Multilayer skin or dermal equivalent having a layer containing mesenchymal stem cells

• US 6,497,875 B1
• Filed: 10/26/1998
• Issued: 12/24/2002
• Est. Priority Date: 04/26/1996
• Status: Expired due to Term

• Current Assignee: Case Western Reserve University
• Original Assignee: Case Western Reserve University
• Inventors: Sorrell, J. Michael, Caplan, Arnold I.
• Primary Examiner(s): Naff, David M.
• Application Number:
  US09/171,445
• Time in Patent Office: 1,520 Days
• Field of Search: 424/93.7, 435/174, 435/325, 435/395, 435/366, 435/177, 435/1.1
• US Class Current: 424/93.7
• 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 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 27/3834 Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
• CPC Class Codes: A61L 27/3886 comprising two or more cell types
• CPC Class Codes: A61L 27/3891 as distinct cell layers
• 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 2502/1358 Bone marrow mesenchymal stem cells (BM-MSC)
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 5/0656 Adult fibroblasts
• CPC Class Codes: C12N 5/0698 Skin equivalents

Abstract

A multilayer skin equivalent is formed having a scaffold layer containing dermis-forming cells, and a keratinocyte layer. The dermis-forming cells and keratinocytes are preferably autologous, and the dermis-forming cells can be human mesenchymal stem cells (MSCs), dermal fibroblasts (e.g., papillary or reticular dermal fibroblasts) or mixtures thereof. The scaffold is preferably type I collagen alone, or types I and II collagen in combination. Also formed is a multilayer skin equivalent having a scaffold layer containing a layer of extracellular matrix component containing papillary dermal fibroblasts in laminar relationship with a layer of extracellular matrix component containing reticular dermal fibroblasts, and a keratinocyte layer. A multilayer dermal equivalent is provided having a layer of extracellular matrix component containing papillary dermis-forming cells and a layer of extracellular matrix component containing reticular dermis-forming cells. In another embodiment, the dermal equivalent has a layer containing MSCs and a layer selected from a layer of extracellular matrix component containing papillary dermis-forming cells and a layer of extracellular matrix component containing reticular dermis-forming cells, and optionally a keratinocyte layer. In the skin and dermal equivalents, at least one layer may contain an agent that promotes adhesion or angiogenesis. Also present may be a bioactive agent that enhances proliferation, commitment or differentiation of mesenchymal stem cells into dermal components, either in vitro or in vivo. An injectionable composition is also provided containing dermis-forming cells and an extracellular matrix component in a pharmaceutically acceptable injectable carrier.

Claims

1. A multilayer skin equivalent consisting essentially of:

(i) a scaffold layer incorporated with dermis-forming cells consisting essentially of mesenchymal stem cells and

(ii) a keratinocyte layer.

2. The multilayer skin equivalent of claim 1 wherein the mesenchymal stem cells are human.

3. The multilayer skin equivalent of claim 1, wherein the mesenchymal stem cells are autologous.

4. The multilayer skin equivalent of claim 1 wherein the scaffold layer contains at least one extracellular matrix component.

5. The multilayer skin equivalent of claim 4 wherein the extracellular matrix component is selected from the group consisting of collagen, elastin, intercellular adhesion molecules, neural cell adhesion molecule, laminin, heparin binding growth factors, fibronectin, proteoglycans, tenascin, E-cahedrin, and fibrillin.

6. The multilayer skin equivalent of claim 1 wherein the mesenchymal stem cells are proliferated and further differentiated on the scaffold layer.

7. The multilayer skin equivalent of claim 1 wherein at least one of said layers further includes at least one pharmaceutical agent which promotes adhesion or angiogenesis of the skin equivalent.

8. The multilayer skin equivalent of claim 1 wherein the keratinocytes are obtained from an individual to be treated with the multilayer skin equivalent.

9. The multilayer skin equivalent of claim 1 wherein the keratinocytes are proliferated on the scaffold layer.

10. The multilayer skin equivalent of claim 1 wherein the keratinocytes are proliferated on the scaffold layer in Rheinwald Green medium.

11. A multilayer dermal equivalent consisting essentially of at least one dermis-forming layer consisting essentially of mesenchymal stem cells and a layer selected from the group consisting of

(i) a layer of at least one extracellular matrix component containing papillary dermis forming cells;

and

(ii) a layer of at least one extracellular matrix component containing reticular dermis forming cells.

12. The multilayer dermal equivalent of claim 11 wherein at least two layers are in laminar relationship.

13. The multilayer dermal equivalent of claim 11 wherein the extracellular matrix component is selected from the group consisting of collagen, elastin, intercellular adhesion molecules, neural cell adhesion molecule, laminin, heparin binding growth factors, fibronectin, proteoglycans, tenascin, E-cahedrin, and fibrillin.

14. The multilayer dermal equivalent of claim 11 further comprising a keratinocyte layer.

15. The multilayer dermal equivalent of claim 11 wherein at least one of said layers further includes a bioactive factor which enhances proliferation, commitment or differentiation of mesenchymal stem cells into dermal components, either in vitro or in vivo.

16. The multilayer dermal equivalent of claim 11 wherein at least one of said layers further includes at least one pharmaceutical agent which promotes adhesion or angiogenesis of the dermal skin equivalent.

17. The multilayer dermal equivalent of claim 11 wherein the papillary and reticular dermis forming cells are from the same individual.

18. The multilayer dermal equivalent of claim 11 wherein the papillary and reticular dermis forming cells are from an individual to whom the multilayer dermal equivalent is to be administered.