Cultured epidermal cell sheet, laminated cultured skin sheet and process for producing the same

• US 20040028657A1
• Filed: 06/16/2003
• Published: 02/12/2004
• Est. Priority Date: 07/21/2000
• Status: Active Grant

• Current Assignee: CellSeed Inc.
• Original Assignee: CellSeed Inc.
• Inventors: Utsumi, Mika, Kushida, Ai, Konno, Chie, Okano, Teruo, Kikuchi, Akihiko, Yamato, Masayuki
• Granted Patent: US 8,889,417 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• CPC Class Codes: A61L 27/362 Skin, e.g. dermal papillae
• CPC Class Codes: A61L 27/3804 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
• CPC Class Codes: A61L 27/3891 as distinct cell layers
• CPC Class Codes: A61L 27/60 Materials for use in artificial skin
• CPC Class Codes: C12N 2502/094 keratinocytes
• CPC Class Codes: C12N 2502/1323 Adult fibroblasts
• CPC Class Codes: C12N 2539/10 Coating allowing for selective detachment of cells, e.g. thermoreactive coating
• CPC Class Codes: C12N 5/0698 Skin equivalents

Abstract

Cells are cultured on a cell culture support having a substrate surface coated with a temperature-responsive polymer whose upper or lower critical solution temperature in water is 0˜80° C., optionally forming multiple cultured cell layers by a conventional technique, and subsequently:

(1) the temperature of the culture solution is brought to above the upper critical solution temperature or below the lower critical solution temperature;

(2) the cultured epidermal cell sheet or multi-layered skin sheet is brought into close contact with the polymer membrane; and

(3) the sheet is peeled off together with the polymer membrane. The thus obtained epidermal cultured cell sheet and multi-layered cultured skin sheet do not degrade E-cadherin or laminin 5 as contrasted with Dispase treatment and they still involve very few structural defects; these sheets hence hold great promise in clinical applications such as skin grafting.

Claims

1. An epidermal cultured cell sheet or a multi-layered cultured skin sheet that has only a few structural defects as they are recovered with intercellular desmosome structures and the cell-to-substrate basal membrane protein being kept intact.

2. The epidermal cultured cell sheet or multi-layered cultured skin sheet according to claim 1, which has been peeled off from a substrate without being treated with a proteolytic enzyme.

3. The epidermal cultured cell sheet or multi-layered cultured skin sheet according to claim 1 or 2, which are overlaid with a fibroblast sheet and/or a vascular endothelial cell sheet.

11. The epidermal cultured cell sheet or multi-layered cultured skin sheet according to any one of claims 1-3 and 10, which is adapted for use in the treatment of a burn and/or a wound that are gouged deep into a skin tissue.

12. A therapeutic method in which the epidermal cultured cell sheet or multi-layered cultured skin sheet according to any one of claims 1-3 and 10 is grafted to a burn and/or a wound that are gouged deep into a skin tissue.

4. A process for producing an epidermal cultured cell sheet or a multi-layered cultured skin sheet, which comprises culturing cells on a cell culture support having a substrate surface coated with a temperature-responsive polymer whose upper or lower critical solution temperature in water is 0˜

80°

C., optionally forming multiple cultured cell layers by a conventional technique, and subsequently;

(1) bringing the temperature of the culture solution to above the upper critical solution temperature or below the lower critical solution temperature;

(2) bringing the cultured epidermal cell sheet or multi-layered skin sheet into close contact with the polymer membrane; and

(3) peeling the sheet off together with the polymer membrane.

5. A process for producing a multi-layered cultured skin sheet, in which the epidermal cultured cell sheet or multi-layered cultured skin sheet in close contact with the polymer membrane as obtained in claim 4 is again allowed to adhere to a cell culture support, a cell culture support coated with a temperature-responsive polymer, a polymer membrane, other cellular sheet, etc. and the polymer membrane in close contact is thereafter peeled off to form multiple cultured cell layers.

6. The process for producing an epidermal cultured cell sheet or multi-layered cultured skin sheet according to claim 4 or 5, wherein the step of peeling off does not involve treatment with a proteolytic enzyme.

7. The process for producing an epidermal cultured cell sheet or multi-layered cultured skin sheet according to claim 4 or 5, wherein the temperature-responsive polymer is poly(N-iospropylacrylamide).

8. The process for producing an epidermal cultured cell sheet or multi-layered cultured skin sheet according to claim 4 or 5, wherein the polymer membrane is hydrophilized polyvinylidene difluoride.

9. The process for producing a multi-layered cultured skin sheet according to claim 5, wherein the other cellular sheet is at least one member of the group consisting of an epidermal cultured cell sheet, a multi-layered cultured skin sheet, as well as a fibroblast sheet and a vascular endothelial cell sheet that are prepared by the process according to claim 4.

10. An epidermal cultured cell sheet or a multi-layered cultured skin sheet that are produced by the process according to any one of claims 4 to 9.