CN105238740A - In-vitro screening and culturing method for tissue engineering skin seed epidermal keratinocyte high-simulation in-vivo cell extracellular matrix attachment - Google Patents
In-vitro screening and culturing method for tissue engineering skin seed epidermal keratinocyte high-simulation in-vivo cell extracellular matrix attachment Download PDFInfo
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Abstract
The invention discloses the culturing, screening and amplification technology for keratinocytes. The bottlenecks are always hot spots and difficulty in biological cell field research. At present, separation and purification are conducted through basilar membrane significant adhesion characteristics, and the keratinocytes are obtained through a three-dimensional culturing and amplification means. The technology is suitable for depending on amplified anchorage-dependent cells and providing a three-dimensional high-simulation in-vivo cell extracellular matrix system for screening and separation of target cell levels. The in-vivo attachment environment is highly simulated, and a novel biological cell culturing technology for culturing adult (embryo skin) skin cells is provided. Adhering, screened and cultured target keratinocytes in the environment of high-simulation substrates have high passage capacity and multiplication capacity and can form a cortex structure under the in-vitro environment. Immunogenicity cells are screened out to safely obtain immune escape, transplanting time is prolonged, tissue engineering clinical application is improved, wound healing is promoted, skin diseases are treated, and a clinical treatment-grade cell solution with absolute advantages is provided.
Description
One, technical field:
The invention belongs to bioengineered tissue skin seed keratinocyte (KC) technology, field of tissue engineering technology.Relate in a kind of external structure height analogue body and attach environment separation screening, skin keratin forms cultivation, the amplification technique of (KC) cell.The KC cell that the present invention obtains is applied to the spike daughter cell in organization engineering skin constructing technology.
Two, background technology:
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 (extracellularmatrix, ECM) is extensively present in the basic filling component in intercellular substance, is cell metabolism, growth, breeding, differentiation, expression, transmission, reciprocal relied on and important place.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.But the phenomenon that ECM affects cell behavior is paid close attention to and is studied not shaping theoretical basis evidence ECM and the substantial connection of cell widely.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 that cell epimatrix material utilizes is just reverse theory, and instruct the understanding that we carry out in a deep going way ECM, also retroaction obtains our available stem cell simultaneously, and this research mode promotes that subject moves ahead and develops.
Keratinocyte is at normal skin tissue's epidermal area, and vitro culture can form skin layer tissue, and vitro culture has long term growth potential and can form 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, security filtering is carried out to KC cell and reports less.
The keratinocyte (KC) that the present invention relates to is epidermic cell chief component, and the atomization different according to it and cellular form are divided into usually: basal cell layer, stratum spinosum epidermidis, granular cell layer and stratum corneum.The culture technique of KC has been widely used in the aspects such as dermatopathology, beautifying skin reduction, physiology, pharmacology, drug toxicology, organizational engineering and gene therapy.But people also do not obtain desirable result in the cultivation of clinical treatment level KC.
Keratinocyte (KC) derives from epiblast, belong to the born of the same parents that often attenuate, continuous self, in body development course, be differentiated to form epidermis, follicular epithelium main composition cell gradually, in epidermis, more than 90% belongs to this cell, and remainder is made up of melanophore, youth Ge Hansi, Merkel's cell.
Be most commonly used to the reparation of skin injury, as large-area burns, skin grafting etc., this cell can be applied and be combined with biologic bracket material, as the organization engineering skin of cutaneous integument or surrogate.As the common essential species sub-element of molded tissue block basic building.
Epidermal keratinocytes is present in epidermal area, and Peripheral blood is for good, and nutritious, residing microenvironment has superiority; Under damage or the factor such as growth-stimulating can self multiplication capacity strong; Body outer clone ability is outstanding waits predominant cell feature.Important healing cell is rebuild in the regeneration of trauma repair.
In prior art tradition method for sieving, because skin cells has stronger adhesivity to attach habit to basilar membrane, so utilize extracellular matrix this kind of characteristic to carry out cultivation screening.Skin cells screening traditional is at present following sets forth:
Method one (3T3 trophoderm method): the historical background in human skin cell vitro culture research existing 1st 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 stem cell to be subject to the impact of heterologous species.Deactivation 3T3 trophocyte can urge as the trophoderm cultivating epidermic cell.Enter attaching and the growth of the screening of shape epidermal keratinocytes, and have not by the function of inhibition contact fibroblastic growth proved.The individual 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): GraziellaP scholar's research Fibrinogen and zymoplasm are as matrix separate skin cell;
Method three (IV Collagen Type VI sticks method): JonePH etc. utilize aforesaid method from adult foreskin or cadaver skin through digestion, utilize IV Collagen Type VI to do substrates and be separated epidermal keratinocytes, 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 VanRossumMMj and KaurP etc. obtains epithelial cells from people's fritter biopsy, utilize integrin to mark different in nature mark and utilize flow cytometric sorting stem cell, 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 skin cells is screened, but marker label retaining cell can be left cannot effectively remove, exist and whether affect stem cells hyperplasia, differentiation, variability etc.This method is applicable to Marker Identification and measures skin cells, and is not suitable for the skin cells enriching method of clinical application;
Method five (xenogenesis becomes fiber suspension sieve method): scholar HagarB utilizes DMEM, low calcium ion mouse fibroblast cell conditioned medium as substratum, without trophocyte.But having research report skin cells and basilar membrane to depart to bring out and enter the differentiation cycle, be divided into transitional expansion cell, is likely the possible condition maintaining skin cells control differentiation characteristic according to the high-adhesiveness guessing skin cells and basilar membrane.If so do not utilize trophoderm, likely in long-term cultivation process, skin cell differentiation 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 HagarB 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 purported skin cell, skin cells has the same originality of consubstantiality in theory, another technical problem is, two kinds of cells are in total same culture environment, the complicacy of consideration nutrient solution, the phenomenons such as emulative growing multiplication suppression may be there is in two kinds of cells, there is technical difficulty in later separation target cell, 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 purported skin cell yield, or deactivation becomes in fiber lay down ware confluent monolayer cells or culturing process to add essential gene element.
In sum, the adhesion properties of epidermal keratinocytes extracellular matrix, it is fast-developing that the human epidermic keratinocyte that in-vitro screening is cultivated is able to method specificity at home and abroad.Adopt screening and culturing of the present invention can accomplish to promote function, avoid potential deleterious gene.Separately all there is the important directive significance do not replaced of period of history in basic scientific research, clinical treatment, cell research direction, skin regeneration injury repairing, the high structure intending organization engineering skin etc.
It is that work starts from this century (Harrison1907, Carrel1912) that research reports the foundation of regenerating tissues engineering cell, and instruct and lead biology, each large linking fields such as clinical medicine material, are called 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 YannasI.V. of JohnF.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, and collaborate solves trauma skin reparation and builds, and real has cooperated at the EugenenBellhe of Ha Fu university HonwardGreen 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 (OrganogenesisInc) 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 AdvancedBionhealing 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 OrganogenesisInc manufactures also obtains FDA approval, utilizes 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 strict above has class skin tissue engineering artificial skin, is have integral skin dermal cell to be structured in degradable holostrome artificial skin on tissue engineering bracket containing active 4 ~ 8 passage cells plantations.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, present inventor plans to build vertical organization engineering skin stem cell-height and intends cells in vivo epimatrix, matrix attachment in-vitro screening method, design contains the holostrome tissue engineering artificial skin industrialization product of epidermal keratinocytes and other attached skin cells further.
Prior art related to the present invention and reference:
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Summary of the invention
The object of the invention is to the defect overcoming prior art existence, provide tissue engineering skin seed epidermal keratinocytes height to intend the outer screening and culturing method of cells in vivo epimatrix attachment.
Object of the present invention is that technical scheme realizes under passing through:
One, the invention provides following height and intend cells in vivo epimatrix kind
(1.1) it is characterized in that: for the current situation of the technology of the present invention, the object of this invention is to provide a kind of high body entocuticle layer of intending to attach for screening as extracellular matrix, the method of adult or embryo skin epidermal keratinocytes screening and separating and vitro culture, and the high purity epidermal keratinocytes making acquisition.
(1.2) it 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 epidermal keratinocytes screening and separating and vitro culture, and the high purity epidermal keratinocytes making acquisition.
(1.3) it 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 cells screening and separating and vitro culture, and the high purity purported skin cell making acquisition.Skin cells comprises: (the attached relevant stem cell of epidermal keratinocytes, hair follicle stem cells, dermal multipotent stem cells and other appendages of skin and skin cells).
The method of this screening and separating and vitro culture, the epidermal keratinocytes of acquisition overcomes the defect of above traditional six kinds of methods.
The invention belongs to biological adult (fetus) skin born of the same parents' cell technology field, relate to the separation and ientification method of several human skin cell.Method feature of the present invention comprises: consubstantiality epidermis takes off Cuticle of cell, consubstantiality corium acellular dermal substrate, holostrome take off cell substrate preparation; The separation of consubstantiality keratinocyte; Utilize high screening and the cultivation of intending attaching substratum keratinocyte in body.Subculture has been stablized in vitro more than 8 ~ 13 (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 epidermal area 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-tissue 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 III Collagen Type VI 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 epidermal keratinocytes adhere fast, propagation expansion provides strong support.Therefore, high substrate of intending is the solution that current optimal organization engineering skin stem cell and skin cells screen.
The invention provides skin cells height and intend the concrete implementation step of cells in vivo matrix composition and method.
Of the present inventionly to have the following advantages:
The invention discloses the cultivation of skin cells keratinocyte, screening, amplification technique, these bottlenecks are biological cell area research focus and difficult point always, utilize the remarkable adhesion properties of basilar membrane to carry out separation and purification at present, adopt three-dimensional dimension to cultivate amplification means and 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 biomass cells of vitro culture adult (embryo skin) keratinocyte.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 skin cells of matrix substrates screening; Corresponding epidermis dermal cell height simulation substrate place environment sticks screening and culturing target keratinocyte.With the cell that the method obtains, there is high passage capacity, high proliferation ability, and cortex construction, this method can be formed in vitro under environment have more Scientific Research and Teaching clinical application than traditional cell screening and further investigation keratinocyte growth and proliferation of cell 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 stem cell, and preparation simply can Reusability; Build world's the first containing epidermal keratinocytes 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 epidermal keratinocytes 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, epidermal keratinocytes scientific research value: the cytophyletic research of epiderm skin keratinocyte, further investigated research epidermal keratinocytes is sticked, and the Scientific basis such as growth, propagation, Immune expression is studied;
5, the method can be the regeneration of other organ vitro culture and provides good scientific research theoretical basis.
Accompanying drawing explanation
Fig. 1 is the preparation flow that skin cells of the present invention height intends cells in vivo epimatrix.
Fig. 2 is that keratinocyte of the present invention height intends cells in vivo screening and culturing method flow.
Embodiment
Embodiment 1 the present invention height intends the concrete implementation step of preparation and the method for cells in vivo epimatrix:
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:
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:
The above method height prepared as described in above-mentioned (1.1), (1.2), (1.3) kind all capable of being combined intends cells in vivo epimatrix, carries out cell screening.
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 ".
Embodiment 2 height of the present invention is intended cells in vivo epimatrix screening epidermal keratinocytes culture method and is comprised:
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.
Skin cells screening and culturing step:
Because epidermal keratinocytes has stronger adhesivity to basilar membrane, to adhesion differential and the adhesion growth amplification habit of cell extracellular matrix, so utilize this kind of characteristic to carry out cultivation screening.
Epidermal keratinocytes screening and culturing 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, CaCl
20.2 ~ 0.3mmol/l, cholera mycin 2.5 ~ 4.5u/L, EGF5 ~ 25ng/ml, hydrocortisone 0.4 μ g/ml, commercial substratum DMEM/F12 (3:1) or (K-SFM service condition adjustment EGF5 ~ 8ng/ml) constant volume are to 500ml.
A.1: preparation nutrient solution A embodiment
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, Regular Insulin 2 ~ 3 μ g/ml, EGF5 ~ 25ng/ml, ox pituitary gland extract (BPE) 1 ~ 3mg/L, CaCl
20.1 ~ 0.3mmol/l, hydrocortisone 0.3 μ g/ml, cholera mycin 2.5 ~ 4.5u/L, vitamins C 1 ~ 3 μ g/ml, commercial substratum DMEM and commercial substratum F12 (1:1) or (K-SFM service condition adjusts EGF5 ~ 6ng/ml) constant volume are to 500ml.PH controls 7.2 ~ 7.4
B.1: preparation nutrient solution B embodiment
Preparation nutrient solution C: inactivated fetal bovine serum 30ml, Transferrins,iron complexes 6 ~ 17 μ g/ml, adenosine 10 ~ 25mg/ml, adenine phosphate 0.5 ~ 1 × 10
-4/ L, vitamins C 1 ~ 3 μ g/ml, Regular Insulin 4 ~ 6 μ g/ml, EGF5 ~ 25ng/ml, bFGF10 ~ 18ng/ml, ox pituitary gland extract (BPE) 1.5 ~ 3mg/L, CaCl
20.2 ~ 0.4mmol/l, stem cell factor (SCF) 5 ~ 15ng/ml, hydrocortisone 0.3 μ g/ml, commercial substratum DMEM and commercial substratum F12 (1:1) or (K-SFM service condition adjusts EGF5ng/ml) 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 EGF, bEGF consumption to make it break up when building tissue engineering epidermis layer and set up epidermal structure.Other basic components is constant.
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 0.25% pancreatin and 0.02%EDTA (1:1) without Ca
2+, Mg
2+, serum-free medium is solvent, obtain solution.
Digestive system preparation C: containing solute 0.25% pancreatin and 0.01%EDTA (1:1) without Ca
2+, Mg
2+, serum-free medium is solvent, obtain solution.
Skin and the preparation of screening cell freeze thawing conserving liquid:
Freeze proof solute component comprises: adenine phosphate 5 ~ 15 μ g/ml, Catergen ~ 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.
Skin sample irradiates low-temperature storage immunologic escape antigenicity cellular processes:
1. immunologic escape method rays method: epidermis sample disposal: get the skin described in five, is immersed in nutrient solution A and irradiates 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.
2. immunologic escape method freeze-thaw method:
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.
Tissue engineering skin seed epidermal keratinocytes screening step method:
Step 1, 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 ~ 18h in Digestive system preparation A and take out samples, peel off epidermis, skin corium; Collect epidermis skin graft, put in Digestive system preparation B or C.37 DEG C of 10 ~ 30min digestion, stop digestion with preparation nutrient solution A, a little after piping and druming, filter, collecting cell suspension.Centrifugal 5 ~ the 10min of 600 ~ 1000r/min, abandons or adopts supernatant liquor, and add preparation nutrient solution A nutrient solution, cell counting, re-suspended cell, cell concn is adjusted to l × 10
3~ 1 × 10
5/ ml.
Step 2, get and high intend (1.1) described in the choosing of cells in vivo epimatrix, also optional height intends (1.2), high (1.3) of intending described in the choosing of cells in vivo epimatrix described in the choosing of cells in vivo epimatrix, 3 times are rinsed with containing antibiotic PBS, 3 times are rinsed again with preparation nutrient solution A, in 37 DEG C, be immersed in nutrient solution A the 10 ~ 12h that recovers for subsequent use.
The screening of step 3, keratinocyte and cultivation: (1.1) of getting described in the choosing of ready high plan cells in vivo epimatrix are put into plate and added preparation nutrient solution B, add cell concn l × 10
3~ 1 × 10
5/ ml epidermal cell suspension, 37 DEG C, gas phase 5%CO2 incubator is cultivated 3 ~ 5h and is taken out, microscopy keratinocyte is adherent, put out the feelers, take out high (1.1) of intending described in the choosing of cells in vivo epimatrix of transfer, PBS or nutrient solution B cleans 3 ~ 6 times, and target cutin forms cell attachment and adheres on high (1.1) of intending described in the choosing of cells in vivo epimatrix.Change ware and add preparation nutrient solution C cultivation, rear every 2 ~ 3d changes liquid and once continues amplification cultivation.
Step 4, digestion enrichment Secondary Culture:
Above-mentioned keratinocyte is after amplification, and add high (1.1) of intending described in the choosing of cells in vivo epimatrix of Digestive system preparation B or C digestion, 37 DEG C of digestion 4 ~ 8min, nutrient solution B stop digestion; Blow and beat a little, collecting by filtration cell suspension transfers to centrifuge tube, and the centrifugal 4 ~ 6min of 600 ~ 1000r/min, abandons supernatant liquor, and 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 ~ 3d changes nutrient solution C processed and once continues amplification cultivation.
Tissue engineering skin seed epidermal keratinocytes biological characteristics and immunohistochemical method inspection:
The keratinocyte KC that under microscope, this combined method is cultivated, all in cloning growth, in polygon or irregular shape, as paving stone sample grows, morphology is accredited as keratinocyte.
Detect according to people's antikeratin antibody (AKA) immunohistochemical kit experimental procedure.
Cell antikeratin antibody (AKA) stained positive that fluorescence microscopy Microscopic observation obtains.
In the present invention, in lifting target cell purity, epidermal keratinocytes specific implementation method 3 step can be repeated and obtain more highly purified epidermal keratinocytes.Also can choose (1.2) simultaneously, (1.3) height intends cells in vivo epimatrix, screen.
Claims (10)
1. one kind is combined with the substratum of cultured tissue engineering skin seed coat keratinocyte for screening, and it is characterized in that, it comprises:
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, CaCl
20.2 ~ 0.3mmol/l, cholera mycin 2.5 ~ 4.5u/L, EGF5 ~ 25ng/ml, hydrocortisone 0.4 μ g/ml, commercial substratum DMEM/F12 (3:1) or K-SFM, constant volume is to 500ml; Wherein, when adopting K-SFM, described EGF is 5 ~ 8ng/ml;
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, Regular Insulin 2 ~ 3 μ g/ml, EGF5 ~ 25ng/ml, ox pituitary gland extract (BPE) 1 ~ 3mg/L, CaCl
20.1 ~ 0.3mmol/l, hydrocortisone 0.3 μ g/ml, cholera mycin 2.5 ~ 4.5u/L, vitamins C 1 ~ 3 μ g/ml, commercial substratum DMEM and commercial substratum F12 (1:1) or K-SFM, constant volume controls 7.2 ~ 7.4 to 500ml, PH; Wherein, when adopting K-SFM, described EGF is 5 ~ 6ng/ml;
Nutrient solution C: inactivated fetal bovine serum 30ml, Transferrins,iron complexes 6 ~ 17 μ g/ml, adenosine 10 ~ 25mg/ml, adenine phosphate 0.5 ~ 1 × 10
-4/ L, vitamins C 1 ~ 3 μ g/ml, Regular Insulin 4 ~ 6 μ g/ml, EGF5 ~ 25ng/ml, bFGF10 ~ 18ng/ml, ox pituitary gland extract B PE1.5 ~ 3mg/L, CaCl
20.2 ~ 0.4mmol/l, stem cell factor SCF5 ~ 15ng/ml, hydrocortisone 0.3 μ g/ml, commercial substratum DMEM and commercial substratum F12 (1:1) or K-SFM, constant volume to 500ml, PH7.2 ~ 7.4, wherein, when adopting K-SFM, described EGF is 5ng/ml.
2. prepare the high method intending cells in vivo epimatrix, it is characterized in that, it comprises step:
A) To body material and skin degerming process is got; B) de-cell process; Low temperature, dehydration, irradiation sterilization process is carried out further by sterilization or through the skin of de-cell process;
Described step b) in 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. by method according to claim 2, it is characterized in that, wherein cold enzyme Crosslink bond type comprises: 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, crosslinked 4 ~ 6h, ultrapure water or water for injection rinse 8 ~ 10 times.
4. by method according to claim 2, it is characterized in that, wherein enzyme-nonionogenic tenside-complexing agent integrated process comprises: 0.1 ~ 0.25% neutral protease and 0.01 ~ 0.02%EDTA, the TritonX-100 solution of 0.3 ~ 0.6%, acts on 24 ~ 48h under room temperature.
5. by method according to claim 2, it is characterized in that, its complexing agent salt adding-anion surfactant method comprises: 0.01 ~ 0.02%EDTA, containing 0.6 ~ 0.8mol/L sodium chloride solution, acts on 12 ~ 24h at 37 DEG C, after use 0.2 ~ 0.5%SDS solution, under room temperature act on 30 ~ 60min.
6. by method according to claim 2, it is characterized in that, wherein pancreatin associating EDTA crosslinked fluid comprises: 0.25 ~ 0.40% trypsinase of 1:2 and 0.02 ~ 0.04%EDTA, 37 DEG C of rapid digestion 10 ~ 60min, crosslinked 4 ~ 6h in crosslinked fluid, rinses 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 height according to claim 7 intends cells in vivo epimatrix in the purposes for screening in epidermal stem cells.
9. utilize the substratum described in claim 1 to combine and the high method intended the screening of cells in vivo epimatrix and cultivate epidermal stem cells according to claim 7, it is characterized in that, it comprises:
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, adds nutrient solution A after centrifugal;
B) screening of tissue engineering skin seed epidermal keratinocytes 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, destination organization engineering skin seed coat keratinocyte 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;
The process of immunologic escape method draw materials before skin or obtain target cell.
10., by method according to claim 9, it is characterized in that, described immunologic escape method is rays method or low temperature freeze-thaw method.
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