Materials and methods for nerve grafting, selection of nerve grafts, and in vitro nerve tissue culture
View Patent ↗The subject invention pertains to compositions and methods for culturing nerve tissue in vitro and nerve grafts produced using such methods. The compositions and methods of the subject invention can be employed to restore the continuity of nerve interrupted by disease, traumatic events or surgical procedures. The invention also concerns methods for promoting repair of damaged nerve tissue using the present compositions and nerve tissue treated according to such methods.
1. A method for preparing a nerve graft suitable for subsequent implantation, the method comprising:
degrading, by in vitro culturing, chondroitin sulfate proteoglycan of a nerve graft comprising a nerve tissue segment while maintaining an intact basal lamina tube structure of the nerve graft, thereby enhancing post-implantation axonal traversal of an interface between the nerve graft and host nerve tissue relative to a nerve graft in which chondroitin sulfate proteoglycan was not degraded; and
rendering the nerve graft acellular by killing cells in the nerve graft.
2. The method according to claim 1 , wherein said culturing of the nerve graft in vitro is for a period of time that achieves an increase in post-implantation axon ingress and extent of growth within the nerve graft relative to the nerve graft in which chondroitin sulfate proteoglycan was not degraded.
3. The method according to claim 1 , wherein said culturing of the nerve graft in vitro is for a period of time within the range of about 24 hours to about 96 hours.
4. The method according to claim 1 , wherein said culturing of the nerve graft in vitro is for a period of time within the range of about 24 hours to about 72 hours.
5. The method according to claim 1 , wherein said culturing of the nerve graft in vitro is for a period of time of about 48 hours.
6. The method according to claim 1 , wherein said culturing of the nerve graft in vitro is conducted at a temperature within the range of about 10° C. to about 37° C.
7. The method according to claim 1 , wherein said culturing of the nerve graft in vitro is conducted at a temperature within the range of about 30° C. to about 37° C.
8. The method according to claim 1 , wherein said culturing of the nerve graft in vitro is conducted at a temperature of about 37° C.
9. The method according to claim 1 , wherein the nerve graft is an explant.
10. The method according to claim 1 , wherein the nerve graft is mammalian tissue.
11. The method according to claim 1 , wherein the nerve graft is mammalian tissue selected from the group consisting of human tissue, non-human primate tissue, porcine tissue, rodent tissue, and bovine tissue.
12. The method according to claim 1 , wherein the nerve graft is human tissue.
13. The method according to claim 1 , wherein the nerve graft is an autograft.
14. The method according to claim 1 , wherein the nerve graft is an allograft.
15. The method according to claim 1 , wherein the nerve graft is a xenograft.
16. The method according to claim 1 , wherein rendering the nerve graft acellular by killing cells in the nerve graft occurs after culturing.
17. The method according to claim 1 , wherein rendering the nerve graft acellular by killing cells in the nerve graft comprises a process selected from the group consisting of freeze-killing and chemical treatment.
18. The method according to claim 1 , wherein said method further comprises freezing the nerve graft for storage.
19. The method according to claim 18 , wherein said freezing is carried out after said culturing in vitro.
20. The method according to claim 1 , wherein the nerve graft comprises peripheral nerve tissue.
21. The method according to claim 1 , wherein said culturing comprises placing the nerve graft in contact with culture medium.
22. The method according to claim 21 , wherein the culture medium comprises a defined medium.
23. The method according to claim 21 , wherein the culture medium comprises a defined medium supplemented with serum.
24. The method according to claim 21 , wherein the culture medium comprises undefined medium.
25. The method according to claim 21 , wherein the culture medium comprises dulbecco's modified eagles' medium.
26. The method according to claim 1 , wherein said method further comprises isolating the nerve graft from a mammal prior to said culturing of the nerve graft in vitro.
27. The method according to claim 1 , wherein said method further comprises applying a tissue adhesive to the nerve graft.
28. A method for enhancing the regenerative potential of a nerve graft suitable for subsequent implantation, the method comprising:
degrading, by in vitro culturing, chondroitin sulfate proteoglycan of a nerve graft comprising a nerve tissue segment while maintaining an intact basal lamina tube structure of the nerve graft, thereby enhancing post-implantation axonal traversal of an interface between the nerve graft and host nerve tissue relative to a nerve graft in which chondroitin sulfate proteoglycan was not degraded, wherein culturing conditions comprise a temperature within the range of about 10° C. to about 37° C. for a period of time within the range of about 24 hours to about 96 hours; and
rendering the nerve graft acellular by killing cells in the nerve graft.
29. The method according to claim 28 , wherein said culturing of the nerve graft in vitro is for a period of time within the range of about 24 hours to about 72 hours.
30. The method according to claim 28 , wherein said culturing of the nerve graft in vitro is for a period of time of about 48 hours.
31. The method according to claim 28 , wherein said culturing of the nerve graft in vitro is conducted at a temperature within the range of about 30° C. to about 37° C.
32. The method according to claim 28 , wherein said culturing of the nerve graft in vitro is conducted at a temperature of about 37° C.
33. The method according to claim 28 , wherein said culturing comprises placing the nerve graft in contact with culture medium.
34. The method according to claim 33 , wherein the culture medium comprises defined medium.
35. The method according to claim 33 , wherein the culture medium comprises defined medium supplemented with serum.
36. The method according to claim 33 , wherein the culture medium comprises undefined medium.
37. The method according to claim 28 , wherein rendering the nerve graft acellular by killing cells in the nerve graft occurs after culturing.
38. The method according to claim 28 , wherein rendering the nerve graft acellular by killing cells in the nerve graft comprises a process selected from the group consisting of freeze-killing and chemical treatment.
39. The method according to claim 28 , wherein the nerve graft is mammalian tissue.
40. The method according to claim 28 , wherein the nerve graft is mammalian tissue selected from the group consisting of human tissue, non-human primate tissue, porcine tissue, rodent tissue, and bovine tissue.
41. The method according to claim 28 , wherein the nerve graft is human tissue.
42. The method according to claim 28 , wherein the nerve graft comprises peripheral nerve tissue.
43. The method according to claim 28 , wherein the nerve graft is an autograft.
44. The method according to claim 28 , wherein the nerve graft is an allograft.
45. The method according to claim 28 , wherein the nerve graft is a xenograft.
46. The method according to claim 1 , wherein the nerve graft comprises central nervous system tissue.
47. The method according to claim 28 , wherein the nerve graft comprises central nervous system tissue.
48. The method according to claim 28 , wherein the nerve graft is an explant.
49. The method according to claim 28 , wherein said method further comprises freezing the nerve graft for storage.
50. The method according to claim 49 , wherein said freezing is carried out after said culturing in vitro.
51. The method according to claim 28 , wherein said method further comprises isolating the nerve graft from a mammal prior to said culturing of the nerve graft in vitro.
52. The method according to claim 28 , wherein said method further comprises applying a tissue adhesive to the nerve graft.