SOLUTIONS FOR TISSUE ENGINEERING AND METHODS OF USE

• US 20100035344A1
• Filed: 06/11/2009
• Published: 02/11/2010
• Est. Priority Date: 06/11/2008
• Status: Active Grant

• Current Assignee: Children's Mercy Hospital
• Original Assignee: Children's Mercy Hospital
• Inventors: Hopkins, Richard
• Granted Patent: US 9,682,173 B2
• Time in Patent Office: Days
• US Class Current: 435/395
• 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 27/3695 characterised by the function or physical properties of the final product, where no specific conditions are defined to achieve this A61L27/3687, A61L27/3691 take precedence
• CPC Class Codes: C12N 2521/00 Culture process characterised by the use of hydrostatic pressure, flow or shear forces
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06

Abstract

The present invention provides for solutions and methods of preparing a decellularized tissue for recellularization. The solutions provide collagen conditioning to restore collagen triple helix structure, strengthening of the collagen structure of the tissue, and biologically preparing the decellularized tissue by placing it in an environment that promotes recellularization. Methods and solutions for recellularization of a decellularized tissue, in accordance with the present invention, are also provided.

Claims

1. A method of preparing a decellularized tissue for recellularization comprising the steps of conditioning the collagen in said decellularized tissue and biologically preparing said decellularized tissue by placing said decellularized tissue in an environment that promotes the recellularization of said tissue.

2. The method of claim 1, wherein said collagen conditioning comprises the steps of

(a) contacting said tissue with a solution comprising saline and a sugar;

and

(b) contacting said tissue with a solution comprising one or more alcohols, lubricants, and ECM proteins.

3. The method of claim 2, wherein said sugar is a sugar alcohol.

4. The method of claim 2, wherein said one or more alcohols are selected from the group of solvent alcohols, fatty alcohols, and combinations thereof.

5. The method of claim 2, further comprising the step of contacting said tissue with a surfactant.

6. The method of claim 2, further comprising the step of contacting said tissue with a lubricant or surfactant having lubricant properties.

7. The method of claim 6, wherein said lubricant or surfactant having lubricant properties is selected from the group consisting of hyaluronan, oils, non-sulfated GAGs, fibronectin, chondroiton SO₄, heparin SO₄, keratin SO₄, proteoglycans, decorin, serglycin, syndeum, aggrecon, heparin, and combinations thereof.

8. The method of claim 1, wherein said method further comprises a structural strengthening step.

9. The method of claim 8, wherein said structural strengthening step comprises the steps of:

(a) contacting said tissue with a collagen or elastin cross-linking agent; and

(b) contacting said tissue with saline and an anti-mineralization agent.

10. The method of claim 9, wherein said contacting with a collagen or elastin cross-linking agent step further comprises contacting said tissue with H₂O.

11. The method of claim 1, wherein said biological environment comprises at least 7 components selected from the group consisting of traditional cell culture media, serum, insulin, growth promoters, myofibroblast phenotype maintenance agents, L-glutamine, buffer, heparin, steroid-like compounds, Chondroitin sulfate, ethyl pyrubate, ascorbic acid, ascorbate, collagen cross-linking agent, co-enzyme, and antibiotic agents.

13. A solution for the treatment of tissue comprising ethyl alcohol, lauryl alcohol, myristyl alcohol, and hyaluronan HMW salt.

14. The solution of claim 13, further comprising TWEEN 20.

15. The solution of claim 13, further comprising glycerol.

16. A solution for the treatment of tissue comprising one or more solvent alcohols, one or more fatty alcohols, one or more lubricants, one or more ECM protein restoration agents, and combinations thereof.

22. A method of preparing a tissue for engineering applications comprising treatment with the solution of claim 16 and the solution of claim 17.

23. The method of claim 22, further comprising contacting said tissue with citric acid.

17. A solution for the treatment of tissue comprising:

(a) A serum solution comprising serum, cell culture medium, insulin, and buffer;

(b) A growth factor solution comprising one or more growth factors and at least one myofibroblast phenotype maintenance agent;

(c) A nutrient solution to accelerate cell seeding comprising steroid-like compounds, a collagen cross-linking agent; and

(d) At least one antibiotic.

12. The method of claim 30, wherein said biological preparation step comprises protein insudation.

18. The solution of claim 17, wherein said serum solution comprises serum specific to the donor or recipient species, DMEM+F12, Insulin, BFGF, L-glutamine, Hepes buffer, and heparin.

19. The solution of claim 17, wherein said growth factor solution comprises Gensenoside Rg1, FGF-2, Co-enzyme Q10, and TGF-B1.

20. The solution of claim 17, wherein said nutrient solution comprises Chondroitin SO₄, Ethyl pyruvate, Sphenogosine-1-phosphate, Ascorbic acid-2-phosphate and Ascorbate.

21. The solution of claim 17, wherein said antibiotic mixture comprises amphoterian-B, vancomycin, and gentamicin.

29. The method of claim 24, wherein said cell differentiation and migration phase comprises the following steps:

(a) Placing a decellularized tissue in a bioreactor with bone marrow cells;

(b) adding the solution of claim 17;

(c) creating a hyperoxic environment within said bioreactor;

(d) introducing a mechanical environment; and

(e) allowing said decellularized tissue to incubate.

30. The method of claim 29, wherein said mechanical environment comprises cyclic pressure and cyclic flow.

24. A method of reseeding a decellularized tissue comprising the steps of

(a) A seeding phase;

and

(b) A cell differentiation and migration phase.

25. The method of claim 24, wherein said seeding phase comprises

(a) placing a decellularized tissue into a bioreactor;

(b) adding mesenchymal stem cell proliferating media into said bioreactor;

(c) adding bone marrow from said decellularized tissue recipient;

to said bioreactor and

(d) allowing said bioreactor to incubate.

26. The method of claim 25, wherein said bioreactor is incubated for 4-6 days.

27. The method of claim 25, wherein a hypoxic environment is created within said bioreactor.

28. The method of claim 25, wherein said bioreactor is coated with a non-adherent surface.

31. The method of claim 24, further comprising a cell proliferation phase.

32. The method of claim 31, wherein said cell proliferation phase comprises the steps of

(a) Harvesting bone marrow from a patient receiving said decellularized tissue;

(b) Placing said bone marrow into a bioreactor;

(c) Adding mesenchymal stem cell proliferating media;

(d) Allowing the cells to incubate in said bioreactor.

33. The method of claim 32, wherein a hypoxic environment is created within said bioreactor.

34. The method of claim 32, wherein said bioreactor is coated with a non-adherent surface.

35. The method of claim 32, further comprising nutrients to supplement said mesenchymal stem cell proliferating media.

36. The method according to claim 32, wherein 1 to 3 said bioreactors are utilized.