CN1115957C - Method of preparing blood vessel graft - Google Patents
Method of preparing blood vessel graft Download PDFInfo
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- CN1115957C CN1115957C CN00120209A CN00120209A CN1115957C CN 1115957 C CN1115957 C CN 1115957C CN 00120209 A CN00120209 A CN 00120209A CN 00120209 A CN00120209 A CN 00120209A CN 1115957 C CN1115957 C CN 1115957C
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- described method
- tissue
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- alkaline solution
- animal
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- 238000000034 method Methods 0.000 title claims abstract description 30
- 210000004204 blood vessel Anatomy 0.000 title claims description 5
- 210000001519 tissue Anatomy 0.000 claims abstract description 45
- 210000004027 cell Anatomy 0.000 claims abstract description 30
- 210000002744 extracellular matrix Anatomy 0.000 claims abstract description 19
- 241001465754 Metazoa Species 0.000 claims abstract description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 21
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 claims description 14
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 claims description 14
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 14
- 239000012670 alkaline solution Substances 0.000 claims description 12
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 9
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 7
- 235000001014 amino acid Nutrition 0.000 claims description 5
- 150000001413 amino acids Chemical class 0.000 claims description 5
- 239000000834 fixative Substances 0.000 claims description 5
- 239000004471 Glycine Substances 0.000 claims description 4
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 4
- 239000003513 alkali Substances 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 4
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 3
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical group O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 3
- 235000013922 glutamic acid Nutrition 0.000 claims description 3
- 239000004220 glutamic acid Substances 0.000 claims description 3
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 claims description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 2
- 241001494479 Pecora Species 0.000 claims description 2
- 150000008044 alkali metal hydroxides Chemical class 0.000 claims description 2
- 150000001412 amines Chemical class 0.000 claims description 2
- 239000000908 ammonium hydroxide Substances 0.000 claims description 2
- 230000012447 hatching Effects 0.000 claims description 2
- 230000007935 neutral effect Effects 0.000 claims 2
- 230000002792 vascular Effects 0.000 claims 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims 1
- 238000002054 transplantation Methods 0.000 abstract description 4
- 239000000126 substance Substances 0.000 abstract description 3
- 210000003850 cellular structure Anatomy 0.000 abstract 1
- 238000005406 washing Methods 0.000 description 8
- 239000000463 material Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 230000004069 differentiation Effects 0.000 description 4
- 230000008520 organization Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 102000008186 Collagen Human genes 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 210000000845 cartilage Anatomy 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 230000001079 digestive effect Effects 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- MNEIPOIFQCXXGM-UHFFFAOYSA-M O.O.O.[OH-].[Na+] Chemical compound O.O.O.[OH-].[Na+] MNEIPOIFQCXXGM-UHFFFAOYSA-M 0.000 description 1
- 229920001963 Synthetic biodegradable polymer Polymers 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- -1 asparatate Chemical class 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000002308 calcification Effects 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000007797 corrosion Effects 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 229920001971 elastomer Polymers 0.000 description 1
- 239000000806 elastomer Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000003037 histogenic effect Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000006651 lactation Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000005445 natural material Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 231100000241 scar Toxicity 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
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- Materials For Medical Uses (AREA)
- Prostheses (AREA)
Abstract
The present invention relates to tissue obtained from a donor of a human being or an animal. The tissue can be fixed through a fixing method, cell components capable of causing rejection reaction can be removed from the tissue through chemical treatment, and thus, extracellular matrixes (ECM) reserve the characteristics of original shape, original biological structure, original ultrastructure, positioning and durability. Live cells are grown on the extracellular matrixes (ECM) as cell frames, and then a live transplant used for transplantation or homologous transplantation is formed.
Description
The present invention relates to from the method for the resulting tissue preparation biological cell of human or animal tissues epimatrix.
One of problem that the damage of organ or tissue or disappearance are the most frequent generations in the human health, tool is calamitous and cost is the most expensive.Therefore, organizational engineering is just arisen at the historic moment as a new field, and it is to utilize biology and engineering science principle to develop the function replacement thing of damaged tissue.
Organizational project be between the normal and pathological state lactation tissue of an application project and the research of life science principle structure-functional relationship and and then develop and can recover, keep or improve the field of function of organization.It comprises and living cells and natural or artificial extracellular matrix one is used from development is used to recover or the transplant of alternative function of organization.
For one of basic design of new three kinds that tissue substituent taked of preparation be cell is placed on the matrix or among.This matrix is from natural material or synthetic biodegradable polymer fiber framework, and it is used as ECM.
Although developed many artificial or synthetic grafts, these tissue substituents still can not replace the structure of natural high complexity rightly.The challenge that faced today in tissue transplantation is to overcome the immune system of body and improve the function of graft as possible.Although obtained remarkable progress in this area, present tissue transplantation still has many problems, and this comprises inflammation, degraded, and scar infects calcification, interlock and repulsion.Therefore, medical biotechnology investigation of materials extensively and profoundly is towards the engineering of improving the transplanted tissue graft at present, but desirable artificial graft's thing is not also developed.
United States Patent (USP) 4,801, thus 299 put down in writing and a kind ofly obtained method for the extracellular matrix of transplanting usefulness by remove cell composition in the tissue with washing agent.But fixing with fixative in advance as this tissue, then this washing agent can not be used for the preparation of this tissue.
The inventor unexpectedly finds under study for action by fixing with fixer earlier from the tissue of human or animal's donor, the alkaline solution that is used as digestive juice then removes that cell obtains extracellular matrix in the fixing organization, this extracellular matrix has good tissue integrity and as other character that graft possessed, and make the source of donor wider, thereby provide extensive source for donor.Described animal can be pig, ox or sheep.The present invention is based on above-mentioned discovery is accomplished.
The purpose of this invention is to provide the method for a kind of processing from human or animal's biological tissue, it is to prepare the ECM based on collagen that takes off cellization by chemical treatment, and resulting biological tissue is used for the transplanting of the mankind or animal.The invention provides the effective ways of the improved ECM of preparation, this ECM generally has original shape of this tissue and has kept needed biological structure simultaneously.This tissue can be obtained by known method, and this tissue be the host's or obtain from corpse or donor.This takes off cell after being organized in and fixing.
After obtaining this tissue, it is fixed, take off cell then, wherein take off the digestive juice that cell uses and be selected from alkali metal hydroxide or its carbonate or bicarbonate, ammonium hydroxide, alkali alcoholate, organic amine or their mixture.Can adopt oxygen sodium hydroxide or potassium hydroxide solution.With resulting ECM washing, hatch and preserve then.
Before transplanting, can be used to cell seeding from host's (self cell) or other people as the ECM of cell framework.The ECM of this body tissue also can be used as host material and is used for tissue substitute.
The present invention includes by fixing and handle, remove cell, sterilization and cell reconstruction procedures obtain biological tissue, thus the biological substitution tissue that is provided for transplanting.
What obtained in preferred embodiments, organizes preferably identical with the graft of implanting subsequently tissue.For example skin is used to replace skin, and blood vessel is used to replace blood vessel, and cartilage is used to replace cartilage etc.
The biological tissue that the corpse of personnel selection or animal donor obtain has 1cm
3Volume or bigger, it obtains the back and places the fixer that has fixative, as glutaraldehyde or formaldehyde at once.This fixer can be contained in 2% formaldehyde and 1.5% glutaraldehyde of (PBS) in buffer solution such as the sodium phosphate buffer.This was organized in the fixer minimum retention time about 2 hours, or was fixed fully up to this tissue.After the fixation of tissue, with appropriate materials several times, as use distilled water to its washing.
In preferred embodiments, this tissue is placed in NaOH or KOH solution or any other alkaline solution then carrying out hatching of this tissue, thereby removes cell in the fixing organization.This is organized in the retention time of room temperature in this solution is about 12 hours-5 days.Solution preferably contains the alkaline solution of 1.5-2.0N NaOH or KOH.Concentration range is 0.1N-5N, and temperature range is about 4 ℃-60 ℃, and this depends on the type of the donor tissue of processing and obtain this and takes off the required time span of cell ECM.Higher concentration, higher temperature or longer digestion will damage this result.In the practice, handle the digestion that the longer time can cause elastomer with NaOH or KOH in room temperature.Gained ECM can arrive by microscope and sem observation.
After tissue takes off cellization, the washing of organizing.The solution of washing tissue can be distilled water and buffer solution, as sodium phosphate.
In this step, can change wash solution, be preferably about 30 minutes, and continue washing up to the ECM shape that is translucent.Behind the washing ECM, ECM in amino acid such as asparatate, is hatched in glutamic acid or the glycine solution to eliminate remaining free fixative and to reduce pH.Concrete amino acid is 0.5% asparatate or 0.1M glycine solution.ECM is hatched preferably and is taken cleaning solution pH to about 7.4 in the buffer solution.After the processing, can before transplanting, not damage any way of ECM structure and place and store this cell free ECM.
This ECM structure provides and has been used for cell adhesion, the suitable effective biological substrate of growth and differentiation.This ECM can be before transplanting plants living cells thereon by cell culture technology to be become alternate sets as the cell framework and knits, or by various medical science steps directly as in the body or the template of external implantation.
The invention provides a kind of tissue products, it comprises good corrosion-resistant ECM.This ECM regulate play a part on the cell behavior that is in contact with it active and comprehensive.
The present invention is exactly the extracellular matrix of an intact reservation of preparation, the various actions of at present a large amount of research is verified extracellular matrix pair cell contacted with it play very important regulating action, being centered around extracellular matrix is a kind of relatively very stable structure material, but only think and be centered around the inertia of support and framework extracellular matrix only plays a part to(for) cell, this view has become in the past, each composition that constitutes extracellular matrix for the growth of its contacted cell, differentiation all has special function, the extracellular matrix of the three-dimensional space that constitutes by their combinations, growth for cell, differentiation, metabolism, regulate the irreplaceable effect that all plays, produced by cell owing to these extracellular matrixs conversely, so cell plays such or such role and influence for the formation that is centered around its extracellular matrix on every side, on the surface of cell the special acceptor that is connected with ECM is arranged, cytostromatic like this constituent and space structure are for the metabolism of cell, the form Growth and Differentiation has closely-related effect.
Therefore, for tissue by this technology specially treated, because all can be identified by the host the antigenic cellular material of host expresses histogenic immunity is removed, and complete its form of the ECM that remains, institutional framework, ultra microstructure, the location is all the same with former matrix in a organized way with tolerance etc.
When comparing with the graft of existing market, the prepared ECM of the present invention has the natural biological structure that is difficult to imitate and have superior function in application.This ECM compares with the transplant of being made up of the chemical polymerization thing substrate of ECM composition such as collagen or fibrin coating, and it is definitely nontoxic, can not cause bad irritated activity and can promote cell proliferation.When the ECM of the present invention and United States Patent (USP) 4,801,299 and method relatively the time, the present invention is by simpler and easy, and the method for low cost prepares ECM in the shorter time, kept required natural structure simultaneously, and the source of donor is more extensive, and is convenient.
Claims (20)
1. one kind by having removed the cell composition, keeps extracellular matrix and prepare the method for blood vessel graft, and it may further comprise the steps:
A. obtain vascular tissue from human or animal's donor,
B. the vascular tissue of gained is fixed with fixative,
C. will be selected from alkali metal hydroxide or its carbonate or bicarbonate, ammonium hydroxide, alkali alcoholate, the alkaline solution of organic amine or their mixture is used for described tissue,
D. with buffer solution tissue is washed,
E. the extracellular matrix that is obtained is being selected from asparatate, the step of hatching in the Freamine of glutamic acid or glycine is so that eliminate residual fixer composition and reduce the pH value of extracellular matrix.
2. the described method of claim 1, amino acid wherein is asparatate.
3. the described method of claim 1, amino acid wherein is glutamic acid.
4. the described method of claim 1, wherein amino acid is glycine.
5. the described method of claim 1, alkaline solution wherein is a sodium hydroxide solution.
6. the described method of claim 2, wherein alkaline solution is a sodium hydroxide solution.
7. the described method of claim 3, wherein alkaline solution is a sodium hydroxide solution.
8. the described method of claim 4, wherein alkaline solution is a sodium hydroxide solution.
9. the described method of claim 1, wherein alkaline solution is a potassium hydroxide solution.
10. the described method of claim 2, wherein alkaline solution is a potassium hydroxide solution.
11. the described method of claim 3, wherein alkaline solution is a potassium hydroxide solution.
12. the described method of claim 4, wherein alkaline solution is a potassium hydroxide solution.
13. the described method of claim 1, wherein fixative is selected from glutaraldehyde and/or formaldehyde among the b.
14. the described method of claim 1, wherein the concentration of c neutral and alkali solution is 0.1N-5N.
15. the described method of claim 1, the temperature that wherein is used for described tissue among the c are 4 ℃-60 ℃.
16. the described method of claim 1, wherein to be used for the time of described tissue be 12 hours to 5 days to c neutral and alkali solution.
17. the described method of claim 1, wherein the value of pH described in the e is 7.4.
18. the arbitrary desired method of claim 1-17, wherein prepared ECM can be used as the transplanting that the template of transplanting usefulness is directly used in the human or animal.
19. the arbitrary desired method of claim 1-17, wherein prepared ECM can be gone up from host or other human or animal's living cells by plantation and the transplant of transplanting as the human or animal.
20. the described method of claim 1, wherein said animal can be pig, ox, sheep.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN00120209A CN1115957C (en) | 2000-07-13 | 2000-07-13 | Method of preparing blood vessel graft |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN00120209A CN1115957C (en) | 2000-07-13 | 2000-07-13 | Method of preparing blood vessel graft |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN96104550A Division CN1061812C (en) | 1996-04-01 | 1996-04-01 | Preparation method of cell epimatrix used as cell planting frame and implant |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1284265A CN1284265A (en) | 2001-02-21 |
| CN1115957C true CN1115957C (en) | 2003-07-30 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN00120209A Expired - Lifetime CN1115957C (en) | 2000-07-13 | 2000-07-13 | Method of preparing blood vessel graft |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1115957C (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104095692B (en) * | 2013-04-08 | 2017-02-22 | 天津市塑料研究所有限公司 | Method for manufacturing bovine jugular vein valved conduit |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3649163A (en) * | 1968-03-25 | 1972-03-14 | American Cyanamid Co | Method for removing noncollagenous matter from mammalian gut |
| EP0128706A2 (en) * | 1983-06-10 | 1984-12-19 | University Patents, Inc. | Body implants of extracellular matrix and means and methods of making and using such implants |
| EP0358506A2 (en) * | 1988-09-08 | 1990-03-14 | MARROW-TECH INCORPORATED (a Delaware corporation) | Three-dimensional cell and tissue culture system |
-
2000
- 2000-07-13 CN CN00120209A patent/CN1115957C/en not_active Expired - Lifetime
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3649163A (en) * | 1968-03-25 | 1972-03-14 | American Cyanamid Co | Method for removing noncollagenous matter from mammalian gut |
| EP0128706A2 (en) * | 1983-06-10 | 1984-12-19 | University Patents, Inc. | Body implants of extracellular matrix and means and methods of making and using such implants |
| US4801299A (en) * | 1983-06-10 | 1989-01-31 | University Patents, Inc. | Body implants of extracellular matrix and means and methods of making and using such implants |
| EP0358506A2 (en) * | 1988-09-08 | 1990-03-14 | MARROW-TECH INCORPORATED (a Delaware corporation) | Three-dimensional cell and tissue culture system |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1284265A (en) | 2001-02-21 |
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