CN104263698A - Screening and culturing methods for large-scale preparation of human extracellular matrix from fibroblasts for clinical treatment level cellular therapy - Google Patents

Abstract

The invention discloses a culture, screening and amplification technology of skin clinical treatment level fibroblasts. These bottlenecks are always hot and difficult problems of research in the field of biological cells; at present, separation and purification are carried out by using the remarkable adhesion characteristic of a basement membrane, and the skin clinical treatment level fibroblasts are obtained by adopting a three-dimensional culture and amplification means. The technology is applicable to the screening and separation of a target cell level by depending on amplifying adherent type cells and providing a three-dimensional high-simulation in-vivo extracellular matrix system. The technology is a novel biological cell culture technology, and adult (embryo skin) cells are subjected to in-vitro culture in a high-simulation in-vivo attached environment. The environment where a high-simulation substrate is located adheres, screens and cultures target fibroblasts, so that the passage capability is high, the multiplication capacity is high, and a cortical layer structure can be formed in an in-vitro environment. Immunogenic cells are screened out, so that immunologic escape is safely obtained, the transplantation time is prolonged, the clinical application of tissue engineering is improved, the healing of a surface of wound is promoted, and a clinical treatment level cellular solving scheme with absolute advantages is provided for skin diseases.

Description

Human cell's epimatrix screening and culturing method is prepared in the inoblast mass-producing of clinical treatment level cell therapy

One, technical field:

The invention belongs to biomass cells technology, field of tissue engineering technology.Relate in a kind of external structure height analogue body and attach environment separation screening, the fibroblastic cultivation of skin cells, amplification technique.Acquisition clinical treatment level inoblast is applied to the spike daughter cell in organization engineering skin constructing technology.

Two, background technology introduction:

Skin is the maximum organ of human body, participate in resisting biotic intrusion, ultraviolet radiation and prevent loss of moist, regulate body temperature maintains the aspects such as individual appearance and plays very important physiological action, it is the important part that human immune system forms, and skin cells is the elementary cell forming this organ, skin cells fundamental research field is the important research approach disclosing skin health, growth, prevention, treatment.

Extracellular matrix (extracellular matrix, ECM) is extensively present in the basic filling component in intercellular substance, is cell metabolism, growth, breeding, differentiation, expression, transmission, reciprocal relied on place of crucial importance.Early stage researcher thinks. epimatrix is a kind ofly organize interior, extracellular simple underwork.Hauschka and Konigsberg finds the collagen of filling tissue space research in 1966 and promotes that sarcoplast transforms the phenomenon of myotube.And after the two years, proved that scholars have just had new Cognize and Ponder to this stable outer material microenvironment.

Hay reported ECM and has important inducing action to embryo development procedure after 11 years.The phenomenon that ECM affects cell behavior is paid close attention to and is studied widely, but the substantial connection of not shaping theoretical basis evidence ECM and cell.Until nineteen eighty-two has scholar to bring forward proof and sets up ECM and the model of " dynamically reciprocal " between cytoskeleton and nuclear matrix.In this mould, ECM molecule and cell surface receptor interact, and then signal are entered in cytoplasm by cytolemma conduction, cause from cytoskeleton to nuclear a series of change, cause the expression of some specific gene.

And prove that the expression product of these genes can be had an effect to ECM again conversely, many researchs of today confirm the reasonableness of this model, and prove cell and ECM interact participated in the sticking of cell directly, grow, migration, differentiation and procedural extremely (apoptosis) process; Epimatrix participates in the activity regulating cytokine, somatomedin; Can also directly activate, signal transduction mechanism in active cell.Due to this adjustment to cell and the conduction to signal.Therefore, we must fully understand the chemistry of extracellular matrix, physics, biological property and function and in the formation of tissue and the effect in repairing, could apply these biomaterials better and be configured to tissue acceptance, and even simulated tissue improves " the biological framework " of regeneration.What the height that we set up intended cell epimatrix material utilization is just reverse theory, instructs us to carry out in a deep going way to obtain our available stem cell to the also retroaction of recognizing of ECM, and this research mode promotes that subject moves ahead and develops.

Skin progenitor cell content in normal skin tissue is few, and it is that one has unlimited updating ability in vivo that stem cell is generally acknowledged, and can be differentiated to form corresponding holostrome tissue differentiation cell, and vitro culture has long term growth potential and can form unlimited clone.The wound healing surface of a wound, the important participant of skin renewal metabolism regeneration.But owing to lacking desirable screening method at present, stem cell is screened and authentication method report less.

Three, skin cells title general introduction

Inoblast (fibroblast), also referred to as fibroblast, is the main cellular of loose connective tissue, is differentiated by the mesenchymal cell (mesenchymal cell) of embryonic stage.Inoblast is comparatively large, and profile is clear, mostly is the fusiform of projection or the flats structure of star, and its nucleus is in the oval of rule, and kernel greatly and obviously.According to difference in functionality active state, cell can be divided into inoblast and fibrocyte, Fibroblast Function activity is vigorous, tenuigenin is addicted to weakly alkaline, the obvious protein synthesis of tool and secretory activity, under certain condition, it can realize with fibrocellular mutual conversion.The reparation of inoblast to cytopathy in various degree, necrosis and tissue defect and bone wound has very important effect.

Four, inoblast general introduction

Inoblast cell space is comparatively large, and the weak basophilia of kytoplasm, the larger ovalize of karyon, chromatin loosens painted shallow, and kernel is obvious.Under Electronic Speculum, the Golgi complex of the rough surfaced endoplasmic reticulum that its kytoplasm enriches as seen, free ribosome and prosperity, shows that it has the function of synthesis and secretory protein.Collegen filament, spandex fiber, reticulin fiber and organic substrate be synthesized and be secreted to inoblast still can.The procollagen molecule of its synthesis is through restriction endonuclease effect, and polymerization and rearrangement, can form the collagen fibril with 64nm cycle band the same as the collagen fibril of scleroblast synthesis secretion, collagen fibril is through formation collegen filament bonded to each other.After testing, the collegen filament of these two kinds of cell synthesis secretions are all type i collagen fibers, identical in form with biochemical structure.Be in the inoblast of ripening stage or title stationary state, cell space diminishes, and in spindle shape, rough surfaced endoplasmic reticulum and Golgi complex are all undeveloped, are called as fibrocyte.Under the factors such as wound stimulate, few fibers cell can change inmature inoblast again into, and its functional activity is also recovered, and participates in the reparation after tissue injury.In addition, in reticular tissue, still maintain the mesenchymal cell on a small quantity with differentiation potential, they can proliferation and differentiation be inoblast in the situations such as trauma repair.

Five, the feature of inoblast application

General trauma repair

Various wound all can cause cytopathy in various degree, necrosis and tissue defect, must carry out tissue repair by the formation of hyperplasia and intercellular matrix.In this repair process, inoblast plays a very important role.For wound healing process, inoblast is bred in a large number by mitotic division, and synthesized from 4 ~ 5 days or 6 days and secrete a large amount of collegen filament and matrix components, granulation tissue is jointly formed with new capillary vessel etc., fill up wound tissue's defect, for the covering of epidermic cell creates conditions.In wound healing, inoblast is mainly derived from dermopapillary local fibroblasts and undifferentiated mesenchymal cell, and circumvascular inoblast and pericyte.During visceral injury, participate in the inoblast of repair process many from interstitial and coating, and under mucous membrane or Subserosal reticular tissue.Someone thinks a large amount of inoblasts that injury is assembled in wound healing process, by division growth by inoblast on the one hand, on the other hand, be to injury more by the differentiation such as contiguous mesenchymal cell, fibrocyte and pericapillary cells or migration.In the later stage of trauma repair, inoblast participates in the reconstruction of repairing rear tissue by secretion collagenase.Under some pathological conditions, be that granulation tissue or hyperplastic tissue's block of main cellular in non-bone tissue, calcification can also occur with inoblast, cause ectopic ossification (ectopic ossification).But for the participation cell of ectopic ossification and mechanism still not fully aware of, the cells that can incorporate into as inducibility osteoprogenitor cells such as undifferentiated mesenchymal cell, inoblast, endotheliocyte and pericapillary cells all likely participate in this process bone wound reparation

Namely the simplest and the most common bone wound is fracture, and its agglutination must through Inflammatory response, cleaning, fiber poroma and 4 stages of osseous callus.The cell bodies that different steps participates in is different.Inoblast just comes across the 3rd day in fracture local hematoma from fracture, and fracturing latter 5 days namely in the gap of machine hemotoncus and fracture site and in a large number to exist around, is the main cellular in participation fiber poroma stage.At this stage inoblast a large amount of division growth on the one hand, synthesize again and secrete large amounts of type i collagen on the one hand, the reticular tissue making granulation tissue progressively become loose, is surrounded bone stump, forms the huge fiber poroma of joint two fracture site.But, this is that the fibrillar connective tissue that main body is formed but can not develop into the scar tissue common when other tissue injury is repaired by countless inoblast and abundant granulation tissue, but constantly deposited therein by calcium salt crystallization, develop into osseous callus gradually, make the reparation of fracture local reach bony union, recover the structure of osseous tissue.Now, union of fracture portion only has osseous tissue and no longer there is inoblast.

Six, fibroblastic separation and discriminating

Basis of microscopic observation inoblast is fusiformis, and radially, swirling grows, and anti-desmin monoclonal antibody and actin monoclonal antibodies immunohistochemical staining are negative, and anti-vimentin monoclonal antibody is positive.

Seven, traditional method for sieving introduction

Because skin cells has stronger adhesivity to basilar membrane, more faster than other cell type to the adhesion speed of cell extracellular matrix, so utilize this kind of characteristic to carry out cultivation screening.Skin progenitor cell screening traditional is at present following sets forth:

Method one (3T3 trophoderm method): the historical background in a human skin cell vitro culture research existing century, set up amplification in vitro culture system in the fibroblast strain 3T3 trophocyte of 3TS-J2 mice embryonic knurl designed by scholar Rheinwald and Green in 1975, and irradiate by gamma-rays lethal dose, prevent cell to be subject to the impact of heterologous species.Deactivation 3T3 trophocyte can promote the attaching that epidermal stem cells screens and growth as cultivating the trophoderm of epidermic cell, and has not by the function of inhibition contact fibroblastic growth proved.Each subbreed that selected by laboratory, function is more stable but the misgivings can't got rid of completely it, also need to continue observe and explore effective alternative method.Although Green study this method 3T3 it be an xenogenesis there is the cell of asking change nature, have many laboratories once to attempt to be replaced with other measures, up to now, its effect is still the most effective in various method, continues to be widely used.From the 1st routine clinical application to nearly 30 years of order, not yet find the adverse consequences caused because of it.

Method two (Fibrinogen and thrombin substrate method): Graziella P scholar's research Fibrinogen and zymoplasm are as matrix separate skin cell;

Method three (IV Collagen Type VI sticks method): Jone PH etc. utilize aforesaid method from adult foreskin or cadaver skin through digestion, IV Collagen Type VI is utilized to do substrates separate skin cell, selected substrate is xenogenesis collagen, there is the possibility of pollution of different genera, and substrate is expensive, be not suitable for industrialized scale or long-term cultivation, be applicable to tentative low dose research;

Method four (flow cytometer sieve method): scholar Van Rossum MMj and Kaur P etc. obtains epithelial cells from people's fritter biopsy, utilize integrin to mark different in nature mark and utilize flow cytometric sorting skin cells, selecting method is complicated and technical requirements is high, substantially apply integrin β 1, cell adhesion molecule CD49, add the multiple marks such as cell nuclear antigen (PCNA) and carry out character separation, although cell is screened, but marker label retaining cell can be left cannot effectively remove, exist and whether affect cell proliferation, differentiation, variability etc.This method is applicable to Marker Identification and measures cell, and is not suitable for the cell enrichment methods of clinical application;

Method five (xenogenesis becomes fiber suspension sieve method): scholar Hagar B utilizes DMEM, low calcium ion mouse fibroblast cell conditioned medium as substratum, without trophocyte.But having research report epidermal stem cells and basilar membrane to depart to bring out and enter the differentiation cycle, be divided into transitional expansion cell, is likely maintain the possible condition that epidermal stem cells controls differentiation characteristic according to the high-adhesiveness guessing epidermal stem cells and basilar membrane.If so do not utilize trophoderm, likely in long-term cultivation process, differentiation of stem cells causes the uncontrollable development of follow-up study;

Method six (consubstantiality becomes fiber to attach sieve method): scholar's banket etc. utilize with reference to improving scholar Hagar B and Dunnwald mouse fibroblast cell, make into donor in advance amplification in vitro inoblast for attachment substrate as trophoderm, cultivate consubstantiality skin cells, but patent is the clear and definite detailed step how preparing trophoderm plate not, the paving ware cycle is short, become the adherent power of fiber low, screen repeatedly PBS clean purge process can lose part cultivate target attach successful inoblast, thus reduce the yield of targeted stem cells, theory of stem cell has the same originality of consubstantiality, another technical problem is, two kinds of cells should consider the complicacy of nutrient solution in total same culture environment, the phenomenons such as emulative growing multiplication suppression may be there is in two kinds of cells, there is technical difficulty in later separation skin cells, common methods in existing six can not be better than, advise that it can make compound into this method and add other and cover effective other the larger screening thing of behavior that sticks of ware with increasing target cell yield, or deactivation becomes fiber lay down ware confluent monolayer cells.

Method seven (methylcellulose gum culture dish attaches sieve method):

Foreign scholar utilizes the DMEN/F12 adding methylcellulose gum B27 to cultivate.

In sum, skin cells extracellular matrix stick strong rapid charater, it is fast-developing that the human fibroblasts that in-vitro screening is cultivated is able to method specificity at home and abroad.And inoblast biology performance, can accomplish to promote function, avoid potential deleterious gene and to fibroblast cells control divergaence time with control different differentiation directions etc., all have the important directive significance do not replaced of period of history in the structure of basic scientific research, clinical treatment, skin cells research direction, skin regeneration injury repairing, high plan organization engineering skin etc.

Eight, organization engineering skin background introduction

It is that work starts from this century (Harrison 1907, Carrel 1912) that regenerating tissues engineering cell is set up, and instructs and leads biology, each large linking fields such as clinical medicine material, becomes one of important basic science.Tissue construction and cell injuring model are vital problems.Get tissue from donor donor live body, the serial specific micro-environment in vitro system such as physiological environment in analogue body, carry out hatching and cultivate, make histocyte healthy growth.

Organization engineering skin is set up to be applied in early days and is repaired burn and ulcer and other skin injury field, and the Yannas I.V. of John F.Burke and the MIT of Massachusetts General Hospital cooperates, and prepares artificial skin surrogate first.But this artificial skin surrogate is not relevant to cytobiology, is under the jurisdiction of scaffold materials of tissue-engineered skin category.Along with the basic subject of cytobiology develops, people start to integrate two large subjects, collaborate solves trauma skin reparation and builds, and real has cooperated at the Eugenen Bellhe of Ha Fu university Honward Green and MIT containing active cells organization engineering skin.Early stage customization dermatoplasty treatment achieves biology medical science and to be situated between concern of attracting attention.

Phase early 1980s timbering material is by FDA license listing, the composite skin graft material be made up of collagen grid is continuously created, internal layer is that bovine collagen and chondroitin sulfate are formed, skin is silica column, after surface of a wound transplanting, internal layer contacts with the surface of a wound and is slowly degraded, after several weeks, silicone layer removes, and covers autotransplantation skin after debridement, and coverture is accepted by clinical.

The scientist Bellhe of MIT creates organization engineering skin materialogy and biology perfect adaptation and establishes first hand organizational project regeneration company (Organogenesis Inc) in the world, is the model of scientific circles businessman.This active mass of family engineering skin pioneer leads company to remain in one of the highest tissue engineering companies of artificial skin share of market.

The TransCyte produced by Advanced Bionhealing company by first tissue engineering product of U.S. FDA approval listing for 1997.For burn wound repairing and treating.This product belongs to the fibrocyte of collagen as tissue engineering scaffold plantation deactivation, becomes business-like first " allosome non-activity cellular system engineering corium therapy ".The Apligraf that Dermagraft and Integra and Organogenesis Inc manufactures also obtains FDA approval, utilize neonatal foreskin through biology vitro culture active cells, the organization engineering skin of preparation, for ulcerous skin and large-area burns regenerative therapy.This product stricti jurise has class skin tissue engineering artificial skin, contains active 4 ~ 8 cells gone down to posterity and have integral skin dermal cell to be structured in degradable holostrome artificial skin on tissue engineering bracket.And part is rejected and is reduced Langerhans cell, reduce the immunological rejection of acceptor, the dead cell of non-activity is absorbed by the surface of a wound through the effect of wound tissue liquid and the relevant humoral factor metabolism small molecule segment basal nutrient healing substances that can be used for wound repair that is decomposed.This product is made and is placed on-70 DEG C of preservations, and 37 DEG C of recoveries rapidly, have the active cells of 50% to have activity after recovery, active maintenance posts to the medical department of specifying in 5 days by express delivery.

The market access of these products has led the brand-new regenerative medicine organization engineering skin epoch to start.

Based on tissue engineering artificial skin present situation both at home and abroad, set up organization engineering skin stem cell-Gao and intend cells in vivo epimatrix, the design of matrix attachment in-vitro screening method is the first containing epidermal stem cells fibroblastic holostrome tissue engineering artificial skin industrialization product.

Summary of the invention

1. one kind is combined for screening and be trained fibrocellular substratum,

Preparation nutrient solution A: inactivated fetal bovine serum 40 ~ 60ml, Transferrins,iron complexes 5 ~ 15 μ g/ml, adenine phosphate 0.5 ~ 1 × 10 ^-4/l, vitamins C 1 ~ 3 μ g/ml, Regular Insulin 2 ~ 4 μ g/ml, cholera mycin 2.5 ~ 4.5u/L, EGF5 ~ 25ng/ml, hydrocortisone 0.4 μ g/ml, DMEM/F12 (3: 1) constant volume are to 500ml.

Preparation nutrient solution B: preparation nutrient solution B: inactivated fetal bovine serum 80ml, Transferrins,iron complexes 6 ~ 17 μ g/ml, adenosine 10 ~ 25mg/ml, adenine phosphate 0.5 ~ 1 × 10 ^-4/ L, cholera mycin 2.5 ~ 3.5u/L, penicillin 50-100u/ml, Regular Insulin 4 ~ 6 μ g/ml, EGF5 ~ 25ng/ml, bFGF10 ~ 18ng/ml, ox pituitary gland extract (BPE) 1 ~ 3mg/L, stem cell factor (SCF) 5 ~ 15ng/ml, hydrocortisone 0.3 μ g/ml, DMEM/F12 (1: 1) constant volume are to 500ml, pH7.2 ~ 7.4.

Preparation nutrient solution C: inactivated fetal bovine serum 60ml, Transferrins,iron complexes 6 ~ 17 μ g/ml, adenosine 10 ~ 25mg/ml, adenine phosphate 0.5 ~ 1 × 10 ^-4/ L, Regular Insulin 4 ~ 6 μ g/ml, bFGF10 ~ 20ng/ml, ox pituitary gland extract (BPE) 2 ~ 3mg/L, stem cell factor (SCF) 10 ~ 30ng/ml, cholera mycin 2.5 ~ 3.5u/L, Streptomycin sulphate 50 ~ 100 μ g/ml, hydrocortisone 0.3 μ g/ml, DMEM/F12 (1: 1) constant volume are to 500ml, pH7.2 ~ 7.4.

2. prepare the high method intending cells in vivo epimatrix, comprise the following steps: a) draw materials and skin degerming process; B) de-cell process; Preferably, also comprise skin sterilization or warp being taken off cell process further and carry out low temperature, dehydration, irradiation sterilization process further; More preferably step b) method for removing cells be selected from cold enzyme Crosslink bond type, enzyme-nonionogenic tenside-complexing agent integrated process, complexing agent salt adding-anion surfactant method or pancreatin associating EDTA crosslinked fluid method.

3. method described in claim 2, wherein cold enzyme Crosslink bond type is: add the cold 12 ~ 48h that disappears of 0.1 ~ 0.25% proteolytic enzyme ice bath.With containing 0.25% glutaraldehyde phosphate buffer solution, pH is between 7.20 ~ 7.40, and crosslinked 4 ~ 6h, ultrapure water or water for injection rinse 8 ~ 10 times.

4. method described in claim 2, wherein enzyme-nonionogenic tenside-complexing agent integrated process is: 0.1 ~ 0.25% neutral protease and 0.01 ~ 0.02%EDTA, TritonX-100 (0.3 ~ 0.6%) solution, acts on 24 ~ 48h under room temperature.

5. method described in claim 2, its complexing agent salt adding-anion surfactant method is: 0.01 ~ 0.02%EDTA is containing 0.6 ~ 0.8mol/L sodium chloride solution, 12 ~ 24h is acted on, rear use (0.2 ~ 0.5%SDS solution acts on 30 ~ 60min under room temperature) at 37 DEG C.

6. method described in claim 2, wherein pancreatin associating EDTA crosslinked fluid is: 0.25 ~ 0.40% trypsinase and 0.02 ~ 0.04%EDTA (1: 2), 37 DEG C of rapid digestion 10 ~ 60min.Crosslinked 4 ~ 6h in crosslinked fluid.Rinse 8 ~ 10 times with ultrapure water or water for injection after crosslinked.

7. the height prepared by either method described in claim 2-6 intends cells in vivo epimatrix.

8. the height that prepared by either method described in claim 2-6 intends cells in vivo epimatrix or high plan cells in vivo epimatrix according to claim 7 is being filtered into the purposes in fibrocyte.

9. utilize the combination of the substratum described in claim 1 and height according to claim 7 intend the screening of cells in vivo epimatrix and are trained a fibrocellular method, comprising:

A) prepare epidermal cell suspension: draw materials, digestion, peel off epidermis and collect skin graft, digestion, stop digestion with nutrient solution A according to claim 1, collecting cell suspension.Nutrient solution A nutrient solution is added after centrifugal;

B) fibroblastic screening and cultivation: height according to claim 7 is intended cells in vivo epimatrix and adds nutrient solution B according to claim 1, add the cell suspension that step a) is prepared, cultivate, cleaning, target epidermal stem cells is adherent adheres to high plan cells in vivo epimatrix, adds nutrient solution C according to claim 1 and carries out amplification cultivation;

C) enrichment Secondary Culture is digested: digestion, adds nutrient solution C according to claim 1 and carry out amplification cultivation after centrifugal.Preferably, the process of immunologic escape method draw materials before skin or obtain cell, immunologic escape method can be rays method, low temperature freeze-thaw method

Accompanying drawing explanation

Fig. 1 height intends cells in vivo epimatrix preparation flow

Fig. 2 cell screening is cultivated

One, high plan cells in vivo epimatrix kind

(skin related to is organ donation, and in peritomize skin and medical treatment process, skin-grafting is got skin and discarded skin histology)

1.1 is characterized in that: for the current situation of the technology of the present invention, the object of this invention is to provide skin corium in a kind of high plan body to attach for screening as extracellular matrix, the method of adult or embryo skin inoblast screening and separating and vitro culture, and the high purity inoblast making acquisition.

1.2 is characterized in that: for the current situation of the technology of the present invention, the object of this invention is to provide full thickness skin in a kind of high plan body to attach for screening as extracellular matrix, the method of adult or embryo skin skin progenitor cell screening and separating and vitro culture, and the high purity skin progenitor cell making acquisition.Skin cells comprises: (the attached relevant stem cell of inoblast, hair follicle stem cells, epidermal stem cells and other appendages of skin and skin relevant cell).

The method of this screening and separating and vitro culture, the inoblast cell of acquisition overcomes the defect of above traditional seven kinds of methods.

Invention belongs to biological adult (fetus) skin cells cell technology field, relates to fibroblastic separation and ientification method.Method feature comprises: consubstantiality epidermis takes off the preparation of Cuticle of cell substrate; The separation of consubstantiality dermal cell; Utilize high screening and the cultivation of intending attachment stromal fibroblast cells in body.Subculture has been stablized in vitro more than 43 ~ 60 (generation) at present by this method, the acellular poison of this cell is confirmed through experiment in vivo and vitro, there is strong multiplication capacity, and the holostrome differentiation corium cortex construction that can form maturation is about skin cells plays immeasurable effect in scientific research, teaching and clinical application, breeds huge academic direction and Social benefit and economic benefit simultaneously.The academic Foundation of other organ cells of China is advanced by this kind of theoretical method basis.

Two, high cells in vivo matrix of intending is formed:

Contain multiple collagen by analysis and comprise IV type, wherein type i collagen content maximum (ratio of I and type III collagen is 10: 1), also retains complete basement membrane structure simultaneously.

Three, height is intended cells in vivo matrix substrates porosity and is sticked surface characteristic:

High plan substrate electron microscopic observation, the basilar membrane pore diameter measuring the substrate tissue regeneration that epidermis sticks is 32 ~ 145 μm, promotes that sticking dermal tissue regeneration substrate basilar membrane porous pore diameter is 72 ~ 191 μm.This porosity purchase the space built and stick all favourable Stem Cells adhesion apposition growth in surface, propagation, differentiation.

By above preparation method; Effectively remain the microcosmic framework of skin cells epimatrix and complete basement membrane structure, its main component comprises the insoluble matrix compositions such as each Collagen Type VI, elastin, protein-polysaccharide and glycosaminoglycan, can be the adhere fast such as corium stem cell, epidermal stem cells, propagation expansion strong support is provided.Therefore, high plan substrate is the solution of current optimal organization engineering skin stem cell screening.

Four, skin cells height intends the concrete implementation step of cells in vivo matrix composition and method:

One, following two step kind methods are comprised:

Step one: skin degerming treating processes: get donor dermal and comprise following (embryo skin, adult foreskin outside plate, scalp, armpit skin and body skin) partial thickness skin containing epidermis dermis structure, cut into tissue block and be immersed in normal saline dilution containing 10 ~ 15min in 0.05 ~ 0.1% benzalkonium bromide solution, disinfect, with stroke-physiological saline solution cleaning and removing residual Morpan BB repeatedly.

Step 2: low temperature-60 ~-80 DEG C of lyophilizes, vacuum tightness reaches 20 ~ 110pa, sloughs tissue block more than 90 ~ 95% moisture, takes out sealing and vacuum packaging, through irradiation 25 ~ 60KGY doses of sterilization.Whole product can be preserved as long as five years at room temperature.Start and only need soak 37 DEG C, PBS liquid or below described nutritive medium recovery 15 ~ 30min can apply, and it is better that immersion extends to 10 ~ 12h effect.Use rear and the de-cell process of cell rinsing liquid (mentioning applicable de-enchylema see corresponding in lower method) should be spent, again through the feature of said process immortality Reusability.

1: step 2 embodiment:

Described in above-mentioned mentioned de-enchylema is prepared as follows:

Two, method:

Case method 1: skin graft adds the cold 12 ~ 48h that disappears of 0.1 ~ 0.25% proteolytic enzyme ice bath after treatment.Between 7.20 ~ 7.40, tissue block is rinsed 3 ~ 6 times with crosslinked fluid with regulating PH containing 0.25% glutaraldehyde phosphate buffer solution, finally put into the crosslinked 4 ~ 6h of crosslinked fluid, terminate rear ultrapure water (or water for injection) and repeatedly rinse the residual glutaraldehyde of 8 ~ 10 removals, can freeze-drying and dehydrating encapsulation be carried out.

1.1: case method 1 embodiment:

Method one feature: linking agent can effectively connective tissue protein not capacitive be cross-linked framework, strengthen the anti-degradation property of substrate, keep substrate complete, protease treatment substrate can make its soft wilfulness strengthen.

Skin epidermis includes four-layer structure, and each confluent monolayer cells amount is 10 ~ 30%, and wherein malpighian layer is called that basal cell layer is the attachment Differentiation place that cell enlivens the most, has and reaches 9 ~ 15 layers more than; Granular layer structure contains a large amount of gaps nonwoven fabric from filaments, and cutinized layer is many in netted braided, the three-dimensional complicated capable structure of space IPN, and external supporting material is difficult to simulation and imitates.So class substrates is best suited for the ideal microenvironment place needed for cell adhesion proliferate differentiation.

Case method 2: enzyme-nonionogenic tenside-complexing agent integrated process

0.1 ~ 0.25%DisPase neutral protease and 0.01 ~ 0.02%EDTA simultaneous digestion, neutral protease acts on basilar membrane, optionally decompose fibronectin and IV Collagen Type VI (acting on 48h at 4 DEG C), then applying nonionic surface active agent makes cytolysis destroy TritonX-100 (Triton X-100) (0.3 ~ 0.6% solution, 24 ~ 48h is acted under room temperature) form mixture with the lipid binding such as phosphatide in microbial film and be dissolved in solution, oleophylic cardinal extremity simultaneously in TritonX-100 also can with cell outer membrane protein decohesion microbial film, thus reach de-and abandon cytosis.

After digestion, alternative stripping epidermis dermis, carries out glutaraldehyde cross-linking step through method 1, can carry out freeze-drying and dehydrating encapsulation.

2.1: case method 2 embodiment:

Case method 3: complexing agent salt adding-anion surfactant method

(0.01 ~ 0.02%EDTA is containing 0.6 ~ 0.8mol/L sodium chloride solution to utilize high permeating sodium chloride solution, 12 ~ 24h is acted at 37 DEG C) hemidesmosome of anchor-shaped filament and epidermal basal cell is separated, thus complete removal epidermis, rupture of membranes agent sodium laurylsulfonate (0.2 ~ 0.5%SDS solution acts on 30 ~ 60min under room temperature) is used cellular constituent to be removed from corium again.

Above step alternative peels off epidermis dermis, retains holostrome and carries out glutaraldehyde cross-linking step through method 1, can carry out freeze-drying and dehydrating encapsulation.

3.1: case method 3 embodiment:

Case method 4: by 0.25 ~ 0.40% trypsinase and 0.02 ~ 0.04%EDTA (1: 2) mixed solution simultaneous digestion tissue block, 37 DEG C, rapid digestion 10 ~ 60min, rinsing takes off cell repeatedly, finally put into the crosslinked 4 ~ 6h of crosslinked fluid, terminate rear ultrapure water (or water for injection) and repeatedly rinse the residual glutaraldehyde of 8 ~ 10 removals, can freeze-drying and dehydrating encapsulation be carried out.

4.1: case method 4 embodiment:

Above method is all capable of being combined prepares high plan described in cells in vivo epimatrix: 1.1,1.2 carry out screening skin cells.

The obtained slightly difference of aforesaid method, cost had time different but all can effectively make cellular constituent be eliminated, the structural integrity of matrix is retained, content containing elastin, keratan sulfate, laminin, IV Collagen Type VI, fibronectin, desmin, HLA-DR is more, and basement membrane structure composition all can completely retain.

Seed skin cells for tack provides favourable microenvironment, and make it express, grow, breed, differentiation etc. is played and important, and is better than above-mentioned 6 kinds of conventional screening assays irreplaceable height simulation attachment state space.We are referred to as " the black edatope of cell seeding ".

High cells in vivo epimatrix of intending screens skin flbroblast culture method

Five, skin is drawn materials screening:

Skin cells screening position comprises, fetal skin, foreskin outside plate, scalp, armpit area skin.

Draw materials in high cells in vivo epimatrix position of intending: fetal skin, foreskin outside plate, scalp, armpit area skin, other area skin.

Fully soak 3 ~ 5min with the PBS containing microbiotic (penicillin 80 ~ 100u/ml and 80 ~ 100 μ g/ml Streptomycin sulphates) when receiving, finishing sample removes subcutis as far as possible.

Six, inoblast screening and culturing step

Because inoblast has adhesivity to basilar membrane, to the adhesion growth amplification demand of cell extracellular matrix, so utilize this kind of characteristic to carry out cultivation screening.

Seven, skin cells screening and culturing claimed method:

1, nutrient solution preparation:

Preparation nutrient solution A: inactivated fetal bovine serum 40 ~ 60ml, Transferrins,iron complexes 5 ~ 15 μ g/ml, adenine phosphate 0.5 ~ 1 × 10 ^-4/l, vitamins C 1 ~ 3 μ g/ml, Regular Insulin 2 ~ 4 μ g/ml, cholera mycin 2.5 ~ 4.5u/L, EGF5 ~ 25ng/ml, hydrocortisone 0.4 μ g/ml, commercial substratum DMEM/F12 (3: 1) constant volume are to 500ml.

A.1: preparation nutrient solution A embodiment

Preparation nutrient solution B: preparation nutrient solution B: inactivated fetal bovine serum 80ml, Transferrins,iron complexes 6 ~ 17 μ g/ml, adenosine 10 ~ 25mg/ml, adenine phosphate 0.5 ~ 1 × 10 ^-4/ L, cholera mycin 2.5 ~ 3.5u/L, penicillin 50-100u/ml, Regular Insulin 4 ~ 6 μ g/ml, EGF5 ~ 25ng/ml, bFGF10 ~ 18ng/ml, ox pituitary gland extract (BPE) 1 ~ 3mg/L, stem cell factor (SCF) 5 ~ 15ng/ml, hydrocortisone 0.3 μ g/ml, commercial substratum DMEM and commercial substratum F12 (1: 1) constant volume are to 500ml.PH7.2～7.4。

B.1: preparation nutrient solution B embodiment

Preparation nutrient solution C: inactivated fetal bovine serum 60ml, Transferrins,iron complexes 6 ~ 17 μ g/ml, adenosine 10 ~ 25mg/ml, adenine phosphate 0.5 ~ 1 × 10 ^-4/ L, Regular Insulin 4 ~ 6 μ g/ml, bFGF10 ~ 20ng/ml, ox pituitary gland extract (BPE) 2 ~ 3mg/L, stem cell factor (SCF) 10 ~ 30ng/ml, cholera mycin 2.5 ~ 3.5u/L, Streptomycin sulphate 50 ~ 100 μ g/ml, hydrocortisone 0.3 μ g/ml, commercial substratum DMEM and commercial substratum F12 (1: 1) constant volume are to 500ml.PH7.2～7.4。

C.1: preparation nutrient solution C embodiment

Illustrate that this formula is original cuiture, building is stablize follow-up resuming for reducing foetal calf serum usage quantity, cultivates domestication cell non-serum and cultivates habit.Other carry out descending domestication and cultivate as Regular Insulin, hydrocortisone are corresponding.Regulate bEGF, SCF, BPE consumption to make it break up when building tissue engineering epidermis layer and set up dermis.Other basic components are constant.

2, Digestive system preparation method:

Digestive system preparation A: containing solute neutral protease 0.1% without Ca ²⁺, Mg ²⁺, serum-free medium is solvent, obtain solution.

Digestive system preparation B: containing solute 0.15% pancreatin and 0.01%EDTA type i collagen enzyme 0.1% collagenase (1: 1: 1) without Ca ²⁺, Mg ²⁺, serum-free medium is solvent, obtain solution.

3, skin and the preparation of screening cell freeze thawing conserving liquid:

Freeze proof solute component comprises: adenine phosphate 5 ~ 15 μ g/ml, vitamins C 2 ~ 6 μ g/ml, chondroitin sulfate 3 ~ 6%, polyoxyethylene glycol 4ml, deactivation calf serum 10 ~ 15%, glycerine 20%, N.F,USP MANNITOL 3ml, cytochalasin 2.7 μ g/ml, dimethyl sulfoxide (DMSO) (DMSO) 10%, conserving liquid with DMEM/F12 (1: 1) 46 ~ 50% for antifreeze solution prepared by solvent.PH controls 7.3 ~ 7.4,0.22 μm of micropore Entkeimung.

3.1:3 skin and screening cell freeze thawing conserving liquid prepare embodiment:

Illustrate, it is characterized in that: this refrigerating fulid not only, formulating of recipe active by freezing long-term preservation skin and skin cells can also have and suppress Langerhans cell present function and play the immunogenicity that can reduce skin.

4, skin sample irradiates low-temperature storage immunologic escape antigenicity cellular processes:

3.1 immunologic escape method rays methods: epidermis sample disposal: get the skin described in five, are immersed in nutrient solution A and irradiate 10 ~ 30min through UV-B/C (UVB middle-ultraviolet lamp, UVC far ultraviolet rays yue).

Illustrate: uviolizing, gamma-rays can reduce immunological rejection, uviolizing effectively can reduce this cell quantity of Netac's Chinese, significantly suppress the antigen presentation capability of Langerhans cell.

At present disclose to irradiate how to affect APC function, ray can affect epidermic cell produce as some immunosuppression things such as the uridylic acid (cis urocanic acid) in IL-10, stratum corneum with this reduce transplanting the immunological rejection effect that produces.Clinical treatment is approved by extensive scholar doctor through postradiation skin graft survival time significant prolongation.

3.2 immunologic escape method freeze-thaw methods:

Epidermis sample disposal: get the skin described in five, soaks skin freeze thawing conserving liquid through anti frozen liquid process, is placed on 12 ~ 48h in-40 ,-60 ,-90 ,-100 ,-196 DEG C of environment respectively.

Illustrate: to express change not obvious for Langerhans cell cytolemma HLA-DR after cryopreservation, but its costimulatory molecules CD86 significantly reduces, and transplants such skin, accepted the time by acceptor and obviously extend.Low temperature makes the disappearance of the function limitation of Langerhans cell, cytoplasmic process, quantity minimizing, smaller volume, shows that these temperature spots can obviously reduce transplant rejection phenomenon through test mice transplant experiment simultaneously, and it is more obvious that trend presents temperature lower immunologic escape phenomenon.

5, inoblast screening step method:

Step 1, get skin cells screening in be separated skin corium skin histology sample (get the skin histology sample of 0.5 × 1.0cm, 1 × 2cm size or other specification, to rinse 3 ~ 6 times containing antibiotic PBS, put 4 DEG C of cold digestion 12 ~ 24h in Digestive system preparation A and take out sample, peel off epidermis, skin corium), to rinse 3 ~ 6 times containing antibiotic PBS, by corium skin graft, put in Digestive system preparation B.37 DEG C of 40 ~ 60min digestion, stop digestion with preparation nutrient solution B, the centrifugal 5 ~ 10min of 600 ~ 1000r/min, abandons or adopts supernatant liquor, and add preparation nutrient solution A, cell concn is adjusted to 1 × 10 ³~ 1 × 10 ⁵/ ml.

Step 2, get high cells in vivo epimatrix of intending and select 1.1, also optional standby 1.2, rinse 3 times with containing antibiotic PBS, then rinse 3 times with preparing nutrient solution B, in 37 DEG C, be immersed in recovery 10 ~ 12h in nutrient solution B for subsequent use.

Step 3, fibroblastic screening and cultivation: get ready 1.1 and put into plate and add preparation nutrient solution B, add cell concn 1 × 10 ³~ 1 × 10 ⁴/ ml dermal cell suspension, 37 DEG C, gas phase 5%CO ₂incubator is cultivated 10 ~ 30h and is taken out, and taking-up transfer 1.2, PBS or nutrient solution B clean 3 ~ 6 times, and target inoblast is adherent to adhere on 1.1.Change ware and add preparation nutrient solution B cultivation, rear every 2 ~ 4d changes liquid and once continues amplification cultivation.

Step 4, digestion enrichment Secondary Culture:

Above-mentioned inoblast is after amplification, and add Digestive system preparation B and digest 1.2 matrix, 4 DEG C of digestion 24h, nutrient solution B stop digestion; Suction pipe is is fully blown and beaten, and cross the cell sieve in 40 μm of apertures, collect filtrate, 4 DEG C leave standstill 20 ~ 30min; Then, with the centrifugation 10min of 800 ~ 1000r/min.Abandon supernatant liquor, add the nutrient solution C of preparation, re-suspended cell, with 1 ~ 3 × 10 ⁶the density transfer of/ml is cultivated containing in trophoblastic ware, puts 37 DEG C, gas phase 5%CO ₂incubator is cultivated, and every 2 ~ 4d changes nutrient solution C processed and once continues amplification cultivation.

Step 5, morphology and immunohistochemical method are examined:

3 generation cell observation microscopy be fusiformis, swirling grow

Above cultural method carries out difference detected result:

The inoblast that equal basis of microscopic observation is cultivated is fusiformis, and radially, swirling grows.

Anti-desmin monoclonal antibody and actin monoclonal antibodies immunohistochemical staining are all negative, and anti-vimentin monoclonal antibody is all positive.

The method obtains inoblast, can keep Secondary Culture 43 ~ 60 generation passage capacity.

To sum up fibroblastic through immunohistochemical method qualification, on the basis of quick wall attaching, subculture in vitro separately multiplication capacity is strong, and the cell obtained is target inoblast.

Conclusion: high plan obtains inoblast in the dermal tissue of cells in vivo epimatrix 1.1 and 1.2 sieve method enrichment.

What eight, clinical treatment level inoblast was invented has the following advantages:

The invention discloses the cultivation of skin cells, screening, amplification technique, these bottlenecks are biology inoblast area research focus and difficult point always, utilize the remarkable adhesion properties of basilar membrane to carry out separation and purification at present, adopt dimensional culture amplification means to obtain.

This invention is applicable to relying on the adherent type cell of amplification and provides three-dimensional high to be intended screening that cells in vivo epimatrix system carries out target cell level and is separated.

The invention belongs in high analogue body and attach environment, the novel culture technique of vitro culture adult (embryo skin) fibroblastic biomass cells.It is characterized in that: cell matrix substrate is that donor takes off Cuticle of cell, corium is the best preparation method of sticking place screening stem cell of matrix substrates screening; Corresponding epidermis dermal cell height simulation substrate place environment sticks screening and culturing target cell.With the inoblast that the method obtains, there is high passage capacity, high proliferation ability, and skin corium structure can be formed under environment in vitro, this method has more Scientific Research and Teaching clinical application and further investigation fibroblastic growth than traditional skin cells screening, breed, provides immeasurable scientific research learning value, start external height and intend new era that in body, adherent attachment is cultivated, this method proposes as to initiate both at home and abroad.

By the screening of this high target pattern, the how safe adaptive immune of conjunctive tissue engineering immunogenicity cell is escaped, and extend transplant time and increase organizational project clinical application, wound healing and dermatosis provide the solution of absolute predominance.

1, organization engineering skin; Consubstantiality-Gao intends cells in vivo epimatrix, and it is that current any method is not replaced that matrix attachment in-vitro screening method carries out the microenvironment consistence of screening with in body to seed inoblast, and preparation simply can Reusability; Build world's the first containing inoblast all layers of skin used by tissue engineering basis;

2, high specificity, by immunohistochemical methods detection, transmission electron microscope observing and flow cytomery, result shows that cell purity of the present invention is high;

3, the application in trauma repair, in gene studies, beauty and shaping reparation, oral disease, dermatological field;

4, stem cell scientific research value: the cytophyletic research of inoblast, further investigated research cell adhesion, the Scientific basis researchs such as growth, propagation, differentiation, Immune expression;

5, the method can be the regeneration of other organ vitro culture and provides good scientific research theoretical basis.

6, fibroblastic mass-producing industrialization preparation amplification.

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Claims (9)

1. one kind is combined for screening and be trained fibrocellular substratum,

Preparation nutrient solution A: inactivated fetal bovine serum 40 ~ 60ml, Transferrins,iron complexes 5 ~ 15 μ g/ml, adenine phosphate 0.5 ~ 1 × 10 ^-4/l, vitamins C 1 ~ 3 μ g/ml, Regular Insulin 2 ~ 4 μ g/ml, cholera mycin 2.5 ~ 4.5u/L, EGF5 ~ 25ng/ml, hydrocortisone 0.4 μ g/ml, DMEM/F12 (31) constant volume are to 500ml.

Preparation nutrient solution B: preparation nutrient solution B: inactivated fetal bovine serum 80ml, Transferrins,iron complexes 6 ~ 17 μ g/ml, adenosine 10 ~ 25mg/ml, adenine phosphate 0.5 ~ 1 × 10 ^-4/ L, cholera mycin 2.5 ~ 3.5u/L, penicillin 50-100u/ml, Regular Insulin 4 ~ 6 μ g/ml, EGF5 ~ 25ng/ml, bFGF10 ~ 18ng/ml, ox pituitary gland extract (BPE) 1 ~ 3mg/L, stem cell factor (SCF) 5 ~ 15ng/ml, hydrocortisone 0.3 μ g/ml, DMEM/F12 (1: 1) constant volume are to 500ml, pH7.2 ~ 7.4.

Preparation nutrient solution C: inactivated fetal bovine serum 60ml, Transferrins,iron complexes 6 ~ 17 μ g/ml, adenosine 10 ~ 25mg/ml, adenine phosphate 0.5 ~ 1 × 10 ^-4/ L, Regular Insulin 4 ~ 6 μ g/ml, bFGF10 ~ 20ng/ml, ox pituitary gland extract (BPE) 2 ~ 3mg/L, stem cell factor (SCF) 10 ~ 30ng/ml, cholera mycin 2.5 ~ 3.5u/L, Streptomycin sulphate 50 ~ 100 μ g/ml, hydrocortisone 0.3 μ g/ml, DMEM/F12 (1: 1) constant volume are to 500ml, pH7.2 ~ 7.4.

2. prepare the high method intending cells in vivo epimatrix, comprise the following steps: a) draw materials and skin degerming process; B) de-cell process; Preferably, also comprise skin sterilization or warp being taken off cell process further and carry out low temperature, dehydration, irradiation sterilization process further; More preferably step b) method for removing cells be selected from cold enzyme Crosslink bond type, enzyme-nonionogenic tenside-complexing agent integrated process, complexing agent salt adding-anion surfactant method or pancreatin associating EDTA crosslinked fluid method.

3. method described in claim 2, wherein cold enzyme Crosslink bond type is: add the cold 12 ~ 48h that disappears of 0.1 ~ 0.25% proteolytic enzyme ice bath.With containing 0.25% glutaraldehyde phosphate buffer solution, pH is between 7.20 ~ 7.40, and crosslinked 4 ~ 6h, ultrapure water or water for injection rinse 8 ~ 10 times.

4. method described in claim 2, wherein enzyme-nonionogenic tenside-complexing agent integrated process is: 0.1 ~ 0.25% neutral protease and 0.01 ~ 0.02%EDTA, TritonX-100 (0.3 ~ 0.6%) solution, acts on 24 ~ 48h under room temperature.

5. method described in claim 2, its complexing agent salt adding-anion surfactant method is: 0.01 ~ 0.02%EDTA is containing 0.6 ~ 0.8mol/L sodium chloride solution, 12 ~ 24h is acted on, rear use (0.2 ~ 0.5%SDS solution acts on 30 ~ 60min under room temperature) at 37 DEG C.

6. method described in claim 2, wherein pancreatin associating EDTA crosslinked fluid is: 0.25 ~ 0.40% trypsinase and 0.02 ~ 0.04%EDTA (1: 2), 37 DEG C of rapid digestion 10 ~ 60min.Crosslinked 4 ~ 6h in crosslinked fluid.Rinse 8 ~ 10 times with ultrapure water or water for injection after crosslinked.

7. the height prepared by either method described in claim 2-6 intends cells in vivo epimatrix.

8. the height that prepared by either method described in claim 2-6 intends cells in vivo epimatrix or high plan cells in vivo epimatrix according to claim 7 is being filtered into the purposes in fibrocyte.

9. utilize the combination of the substratum described in claim 1 and height according to claim 7 intend the screening of cells in vivo epimatrix and are trained a fibrocellular method, comprising:

A) prepare epidermal cell suspension: draw materials, digestion, peel off epidermis and collect skin graft, digestion, stop digestion with nutrient solution A according to claim 1, collecting cell suspension.Nutrient solution A nutrient solution is added after centrifugal;

B) fibroblastic screening and cultivation: height according to claim 7 is intended cells in vivo epimatrix and adds nutrient solution B according to claim 1, add the cell suspension that step a) is prepared, cultivate, cleaning, target epidermal stem cells is adherent adheres to high plan cells in vivo epimatrix, adds nutrient solution C according to claim 1 and carries out amplification cultivation;

C) enrichment Secondary Culture is digested: digestion, adds nutrient solution C according to claim 1 and carry out amplification cultivation after centrifugal.Preferably, the process of immunologic escape method draw materials before skin or obtain cell, immunologic escape method can be rays method, low temperature freeze-thaw method.