CN1061812C

CN1061812C - Preparation method of cell epimatrix used as cell planting frame and implant

Info

Publication number: CN1061812C
Application number: CN96104550A
Authority: CN
Inventors: 胡杰
Original assignee: Individual
Current assignee: Individual
Priority date: 1996-04-01
Filing date: 1996-04-01
Publication date: 2001-02-14
Anticipated expiration: 2016-04-01
Also published as: CN1160480A

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 method for preparing the dermatoplasty body

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 one 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 required other character that possesses of graft, and make the source of donor wider, thereby provide extensive source for donor.Wherein said 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 EGM 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 per approximately 30 minutes, and continue wash the shape that is translucent up to ECM.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 that constitutes by their combinations with three-dimensional space, 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 composition 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

1. one kind by having removed the cell composition, keeps extracellular matrix and prepare the method for dermatoplasty body, and it may further comprise the steps:

A. obtain skin histology from the human or animal,

B. the skin 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 fixing skin histology, removing the cell composition in the skin histology,

D. with buffer solution the Skin Cell epimatrix is washed.Wherein the Skin Cell epimatrix is meant the skin histology of being handled by above-mentioned steps c except the cell composition,

E. the Skin Cell epimatrix that is obtained is hatched in hatching solution so that eliminate residual fixer composition and reduce the pH value of Skin Cell epimatrix.

2. the described method of claim 1, Incubating Solution wherein is an asparatate.

3. the described method of claim 1, Incubating Solution wherein is a glutamic acid.

4. the described method of claim 1, wherein incubating clothing liquid 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 3, wherein alkaline solution is a sodium hydroxide solution.

9. the described method of claim 1, alkaline solution wherein 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 arbitrary desired method of claim 1-12, the template that wherein prepared ECM can be used as the value of moving body is directly used in human or animal's transplanting.

14. the arbitrary desired method of claim 1-12, 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.

15. the desired method of claim 1, wherein said animal can be pig, ox, sheep.