Method for chemically acellularizing a biological tissue sample

• US 20010049138A1
• Filed: 06/29/2001
• Published: 12/06/2001
• Est. Priority Date: 09/15/1998
• Status: Active Grant

• 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.
• Granted Patent: US 6,448,076 B2
• Time in Patent Office: Days
• 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 biological tissue sample, the method comprising:

disrupting the cell membranes of the biological tissue sample; and

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.

2. The method according to

claim 1

, wherein disrupting the cell membrane includes submersing the biological tissue sample in a solution including glycerol.

3. The method according to

claim 1

, wherein denaturing and removing intracellular proteins includes submersing the biological tissue in at least one detergent solution.

4. The method according to

claim 3

, wherein the at least one detergent solution includes sodium deoxycholate.

5. The method according to

claim 3

, wherein the at least one detergent includes sodium dodecyl sulfate.

6. The method according to

claim 3

, wherein the at least one detergent solution includes TRITON®

X-100.

7. The method according to

claim 1

, further including harvesting the tissue sample from a suitable donor.

8. The method according to

claim 1

, further including submersing the tissue sample in a balanced salt solution prior to disrupting the cell membranes.

9. The method according to

claim 8

, wherein the balanced salt solution includes Dulbecco'"'"'s phosphate buffered saline.

10. The method according to

claim 1

, further including storing the acellularized tissue construct in a physiologic saline solution.

11. The method according to

claim 1

, further including implanting the acellularized tissue construct in a suitable recipient.

12. A method for producing a chemically acellularized tissue construct, the method comprising:

harvesting a biological tissue sample from a donor;

submersing the biological tissue sample in a glycerol solution for disrupting the cell membranes of the tissue; and

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

13. The method according to

claim 12

, wherein the at least one detergent solution includes a nonionic detergent.

14. The method according to

claim 13

, wherein the nonionic detergent includes TRITON®

100.

15. The method according to

claim 12

, wherein the at least one detergent solution includes an ionic detergent.

16. The method according to

claim 15

, wherein the ionic detergent includes sodium deoxycholate.

17. The method according to

claim 15

, wherein the ionic detergent includes sodium dodecyl sulfate.

18. The method according to

claim 12

, wherein submersing the biological tissue 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.

19. The method according to

claim 18

, wherein the succession of solutions includes a sodium deoxycholate solution, a sodium dodecyl sulfate solution, and a TRITON®

X-100 solution.

20. The method according to

claim 12

, further including rinsing the biological tissue sample with distilled water between each solution change.

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

harvesting a peripheral nerve segment from a donor;

disrupting the cell membranes of the peripheral nerve segment;

denaturing intracellular proteins within the cells of the peripheral nerve segment; and

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

22. The method according to

claim 21

, further comprising grafting the peripheral nerve construct within a recipient nerve.