Method of providing a substrate with a ready-to-use, uniformly distributed extracellular matrix

• US 7,541,187 B2
• Filed: 06/06/2002
• Issued: 06/02/2009
• Est. Priority Date: 06/06/2001
• Status: Expired due to Term

• Current Assignee: Corning Incorporated
• Original Assignee: Becton, Dickinson & Co
• Inventors: Ilsley, Stephen R., Myles, Arthur, Mannuzza, Frank J.
• Primary Examiner(s): Weber; Jon P
• Assistant Examiner(s): Srivastava; Kailash C
• Application Number:
  US10/164,486
• Publication Number:
  US 20020197718A1
• Time in Patent Office: 2,553 Days
• Field of Search: 435/70.1, 435/366, 435/368, 435/371, 435/395
• US Class Current: 435/395
• CPC Class Codes: C12M 25/14 Scaffolds; Matrices in general C12N5/0068
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06

Abstract

Method of providing a substrate with a ready-to-use uniformly distributed extracellular matrix is disclosed. This method includes applying extracellular matrix components to a substrate area; incubating the extracellular matrix components to allow polymerization thereof; freezing the polymerized extracellular matrix on the substrate area; lyophilizing the frozen extracellular matrix on the substrate area; and allowing the lyophilized extracellular matrix to warm to room temperature. Also contemplated is a cell culture apparatus having a dried uniformly distributed extracellular matrix formed by the above-mentioned method.

Claims

1. A method of preparing a cell culture substrate with a uniformly distributed extracellular matrix comprising:

a) raising the pH of an acidic solution of an extracellular matrix comprising collagen fibrils to 6-8 by the addition of a base;

b) applying the solution of the extracellular matrix of step a) to a porous substrate area;

c) polymerizing said applied extracellular matrix on said porous substrate area, wherein said polymerizing comprises incubating said solution at 15°

C. to 40°

C.;

d) freezing said polymerized extracellular matrix on said porous substrate area;

e) lyophilizing said frozen extracellular matrix on said porous substrate area, thereby obtaining a cell culture substrate with a uniformly distributed extracellular matrix.

2. The method according to claim 1, wherein said polymerizing comprises incubating said applied solution of extracellular matrix for 0.5 to 3 hours in the presence of carbon dioxide.

3. The method according to claim 1, wherein said polymerized extracellular matrix is frozen at a temperature no warmer than −

30°

C.

4. The method according to claim 1, wherein said extracellular matrix further comprises a component selected from the group consisting of laminin, entactin, heparan sulfate proteoglycan, growth factors and combinations thereof.

5. The method according to claim 1, wherein said porous substrate area is a membrane material selected from the group consisting of porous membrane, etched membrane, cast membranes and combinations thereof.

6. The method of claim 1, wherein said porous substrate area includes natural or synthetic polymers selected from the group consisting of cellulose membranes, porous polycarbonate, porous polytetrafluoroethylene, nylon membranes and meshes, glass filters, porous polyethyleneterephthalate, polymethylpentane, polypropylene, polyethylene and combinations thereof.

7. The method according to claim 2, wherein said polymerizing comprises incubating said applied solution of extracellular matrix at a temperature of about 37°

C.

8. The method according to claim 2, wherein said carbon dioxide concentration is 5%.

9. The method according to claim 5, wherein said membrane material has pores from about 0.5 to about 30 microns.