Methods and compositions for organ decellularization

• US 6,753,181 B2
• Filed: 03/05/2002
• Issued: 06/22/2004
• Est. Priority Date: 12/29/1999
• Status: Expired due to Term

• Current Assignee: Children's Medical Center Corporation
• Original Assignee: Children's Medical Center Corporation
• Inventors: Atala, Anthony
• Primary Examiner(s): Lankford, Jr., Leon B.
• Application Number:
  US10/091,665
• Publication Number:
  US 20020102727A1
• Time in Patent Office: 840 Days
• Field of Search: 435/376, 435/1.1, 435/379, 435/395, 530/536
• US Class Current: 435/376
• CPC Class Codes: A61L 2430/26 for kidney reconstruction
• CPC Class Codes: A61L 2430/40 Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
• CPC Class Codes: A61L 27/3604 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
• CPC Class Codes: A61L 27/3687 characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
• CPC Class Codes: A61L 27/3691 characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
• CPC Class Codes: A61L 27/38 containing added animal cells organs or tissue containing native cells A61L27/36

Abstract

The invention is directed to methods for producing a decellularized organ or part of an organ. A decellularized organ is produced using an isolated organ mechanically agitated to remove cellular membranes surrounding the isolated organ without destroying the interstitial structure of the organ. After the cellular membrane is removed, the isolated organ is exposed to a solubilizing fluid that extracts cellular material without dissolving the interstitial structure of the organ. A washing fluid is used to remove the solubilized components, leaving behind a decellularized organ.

Claims

1. A method for producing a decellularized organ scaffold comprising:

mechanically agitating an isolated untreated organ in membrane stripping fluid to disrupt cell membranes while maintaining the interstitial structure of the organ;

treating the isolated organ in a solubilizing fluid at a concentration effective to extract cellular material from the organ while maintaining the interstitial structure of the organ; and

washing the isolated organ in a washing fluid to remove cellular debris while maintaining the interstitial structure of the organ until the isolated organ is substantially free of cellular material, to thereby produce a decellularized organ scaffold.

2. The method of claim 1, further comprising equilibrating the decellularized organ scaffold in an equilibrating fluid.

3. The method of claim 2, further comprising drying the decellularized organ scaffold.

4. The method of claim 1, wherein the step of mechanically agitating the isolated organ comprises placing the isolated organ in a stirring vessel having a paddle which rotates at a speed ranging from about 50 revolutions per minute (rpm) to about 150 rpm.

5. The method of claim 1, wherein the step of mechanically agitating the isolated organ in membrane stripping fluid occurs in a non-detergent membrane stripping fluid.

6. The method of claim 5, wherein the step of mechanically agitating the isolated organ occurs in a non-detergent membrane stripping fluid selected from the group consisting of distilled water, physiological buffer and culture medium.

7. The method of claim 1, wherein the step of treating the isolated organ in the solubilizing fluid also occurs in a stirring vessel.

8. The method of claim 7, wherein the step of treating comprises using a solubilizing fluid that is an alkaline solution having a detergent.

9. The method of claim 8, wherein the step of treating comprises treating the isolated organ in an alkaline solution selected from the group consisting of sulphates, acetates, carbonates, bicarbonates and hydroxides, and a detergent selected from the group consisting of Triton X-100, Triton N-101, Triton X-114, Triton X-405, Triton X-705, and Triton DF-16, monolaurate (Tween 20), monopalmitate (Tween 40), monooleate (Tween 80), polyoxethylene-23-lauryl ether (Brij 35), polyoxyethylene ether W-1 (Polyox), sodium cholate, deoxycholates, CHAPS, saponin, n-Decyl β

-D-glucopuranoside, n-heptyl β

-D glucopyranoside, n-Octyl α

-D-glucopyranoside and Nonidet P-40.

10. The method of claim 9, wherein the step of treating comprises treating the isolated organ in an ammonium hydroxide solution having Triton X-100.

11. The method of claim 1, wherein the step washing the isolated organ also occurs in a stirring vessel.

12. The method of claim 1, wherein the step of washing comprises washing the isolated organ in a washing fluid selected from the group consisting of distilled water, physiological buffer and culture medium.

13. The method of claim 2, wherein the step of equilibrating comprises equilibrating the decellularized organ scaffold in an equilibrating fluid selected from the group consisting of distilled water, physiological buffer and culture medium.

14. A method for producing a decellularized blood vessel scaffold comprising:

mechanically agitating an isolated untreated blood vessel in membrane stripping fluid to disrupt the outer part of the blood vessel while maintaining the interstitial structure of the blood vessel;

treating the isolated blood vessel in a solubilizing fluid at a concentration effective to extract cellular material from the blood vessel while maintaining the interstitial structure of the blood vessel; and

washing the isolated blood vessel in a washing fluid to remove cellular debris while maintaining the interstitial structure of the blood vessel until the isolated blood vessel is substantially free of cellular material, to thereby produce a decellularized blood vessel scaffold.

15. The method of claim 14, further comprising equilibrating the decellularized blood vessel scaffold in an equilibrating fluid.

16. The method of claim 15, further comprising drying the decellularized blood vessel scaffold.

17. The method of claim 14, wherein the step of mechanically agitating the isolated blood vessel comprises placing the isolated blood vessel in a stirring vessel having a paddle which rotates at a speed ranging from about 50 revolutions per minute (rpm) to about 150 rpm.

18. The method of claim 14, wherein the step of mechanically agitating the isolated blood vessel in membrane stripping fluid occurs in a non-detergent membrane stripping fluid.

19. The method of claim 18, wherein the step of mechanically agitating the isolated blood vessel occurs in a non-detergent membrane stripping fluid selected from the group consisting of distilled water, physiological buffer and culture medium.

20. The method of claim 14, wherein the step of treating the isolated blood vessel in the solubilizing fluid also occurs in a stirring vessel.

21. The method of claim 20, wherein the step of treating comprises using a solubilizing fluid that is an alkaline solution having a detergent.

22. The method of claim 21, wherein the step of treating comprises treating the isolated blood vessel in an alkaline solution selected from the group consisting of sulphates, acetates, carbonates, bicarbonates and hydroxides, and a detergent selected from the group consisting of Triton X-100, Triton N-101, Triton X-114, Triton X-405, Triton X-705, and Triton DF-16, monolaurate (Tween 20), monopalmitate (Tween 40), monooleate (Tween 80), polyoxethylene-23-lauryl ether (Brij 35), polyoxyethylene ether W-1 (Polyox), sodium cholate, deoxycholates, CHAPS, saponin, n-Decyl β

-D-glucopuranoside, n-heptyl β

-D glucopyranoside, n-Octyl α

-D-glucopyranoside and Nonidet P-40.

23. The method of claim 22, wherein the step of treating comprises treating the isolated organ in an ammonium hydroxide solution having Triton X-100.

24. The method of claim 14, wherein the step of washing the isolated blood vessel also occurs in a stirring vessel.

25. The method of claim 14, wherein the step of washing comprises washing the isolated blood vessel in a washing fluid selected from the group consisting of distilled water, physiological buffer and culture medium.

26. The method of claim 15, wherein the step of equilibrating comprises equilibrating the decellularized blood vessel scaffold in an equilibrating fluid selected from the group consisting of distilled water, physiological buffer and culture medium.