Skin-equivalent prepared by the use of punch biopsy

• US 4,604,346 A
• Filed: 10/09/1984
• Issued: 08/05/1986
• Est. Priority Date: 10/09/1984
• Status: Expired due to Term

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Dubertret, Louis, Bell, Eugene
• Primary Examiner(s): Rosen, Sam
• Application Number:
  US06/658,499
• Time in Patent Office: 665 Days
• Field of Search: 435/1, 435/240, 424/95
• US Class Current: 606/132
• CPC Class Codes: A61F 2310/00365 Proteins; Polypeptides; Degradation products thereof
• CPC Class Codes: A61L 27/24 Collagen
• CPC Class Codes: A61L 27/60 Materials for use in artificial skin

Abstract

An improvement in the preparation of living skin-equivalents from contracted hydrated collagen lattices having keratinocyte cells on the surface is disclosed herein. In this improvement, punch biopsies of skin are employed to provide a source of keratinocyte cells to the lattices.

Claims

1. A method of forming a skin-equivalent, comprising:

a. forming an acidic solution of collagen;

b. combining contractile cells and nutrient medium with said acidic solution of collagen;

c. raising the pH of said solution of collagen to a level sufficient to precipitate collagen fibrils into a hydrated collagen lattice containing said contractile cells;

d. incorporating one or more punch biopsies of skin into said hydrated collagen lattice; and

e. maintaining said hydrated collagen lattice containing said punch biopsies under conditions sufficient for the contractile cells to contract the hydrated collagen lattice into a dermal-equivalent and sufficient to allow keratinocyte cells from said punch biopsies to overgrow the surface of said dermal equivalent thereby producing a skin-equivalent.

2. A method of claim 1 wherein steps b and c are done simultaneously.

3. A skin-equivalent formed by the method of claims 1 or 2.

4. In a method of forming skin-equivalent, including the steps of:

(a) forming a hydrated collagen lattice incorporating a living cellular contractile agent therein;

(b) maintaining said lattice and said contractile agent under conditions sufficient for said agent to contract said collagen lattice to form living dermal-equivalent;

(c) overgrowing keratinocyte cells upon said dermal equivalent; and

(d) maintaining said dermal equivalent under conditions sufficient for growth of said living cellular contractile agent and said keratinocyte cells to thereby produce a skin-equivalent;

the improvement wherein keratinocyte cells are grown on the surface of said dermal-equivalent by incorporating one or more punch biopsies of skin into said dermal-equivalent and maintaining said dermal-equivalent with the punch biopsies therein under conditions sufficient for keratinocyte cells from said skin biopsies to overgrow the surface of said dermal-equivalent.

5. A method of treating a wound to or disease of the skin of a recipient, comprising:

a. forming a hydrated collagen lattice incorporating a living cellular contractile agent therein;

b. removing one or more punch biopsies of skin from a donor;

c. incorporating said punch biopsies removed from the donor into said hydrated collagen lattice;

d. maintaining the hydrated collagen lattice containing said punch biopsies under conditions sufficient for the contractile cells to contract the hydrated collagen lattice and sufficient to allow keratinocyte cells from said punch biopsies of skin to overgrow the surface of said hydrated collagen lattice thereby producing a skin-equivalent for said recipient; and

e. applying said skin-equivalent to the wound of said recipient.

6. A method of claim 5 wherein said recipient is a human being.

7. A method of claim 6 wherein said donor is a human being.

8. A method of claim 5 wherein said recipient and said donor comprise the same human being.

9. A method of claims 5 or 8 wherein said contractile agent comprises fibroblast cells.

10. A method of preparing skin-equivalent, comprising:

a. forming a hydrated collagen lattice incorporating a living cellular contractile agent therein;

b. maintaining the hydrated collagen lattice and said contractile agent under conditions sufficient for said agent to contract said collagen lattice to form living dermal-equivalent;

c. growing keratinocyte cells upon the surface of said dermal equivalent to form a skin-equivalent;

d. removing one or more punch biopsies from said skin-equivalent;

e. incorporating said punch biopsies into another hydrated collagen lattice containing a living cellular contractile agent;

f. maintaining said another hydrated collagen lattice containing a living cellular contractile agent and said punch biopsies under conditions sufficient for formation of another skin-equivalent therefrom; and

g. optionally repeating steps d-f to produce further skin-equivalents.