Method for decellularization of tissue grafts
View Patent ↗The subject invention pertains to materials and methods for processing tissue to produce a natural, acellular replacement tissue that is immunocompatible with a recipient. According to the subject invention, harvested tissue is subjected to wash solutions wherein only amphoteric detergent(s) are used (e.g., anionic detergents are excluded). Following extraction by amphoteric detergent(s), the tissue is washed with a buffer system to facilitate the clearance of cellular components and detergent. In one embodiment, the subject invention pertains to a replacement tissue that is a nerve graft that supports axonal regeneration, guides axons toward the distal nerve end and/or is immunologically tolerated. Preferably, the nerve graft retains essential extracellular matrix scaffolding as well as biological components that promote nerve regeneration through the nerve graft.
1. A method for decellularizing a tissue graft wherein said method comprises contacting a tissue graft with an extraction composition that comprises an amphoteric detergent at a concentration, and for a contact time, that is sufficient to rupture cells in the tissue, and wherein the method is performed in the absence of an anionic detergent.
2. The method, according to claim 1 , wherein after decellularization the extracellular matrix structures remain intact and preservation is measurably equal to or better than when decellularized by methods that include an anionic detergent.
3. The method, according to claim 1 , wherein no anionic detergent is applied to the tissue.
4. The method, according to claim 1 , wherein Triton X-200™ is not applied to the tissue.
5. The method, according to claim 1 , wherein the concentration of the amphoteric detergent(s) is at least the critical micelle concentration.
6. The method, according to claim 1 , wherein the extraction composition has physiologic, or greater, salinity.
7. The method, according to claim 1 , further comprising physically removing non-structural debris from the tissue graft.
8. The method, according to claim 1 , further comprising freezing the tissue graft either before or after decellurization.
9. The method, according to claim 1 , further comprising introducing into the tissue graft one or more bioactive molecules or cells.
10. The method, according to claim 1 , wherein the tissue graft's neurite-promoting activity is retained after decellularization compared to nerve tissue treated with buffer wash only.
11. The method, according to claim 1 , wherein after decellularization the tissue graft's neurite-promoting activity is retained and preservation is measurably equal to or better than when decellularized by methods that include an anionic detergent.
12. The method, according to claim 1 , wherein the amphoteric detergent is selected from the group consisting of sulfobetaine-10 (SB-10), sulfobetaine-16 (SB-16), and a combination thereof.
13. The method, according to claim 12 , wherein the tissue graft is contacted with a first extraction composition consisting of SB-10 and then contacted with a second extraction composition consisting of SB-16.
14. The method, according to claim 1 , further comprising at least one rinse step comprising contacting the tissue with a solution having less than physiologic salinity.
15. The method, according to claim 14 , wherein the rinse solution has no salinity.