Method for chemically acellularizing a biological tissue sample

• US 6,448,076 B2
• Filed: 06/29/2001
• Issued: 09/10/2002
• Est. Priority Date: 09/15/1998
• Status: Expired due to Term

• Current Assignee: Board of Regents of the University of Michigan (University of Michigan)
• Original Assignee: Board of Regents of the University of Michigan (University of Michigan)
• Inventors: Cederna, Paul S., Kuzon, William M. Jr., Dennis, Robert G.
• Primary Examiner(s): Lankford, Jr., Leon B.
• Application Number:
  US09/896,651
• Publication Number:
  US 20010049138A1
• Time in Patent Office: 438 Days
• Field of Search: 435/325, 435/368, 435/395
• 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 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 2533/92 Amnion; Decellularised dermis or mucosa
• CPC Class Codes: C12N 5/0658 Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts

Abstract

A method for chemically acellularizing a biological tissue sample, such as a peripheral nerve, is provided. The method includes disrupting the cell membranes of the biological tissue sample, and then denaturing intracellular proteins within the cells of the tissue sample and removing the denatured proteins from the cells while preserving the extracellular matrix to produce an acellularized tissue construct.

Claims

1. A method for chemically acellularizing a peripheral nerve sample to produce an acellularized peripheral nerve construct, the method comprising:

disrupting the cell membranes of the peripheral nerve sample by submersing the sample in a solution including glycerol; and

denaturing intracellular proteins within the cells of the sample and removing the denatured proteins from the cells while preserving the extracellular matrix to produce the acellularized peripheral nerve construct.

2. The method according to claim 1, wherein denaturing and removing intracellular proteins includes submersing the biological tissue in at least one detergent solution.

3. The method according to claim 2, wherein the at least one detergent solution includes sodium deoxycholate.

4. The method according to claim 3, wherein the at least one detergent includes sodium dodecyl sulfate.

5. The method according to claim 2, wherein the at least one detergent solution includes TRITON®

X-100.

6. The method according to claim 1, further including harvesting the tissue sample from a suitable donor.

7. The method according to claim 1, further including submersing the tissue sample in a balanced salt solution prior to disrupting the cell membranes.

8. The method according to claim 7, wherein the balanced salt solution includes Dulbecco'"'"'s phosphate buffered saline.

9. The method according to claim 1, further including storing the acellularized peripheral nerve construct in a physiologic saline solution.

10. The method according to claim 1, further including implanting the acellularized peripheral nerve construct in a suitable recipient.

11. A method for producing a chemically acellularized peripheral nerve construct, the method comprising:

harvesting a peripheral nerve sample from a donor;

submersing the peripheral nerve sample in a glycerol solution for disrupting the cell membranes of the peripheral nerve; and

submersing the peripheral nerve sample in at least one detergent solution for denaturing the intracellular proteins of the cells of the peripheral nerve and removing the denatured proteins while leaving the extracellular matrix intact to produce the acellularized peripheral nerve construct without using mechanical agents.

12. The method according to claim 11, wherein the at least one detergent solution includes a nonionic detergent.

13. The method according to claim 12, wherein the nonionic detergent includes TRITON®

X-100.

14. The method according to claim 11, wherein the at least one detergent solution includes an ionic detergent.

15. The method according to claim 14, wherein the ionic detergent includes sodium deoxycholate.

16. The method according to claim 14, wherein the ionic detergent includes sodium dodecyl sulfate.

17. The method according to claim 11, wherein submersing the peripheral nerve sample in at least one detergent solution includes submersing the sample in a succession of solutions including at least one ionic detergent solution and at least one nonionic detergent solution.

18. The method according to claim 17, wherein the succession of solutions includes a sodium deoxycholate solution, a sodium dodecyl sulfate solution, and a TRITON®

X-100 solution.

19. The method according to claim 11, further including rinsing the peripheral nerve sample with distilled water between each solution change.

20. A method for producing a chemically acellularized peripheral nerve construct, comprising:

harvesting a peripheral nerve segment from a donor;

disrupting the cell membranes and removing lipid-soluble cell structures of the peripheral nerve segment using a glycerol solution;

denaturing intracellular proteins within the cells of the peripheral nerve segment using at least one ionic detergent; and

removing the denatured proteins from the cells while leaving the extracellular matrix intact using ionic and nonionic detergents to produce the acellularized peripheral nerve construct without using mechanical agents.

21. The method according to claim 20, further comprising grafting the acellularized peripheral nerve construct within a recipient nerve.