CN101352586B - Method for preparing full-thickness skin for toxicity test by stem cell raft type cultivation - Google Patents
Method for preparing full-thickness skin for toxicity test by stem cell raft type cultivation Download PDFInfo
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Abstract
The invention relates to a method for preparing full-thickness skin used in toxicity testing by adopting stem cell raft culture, which comprises: 1. preparing and modifying a polymeric dermal scaffold; 2. preparing epidermal stem cells from embryonic stem cells and skin tissues; 3. preparing pancreatic islet cells; 4. preparing a special medium for the full-thickness skin; 5. and constructing the full-thickness skin. By utilizing the characteristics of strong differentiation and proliferation capacity of the epidermal stem cell, the skin morphology, organizational structure and functional activity of the constructed full-thickness skin of the invention can meet the demands of sub-chronic toxicity testing; the synthetic scaffold material has high degree of standardization and small batch-to-batch variation; as a serum free medium is adopted through the skin construction process, the factors influencing tissue construction are reduced, thus providing a foundation for the future use in toxicity testing. The all-thickness skin prepared by the method of the invention is closer to a natural skin, the application of which in the mode of toxicity testing and detection index agrees with practical situation better, thus being able to replace animals to be directly applied to the skin toxicity testing of chemicals, cosmetics, medicines and other health-related products.
Description
Technical field
The present invention relates to a kind of stem cell tissue engineering technique that utilizes and make up the method that holostrome substitutes skin, can be used for substituting living animal (or human body) skin and be used for the toxicology test field.
Background technology
Skin is the maximum organ of human body, is the major organs of external source chemical factor (chemical, medicine, makeup etc.) or physical mechanical factor (ultraviolet ray, microwave etc.) toxic action.Utilizing animal or human's body skin to carry out the dermal toxicity test is the conventional sense project of makeup, medicine, foodstuff additive and raw material, agricultural chemicals, biological products, the healthy and safe evaluation of chemical.Traditional cutaneous safety evaluation experimental not only requires some amount and high-quality laboratory animal, and the test period is long, and cost is high, and some reaction meeting causes great misery to animal.The welfare problem of laboratory animal is paid much attention in countries in the world, the as above animal welfare method of Century European various countries, U.S. promulgation, " the management of laboratory animal regulations " of China etc.Rise along with international animal protection and animal welfare motion; Abroad proposed at first before more than 40 year to be " the 3R principle " of core content with " optimizing (refine) ", and to carry out the research of experimentation on animals alternative method on this basis energetically with " reducing (reduce) ", " substituting (replace) ".Particularly nearly 20 years, no longer be that the toxicology system on basis is adopted by scientist, and accepted by the government of many countries, Europe and international regulations with the zootype.On November 7th, 2002, European Parliament and EU Council reached an agreement in Brussels; Decision " in European Union's scope, banning use of animal to carry out makeup toxicity and allergic experiment from March 11st, 2009 " " does not allow member states to carry out zooperal makeup from foreign import and sale " yet.European Union's new chemical rules REACH (registration of chemical, assessment, permission and restriction) also proposes the development and application of encourage animals test alternative method.It is one of essential domain of animal experiment substitute technology that the organization engineering skin of employing external structure carries out toxicity assessment.
Tissue engineering skin is divided into several types of tissue-type epiderm substitute, dermal substitute and organotypic holostrome Graftskins.1975; Rheinwald and Green adopt the 3T3 inoblast to make trophoderm; Vitro culture people is from body surface chrotoplast achieving success (Rheinwald JG; Green H.Serial cultivation of strains of humanepidermal keratinozytes:the formation of keratinizing colonies from singlecells.Cell.1975,6:331-344).The artificial dermis of the commodity that U.S. Advanced Tissue Science company produces Dermagraft by name is the basis with macromolecular material PGA, PGL net; Implant the worker's dermal substitute of bringing out behind the newborn infant inoblast; In conjunction be used for clinical transplantation (Hansbrough JF from the body surface leather diaphragm; Dore C, Hansbrough WB.Clinical trails of a living dermal tissue replacement placedbeneath mesh, split-thickness skin grafts on excised burn wounds.J Burn CareRehabil; 1992,13:519-529).The Testskin artificial skin model that U.S. Organogenesis company produces; Its epidermal area is differentiated to form by human keratinized cell; Skin corium is planted to prop up in I type bovine collagen by the human fibroblasts and is configured to (Rodriguez H, O ' Connell C, Barker PE; Et al.Measurement of DNAbiomarkers for the safety of tissue-engineered medical products; Usingartificial skin as a model.Tissue Eng, 2004,10 (9-10): 1332-1345).February 6 calendar year 2001, people such as Ri Wujinjin applied for relevant patent (one Chinese patent application number 01107099.4), and this invention comprises the preparation of collagen gel and the dimensional culture of cell etc.On September 2nd, 2002, people such as banket applied for relevant patent (one Chinese patent application number 02139398.2), and this invention comprises the preparation of collagen gel and the contents such as cultivation of holostrome skin.But these several kinds of Graftskins are mainly used in burn and skin injury patients' such as ulcer, wound clinical treatment, can't substitute living animal skin maturation and be used for the dermal toxicity check.Have following shortcoming as organization engineering skin: artificial epidermis also is not an artificial skin truly owing to do not contain skin corium yet; Be still waiting in the double-deck artificial skin histological structure to improve existing containing; Culture cycle is long, and skin function is not suitable for chronic toxicity test at the external weak point of holding time.Therefore, the skin that is used for toxicity inspection not only requires form and weave construction and human body skin approaching, and requires physiological function with active similar with skin of living body, the skin good reproducibility of structure, and differences between batches are little, are convenient to the statistical analysis of toxic effect.
Summary of the invention
Technical problem to be solved by this invention, just provide a kind of utilization induce differentiation epidermal stem cells or former generation isolating epidermal stem cells, adopt raft formula culture technique; Prepare the method for toxicity inspection with holostrome skin; This method culture cycle is short, and skin contains epidermis and corium bilayer structure, and tissue is similar with normal skin with structure; Function is held time longlyer with active external, can satisfy the needs in toxicity inspection field.
Realize that the present invention comprises following step successively:
1, the preparation of polymer dermis scaffold and modification
1) PHBV support preparation:
(a) with 3-hydroxybutyric acid-co-3-hydroxyl pentanoate copolymer (poly [3-hydroxybutyric acid-co-3-hydroxyvaleric acid]; PHBV) be dissolved in and process the solution that concentration is 120-130g/L in the chloroform; The sodium-chlor (NaCl) of every liter of solution adding 930-950g is pore-creating agent then; Fully mixing obtains underflow liquid;
(b) pouring underflow liquid into aperture is in 0.8～1.0cm circular metal mould; After treating the about 70-80% of chloroform solvent volatilization; From mould, take out shaped bracket; Place stink cupboard to dry in interior room temperature 48-54 hour again, under 25 ℃ of-35 ℃ of temperature, 1000～1300Pa vacuum range inner drying was removed residual solvent in 2 hours～3 hours;
(c) shaped bracket is immersed in the deionized water; Every changed water 1 time at a distance from 6-8 hour, change water 6-8 time in 48-52 hour until the pore-creating agent filtering is clean, room temperature is dried; Under 25 ℃ of-35 ℃ of temperature, 1000～1300Pa vacuum range inner drying was processed PHBV porous three-dimensional support in 24-48 hour;
(d) with PHBV porous three-dimensional support with soaked in absolute ethyl alcohol 1-2h, respectively shine 1.5-2h with the ultraviolet pros and cons of wavelength 254nm, 36W, aseptic PBS rinsing 3-4 time, each 20-30min dries under the aseptic condition naturally;
2) preparation collagen-chitose-CHS-hyaluronic acid decorated liquid (composite collagen decorating liquid):
By mass ratio be that 14～16:2～4:1:1 prepares the raw material type i collagen, takes off the acetyl chitose, 6-CHS, mucinase A; Under the 36W ultra violet lamp; Add chitose then with 0.1% acetate stirring and dissolving type i collagen; After treating that chitin dissolves fully, again with 15-30 drip/minute flow velocity add good 6-mucinase and the chondroitin sulfate A (CSA) solution of dissolving in advance, continuation stirring and dissolving 3 hours; Make collagen, take off the acetyl chitose, 6-CHS, mucinase A ultimate density be respectively 7-8mg/ml, 1-2mg/ml, 0.5mg/ml and 0.5mg/ml; Under condition of ice bath, add 0.5ml10 * DMEM nutrient solution then, with the fast velocity modulation of the NaOH pH to 7.2-7.3 of 1mol/L;
3) modify the PHBV support:
With 2) the composite collagen decorating liquid of step preparation evenly coats 1) the PHBV porous three-dimensional rack surface of step preparation; Leave standstill 20-30min under the room temperature condition; With this face is the corium face; At the another side of PHBV porous three-dimensional support, use concentration is 0.4% people source type albumen immersion 20-30min, and this face is an epidermis side;
2, preparation epidermal stem cells
Adopt two kinds of approach to obtain epidermal stem cells (ESC), promptly induce differentiation preparation ESC and prepare ESC from human skin separation and Culture of former generation from human embryo stem cell for directional.
1) induces differentiation preparation epidermal stem cells from embryo stem cell for directional
Prepare membrane film earlier: under the aseptic condition, get the caesarean amnion of full-term pregnancy, chorion is removed in the passivity separation, and with containing 100U/ml penicillium mould, the PBS of 100ug/ml Streptomycin sulphate rinses blood stains well, transfers in the DMEM liquid again; Then under gnotobasis, be cut into the membrane film of circle or semicircle by big young pathbreaker people's amnion in 6 well culture plates or 12 well culture plate apertures, again the amnion epithelial surface is distributed in the hole of 6 orifice plates or 12 orifice plates to the upper berth at the bottom of; Add the embryonic stem cell nutrient solution of no LIF (LIF) at last, it is subsequent use to collect amniotic secretion liquid.
From commercial sources purchaser embryonic stem cell (hES), go down to posterity and cultivate after 48 hours, be that trypsinase+0.01%EDTA Digestive system digestion back of 0.125% is by 5 * 10 with final concentration
4Individual cell/cm
2Density is inoculated in the shop and is furnished with in 6 holes or 12 well culture plates of amnion, cultivates with the ES cell culture fluid of unmanned LIF; Every partly measuring at a distance from 1-2 days changed liquid; After inducing differentiation culture through 4 days, collect the epidermal stem cells (ESC) that sticks to the amnion epithelial surface;
2) prepare epidermal stem cells from human skin separation and Culture of former generation
The PBS damping fluid of the fresh foreskin of child under the surgical operation ring cutting with the 0.1mol/L that contains 100U/mL penicillium mould, 100ug/mL Streptomycin sulphate thoroughly cleaned 2 times; Go down to remove subcutaneous fat and reticular tissue at aseptic condition then, foreskin is sheared the skin graft of written treaty 2.0mm * 3.0mm; Lyase (Dispases) with mass concentration 0.25% separates epidermis and corium, cleans 3-5 time with saline water or D-Hanks liquid then;
Epidermis is partly used mass concentration 0.25% pancreatin+0.02%EDTA (by 1: 1 mixed), and 4 ℃ of digestion 2 ± 0.5 hours is digested to single cell suspension, and being inoculated in the shop then, to be furnished with mass concentration be in the petridish that encapsulates of 0.4% IV Collagen Type VI, puts 5% CO
2, stick 10-15min fast in 37 ℃ of incubators after, discard not adherent cell.Adherent cell piping and druming is broken away from the cultivation wall, by 1 * 10
4Individual/cm
2Placing for the 2nd step 1) shop of preparation is furnished with and continues on the petridish of people's amnion to cultivate, and substratum is the ES perfect medium of no LIF; Or with cell inoculation continuation cultivation on the mouse 3T3 inoblast trophoderm of handling with 5 μ g/ml ametycins, used substratum is a holostrome skin special culture media.
It is one anti-that the epidermal stem cells that adopts above-mentioned two kinds of methods preparation is integrated plain monoclonal antibody with mouse-anti human keratinous 19 (CK19) monoclonal antibody and mouse-anti people β 1, adopts the immunocytochemistry identification of cell to be that CK19 is positive to integrate the element positive with β 1.
3, the former generation human fibroblasts of preparation: get the prepuce tissues that the operation of surgery ring cutting is obtained; With the 2nd step 2) said method separate skin corium part; Obtain former being commissioned to train with 0.125% trysinization and support the HSF; Put into then and contain high sugared DMEM (H-DMEM) culture medium culturing of 10% NBCS, half amount was changed liquid in every 24-36 hour.Cultivated 3～4 days, treated cytogamy at about 80% o'clock, the trysinization collecting cell, preparation is used for the preparation of holostrome skin with the resuspended inoblast of KSM substratum.
4, holostrome skin special culture media preparation
Got for the 2nd step 1) people's amniotic secretion liquid of collecting, behind 0.22 μ m micropore group membrane filtration, with keratinocyte serum free medium (KSM) with mix at 1.5～2: 8～8.5 by volume, add crucial somatomedin 5 * 10 again
-10M Toxins,exo-, cholera, 10ng/mL people recombinate Urogastron (EGF), 5 μ g/mL Regular Insulin, 0.5 μ g/mL HYDROCORTISONE INJECTIONS, 30 μ g/mL qingfengmeisu qiongs, 15ng/mL B fungizone, 30 μ g/mL Niu Chuiti extracts, 5 μ g/ml Transferrins,iron complexess, 1 * 10
-10M thyroxine T3,24.3ug/mL VITAMIN B4 isoreactivity composition, the calcium final concentration is 0.10mM in the substratum;
5, holostrome skin preparation
The PHBV porous three-dimensional of the 1st step gained is propped up and is placed on 6 holes or 12 well culture plates; The corium of modifying through composite collagen is towards last; Support the HSF with minimum capacity 100-200 μ l inoculation with former being commissioned to train that people KSM substratum suspends, cell density is 1 * 10
5Cel l/cm
2-2 * 10
5Cell/cm
2, treat that the 4-8 hour cell adheres to after, it is submerged culture to add 1.5-2ml capacity keratinocyte serum free medium, half amount was changed liquid in every 36-48 hour, cultivated 5-7 days, completion holostrome dermis of skin partly makes up;
With PHBV support counter-rotating, inoculated for the 2nd step in epidermis side) or 2) human epidermal stem cell of preparation, inoculating cell density is 1 * 10
5Cell/cm
2-2 * 10
5Cell/cm
2, replace containing the H-DMEM substratum of 10% NBCS with the holostrome skin special culture media of the 4th step preparation, after submerged culture 72-96 hour, adopt liquid-vapo(u)r interface to cultivate, half amount was changed liquid in every 48-60 hour, accomplished the preparation of holostrome skin after 10-14 days;
6, detect
In a collection of organization engineering skin, get wherein that a skin is fixed in 4% paraformaldehyde solution with the PBS preparation, conventional ethanol dehydration, paraffin embedding, slice thick 5 μ m-6 μ m, Hematorylin-Yihong (H.E) dyeing with 12 well culture plates preparations; Microscopically is observed the weave construction of artificial skin: the removal skin texture is imperfect, skin lamination is not obvious or lack layering, keratinocyte content is less; Skin corium inoblast content is few, collagen arrangement disorder person, promptly gets epidermal area and contains differentiation structures such as stratum basale, spinous layer, granular layer and cutinized layer; Skin corium contains a large amount of inoblasts, and good with PHBV support amalgamation, collegen filament have series arrangement; The epidermis dermis tangible organization engineering skin product of demarcating can be used for dermal toxicity and detects test.
Described with mass concentration 0.25% lyase (Dispases) separation epidermis and corium, be that epidermis and corium were digested 3 ± 0.5 hours through mass concentration 0.25% lyase under 37 ℃ temperature; Or under 4 ℃ temperature, digested 12-18 hour through mass concentration 0.25% lyase.
Described embryonic stem cell nutrient solution is a kind of ordinary method, and it adopts the 80%DMEM basic culture solution, adds 20% foetal calf serum then; 1mML-Stimulina, 0.1mM β-thin basic ethanol; 1000U/ml human leukemia inhibitory factor (LIF), 100U/ml penicillium mould, 100ug/ml Streptomycin sulphate.
Described human embryo stem cell (ES cell) can be bought from commercial sources for the myeloid-lymphoid stem cell of a kind of people embryo inner cell mass that derives from or primordial germ ridge.
Described mouse 3T3 inoblast is a kind of inoblast strain that derives from the SWISS mouse, can buy from commercial sources.
Described collagen is commodity, and major ingredient is the type i collagen powder that derives from ox tendon or pigskin or mouse tail.
Described keratinocyte serum free medium KSM (Keratinocyte Serum-Free Medium) is that commercialization is available from offshore company, like the KBM (Keratinocyte Basal Medium) of U.S. BioWhittaker company or the DK-SFM of GIBCO company (Defined Keratinocyte Serum Free Medium).
Beneficial effect: the present invention is with epidermal stem cells cultured tissue engineering holostrome skin, and the cell fission multiplication capacity is strong, and it is longer that the form and the weave construction of skin is complete, function and active body are held time outward, can satisfy the needs in inferior toxicity check field; The timbering material standardization level of synthetic is high, differences between batches are little; Whole process using serum free medium in the skin building process, the clear and definite influence factor of tissue construction reduces to disturb and lays a good foundation for being used for toxicity test later on.The organizational project holostrome skin good reproducibility of preparation, stability are by force thus.Holostrome skin through the present invention's preparation more approaches natural skin on dissection and weave construction; It is applied to the mode of toxicity inspection and detects the practical situation that index more meets the dermal toxicity effect; Can replace whole animal, directly apply to the skin toxicology test of healthy related prodss such as chemical, makeup, medicine, agricultural chemicals.
Description of drawings
Fig. 1 cultivates the mode chart of toxicity inspection with organizational project holostrome skin for the raft formula;
Fig. 2 PHBV and epidermal stem cells make up the organization chart that organizational project substitutes skin.HE dyeing, biology microscope sem observation, 200 times of magnifications.
Embodiment
Embodiment 1
Comprise following step successively:
1, the preparation of polymer dermis scaffold and modification
1) preparation of PHBV support:
(a) an amount of PHBV powder is dissolved in processes the solution that concentration is 120-130g/L in the chloroform, every liter of solution sodium-chlor (NaCl) of adding 940g be pore-creating agent then, and abundant mixing obtains underflow liquid;
(b) pouring underflow liquid into aperture is in 0.8～1.0cm circular metal mould, treat the volatilization of about 80% chloroform solvent after, from mould, take out shaped bracket, place in the stink cupboard room temperature to dry in 48 hours again, residual solvent is removed in vacuum-drying under the room temperature;
(c) shaped bracket is immersed in the deionized water; Whenever changed water 1 time at a distance from 6 hours, change water 8 times in 48 hours until the pore-creating agent filtering is clean, room temperature is dried final vacuum and was processed PHBV porous three-dimensional support in dry 24 hours; The porosity of the support of processing＞90%, pore size are 80-200um;
(d) with support with soaked in absolute ethyl alcohol 1-2h, respectively shine 1.5-2h with the ultraviolet pros and cons of wavelength 254nm, 36W, aseptic PBS rinsing 3-4 time, each 20-30min dries under the aseptic condition naturally;
2) preparation composite collagen decorating liquid: (collagen is commodity, and major ingredient is the type i collagen powder that derives from ox tendon, pigskin or mouse tail)
Under the 35W ultra violet lamp; Add then with 0.1% acetate stirring and dissolving mouse tail collagen and to take off the acetyl chitose; After treating that chitosan dissolves fully, again with 15-30 drip/minute flow velocity add good mucinase A and the 6-chondroitin sulfate cellulose solution of dissolving in advance, continuation stirring and dissolving 3 hours; Mouse tail collagen, to take off acetyl chitose, 6-CHS, mucinase A net weight ratio be 15:3:1:1, and the ultimate density of four kinds of materials is respectively 7.5mg/ml, 1.5mg/ml, 0.5mg/ml and 0.5mg/ml; Under condition of ice bath, add 0.5ml10 * DMEM nutrient solution then, with the fast velocity modulation of the NaOH solution pH to 7.2 of 1mol/L;
3) modify the PHBV support
With 2) the composite collagen decorating liquid of step preparation evenly coats 1) rack surface of step preparation, room temperature leaves standstill 20-30min, is the corium face with this face, and at the support another side, using concentration is that 0.4% people source type albumen soaks 20-30min, and this face is an epidermis side.
2, induce differentiation preparation epidermal stem cells from human embryo stem cell for directional
1) preparation membrane film: under the aseptic condition, get the caesarean amnion of full-term pregnancy, chorion is removed in the passivity separation, and with containing 100U/ml penicillium mould, the PBS of 100ug/ml Streptomycin sulphate rinses blood stains well, transfers in the DMEM liquid again; Then under gnotobasis, be cut into the membrane film of circle or semicircle by big young pathbreaker people's amnion in 6 well culture plate apertures, again the amnion epithelial surface is distributed in the hole of 6 orifice plates to the upper berth at the bottom of; The embryonic stem cell nutrient solution that adds no LIF (LIF) at last, it is subsequent use to collect amniotic secretion liquid, but spreads inoculating cell in the good membrane film of cloth 3 days;
Human embryo stem cell nutrient solution wherein is a kind of ordinary method, adopts the 80%DMEM basic culture solution, adds 20% foetal calf serum then; 1mM L-glutaminate, 0.1mM β-thin basic ethanol; 1000U/ml human leukemia inhibitory factor (LIF), 100U/ml penicillium mould, 100ug/ml Streptomycin sulphate.Human embryo stem cell (ES cell) is a kind of myeloid-lymphoid stem cell that derives from embryo's inner cell mass or primordial germ ridge, buys from commercial sources.
2) embryo stem cell for directional is induced differentiation preparation epidermal stem cells: getting the ES cell that goes down to posterity and cultivated 48 hours, is that 0.125% trypsinase+0.01%EDTA Digestive system digestion back is by 5 * 10 with final concentration
4Cell/ml density is inoculated in 6 well culture plates of spreading the good amnion of cloth, cultivates with the ES cell culture fluid of no LIF; Every partly measuring at a distance from 1-2 days changed liquid; After inducing differentiation culture through 4 days, collect and stick to amnion epithelial surface epidermal stem cells.
It is one anti-that the epidermal stem cells that obtains is integrated plain monoclonal antibody with mouse-anti human keratinous 19 (CK19) monoclonal antibody and mouse-anti people β 1, adopts the immunocytochemistry identification of cell to be that CK19 is positive to integrate the element positive with β 1.
3, preparation inoblast of former generation: use the PBS damping fluid of the 0.1mol/L that contains penicillium mould, Streptomycin sulphate thoroughly to clean the fresh foreskin of child that rings is downcut; Go down except that subcutis at aseptic condition then, foreskin is sheared the skin graft of written treaty 1.0mm * 2.0mm; Separated epidermis and corium in 3 hours with 37 ℃ of following digestion of mass concentration 0.25% lyase (Dispases).The corium part is obtained former being commissioned to train with 0.125% trysinization and is supported the HSF, puts into to contain high sugared DMEM (H-DMEM) culture medium culturing of 10% NBCS, and half amount was changed liquid in every 24-36 hour.Cultivated 3～4 days, treated cytogamy at about 80% o'clock, the trysinization collecting cell, preparation is used for the preparation of holostrome skin with the resuspended inoblast of KSM substratum.
4, organizational project holostrome skin special culture media preparation
Got for the 2nd step 1) people's amniotic secretion liquid of collecting, behind 0.22 μ m micropore group membrane filtration, with keratinocyte serum free medium (KSM) with mix at 1.5～2: 8～8.5 by volume, add crucial somatomedin 5 * 10 again
-10M Toxins,exo-, cholera, 10ng/mL people recombinate Urogastron (EGF), 5 μ g/mL Regular Insulin, 0.5 μ g/mL HYDROCORTISONE INJECTIONS, 30 μ g/mL qingfengmeisu qiongs, 15ng/mL B fungizone, 30 μ g/mL Niu Chuiti extracts, 5 μ g/ml Transferrins,iron complexess, 1 * 10
-10M thyroxine T3,24.3ug/mL VITAMIN B4 isoreactivity composition.The calcium final concentration is 0.10mM in the substratum.
Keratinocyte serum free medium (KSM) is the DK-SFM (Defined Keratinocyte Serum Free Medium) of commercialization available from GIBCO company;
5, organizational project holostrome skin preparation
The PHBV porous three-dimensional of the 1st step gained is propped up and is placed on 12 well culture plates; The corium of modifying through composite collagen is towards last; Inoculated for the 3rd step with minimum capacity 100-200 μ l and support the HSF with former being commissioned to train that the DMEM substratum suspends, cell density is 1 * 10
5Cell/cm
2, treat that 4 hour cells adhere to after, it is submerged culture to add 2ml capacity keratinocyte serum free medium, half amount was changed liquid in every 36-48 hour, cultivated 5-7 days, completion organization engineering skin corium partly makes up;
With the counter-rotating of PHBV support, inoculate the human epidermal stem cell that derives from human embryo stem cell that the 2nd step prepared in epidermis side, inoculating cell density is 2 * 10
5Cell/cm
2, with submerged culture 96 hours of the organizational project holostrome skin special culture media of the 4th step preparation, adopt liquid-vapo(u)r interface to cultivate, half amount was changed liquid in every 48-60 hour, accomplished the preparation of organizational project holostrome skin products after 10-14 days;
5, detect
In a collection of organization engineering skin, get wherein that a skin is fixed in 4% paraformaldehyde solution with the PBS preparation, conventional ethanol dehydration, paraffin embedding, slice thick 5 μ m-6 μ m, Hematorylin-Yihong (H.E) dyeing with 12 well culture plates preparations; Microscopically is observed the weave construction of artificial skin: the removal skin texture is imperfect, skin lamination is not obvious or lack layering, keratinocyte content is less; Skin corium inoblast content is few, collagen arrangement disorder person, promptly gets epidermal area and contains differentiation structures such as stratum basale, spinous layer, granular layer and cutinized layer; Skin corium contains a large amount of inoblasts, and good with PHBV support amalgamation, collegen filament have series arrangement; The epidermis dermis tangible organization engineering skin product of demarcating can be used for dermal toxicity and detects test.
The artificial skin product of gained is used for skin irritation and detects: solid such as makeup, chemical or liquid are tried thing directly be positioned over the artificial skin surface; The liquid consumption is 10 μ l; The solid consumption is that (10 ± 2) mg and coating are even; After the effect (15 ± 1) minute, remove with aseptic PBS flushing and to be tried thing and hatch (42 ± 1) hour again.From 12 porocyte culture plates, take off organizational project and substitute skin, portion of tissue is fixed in Paraformaldehyde 96, HE dyeing tissues observed pathology morphological structure; Portion of tissue adopts the mtt assay analysis of cells active, or with toxic effect indexs such as enzyme-linked immunosorbent assay (ELISA) detection IL-2.
Described mtt assay is a kind of ordinary method of checking cytoactive, and described ELISA detection method is a kind of ordinary method that detects cytokine.
Embodiment 2
1, the preparation of polymer dermis scaffold and modification
1) preparation of PHBV support:
(a) an amount of PHBV powder is dissolved in processes the solution that concentration is 130g/L in the chloroform, every liter of solution sodium-chlor (NaCl) of adding 930g be pore-creating agent then, and abundant mixing obtains underflow liquid;
(b) pouring underflow liquid into diameter is 0.15-0.2ml in the 1.0cm circular metal mould, treat solvent evaporates after, from mould, take out shaped bracket, place in the stink cupboard room temperature to dry in 54 hours, residual solvent is removed in vacuum-drying;
(c) shaped bracket is immersed in the deionized water, whenever changed water 1 time at a distance from 7 hours, change water 7 times in 49 hours until the pore-creating agent filtering is clean, room temperature is dried final vacuum and was processed PHBV porous three-dimensional support in dry 24 hours;
(d) with PHBV porous three-dimensional support with soaked in absolute ethyl alcohol 2h, respectively shine 2h with the ultraviolet pros and cons of wavelength 25nm, 36W, aseptic PBS rinsing 4 times, each 20min dries under the aseptic condition naturally;
2) preparation composite collagen decorating liquid:
Under ultra violet lamp; Add then with 0.1% acetate stirring and dissolving mouse tail collagen and to take off the acetyl chitose; After treating that chitosan dissolves fully, again with 15-30 drip/minute flow velocity add good mucinase A and the 6 chondroitin sulfate cellulose solutions of dissolving in advance, continued stirring and dissolving 3 hours; Mouse tail collagen, to take off acetyl chitose, 6-CHS, mucinase A net weight ratio be 14:4:1:1, and ultimate density is respectively 7mg/ml, 2mg/ml, 0.5mg/ml and 0.5mg/ml; Under condition of ice bath, add the NaOH fast velocity modulation pH to 7.3 of 0.5ml10 * DMEM nutrient solution mixing then with 1mol/L;
3) modify the PHBV support:
With 2) the composite collagen decorating liquid of step preparation evenly coats 1) rack surface of step preparation, room temperature leaves standstill 30min, is the corium face with this face, and at the support another side, using concentration is that 0.4% people source type albumen soaks 30min, and this face is an epidermis side.
2, human skin separation preparation of former generation epidermal stem cells
1) preparation membrane film: under the aseptic condition, get the caesarean amnion of full-term pregnancy, chorion is removed in the passivity separation, rinses blood stains well with containing two anti-PBS, transfers in the DMEM liquid again; Then under gnotobasis, be cut into the membrane film of circle or semicircle by big young pathbreaker people's amnion in 12 well culture plate apertures, again the amnion epithelial surface is distributed in the hole of 12 orifice plates to the upper berth at the bottom of; Add keratinocyte serum free medium (SFM) at last, it is subsequent use to collect amniotic secretion liquid, but inoculating cell in the good membrane film of shop cloth 3 days;
2) human skin separation preparation of former generation epidermal stem cells: use the PBS damping fluid of the 0.1mol/L that contains penicillium mould, Streptomycin sulphate thoroughly to clean the fresh foreskin of child that rings is downcut; Go down except that subcutis at aseptic condition then, foreskin is sheared the skin graft of written treaty 2.0mm * 2.5mm; With 4 ℃ of digestion of mass concentration 0.25% lyase (Dispases) spend the night (12 hours) separate epidermis and corium, saline water cleans 3 times.Epidermis is partly used mass concentration 0.25% pancreatin+0.01%EDTA (by 1: 1 mixed), and 4 ℃ of digestion 2 hours is digested to single cell suspension, and being inoculated in the shop then, to be furnished with mass concentration be in the petridish that encapsulates of 0.4% IV Collagen Type VI, puts 5% CO
2, stick 15min fast in 37 ℃ of incubators after, discard not adherent cell, adherent cell piping and druming is broken away from cultivates wall, by 1 * 10
4Individual/cm
2Placing for the 2nd step 1) shop of preparation is furnished with and continues on the petridish of people's amnion to cultivate, and used substratum is the keratinocyte serum free medium.It is one anti-that the epidermal stem cells that obtains is integrated plain monoclonal antibody with mouse-anti human keratinous 19 (CK19) monoclonal antibody and mouse-anti people β 1, adopts the immunocytochemistry identification of cell to be that CK19 is positive to integrate the element positive with β 1.
Keratinocyte serum free medium (KSM) is the DK-SFM (Defined Keratinocyte Serum Free Medium) of commercialization available from GIBCO company;
3, the former generation human fibroblasts of preparation: got for the 2nd step 2) isolating dermis of skin part is supported the HSF with 0.125% trysinization through obtaining former being commissioned to train; Put into then and contain high sugared DMEM (H-DMEM) culture medium culturing of 10% NBCS, half amount was changed liquid in every 24-36 hour; Cultivated 3～4 days, treated cytogamy at about 80% o'clock, the trysinization collecting cell, preparation is used for the preparation of holostrome skin with the resuspended inoblast of KSM substratum.
4, organizational project holostrome skin special culture media preparation
Got for the 2nd step 1) people's amniotic secretion liquid of collecting, behind 0.22 μ m micropore group membrane filtration, with keratinocyte serum free medium (KSM) with mix at 1.5～2: 8～8.5 by volume, add crucial somatomedin 5 * 10 again
-10M Toxins,exo-, cholera, 10ng/mL people recombinate Urogastron (EGF), 5 μ g/mL Regular Insulin, 0.5 μ g/mL HYDROCORTISONE INJECTIONS, 30 μ g/mL qingfengmeisu qiongs, 15ng/mL B fungizone, 30 μ g/mL Niu Chuiti extracts, 5 μ g/ml Transferrins,iron complexess, 1 * 10
-10M thyroxine T3,24.3ug/mL VITAMIN B4 isoreactivity composition.The calcium final concentration is 0.10mM in the substratum.
4, organizational project holostrome skin preparation:
The PHBV porous three-dimensional of the 1st step gained is propped up and is placed on 12 well culture plates, and the corium of modifying through composite collagen is towards last, inoculates former being commissioned to train that the 3rd substratum suspends with minimum capacity 100-200 μ l and supports the HSF, and cell density is 1 * 10
5Cell/cm
2, treat that 4 hour cells adhere to after, it is submerged culture to add 2ml capacity keratinocyte serum free medium, amount was changed liquid in per 48 hours half, cultivated 5 days, completion organization engineering skin corium partly makes up;
With PHBV support counter-rotating, epidermis side inoculate the preparation of the 2nd step derive from the human skin tissue and former generation human epidermal stem cell, inoculating cell density is 2 * 10
5Cell/cm
2, with submerged culture 80 hours of the organizational project holostrome skin special culture media of the 4th step preparation, adopt liquid-vapo(u)r interface to cultivate then, amount was changed liquid in per 48 hours half, completion people organization engineering skin product prepn after 12 days;
6, detect
With embodiment 1.
Embodiment 3
1, the preparation of polymer dermis scaffold and modification
1) preparation of PHBV support:
(a) an amount of PHBV powder is dissolved in processes the solution that concentration is 130g/L in the chloroform, every liter of solution sodium-chlor (NaCl) of adding 940g be pore-creating agent then, and abundant mixing obtains underflow liquid;
(b) pouring underflow liquid into diameter is 0.15-0.2ml in the 1.0cm circular metal mould, treat solvent evaporates after, from mould, take out shaped bracket, place in the stink cupboard room temperature to dry in 54 hours, residual solvent is removed in vacuum-drying;
(c) shaped bracket is immersed in the deionized water, whenever changed water 1 time at a distance from 8 hours, change water 7 times in 56 hours until the pore-creating agent filtering is clean, room temperature is dried final vacuum and was processed PHBV porous three-dimensional support in dry 24 hours;
(d) with PHBV porous three-dimensional support with soaked in absolute ethyl alcohol 2h, respectively shine 2h with the ultraviolet pros and cons of wavelength 254nm, 36W, aseptic PBS rinsing 4 times, each 20min dries under the aseptic condition naturally;
2) preparation composite collagen decorating liquid:
Under ultra violet lamp; Add then with 0.1% acetate stirring and dissolving mouse tail collagen and to take off the acetyl chitose; After treating that chitosan dissolves fully, again with 15-30 drip/minute flow velocity add good mucinase A and the 6 chondroitin sulfate cellulose solutions of dissolving in advance, continued stirring and dissolving 3 hours; Mouse tail collagen, to take off acetyl chitose, 6-CHS, mucinase A net weight ratio be 16:2:1:1, and ultimate density is respectively 8mg/ml, 1mg/ml, 0.5mg/ml and 0.5mg/ml; Under condition of ice bath, add 0.5ml10 * DMEM nutrient solution then, with the fast velocity modulation of the NaOH pH to 7.3 of 1moil/L;
3) modify the PHBV support:
With 2) the composite collagen decorating liquid of step preparation evenly coats 1) rack surface of step preparation, room temperature leaves standstill 30min, is the corium face with this face, and at the support another side, using concentration is that 0.4% people source type albumen soaks 30min, and this face is an epidermis side.
2, human skin separation preparation of former generation epidermal stem cells
1) preparation membrane film: under the aseptic condition, get the caesarean amnion of full-term pregnancy, chorion is removed in the passivity separation, rinses blood stains well with containing two anti-PBS, transfers in the DMEM liquid again; Then under gnotobasis, be cut into the membrane film of circle or semicircle by big young pathbreaker people's amnion in 12 well culture plate apertures, again the amnion epithelial surface is distributed in the hole of 12 orifice plates to the upper berth at the bottom of; Add keratinocyte serum free medium (SFM) at last, it is subsequent use to collect amniotic secretion liquid, but inoculating cell in the good membrane film of shop cloth 3 days;
2) human skin separation preparation of former generation epidermal stem cells: use the PBS damping fluid of the 0.1mol/L that contains penicillium mould, Streptomycin sulphate thoroughly to clean the fresh foreskin of child that rings is downcut; Go down except that subcutis at aseptic condition then, foreskin is sheared the skin graft of written treaty 1.5mm * 2.5mm; With 4 ℃ of digestion of mass concentration 0.25% lyase (Dispases) spend the night (12 hours) separate epidermis and corium, saline water cleans 3 times.Epidermis is partly used mass concentration 0.25% pancreatin+0.01%EDTA (by 1: 1 mixed), and 4 ℃ digested 2 hours, were digested to single cell suspension, were inoculated in then in the petridish that encapsulates IV collagen, put 5%CO
2, stick 15min fast in 37 ℃ of incubators after, discard not adherent cell, adherent cell piping and druming is broken away from cultivates wall, be inoculated on the mouse 3T3 one-tenth cytotrophoblast of handling with 5 μ g/ml ametycins.Used substratum is the keratinocyte serum free medium.It is one anti-that the epidermal stem cells that obtains is integrated plain monoclonal antibody with mouse-anti human keratinous 19 (CK19) monoclonal antibody and mouse-anti people β 1, adopts the immunocytochemistry identification of cell to be that CK19 is positive to integrate the element positive with β 1.
Keratinocyte serum free medium (KSM) is the KBM (Keratinocyte Basal Medium) of commercialization available from U.S. BioWhittaker company.
3, the former generation human fibroblasts of preparation: got for the 2nd step 2) isolating dermis of skin part is supported the HSF with 0.125% trysinization through obtaining former being commissioned to train; Put into then and contain high sugared DMEM (H-DMEM) culture medium culturing of 10% NBCS, half amount was changed liquid in every 24-36 hour; Cultivated 3～4 days, treated cytogamy at about 80% o'clock, the trysinization collecting cell, preparation is used for the preparation of holostrome skin with the resuspended inoblast of KSM substratum.
4, organizational project holostrome skin special culture media preparation
Got for the 2nd step 1) people's amniotic secretion liquid of collecting, behind 0.22 μ m micropore group membrane filtration, with keratinocyte serum free medium (KSM) with mix at 1.5～2: 8～8.5 by volume, add crucial somatomedin 5 * 10 again
-10M Toxins,exo-, cholera, 10ng/mL people recombinate Urogastron (EGF), 5 μ g/mL Regular Insulin, 0.5 μ g/mL HYDROCORTISONE INJECTIONS, 30 μ g/mL qingfengmeisu qiongs, 15ng/mL B fungizone, 30 μ g/mL Niu Chuiti extracts, 5 μ g/ml Transferrins,iron complexess, 1 * 10
-10M thyroxine T3,24.3ug/mL VITAMIN B4 isoreactivity composition.The calcium final concentration is 0.10mM in the substratum.
5, organizational project holostrome skin preparation:
The PHBV porous three-dimensional of the 1st step gained is propped up and is placed on 12 well culture plates, and the corium of modifying through composite collagen is towards last, inoculates former being commissioned to train that the 3rd substratum suspends with minimum capacity 100-200 μ l and supports the HSF, and cell density is 1 * 10
5Cell/cm
2, treat that 4 hour cells adhere to after, it is submerged culture to add 2ml keratinocyte serum free medium, amount was changed liquid in per 48 hours half, cultivated 5 days, completion organization engineering skin corium partly makes up;
With PHBV support counter-rotating, epidermis side inoculate the preparation of the 2nd step derive from the human skin tissue and former generation human epidermal stem cell, inoculating cell density is 2 * 10
5Cell/cm
2, with submerged culture 96 hours of the organizational project holostrome skin special culture media of the 4th step preparation, adopt liquid-vapo(u)r interface to cultivate, amount was changed liquid in per 48 hours half, completion people organization engineering skin product prepn after 12 days;
6, detect
With embodiment 1.
Claims (5)
1. one kind is adopted the cultivation of stem cell raft formula to prepare the method for toxicity inspection with holostrome skin, comprises the steps: successively
The preparation of I, polymer dermis scaffold and modification
1) PHBV support preparation:
(a) 3-hydroxybutyric acid-co-3-hydroxyl pentanoate copolymer PHBV is dissolved in processes the solution that concentration is 120-130g/L in the chloroform, every liter of solution sodium-chlor of adding 930-950g is pore-creating agent then, and fully mixing obtains underflow liquid;
(b) pouring underflow liquid into aperture is in 0.8～1.0cm circular metal mould; After treating chloroform solvent volatilization 70-80%; From mould, take out shaped bracket; Place stink cupboard to dry in interior room temperature 48-54 hour again, under 25 ℃ of-35 ℃ of temperature, 1000～1300Pa vacuum range inner drying was removed residual solvent in 2 hours～3 hours;
(c) shaped bracket is immersed in the deionized water; Every changed water 1 time at a distance from 6-8 hour, change water 6-8 time in 48-52 hour until the pore-creating agent filtering is clean, room temperature is dried; Under 25 ℃ of-35 ℃ of temperature, 1000～1300Pa vacuum range inner drying was processed PHBV porous three-dimensional support in 24-48 hour;
(d) with PHBV porous three-dimensional support with soaked in absolute ethyl alcohol 1-2h, respectively shine 1.5-2h with the ultraviolet pros and cons of wavelength 254nm, 36W, aseptic PBS rinsing 3-4 time, each 20-30min dries under the aseptic condition naturally;
2) preparation collagen-chitose-CHS-hyaluronic acid decorated liquid:
By mass ratio is 14～16: 2～4: prepare raw material type i collagen, take off acetyl chitose, 6-CHS, mucinase A at 1: 1; Under the 36W ultra violet lamp; Add then with 0.1% acetate stirring and dissolving type i collagen and to take off the acetyl chitose; After waiting to take off the acetyl chitose and dissolving fully, again with 15-30 drip/minute flow velocity add good 6-CHS and the mucinase A solution of dissolving in advance, continuation stirring and dissolving 3 hours; Make collagen, take off the acetyl chitose, 6-CHS, mucinase A ultimate density be respectively 7-8mg/ml, 1-2mg/ml, 0.5mg/ml and 0.5mg/ml; Under condition of ice bath, add 0.5ml10 * DMEM nutrient solution then, with the fast velocity modulation of the NaOH pH to 7.2-7.3 of 1mol/L;
3) modify the PHBV support:
With 2) the composite collagen decorating liquid of step preparation evenly coats 1) the PHBV porous three-dimensional rack surface of step preparation; Leave standstill 20-30min under the room temperature condition; With this face is the corium face; At the another side of PHBV porous three-dimensional support, use concentration is 0.4% people source IV collagen type immersion 20-30min, and this face is an epidermis side;
II, preparation epidermal stem cells
1) preparation of amniotic secretion liquid
Prepare membrane film earlier: under the aseptic condition, get the caesarean amnion of full-term pregnancy, chorion is removed in the passivity separation, and with containing 100U/ml penicillium mould, the PBS of 100ug/ml Streptomycin sulphate rinses blood stains well, transfers in the DMEM liquid again; Then under gnotobasis, be cut into the membrane film of circle or semicircle by big young pathbreaker people's amnion in 6 well culture plates or 12 well culture plate apertures, again the amnion epithelial surface is distributed in the hole of 6 orifice plates or 12 orifice plates to the upper berth at the bottom of; Add the embryonic stem cell nutrient solution of no LIF LIF at last, it is subsequent use to collect amniotic secretion liquid;
2) prepare epidermal stem cells from human skin separation and Culture of former generation
The PBS damping fluid of the fresh foreskin of child under the surgical operation ring cutting with the 0.1mol/L that contains 100U/mL penicillium mould, 100ug/mL Streptomycin sulphate thoroughly cleaned 2 times; Go down to remove subcutaneous fat and reticular tissue at aseptic condition then, foreskin is sheared the skin graft of written treaty 2.0mm * 3.0mm; Lyase Dispases with mass concentration 0.25% separates epidermis and corium, cleans 3-5 time with saline water or D-Hanks liquid then;
Epidermis partly with mass concentration 0.25% pancreatin+0.02%EDTA by 1: 1 mixed, 4 ℃ of digestion 2 ± 0.5 hours is digested to single cell suspension, being inoculated in the shop then, to be furnished with mass concentration be in the petridish that encapsulates of 0.4% IV Collagen Type VI, puts 5%CO
2, stick 10-15min fast in 37 ℃ of incubators after, discard not adherent cell; Adherent cell piping and druming is broken away from the cultivation wall, by 1 * 104/cm
2Placing for the 2nd step 1) shop of preparation is furnished with and continues on the petridish of people's amnion to cultivate, and substratum is the ES perfect medium of no LIF; Or with cell inoculation continuation cultivation on the mouse 3T3 inoblast trophoderm of handling with 5 μ g/ml ametycins, used substratum is a holostrome skin special culture media;
With the 2nd step 2) to integrate plain monoclonal antibody with mouse-anti human keratinous 19CK19 monoclonal antibody and mouse-anti people β 1 be one anti-for the epidermal stem cells of said method preparation, adopts the immunocytochemistry identification of cell to be that CK19 is positive to integrate the element positive with β 1;
III, the former generation human fibroblasts of preparation:
Get the prepuce tissues that the operation of surgery ring cutting is obtained, with the 2nd step 2) said method separate skin corium part, obtain former being commissioned to train with 0.125% trysinization and support the HSF; Put into then and contain the high sugared DMEM culture medium culturing of 10% NBCS; Half amount was changed liquid in every 24-36 hour, cultivated 3～4 days, when treating cytogamy 80%; The trysinization collecting cell, preparation is used for the preparation of holostrome skin with the resuspended inoblast of KSM substratum;
IV, the preparation of holostrome skin special culture media
Got for the 2nd step 1) people's amniotic secretion liquid of collecting, behind 0.22 μ m micropore group membrane filtration, with keratinocyte serum free medium KSM with mix at 1.5～2: 8～8.5 by volume, add crucial somatomedin 5 * 10 again
-10M Toxins,exo-, cholera, 10ng/mL people recombinate Urogastron EGF, 5 μ g/mL Regular Insulin, 0.5 μ g/mL HYDROCORTISONE INJECTIONS, 30 μ g/mL qingfengmeisu qiongs, 15ng/mL B fungizone, 30 μ g/mL Niu Chuiti extracts, 5 μ g/ml Transferrins,iron complexess, 1 * 10
-10M thyroxine T3 and 24.3ug/mL VITAMIN B4, the calcium final concentration is 0.10mM in the substratum;
V, the preparation of holostrome skin:
The PHBV porous three-dimensional of the 1st step gained is propped up and is placed on 6 holes or 12 well culture plates; The corium of modifying through composite collagen is towards last; Support the HSF with minimum capacity 100-200 μ l inoculation with former being commissioned to train that people KSM substratum suspends, cell density is 1 * 10
5Cell/cm
2-2 * 10
5Cell/cm
2, treat that the 4-8 hour cell adheres to after, it is submerged culture to add 1.5-2ml capacity keratinocyte serum free medium, half amount was changed liquid in every 36-48 hour, cultivated 5-7 days, completion holostrome dermis of skin partly makes up;
With PHBV support counter-rotating, inoculated for the 2nd step 2 in epidermis side) human epidermal stem cell of preparation, inoculating cell density is 1 * 10
5Cell/cm
2-2 * 10
5Cell/cm
2, replace containing the H-DMEM substratum of 10% NBCS with the holostrome skin special culture media of the 4th step preparation, after submerged culture 72-96 hour, adopt liquid-vapo(u)r interface to cultivate, half amount was changed liquid in every 48-60 hour, accomplished the preparation of holostrome skin after 10-14 days;
VI, detection
In a collection of organization engineering skin, get wherein that a skin is fixed in 4% paraformaldehyde solution with the PBS preparation, conventional ethanol dehydration, paraffin embedding, slice thick 5 μ m-6 μ m, Hematorylin-Yihong H.E dyeing with 12 well culture plates preparations; Microscopically is observed the weave construction of artificial skin: the removal skin texture is imperfect, skin lamination is not obvious or lack layering, keratinocyte content is less; Skin corium inoblast content is few, collagen arrangement disorder person, promptly gets epidermal area and contains differentiation structures such as stratum basale, spinous layer, granular layer and cutinized layer; Skin corium contains a large amount of inoblasts, and good with PHBV support amalgamation, collegen filament have series arrangement; The epidermis dermis tangible organization engineering skin product of demarcating can be used for dermal toxicity and detects test.
2. employing stem cell raft formula according to claim 1 is cultivated and is prepared the method for toxicity inspection with holostrome skin; It is characterized in that: described with mass concentration 0.25% lyase Dispases separation epidermis and corium, be that epidermis and corium were digested 3 ± 0.5 hours through mass concentration 0.25% lyase under 37 ℃ temperature; Or with epidermis and corium under 4 ℃ temperature through mass concentration 0.25% lyase digestion 12-18 hour.
3. employing stem cell raft formula according to claim 2 is cultivated and is prepared the method for toxicity inspection with holostrome skin, and it is characterized in that: described mouse 3T3 inoblast is the inoblast strain of a kind of SWISS of deriving from mouse, can buy from commercial sources.
4. employing stem cell raft formula according to claim 3 is cultivated and prepared the method for toxicity inspection with holostrome skin, it is characterized in that: described collagen is for being the type i collagen powder that derives from ox tendon or pigskin or mouse tail.
5. employing stem cell raft formula according to claim 4 is cultivated and prepared the method for toxicity inspection with holostrome skin, it is characterized in that: described keratinocyte serum free medium SFM-KC adopts KBM or DK-SFM.
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| CN103468637A (en) * | 2013-09-26 | 2013-12-25 | 青岛农业大学 | Method for high-efficiency in-vitro separation culture of porcine skin-derived stem cells |
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| CN102462864B (en) * | 2010-11-12 | 2014-06-18 | 中国辐射防护研究院 | Novel method for constructing tissue engineering skin |
| CN102367434B (en) * | 2011-06-03 | 2013-06-05 | 中国人民解放军第二军医大学 | Amniotic membrane microcarrier capable of simulating niche microenvironment for growth of epidermal stem cells and skin substitute thereof |
| CN102294055B (en) * | 2011-08-15 | 2013-11-13 | 沈雁 | Method for preparing bionic skin covering from epidermal stem cell-modified chitin membrane and purpose of covering |
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| CN104548214B (en) * | 2015-02-10 | 2017-01-18 | 广州赛莱拉干细胞科技股份有限公司 | Artificial skin and preparation method thereof |
| CN105132357B (en) * | 2015-07-29 | 2019-12-06 | 苏州诺普再生医学有限公司 | serum-free culture system for epidermal cell culture |
| CN108018256A (en) * | 2016-11-01 | 2018-05-11 | 上海家化联合股份有限公司 | Artificial three-dimensional epidermis model, its construction method and culture medium |
| CN109402047A (en) * | 2017-09-18 | 2019-03-01 | 武汉原生原代生物医药科技有限公司 | Promote tissue adhension and growth bioadhesive and its preparation method and application |
| CN111518743A (en) * | 2020-04-20 | 2020-08-11 | 南昌大学 | Method for constructing human dermal fibroblast in-vitro thermal injury model and application thereof |
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