CN1130971C - Process for preparing biological cell exomatrix used as cell planting frame and implant - Google Patents

Abstract

Translated fromChinese

由人类或动物供体得到的组织。该组织可通过固定方法来固定。可引起排斥的细胞成份可通过化学处理从组织中除去，从而使细胞外基质(ECM)保留了它们原有的形状，生物结构，超微结构，定位和耐久性。将活细胞种植在作为细胞框架的所得ECM上，形成用于移植或同种异体移植的有生命的移植物。Tissue obtained from a human or animal donor. The tissue can be fixed by a fixation method. Cellular components that can cause rejection can be removed from the tissue by chemical treatment, so that the extracellular matrix (ECM) retains its original shape, biological structure, ultrastructure, positioning and durability. Living cells are seeded on the resulting ECM as a cellular framework to form a living graft for transplantation or allografting.

Description

Preparation is as the method for the biological cell epimatrix of cell seeding framework and implant

The present invention relates to from the resulting skin that removes of human or animal tissues, the method for the tissue preparation biological cell epimatrix that blood vessel and cartilage are outer.

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.

The biological tissue that the corpse of personnel selection or animal donor obtain has 1cm³Volume 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)

Translated fromChinese

1.一种通过去除了细胞成份，保留细胞外基质而制备除皮肤、血管和软骨外移植体的方法，其包括以下步骤：1. a kind of by removing cell component, retains extracellular matrix and prepares the method for graft except skin, blood vessel and cartilage, it may further comprise the steps:a.从人或动物供体获得除皮肤、血管和软骨外组织，a. Tissues other than skin, blood vessels and cartilage obtained from human or animal donors,b.将所得的除皮肤、血管和软骨外组织用固定剂固定，b. The obtained tissues are fixed with a fixative except skin, blood vessels and cartilage,c.将选自碱金属氢氧化物或其碳酸盐或碳酸氢盐，氢氧化铵，碱金属醇化物，有机胺或它们的混合物的碱性溶液用于所述组织，c. applying to said tissue an alkaline solution selected from alkali metal hydroxides or carbonates or bicarbonates thereof, ammonium hydroxide, alkali metal alcoholates, organic amines or mixtures thereof,d.用缓冲液对组织进行洗涤，d. Wash the tissue with buffer,e.将所获得的细胞外基质在选自天门冬氨酸，谷氨酸或甘氨酸的氨基酸溶液中进行孵育的步骤以便消除残留的固定液成分并降低细胞外基质的pH值。e. A step of incubating the obtained extracellular matrix in an amino acid solution selected from aspartic acid, glutamic acid or glycine in order to eliminate residual fixative components and lower the pH of the extracellular matrix.2.权利要求1所述的方法，其中的氨基酸是天门冬氨酸。2. The method of claim 1, wherein the amino acid is aspartic acid.3.权利要求1所述的方法，其中的氨基酸是谷氨酸。3. The method of claim 1, wherein the amino acid is glutamic acid.4.权利要求1所述的方法，其中氨基酸是甘氨酸。4. The method of claim 1, wherein the amino acid is glycine.5.权利要求1所述的方法，其中的碱性溶液是氢氧化钠溶液。5. The method of claim 1, wherein the alkaline solution is a sodium hydroxide solution.6.权利要求2所述的方法，其中碱性溶液是氢氧化钠溶液。6. The method of claim 2, wherein the alkaline solution is a sodium hydroxide solution.7.权利要求3所述的方法，其中碱性溶液是氢氧化钠溶液。7. The method of claim 3, wherein the alkaline solution is a sodium hydroxide solution.8.权利要求4所述的方法，其中碱性溶液是氢氧化钠溶液。8. The method of claim 4, wherein the alkaline solution is a sodium hydroxide solution.9.权利要求1所述的方法，其中碱性溶液是氢氧化钾溶液。9. The method of claim 1, wherein the alkaline solution is potassium hydroxide solution.10.权利要求2所述的方法，其中碱性溶液是氢氧化钾溶液。10. The method of claim 2, wherein the alkaline solution is potassium hydroxide solution.11.权利要求3所述的方法，其中碱性溶液是氢氧化钾溶液。11. The method of claim 3, wherein the alkaline solution is potassium hydroxide solution.12.权利要求4所述的方法，其中碱性溶液是氢氧化钾溶液。12. The method of claim 4, wherein the alkaline solution is potassium hydroxide solution.13.权利要求1所述的方法，其中b中固定剂选自戊二醛和/或甲醛。13. The method of claim 1, wherein the fixative in b is selected from glutaraldehyde and/or formaldehyde.14.权利要求1所述的方法，其中c中碱性溶液的浓度为0.1N-5N。14. The method of claim 1, wherein the concentration of the alkaline solution in c is 0.1N-5N.15.权利要求1所述的方法，其中c中用于所述组织的温度为4°-60℃。15. The method of claim 1, wherein the temperature for the tissue in c is 4°-60°C.16.权利要求1所述的方法，其中c中碱性溶液用于所述组织的时间为12小时至5天。16. The method of claim 1, wherein the alkaline solution in c is used for the tissue for 12 hours to 5 days.17.权利要求1所述的方法，其中e中所述pH值为7.4。17. The method of claim 1, wherein the pH value in e is 7.4.18.权利要求1-17任一所要求的方法，其中所制备的ECM可作为移植用的模板直接用于人或动物的移植。18. The method as claimed in any one of claims 1-17, wherein the prepared ECM can be directly used as a template for transplantation in human or animal transplantation.19.权利要求1-17任一所要求的方法，其中所制备的ECM可被种植上来自宿主或其它人或动物的活细胞而用作人或动物移植的移植体。19. The method as claimed in any one of claims 1-17, wherein the prepared ECM can be implanted with living cells from the host or other human or animal for use as a graft for human or animal transplantation.20.权利要求1所述的方法，其中所述的动物可以是猪、牛、羊。20. The method of claim 1, wherein said animal is pig, cow, sheep.