (c) in phosphate buffered saline at 4° to 8° C. for 3 to 4 days; and (e) culturing the living stromal tissue produced in step (d) in nutrient medium at 37° C. for 3 to 5 days.

Claim 11

The method of claim 7 in which the nutrient medium comprises Dulbecco's Modified Eagle's Medium containing ten percent calf serum and 100 μg/ml ascorbic acid.

Claim 12

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° C. for 12 to 13 days; (b) culturing the living stromal tissue produced in step (a) in phosphate buffered saline at 4° to 8° C. for 3 to 4 days; (c) culturing the living stromal tissue produced in step (b) in nutrient medium at 37° C. for 3 to 5 days, (d) culturing the living stromal tissue produced in step (c) in phosphate buffered saline at 4° to 8° C. for 3 to 4 days; (e) culturing the living stromal tissue produced in step (d) in nutrient medium at 37° 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 μg/ml ascorbate, and 0.5 μg/ml hydrocortisone, for 24 to 48 hours; and (g) seeding keratinocytes onto the equilibrated living stromal tissue produced in step (f) and; (h) culturing the product of step (g) in stratification medium.

Claim 13

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° C. for 12 to 13 days; (b) culturing the living stromal tissue produced in step (a) in phosphate buffered saline at 4° to 8° C. for 3 to 4 days; (c) culturing the living stromal tissue produced in step (b) in nutrient medium at 37° C. for 3 to 5 days, (d) culturing the living stromal tissue produced in step (c) in phosphate buffered saline at 4° to 8° C. for 3 to 4 days; (e) culturing the living stromal tissue produced in step (d) in nutrient medium at 37° 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 μg/ml ascorbate, and 0.5 μg/ml hydrocortisone, for 24 to 48 hours; and (g) seeding keratinocytes onto the equilibrated living stromal tissue produced in step (f) and; (h) culturing the product of step (g) in stratification medium.

Claim 14

The method according to claim 9 in which the culture is submerged in stratification medium in step (h) and is allowed to grow for 5 to 7 days, and then is cultured at the air-liquid interface of stratification medium for about 5 to 7 days.

Claim 15

The method according to claim 10 comprising the further step of adding a cholesterol rich lipid supplement to the stratification medium and growing the culture for an additional 12 to 21 days.

Claim 16

The method of claim 11 in which the nutrient medium comprises Dulbecco's Modified Eagle's Medium containing ten percent calf serum and 100 μg/ml ascorbic acid.

Claim 17

The method of claim 10 in which the nutrient medium comprises Dulbecco's Modified Eagle's Medium containing ten percent calf serum and 100 μg/ml ascorbic acid.

Claim 18

The method of claim 9 in which the nutrient medium comprises Dulbecco's Modified Eagle's Medium containing ten percent calf serum and 100 μg/ml ascorbic acid.