Compositions and methods for generating skin

• US 7,560,275 B2
• Filed: 03/03/2003
• Issued: 07/14/2009
• Est. Priority Date: 12/30/1999
• Status: Active Grant

• Current Assignee: VCellTherapeutics, Inc.
• Original Assignee: Vbi Technologies, LLC
• Inventors: Vacanti, Martin P., Vacanti, Charles A.
• Primary Examiner(s): DRISCOLL, LORA E BARNHART
• Application Number:
  US10/379,394
• Publication Number:
  US 20040033598A1
• Time in Patent Office: 2,325 Days
• Field of Search: 435/243
• US Class Current: 435/325
• 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: C12N 5/0602 Vertebrate cells

Abstract

Compositions and methods for growing new skin, which can be used to repair or replace skin that has been damaged or removed by a variety of insults.

Claims

1. A population of isolated mammalian or avian multipotent spore-like cells (MSLCs) isolated or cultured from skin tissue and which can differentiate into two or more mature skin cell type cells.

wherein the MSLCs, when first isolated, are approximately one to ten microns in diameter,

wherein the MSLCs are viable following essentially complete oxygen-deprivation for twenty four hours or more,

wherein the MSLCs are surrounded by an outer membrane that is rich in glycolipids and mucopolysaccharides so that the MSLCs appear to have one or more dark stripes when viewed by transmission electron microscopy, and

wherein nucleic acids account for at least about 50% of the volume of each spore-like cell within the population.

2. The MSLC population of claim 1, wherein the cells have an outer coating containing sufficient glycolipids and mucopolysaccharides that the cell appears to have one or more dark stripes when viewed by transmission electron microscopy.

3. The MSLC population of claim 1, wherein the cells are isolated from the skin of a post-natal mammal.

4. The MSLC population of claim 1, wherein the cells are isolated from a culture of spore-like cells isolated from skin by repeated disaggregation of the skin.

5. The MSLC population of claim 1, wherein the cells fail to express nestin.

6. The MSLC population of claim 1, wherein the cells fail to express keratin, tyrosinase, glutamic acid, glutamate, citrulline, tricohyaline, filagrin, epinephrine, norepinephrine, acetylcholine, or melanin granules.

7. MSLC population of claim 1, wherein the cells are isolated from the dermis of a post-natal mammal and, when placed in the vicinity of a skin wound in an animal, expresses a phenotype substantially similar to that of a sympathetic or parasympathetic neuron, or a cell within a sweat gland, a sebaceous gland, or a hair follicle.

8. The MSLC population of claim 1, wherein the cells are isolated from the epidermis of a post-natal mammal and, when placed in the vicinity of a skin wound in an animal, expresses a phenotype substantially similar to that of a melanocyte, a keratinocyte, or a Merkel cell.

17. A skin construct comprising the MSLC population of claim 1.

18. The skin construct of claim 17, further comprising a support structure.

19. The skin construct of claim 18, further comprising hydrogel, wherein the hydrogel and the MSLC population of claim 1 form a hydrogel-MSLC suspension.

20. A method for generating artificial skin, the method comprising combining hydrogel with the MSLC population of claim 1, to form a hydrogel-MSLC suspension.

21. The method of claim 20, further comprising delivering the hydrogel-cell construct into a permeable, biocompatible support structure.

22. A method of treating damaged skin, the method comprising obtaining a hydrogel-MSLC suspension of claim 19 and applying the suspension to the damaged skin or to an area lying under the damaged skin.

9. A method for isolating a multipotent spore-like cell (MSLC population from a post-natal mammal, the method comprising

passing a piece of skin obtained from the mammal through a series of devices having progressively smaller apertures to isolate a population of MSLCs,

wherein the MSLCs, when first isolated, are approximately one to ten microns in diameter;

and are viable following essentially complete oxygen-deprivation for twenty four hours or more, surrounded by an outer membrane that is rich in glycolipids and mucopolysaccharides, and wherein nucleic acids account for at least about 50% of the volume of each spore-like cell within the population, and wherein the MSLC can differentiate into two or more mature skin cell types.

10. The method of claim 9, wherein the device is a pipette.

11. The method of claim 10, wherein the smallest pipette in the series has an inner bore diameter of approximately 15 microns.

12. The method of claim 9, wherein the device is a filter.

13. The method of claim 9, wherein the filter with the smallest apertures in the series has a pore size of approximately 15 microns.

14. The method of claim 9 wherein the skin is exposed to a digestive enzyme.

15. The method of claim 14, wherein the digestive enzyme is trypsin or collagenase.

16. The method of claim 15, wherein the skin is incubated with approximately 0.05% trypsin at 37°

C. for approximately five minutes.

23. The MSLC population isolated by the method of claim 9.