CN101318030A - Method for preparing organ type artificial skin for toxicity inspection by employing built-in cultivation method - Google Patents
Method for preparing organ type artificial skin for toxicity inspection by employing built-in cultivation method Download PDFInfo
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- CN101318030A CN101318030A CNA2008100292108A CN200810029210A CN101318030A CN 101318030 A CN101318030 A CN 101318030A CN A2008100292108 A CNA2008100292108 A CN A2008100292108A CN 200810029210 A CN200810029210 A CN 200810029210A CN 101318030 A CN101318030 A CN 101318030A
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Abstract
The invention discloses a method for preparing apparatus-type artificial skin used in toxicity checking by adopting an embedded cultivation method, and pertains to the field that the apparatus-type artificial skin replaces the skin of living animals (human body) and is used in a toxicology experiment. The method comprises the following steps: preparing a dermis bracket, preparing human fibroblasts and keratinized cells, preparing substitute for dermis, preparing special culture medium of artificial skin with no serum, cultivating apparatus-type all-layer skin and checking the toxicity of artificial skin; the skin prepared has good repetition and high degree of standardization. The apparatus-type all-layer skin prepared by the invention is closer to natural skin in anatomy and organizational structure, the way thereof applied to toxicity checking and checking indicator more conform with the actual condition of skin toxic effect; the apparatus-type all-layer skin can replace whole animal and can be directly applied to the skin toxicity experiments of the products related to health, such as chemicals, cosmetics, drugs, pesticides, etc.
Description
Technical field
The present invention relates to a kind of method of utilizing the organizational project biotechnology to make up organotypic holostrome skin, can be used for substituting living animal (human body) skin and be used for the toxicology test field.
Background technology
Skin is the organ of human body maximum, is the organ of external source chemical factor (chemicals, medicine etc.) or physical mechanical factor (ultraviolet) toxic action.Utilizing animal or human's body skin to carry out the dermal toxicity test is the test item commonly used of medicine, food additive and raw material, pesticide, biological product, the healthy and safe evaluation of chemicals.Traditional cutaneous safety evaluation experimental not only requires the animal of some, and the test period is long, the cost height, and some reaction meeting causes great misery to animal.Along with the rise of international animal protection and animal welfare motion, abroad proposed at first before more than 40 year to substitute with " minimizing ", " " with " optimization " is the 3R principle of representative, and carries out the research of zoopery alternative method on this basis energetically.Particularly nearly 20 years, no longer adopted by scientist and accepted by the government of many countries, Europe and international regulations based on the toxicology system of zootype.On November 7th, 2002, European Parliament and EU Council reached an agreement in Brussels, decision " banning use of animal to carry out cosmetics toxicity and allergic experiment in European Union's scope from 2009 " " does not allow member state to carry out zooperal cosmetics from foreign import and sale " yet.The new chemicals rules REACH of European Union (registration of chemicals, assessment, permission and restriction) also proposes to encourage the development and application of animal experiment alternative method.It is one of key area of animal experiment substitute technology that the organization engineering skin of employing external structure carries out toxicity assessment.
Tissue engineering skin is divided into tectotype epiderm substitute, dermal substitute and several classes of organotypic holostrome Graftskin.1975, Rheinwald and Green adopt the 3T3 fibroblast to make trophoderm, the In 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 with macromolecular material PGA, the PGL net is the basis, implant the worker's dermal substitute of bringing out behind the neonate fibroblast, 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 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 (Chinese patent application number 01107099.4), and this invention comprises the preparation of collagen gel and the dimensional culture of cell etc.JIUYUE in 2002 people such as banket on the 2nd have applied for relevant patent (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 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; Contain and be still waiting on the double-deck artificial skin histological structure to improve; Cultivation cycle is long, and skin function is not suitable for chronic toxicity test at the external weak point of holding time.
Summary of the invention
Technical problem to be solved by this invention just provides a kind of method that adopts embedded culture method to prepare toxicity inspection usefulness organotypic artificial skin, and this method cultivation cycle is short, skin function and active in the external length of holding time.Can satisfy the needs in toxicity inspection field.
Realize that the present invention comprises following step successively:
1, make the composite collagen dermis scaffold of forming by collagen and extracellular matrix mixture:
1) with chitosan with one of hyaluronic acid, 6-chondroitin sulfate or all be mixed with into the extracellular matrix mixture; Mass ratio is 6.5-7.5: 2.5-3.5 when adopting chitosan to mix with hyaluronic acid; Mass ratio is 7.0-7.5: 2.5-3.0 when adopting chitosan and 6-chondroitin sulfate; Adopt chitosan, hyaluronic acid, mass ratio was 4.5-5.5: 2.5-3.5: 1.5-2.5 when the 6-chondroitin sulfate all mixed;
2) under aseptic condition collagen is soaked in ice-cold-4 ℃, 0.1% acetum, collagen concentration is the 8-10mg/ml scope, regulates pH value and prepares collagen solution to 7.2-7.3, and operation should be carried out at 4 ℃;
3) in collagen solution, add 1) the extracellular matrix mixture of step gained, obtain composite collagen solution, the mass ratio of extracellular matrix mixture and collagen is 2-2.5: 7.5-8 in the composite collagen solution;
4) getting composite collagen solution that 1ml prepares, to place the aperture be circular die or the 6 porocyte culture plates of 3.5cm, with 4 ℃ of covalent cross-linkings of glutaraldehyde solution of the 1%-1.5% of 0.1ml 24 ± 0.5 hours, with 0.1mol/L phosphate buffer (PBS) rinsing 2-3 time, make spongy composite collagen substrate;
5) will be placed on-25 ℃ after the ultraviolet radiation degerming of composite collagen substrate with wavelength 254nm, 36W--30 ℃ of cryogenic refrigerator freeze overnight 12-18 hour, and then at-50 ℃--under 55 ℃, 1Pa condition, freeze dryer vacuum lyophilization 24 ± 1 hours, preparation composite collagen dermis scaffold;
2, former being commissioned to train supported the preparation of human skin fibroblast and horn cell:
1) the fresh foreskin that surgical rings is downcut thoroughly cleans with the 0.1mol/LPBS buffer that contains penicillin, streptomycin, aseptic condition goes down except that subcutaneous tissue, with the skin graft of foreskin shearing into about 1.0mm * 1.5mm, separate epidermis and corium with mass concentration 0.25% lyases (Dispases), mode has 37 ℃ of digestion to spend the night 12-18 hour in 2 ± 0.5 hours or 4 ℃, and normal saline or D-Hanks liquid clean 3-5 time;
2) epidermis partly is digested to single cell suspension with mass concentration 0.25% pancreatin+0.01%EDTA, with horn cell serum-free medium re-suspended cell, is inoculated in the culture dish of IV Collagen Type VI bag quilt, puts 5%CO
2Hatch 10-15min in 37 ℃ of incubators, the sucking-off culture fluid reaches not attached cell, continues to cultivate human keratinized cell with the horn cell serum-free medium, and half amount was changed liquid in every 48-60 hour;
3) the corium part is obtained the former foster human skin fibroblast of being commissioned to train with 0.125% trypsinization, puts into to contain high sugared DMEM (H-DMEM) culture medium culturing of 10% new-born calf serum, and half amount was changed liquid in every 24-36 hour;
The composite collagen dermis scaffold of the 1st step gained is placed on the embedded culture dish in 12 holes, it is moistening to add the H-DMEM culture medium that contains 10% new-born calf serum, inoculated for the 2nd step 3 with minimum capacity 100-200 μ l) the former foster human skin fibroblast of being commissioned to train that culture medium suspends, cell density is 1 * 10
5Cell/cm
2-2 * 10
5Cell/cm
2, treat that the 4-8 hour cell adheres to after, adding the 1.5-2ml capacity, to contain the H-DMEM culture medium of 10% new-born calf serum submerged culture, half amount was changed liquid in every 36-48 hour, cultivated 7-10 days, finished artificial dermis substitute structure; Said embedded culture dish is the Transwell of Corning Costar company or the millcell of millpore company, aperture 0.4 μ m, and the bottom supporting film is transparent polyester film, bag is 0.4% people source IV collagen type by concentration before using;
4, artificial skin special culture media preparation:
Under the aseptic condition, get the caesarean amniotic membrane of full-term pregnancy, chorion is removed in the passivity separation, and with containing the 100U/ml penicillin, the PBS of 100ug/ml streptomycin rinses blood stains well, transfers in the H-DMEM liquid; Under gnotobasis, be cut into the membrane film of circle or semicircle by big young pathbreaker people's amniotic membrane in culture plate aperture, upwards full shop of amniotic membrane epithelial surface or half shop are distributed at the bottom of the hole of 6 orifice plates, add the horn cell serum-free medium then, collect amniotic secretion liquid;
Get horn cell serum-free medium and people's amniotic secretion liquid 8-8.5 by volume: 1.5-2 mixes, and adds 5 * 10 again
-10The M cholera toxin, 2.5 μ g/ml hydrocortisone, 25 μ g/ml insulins, 25 μ g/ml transferrinss and 1 * 10
-10M thyroxine T3,0.001ng/mL people recombinate EGF, 24.3ug/mL adenine active component are mixed with the artificial skin special culture media;
5, organotypic holostrome artificial skin preparation
The 2nd step 2 of inoculation on described artificial dermis substitute of the 3rd step) the former foster human keratinized cell of being commissioned to train, inoculating cell density is 1 * 10
5Cell/cm
2-2 * 10
5Cell/cm
2Artificial skin special culture media with the preparation of the 4th step replaces horn cell serum-free medium and the H-DMEM culture medium that contains 10% new-born calf serum, the employing liquid-vapor interface is cultivated, and half amount was changed liquid in every 48-60 hour, finished the preparation of artificial skin primary product after 8-10 days;
6, detect
In a collection of artificial skin with 12 well culture plates preparations, get wherein that an artificial skin takes off from embedded culture dish, be fixed in 4% paraformaldehyde solution with the PBS preparation, conventional ethanol dehydration, paraffin embedding, slice thick 5 μ m-6 μ m, haematoxylin-Yihong (H.E) dyeing; Microscopically is observed the organizational structure of artificial skin: the removal skin texture is imperfect, skin lamination is not obvious or lack layering, horn cell content is less, skin corium fibroblast content is few, collagen arrangement disorder person, promptly gets epidermal area and contains basal layer, spinous layer and cuticular layer three-decker; Skin corium contains a large amount of fibroblasts, and collagen fiber have series arrangement; The epidermis dermis tangible artificial skin product of demarcating can be used for dermal toxicity and detects test.
Described collagen is commodity, and Main Ingredients and Appearance is the type i collagen powder art that derives from cattle tendon or Corii Sus domestica or Mus tail.
Described horn cell serum-free medium (SFM-KC) is that commercialization is available from offshore company, as the KBM (Keratinocyte Basal Medium) of U.S. BioWhittaker company or the DK-SFM of GIBCO company (Defined Keratinocyte Serum Free Medium).
Described dermis scaffold also can be entrusted commercial company's preparation, to realize the standardization of dermis scaffold.
The artificial skin product of gained is used for skin irritation and detects: solid such as cosmetics, chemicals or liquid are tried thing and directly are 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.Take off the semi-transparent supporting film of embedded culture dish, portion of tissue is fixed in paraformaldehyde, HE dyeing tissues observed pathology morphosis; Portion of tissue adopts mtt assay analysis of cells activity, or with poisonous effect indexs such as enzyme linked immunosorbent assay (ELISA) detection IL-2.
Said mtt assay is a kind of conventional method of checking cytoactive, and said ELISA detection method is a kind of conventional method that detects cytokine.
Beneficial effect: it is short that method of the present invention is cultivated the artificial skin cycle, and skin function and actively hold time longlyer external can satisfy the needs in toxicity inspection field, the artificial skin good reproducibility of preparation, standardization level height.Organotypic holostrome skin by the present invention's preparation more approaches natural skin on dissection and organizational structure, 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 products such as chemicals, cosmetics, medicine, pesticide.
Description of drawings
Fig. 1 is the ideograph of embedded cultivation toxicity inspection with organotypic holostrome artificial skin;
Fig. 2 organotypic holostrome artificial skin organization chart.
The specific embodiment
Referring to Fig. 1 and Fig. 2.
Fig. 1 is the ideograph of embedded cultivation toxicity inspection with the organotypic artificial skin
Figure 2 shows that organotypic holostrome artificial skin organization chart, HE dyeing, biology microscope sem observation, 200 times of amplifications.
The three-dimensional method that makes up the double-layer active artificial skin of the embedded culture method of employing of the present invention comprises following step successively:
Embodiment 1
1, make dermis scaffold:
1) in advance chitosan, hyaluronic acid and 6-chondroitin sulfate are pressed 5: 3: 2 usefulness common modes of mass ratio and mixed, preparation extracellular matrix mixture;
2) under the aseptic condition, the collagen powder that derives from the cattle tendon is soaked in-4 ℃, 0.1% acetum, making collagen concentration is 10mg/ml, regulates pH value to 7.2 preparation collagen solution, and operation should be carried out at 4 ℃;
The extracellular matrix mixture that 3) will be pre-mixed then adds in the bovine collagen solution, makes composite collagen solution, and collagen and extracellular matrix mixture quality ratio are 8: 2;
4) getting composite collagen solution that 1ml prepares, to place the aperture be 6 well culture plates of 3.5cm, with 4 ℃ of covalent cross-linkings of glutaraldehyde solution of 1.5% of 0.1ml 24 hours, with 0.1mol/L phosphate buffer (PBS) rinsing 3 times, make spongy composite collagen substrate;
5) will be placed on-28 ℃ of cryogenic refrigerator freeze overnight 15 hours after the ultraviolet radiation degerming of composite collagen substrate with wavelength 254nm, 36W, and then under-52 ℃, 1Pa condition, freeze dryer vacuum lyophilization 24h, preparation composite collagen dermis scaffold; The gained dermis scaffold is vesicular texture, thick about 3mm ± 1mm, aperture 70 ± 20 μ m; Dermis scaffold also can be entrusted commercial company's preparation, to realize the standardization of dermis scaffold;
2, former being commissioned to train supported the preparation of human skin fibroblast and horn cell
1) the fresh foreskin that surgical rings is downcut thoroughly cleans with the 0.1mol/LPBS buffer that contains penicillin, streptomycin, aseptic condition goes down except that subcutaneous tissue, with the skin graft of foreskin shearing into about 1.0mm * 1.5mm, with mass concentration 0.25% lyases (Dispases)) 4 ℃ of cold digestion 15 hours of spending the night, separate epidermis and corium, normal saline or D-Hanks liquid clean 3 times;
2) epidermis partly is digested to single cell suspension with mass concentration 0.25% pancreatin+0.01%EDTA, with horn cell serum-free medium re-suspended cell, is inoculated in the culture dish of IV Collagen Type VI bag quilt, puts 5%CO
2Hatch 10min in 37 ℃ of incubators, the sucking-off culture fluid reaches not attached cell, continues to cultivate with the horn cell serum-free medium; Described horn cell serum-free medium (SFM-KC) is that commercialization is available from offshore company, as the KBM (Keratinocyte Basal Medium) of U.S. BioWhittaker company or the DK-SFM (Defined Keratinocyte SFM) of GIBCO company, amount was changed liquid in per 48 hours half;
3) the corium part is obtained fibroblast with 0.125% trypsinization, puts into to contain 10% new-born calf serum H-DMEM culture medium culturing, and amount was changed liquid in per 24 hours half;
3, artificial dermis substitute preparation:
The composite collagen dermis scaffold of the 1st step gained is placed on the embedded culture dish in 12 holes, it is moistening to add the H-DMEM culture medium that contains 10% new-born calf serum, inoculated for the 2nd step 3 with minimum capacity 150 μ l) the former foster human skin fibroblast of being commissioned to train that culture medium suspends, cell density is 1.5 * 10
5Cell/cm
2, cell adhesion after 4 hours, adding 1.5ml, to contain the H-DMEM culture medium of 10% new-born calf serum submerged culture, and amount was changed liquid in per 36 hours half, cultivated 8 days, finished the artificial dermis substitute and made up; Said embedded culture dish is the Transwell of Corning Costar company, aperture 0.4 μ m, and the bottom supporting film is preferably transparent polyester film, and bag is 0.4% people source IV type collagen fiber by concentration before using;
4, artificial skin special culture media preparation
Under the aseptic condition, get the caesarean amniotic membrane of full-term pregnancy, chorion is removed in the passivity separation, and with containing the 100U/ml penicillin, the PBS of 100ug/ml streptomycin rinses blood stains well, transfers in the H-DMEM liquid; Under gnotobasis, be cut into the membrane film of circle or semicircle by big young pathbreaker people's amniotic membrane in culture plate aperture, upwards full shop of amniotic membrane epithelial surface or half shop are distributed at the bottom of the hole of 6 orifice plates, add the horn cell serum-free medium then, collect amniotic secretion liquid;
Get the horn cell serum-free medium and mixed in 8: 2 by volume, add 5 * 10 again with people's amniotic secretion liquid
-10The M cholera toxin, 2.5 μ g/ml hydrocortisone, 25 μ g/ml insulins, 25 μ g/ml transferrinss and 1 * 10
-10M thyroxine T3,0.001ng/mL people recombinate EGF, 24.3ug./mL adenine active component are mixed with the artificial skin special culture media;
Described horn cell serum-free medium is KBM (Keratinocyte Basal Medium) the horn cell serum-free medium that U.S. BioWhittaker company is bought in commercialization
5, organotypic holostrome artificial skin is cultivated
The former foster human keratinized cell of being commissioned to train of inoculation on the described artificial dermis substitute that grows on the embedded culture dish of the 3rd step, inoculating cell density is 1 * 10
5Cell/cm
2Artificial skin special culture media with the preparation of the 4th step is cultivated the H-DMEM culture medium that replaces containing serum, and liquid-vapor interface is cultivated, and amount was changed liquid in per 48 hours half, finishes the artificial skin primary product after 8-10 days and makes up;
6, check
The tissue engineering artificial skin that builds is taken off from embedded culture dish, be fixed in 4% paraformaldehyde solution with the PBS preparation, conventional ethanol dehydration, paraffin embedding, slice thick 5 μ m-6 μ m, haematoxylin-Yihong (H.E) dyeing;
Microscopically is observed the organizational structure of the artificial skin of this batch, should contain basal layer, spinous layer and cuticular layer three-decker as epidermal area; Skin corium should contain a large amount of fibroblasts, and collagen fiber have series arrangement; The epidermis dermis boundary obviously.Then this batch artificial skin can be used for toxicity inspection.
Embodiment 2
1, makes dermis scaffold
1) in advance chitosan and hyaluronic acid are pressed mass ratio usefulness common mode mixing in 7.2: 2: 8, preparation extracellular matrix mixture;
2) under the aseptic condition, the collagen powder that derives from Corii Sus domestica is soaked in-4 ℃, 0.1% acetum, making collagen concentration is 8mg/ml, regulates pH value to 7.25 preparation collagen solution, and operation should be carried out at 4 ℃, earlier will be in order to avoid the pig collagen degradation;
The extracellular matrix mixture that 3) will be pre-mixed makes composite collagen solution by being to add in the pig collagen solution at 2.5: 7.5 with pig collagen mass ratio;
4) the composite collagen solution for preparing of 1ml places 6 porocyte culture plates, with 4 ℃ of covalent cross-linkings of glutaraldehyde solution of 1.25% of 0.1ml 24.5 hours, with 0.1mol/L phosphate buffer (PBS) rinsing 3 times, makes spongy composite collagen substrate;
5) will be placed on-25 ℃ of cryogenic refrigerator freeze overnight 18 hours after the ultraviolet radiation degerming of composite collagen substrate with wavelength 254nm, 36W, and then under-53 ℃, 1Pa condition, freeze dryer vacuum lyophilization 23h, preparation composite collagen dermis scaffold; The gained dermis scaffold is vesicular texture, thick about 3mm, aperture 80 ± 20 μ m;
2, the preparation of human fibroblasts and horn cell
1) the fresh foreskin that surgical rings is downcut thoroughly cleans with the 0.1mol/LPBS buffer that contains penicillin, streptomycin, aseptic condition goes down except that subcutaneous tissue, with the skin graft of foreskin shearing into about 1.0mm * 1.5mm, with 37 ℃ of digestion of mass concentration 0.25% lyases (Dispases) 2 hours, separate epidermis and corium, normal saline or D-Hanks liquid clean 5 times;
2) epidermis partly is digested to single cell suspension with mass concentration 0.25% pancreatin+0.01%EDTA, with horn cell serum-free medium re-suspended cell, is inoculated in the culture dish of IV Collagen Type VI bag quilt, puts 5%CO
2Hatch 15min in 37 ℃ of incubators, the sucking-off culture fluid reaches not attached cell, continues to cultivate with the horn cell special culture media, and amount was changed liquid in per 54 hours half;
3) the corium part is obtained fibroblast with 0.125% trypsinization, puts into to contain 10% new-born calf serum H-DMEM culture medium culturing, and amount was changed liquid in per 30 hours half;
3, dermal substitute preparation
The composite collagen dermis scaffold of the 1st step gained is placed on the embedded culture dish in 12 holes, it is moistening to add the H-DMEM culture medium that contains 10% new-born calf serum, inoculated for the 2nd step 3 with minimum capacity 150 μ l) the former foster human skin fibroblast of being commissioned to train that culture medium suspends, cell density is 1.5 * 10
5Cell/cm
2, treat that 6 hour cells adhere to after, adding 2ml, to contain the H-DMEM culture medium of 10% new-born calf serum submerged culture, amount was changed liquid in per 42 hours half, cultivated 8 days, finished the corium structure; Said embedded culture dish is the millcell of millpore company, aperture 0.4 μ m, and the bottom supporting film is transparent polyester film, bag is 0.4% people source IV type collagen fiber by concentration before using;
4, artificial skin serum-free special culture media preparation
The GIBCO DK-SFM of company (Defined Keratinocyte SFM) horn cell serum-free medium is bought in commercialization, and the amniotic secretion liquid with aseptic condition is collected down mixed in 8.2: 1.8 by volume; Add 5 * 10 then
-10The M cholera toxin, 2.5 μ g/ml hydrocortisone, 25 μ g/ml insulins, 25 μ g/ml transferrinss and 1 * 10
-10M thyroxine T3,0.001ng/mLEGF, adenine (24.3ug/mL) isoreactivity composition are mixed with artificial skin serum-free special culture media;
6, organotypic holostrome artificial skin is cultivated
Inoculation human keratinized cell of former generation on the artificial dermis that grows on the embedded culture dish, inoculating cell density is 1 * 10
5Cell/cm
2Cultivate the H-DMEM culture medium that replaces containing serum with the artificial skin special culture media, liquid-vapor interface is cultivated, and amount was changed liquid in per 54 hours half, finishes epidermis after 10 days and makes up;
5, check
The tissue engineering artificial skin that builds is taken off from embedded culture dish, be fixed in 4% paraformaldehyde solution with the PBS preparation, conventional ethanol dehydration, paraffin embedding, slice thick 5.5 μ m, haematoxylin-Yihong (H.E) dyeing;
The demonstration skin texture is imperfect under the removal microexamination, skin lamination is not obvious or lack layering, horn cell content is less, and skin corium fibroblast content is few, the artificial skin of collagen arrangement disorder.Microscopically is observed epidermal area and is contained basal layer, spinous layer and cuticular layer three-decker, and skin corium contains fibroblast, arrangement of collagen fibers is neat, the epidermis dermis tangible artificial skin of demarcating.
Embodiment 3
1, make dermis scaffold:
1) in advance chitosan and 6-chondroitin sulfate are pressed mass ratio usefulness common mode mixing in 7.5: 2.5, preparation extracellular matrix mixture;
2) under the aseptic condition, Mus tail collagen powder is soaked in-4 ℃, 0.1% acetum, making bovine collagen concentration is 9mg/ml, regulates pH value to 7.2 preparation collagen solution, and operation should be carried out at 4 ℃;
Extracellular matrix mixture that 3) will be pre-mixed and Mus tail collagen are to add in Mus tail collagen solution at 2.2: 7.8 by mass ratio, make composite collagen solution;
4) to place the aperture be the circular die of 3.5cm to the composite collagen solution for preparing of 1ml, with 4 ℃ of covalent cross-linkings of glutaraldehyde solution of 1.25% of 0.1ml 24 hours, with 0.1mol/L phosphate buffer (PBS) rinsing 2 times, makes spongy composite collagen substrate;
5) will be placed on-28 ℃ of cryogenic refrigerator freeze overnight 18 hours after the ultraviolet radiation degerming of composite collagen substrate with wavelength 254nm, 36W, and then under-50 ℃, 1Pa condition, freeze dryer vacuum lyophilization 24h, preparation composite collagen dermis scaffold;
2, the preparation of human fibroblasts and horn cell
1) the fresh foreskin that surgical rings is downcut thoroughly cleans with the 0.1mol/LPBS buffer that contains penicillin, streptomycin, aseptic condition goes down except that subcutaneous tissue, with the skin graft of foreskin shearing into about 1.0mm * 1.5mm, with the cold digestion 16 hours of spending the night of 4 ℃ of mass concentration 0.25% lyases (Dispases), separate epidermis and corium, normal saline or D-Hanks liquid clean 5 times;
2) epidermis partly is digested to single cell suspension with mass concentration 0.25% pancreatin+0.01%EDTA, with horn cell serum-free medium re-suspended cell, is inoculated in the culture dish of IV Collagen Type VI bag quilt, puts 5%CO
2Hatch 12min in 37 ℃ of incubators, the sucking-off culture fluid reaches not attached cell, continues to cultivate with the horn cell special culture media, and amount was changed liquid in per 60 hours half;
3) the corium part is obtained fibroblast with 0.125% trypsinization, puts into to contain 10% new-born calf serum H-DMEM culture medium culturing, and amount was changed liquid in per 36 hours half;
3, dermal substitute preparation
The composite collagen dermis scaffold of the 1st step gained is placed on the embedded culture dish in 12 holes, it is moistening to add the H-DMEM culture medium that contains 10% new-born calf serum, inoculated for the 2nd step 3 with minimum capacity 100 μ l) the former foster human skin fibroblast of being commissioned to train that culture medium suspends, cell density is 1 * 10
5Cell/cm
2, treat that 8 hour cells adhere to after, adding 2ml, to contain the H-DMEM culture medium of 10% new-born calf serum submerged culture, amount was changed liquid in per 48 hours half, cultivated 7 days, finished the corium structure; Embedded culture dish is the millcell of millpore company, aperture 0.4 μ m, and the bottom supporting film is transparent polyester film, bag is 0.4% people source IV type collagen fiber by concentration before using;
4, artificial skin serum-free special culture media preparation
Buy the commercialization horn cell serum-free medium KBM (KeratinocyteBasal Medium) of U.S. BioWhittaker company, the amniotic secretion liquid with aseptic condition is collected down mixed in 8.2: 1.8 by volume, and this basis adds 5 * 10 in going up again
-10The M cholera toxin, 2.5 μ g/ml hydrocortisone, 25 μ g/ml insulins, 25 μ g/ml transferrinss and 1 * 10
-10M thyroxine T3,0.001ng/mLEGF, adenine (24.3ug/mL) isoreactivity composition are mixed with artificial skin serum-free special culture media;
6, organotypic holostrome artificial skin is cultivated
Inoculation human keratinized cell of former generation on the artificial dermis that grows on the embedded culture dish, inoculating cell density is 1 * 10
5Cell/cm
2Cultivate the H-DMEM culture medium that replaces containing serum with the artificial skin special culture media, liquid-vapor interface is cultivated, and amount was changed liquid in per 60 hours half, finishes epidermis after 7 days and makes up;
5, check
The tissue engineering artificial skin that builds is taken off from embedded culture dish, be fixed in 4% paraformaldehyde solution with the PBS preparation, conventional ethanol dehydration, paraffin embedding, slice thick 5 μ m, haematoxylin-Yihong (H.E) dyeing; Microscopically is observed the organizational structure of artificial skin, and epidermal area contains basal layer, spinous layer and cuticular layer three-decker; Skin corium should contain a large amount of fibroblasts, and collagen fiber have series arrangement; The epidermis dermis boundary obviously.This batch artificial skin can be used for toxicity inspection.
Claims (4)
1, a kind of method that adopts embedded culture method to prepare toxicity inspection usefulness organotypic artificial skin comprises following step successively:
(1) make the composite collagen dermis scaffold of forming by collagen and extracellular matrix mixture:
1) with chitosan with one of hyaluronic acid, 6-chondroitin sulfate or all be mixed with into the extracellular matrix mixture; Mass ratio is 6.5-7.5: 2.5-3.5 when adopting chitosan to mix with hyaluronic acid; Mass ratio is 7.0-7.5: 2.5-3.0 when adopting chitosan and 6-chondroitin sulfate; Adopt chitosan, hyaluronic acid, mass ratio was 4.5-5.5: 2.5-3.5: 1.5-2.5 when the 6-chondroitin sulfate all mixed;
2) under aseptic condition collagen is soaked in ice-cold-4 ℃, 0.1% acetum, collagen concentration is the 8-10mg/ml scope, regulates pH value and prepares collagen solution to 7.2-7.3, and operation should be carried out at 4 ℃;
3) in collagen solution, add 1) the extracellular matrix mixture of step gained, obtain composite collagen solution, the mass ratio of extracellular matrix mixture and collagen is 2-2.5: 7.5-8 in the composite collagen solution;
4) getting composite collagen solution that 1ml prepares, to place the aperture be circular die or the 6 porocyte culture plates of 3.5cm, with 4 ℃ of covalent cross-linkings of glutaraldehyde solution of the 1%-1.5% of 0.1ml 24 ± 0.5 hours, with 0.1mol/L phosphate buffer (PBS) rinsing 2-3 time, make spongy composite collagen substrate;
5) will be placed on-25 ℃ after the ultraviolet radiation degerming of composite collagen substrate with wavelength 254nm, 36W--30 ℃ of cryogenic refrigerator freeze overnight 12-18 hour, and then at-50 ℃--under 55 ℃, 1Pa condition, freeze dryer vacuum lyophilization 24 ± 1 hours, preparation composite collagen dermis scaffold;
(2) former being commissioned to train supported the preparation of human skin fibroblast and horn cell:
1) the fresh foreskin that surgical rings is downcut thoroughly cleans with the 0.1mol/LPBS buffer that contains penicillin, streptomycin, aseptic condition goes down except that subcutaneous tissue, with the skin graft of foreskin shearing into about 1.0mm * 1.5mm, separate epidermis and corium with mass concentration 0.25% lyases (Dispases), mode has 37 ℃ of digestion to spend the night 12-18 hour in 2 ± 0.5 hours or 4 ℃, and normal saline or D-Hanks liquid clean;
2) epidermis partly is digested to single cell suspension with mass concentration 0.25% pancreatin+0.01%EDTA, with horn cell serum-free medium re-suspended cell, is inoculated in the culture dish of IV Collagen Type VI bag quilt, puts 5%CO
2Hatch 10-15min in 37 ℃ of incubators, the sucking-off culture fluid reaches not attached cell, continues to cultivate human keratinized cell with the horn cell serum-free medium;
3) the corium part is obtained the former foster human skin fibroblast of being commissioned to train with 0.125% trypsinization, puts into to contain 10% new-born calf serum DMEM culture medium culturing;
(3) artificial dermis substitute preparation:
The composite collagen dermis scaffold of the 1st step gained is placed on the embedded culture dish in 12 holes, it is moistening to add the DMEM culture medium that contains 10% new-born calf serum, inoculated for the 2nd step 3 with minimum capacity 100-200 μ l) the former foster human skin fibroblast of being commissioned to train that culture medium suspends, cell density is 1 * 10
5Cell/cm
2-2 * 10
5Cell/cm
2, cultivated 7-10 days, finish the artificial dermis substitute and make up;
(4) artificial skin special culture media preparation:
Under the aseptic condition, get the caesarean amniotic membrane of full-term pregnancy, chorion is removed in the passivity separation, and with containing the 100U/ml penicillin, the PBS of 100ug/ml streptomycin rinses blood stains well, transfers in the DMEM liquid; Under gnotobasis, be cut into the membrane film of circle or semicircle by big young pathbreaker people's amniotic membrane in culture plate aperture, upwards full shop of amniotic membrane epithelial surface or half shop are distributed at the bottom of the hole of 6 orifice plates, add the horn cell serum-free medium then, collect amniotic secretion liquid;
Get horn cell serum-free medium and people's amniotic secretion liquid 8-8.5 by volume: 1.5-2 mixes, and adds 5 * 10 again
-10The M cholera toxin, 2.5 μ g/ml hydrocortisone, 25 μ g/ml insulins, 25 μ g/ml transferrinss and 1 * 10
-10M thyroxine T3,0.001ng/mL people recombinate EGF, 24.3ug/mL adenine active component are mixed with the artificial skin special culture media;
(5) organotypic holostrome artificial skin preparation:
The 2nd step 2 of inoculation on described artificial dermis substitute of the 3rd step) the former foster human keratinized cell of being commissioned to train, inoculating cell density is 1 * 10
5Cell/cm
2-2 * 10
5Cell/cm
2, replace horn cell serum-free medium and the DMEM culture medium that contains 10% new-born calf serum with the artificial skin special culture media of the 4th step preparation, adopt liquid-vapor interface to cultivate, finish the preparation of artificial skin primary product after 8-10 days;
(6) detect:
In a collection of artificial skin with 12 well culture plates preparations, get wherein that an artificial skin takes off from embedded culture dish, be fixed in 4% paraformaldehyde solution with the PBS preparation, conventional ethanol dehydration, paraffin embedding, slice thick 5 μ m-6 μ m, haematoxylin-Yihong (H.E) dyeing; Microscopically is observed the organizational structure of artificial skin: the removal skin texture is imperfect, skin lamination is not obvious or lack layering, horn cell content is less, skin corium fibroblast content is few, collagen arrangement disorder person, promptly gets epidermal area and contains basal layer, spinous layer and cuticular layer three-decker; Skin corium contains a large amount of fibroblasts, and collagen fiber have series arrangement, and the epidermis dermis tangible artificial skin product of demarcating can be used for dermal toxicity and detects test.
2, the embedded culture method of employing according to claim 1 prepares the method for toxicity inspection with the organotypic artificial skin, it is characterized in that:
(1) make dermis scaffold:
1) chitosan and hyaluronic acid, 6-chondroitin sulfate mixing quality ratio are 5: 3: 2;
2) collagen concentration is the 10mg/ml scope, regulates pH value to 7.2;
3) mass ratio of extracellular matrix mixture and collagen is 2: 8;
4) the glutaraldehyde covalent cross-linking is 24 hours, rinsing 3 times, and glutaraldehyde concentration is 1%-1.5%;
5) be placed on-28 ℃ of cryogenic refrigerator freeze overnight after the ultraviolet radiation degerming 15 hours, and then at-52 ℃, freeze dryer vacuum lyophilization 24 hours;
(2) former being commissioned to train supported the preparation of human skin fibroblast and horn cell:
1) 37 ℃ were spent the night and to separate epidermis and corium in 15 hours in following 2 hours or 4 ℃;
2) the KBM horn cell serum-free medium of usefulness U.S. BioWhittaker company;
3) the corium part is obtained the former foster human skin fibroblast of being commissioned to train with 0.125% trypsinization, puts into to contain 10% new-born calf serum DMEM culture medium culturing;
(3) artificial dermis substitute preparation:
With capacity 150 μ l inoculation human skin fibroblast, cell density is 1.5 * 10
5Cell/cm
2, cultivated 8 days;
(4) artificial skin special culture media preparation:
Get the horn cell serum-free medium mixed with people's amniotic secretion liquid in 8: 2 by volume;
(5) organotypic holostrome artificial skin preparation:
Inoculating cell density is 1 * 10
5Cell/cm
2, liquid-vapor interface is cultivated, and finishes the preparation of artificial skin primary product after 9 days.
3, the embedded culture method of employing according to claim 1 and 2 prepares the method for toxicity inspection with the organotypic artificial skin, it is characterized in that: said embedded culture dish is the Transwell of Corning Costar company or the millcell of millpore company, aperture 0.4 μ m, the bottom supporting film is transparent polyester film, and bag is 0.4% people source IV type collagen fiber by concentration before using.
4, the embedded culture method of employing according to claim 3 prepares the method for toxicity inspection with the organotypic artificial skin, and it is characterized in that: described horn cell serum-free medium SFM-KC is that commercialization is available from the KBM (Keratinocyte Basal Medium) of U.S. BioWhittaker company or the DK-SFM (DefinedKeratinocyte Serum Free Medium) of GIBCO company.
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| CN106806942A (en) * | 2015-12-02 | 2017-06-09 | 中国科学院大连化学物理研究所 | A kind of preparation method of cardiac muscle tissue engineering acellular collagen diaphragm |
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