THREE-DIMENSIONAL SKIN MODEL

• US 20090298042A1
• Filed: 06/15/2009
• Published: 12/03/2009
• Est. Priority Date: 05/31/2000
• Status: Active Grant

• Current Assignee: Fraunhofer Gesellschaft Zur Foerderung Der Angewandten Forsching E.V.
• Original Assignee: Fraunhofer Gesellschaft Zur Foerderung Der Angewandten Forsching E.V.
• Inventors: Graeve, Thomas, Noll, Michaela
• Granted Patent: US 8,222,031 B2
• Time in Patent Office: Days
• US Class Current: 435/1.1
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61L 2430/06 for cartilage reconstruction, e.g. meniscus
• CPC Class Codes: A61L 2430/40 Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
• CPC Class Codes: A61L 27/24 Collagen
• 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 27/3813 Epithelial cells, e.g. keratinocytes, urothelial cells
• CPC Class Codes: A61L 27/3817 Cartilage-forming cells, e.g. pre-chondrocytes
• CPC Class Codes: A61L 27/3852 Cartilage, e.g. meniscus
• CPC Class Codes: A61L 27/3895 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
• 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 2503/06 Screening or testing on artificial skin
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 5/0062 General methods for three-dimensional culture
• CPC Class Codes: C12N 5/0655 Chondrocytes; Cartilage
• CPC Class Codes: C12N 5/0698 Skin equivalents
• CPC Class Codes: G01N 33/5082 Supracellular entities, e.g. tissue, organisms

Abstract

The present invention relates to methods for cultivating dermal fibroblasts, methods for preparing in vitro dermis equivalents, methods for preparing three-dimensional in vitro skin equivalents, an in vitro dermis equivalent, a three-dimensional in vitro skin equivalent, and methods for determining the effect of a chemical substance or of an agent on human skin cells using the in vitro dermis equivalent and/or the in vitro skin equivalent.

Claims

1. A method for multiplying dermal fibroblasts, comprising cultivating the dermal fibroblasts in a three-dimensional gel-like biomatrix comprising a collagen solution containing 3.5 mg/ml up to 4.5 mg/ml of rat tail collagen in buffered serum-containing cell culture medium, wherein the collagen solution has a high content of non-denatured and non-lyophilized native collagen of at least 90%.

2. A method according to claim 1, wherein cultivating the dermal fibroblasts comprises an at least one- to two-day submerse culture.

3. A method according to claim 1, wherein the dermal fibroblasts are removed from the biomatrix after cultivation and are further cultivated in a higher cell density wherein an in vitro dermis equivalent is obtained.

4. A method according to claim 1, comprising preparing the biomatrix containing embedded dermal fibroblasts by stirring collagen fibers extracted from rat tail tissue in acidic solution for 3 to 14 days at 2 to 10°

C., removing undissolved collagen parts by centrifugation to obtain a collagen-containing solution with a rat tail collagen content of 3.5 mg/ml to 4.5 mg/ml, mixing the collagen-containing solution with a solution containing dermal fibroblasts, cell culture medium, serum, and buffer at 2 to 10°

C. to form a mixture, and gelling the mixture at a higher temperature.

5. A method according to claim 4, wherein the acidic solution is acetic solution.

6. A method according to claim 4, wherein the solution containing cell culture medium, serum, and buffer is mixed in a ratio of 1:

2 with the collagen-containing solution.

7. An in vitro dermis equivalent, prepared in accordance with a method comprising embedding dermal fibroblasts in a three-dimensional, gel-like biomatrix containing 3.5 mg/ml up to 4.5 mg/ml of collagen in buffered serum-containing cell culture medium, whereby the biomatrix is prepared by gelling a collagen solution that has a high content of at least 90% of fresh collagen which is not denatured and not lyophilized and is prepared from rat tail tendon, and cultivating the dermal fibroblasts in such a way that an in vitro equivalent in which shrinkage in at least one of the vertical and horizontal directions has been prevented and an equivalent which has a defined diameter, uniform surface and defined termination with respect to the edge of the biomatrix is obtained.

8. An in vitro dermis equivalent according to claim 7, wherein the dermal fibroblasts are human dermal fibroblasts.

9. The in vitro dermis equivalent according to claim 7 incorporated into a three dimensional skin equivalent prepared in accordance with a method in which the dermal fibroblasts of said in vitro dermis equivalent are cultivated to an at least one- to two-day submerse culture therein, keratinocytes in a cell culture medium are seeded on the biomatrix, and said keratinocytes are cultivated, whereby said three dimensional skin equivalent is realized.

10. A three-dimensional in vitro skin equivalent according to claim 9, wherein the keratinocytes are human keratinocytes.

11. A method for determining the effect of a chemical substance or of an agent on skin cells, comprising the bringing into contact of the chemical substance or of the agent with a three-dimensional in vitro skin equivalent according to claim 9, and determining the interaction between skin equivalent and the chemical substance or the agent.

12. A method for determining the effect of a chemical substance or of an agent on dermis cells, comprising the bringing into contact of the chemical substance or of the agent with an in vitro dermis equivalent according to claim 7, and for determining the interaction between dermis equivalent and the chemical substance or the agent.

13. A method for studying the function, morphology and/or differentiation status of dermal fibroblasts, comprising introducing the dermal fibroblasts to be studied into a three-dimensional gel-like biomatrix comprising a collagen solution containing at least 3.5 mg/ml up to 4.5 mg/ml of rat tail collagen in buffered serum-containing cell culture medium wherein the collagen solution has a high content of non-denatured native and non-lyophilized collagen of at least 90%, cultivating the dermal fibroblasts, and testing the dermal fibroblasts simultaneously and/or subsequently.