Decellularisation of tissue matrices for bladder implantation
The invention provides an improved method of producing a natural, acellular matrix scaffold for subsequent use in tissue-engineered replacement of tissues such as the bladder. Decellularization is carried out on an expanded or distended bladder and the product retains the strength and compliance of natural material. The invention also provides use of the matrix scaffolds as wound healing material and to investigate tissue structure and function in vitro.
1. A method of decellularisation of an intact bladder, the method comprising:
immersing the bladder in a buffer solution at a mild alkaline pH which includes a proteolytic inhibitor;
(ii) expanding the bladder by introducing a sufficient volume of the same buffer solution into an interior cavity of the bladder so as to stretch and thin the bladder wall; and
(iii) continuing decellularisation of the bladder by replacing and introducing fresh solutions both around the exterior surface of the bladder and into the bladder interior itself so as to maintain expansion of the bladder during decellularisation and to maintain the histoarchitecture of the bladder material.
2. The method of claim 1 , wherein step (ii) is performed prior to step (i).
3. The method of claim 1 , wherein the bladder is clamped before and after each step of fluid replacement so as to prevent fluid loss and to maintain expansion of the bladder.
4. The method of claim 1 , wherein the fluid volume added to the interior cavity of the bladder at each step is between 250 ml and 750 ml.
5. The method of claim 1 , wherein the fluid volume added to the interior cavity of the bladder at each step is about 500 ml.
6. The method of claim 1 , wherein the fluid volume around the exterior of the bladder is sufficient to cover or immerse the whole of the expanded fluid filled bladder.
7. The method of claim 1 , wherein once the process of decellularisation is complete, the bladder is either dissected to form a flattened sheet or remains intact.
8. The method of claim 1 , wherein the intact bladder is a whole bladder.
9. The method of claim 1 , wherein the bladder is derived from a pig or a human.
10. The method of claim 1 , wherein the step of continuing decellularisation comprises:
(i) removing said buffer at a mild alkaline pH which comprises a proteolytic inhibitor from both the interior cavity of the bladder and the surrounding exterior area and replacing it with an anionic detergent at a mild alkaline pH at a concentration which is sufficient to effect decellularisation but which maintains the histoarchitecture of the biological material;
(ii) removing said detergent from the interior of the bladder and its exterior and replacing it with a washing buffer solution at a mild alkaline pH both with and without proteolytic inhibitors so as to wash both the interior and/or exterior surfaces of the bladder;
(iii) removing said washing buffer from both the interior of the bladder and its exterior and replacing it with a solution comprising one or more enzymes selected from the group consisting of DNase Type I, DNase Type II, and RNase; and
(iv) optionally removing the solution comprising one or more enzymes from the interior of the bladder and its exterior and optionally placing the biological material in a cryoprotectant medium or storage medium for later use.
11. The method of claim 1 further including modifying the decellularised bladder with a chemical or enzymic agent to enhance immunoacceptability of the bladder.
12. The method of claim 11 , wherein the enzymic agent is an α-galactosidase or a glycosidase digestion to remove α-gal epitopes (Gal α1-3 Gal β1-4Glc NAC-R).
13. The method of claim 11 , wherein the chemical agent is a cross-linking agent.
14. The method of claim 1 further including the step of recellularisation.
15. The method of claim 14 , wherein recellularisation is enhanced by addition of a suitable agent administered either in vitro or in vivo.
16. The method of claim 15 , wherein the agent is coated directly onto the decellularised bladder prior to implantation to provide recellularisation.
17. The method of claim 1 , wherein the proteolytic inhibitor is ethylene diamine tetraacetic acid (EDTA) in an amount from 1 mM to 100 mM or 0.01 to 1.0% w/v.
18. The method of claim 1 , wherein the proteolytic inhibitor is Aprotinin in an amount of from 1KIU to 100KIU.
19. The method of claim 10 , wherein the proteolytic inhibitor is ethylene diamine tetraacetic acid (EDTA) in an amount from 1 mM to 100 mM or 0.01 to 1.0% w/v.
20. The method of claim 10 , wherein the proteolytic inhibitor is Aprotinin in an amount of from 1 KIU to 100 KIU.