CN101297981B - Tissue engineering skin construction method and tissue engineering skin produced by the same - Google Patents
Tissue engineering skin construction method and tissue engineering skin produced by the same Download PDFInfo
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- CN101297981B CN101297981B CN2008101161081A CN200810116108A CN101297981B CN 101297981 B CN101297981 B CN 101297981B CN 2008101161081 A CN2008101161081 A CN 2008101161081A CN 200810116108 A CN200810116108 A CN 200810116108A CN 101297981 B CN101297981 B CN 101297981B
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Abstract
The present invention discloses a method for constructing tissue engineering skin, which comprises the steps as follows: (1) a tissue engineering skin nutrient solution is prepared; (2) a demic mechanocyte feeder layer is prepared; (3) a gelatin biomaterial is prepared; (4) the tissue engineering skin is constructed and cultivated. The method for constructing the tissue engineering skin adopts the adding of the mechanocytes after proliferation inhibition treatment to complete the interaction with epidermic cells so as to promote the mature and differentiation degree of the epidermic cells, has significant effect on improving the accuracy of compound toxicity detection in vitro to ensure that the prepared tissue engineering skin can more effectively resist the toxic action of the compound on competent cells and the forecast precision of the prepared tissue engineering skin to the compound toxicity is improved so as to create more effective animal substitution detection methods which are based on the tissue engineering skin.
Description
Technical field
The present invention relates to a kind of method of utilizing tissue engineering technique to make up skin.
Background technology
Skin is the organ of human body maximum, and it is that epidermal area, skin corium and hypodermic layer constitute by three parts.Epidermal area is positioned at the outermost layer of skin, and thickness is between 0.06-0.8mm, and its micro structure is made of basal layer, prickle cell layer, granular cell layer and horny layer.Horny layer is the end product of epidermis cell differentiation, and 75%-80% was a protein during it was formed, and 5%-15% is a lipid, is made of the structure of the about 10 μ m of thickness the 10-15 confluent monolayer cells.Horny layer has many important physical functions, can effectively stop extraneous toxic chemical to the skin activity cells injury, particularly potential issuable cytotoxicity in the cosmetic composition is had very important barrier action.
Organization engineering skin has experienced the stage from epiderm substitute, dermal substitute to double-deck organization engineering skin substitute, and its basic research and applied research have had progressive widely.Applied research relates in the body to be used and external application, uses the treatment that concentrates on various skin traumas in the body, and the detection of toxicity etc. is grown in the external toxicity detection that mainly applies to chemical compound as corrosivity, zest, phototoxicity, the dirt of chemical compound.The in vitro toxicity context of detection, the leading checking work that various animal alternative methods are carried out of European alternative method authentication center (ECVAM) is a system engineering, has caused worldwide concern.With the organization engineering skin is example, and the chemical compound corrosivity monitor of carrying out has only EpiDerm and two kinds of products of EpiSkin at present by checking, and zest detects only has EpiSkin to pass through checking at present.Why so few organization engineering skin product quantity by checking is, is because due to the horny layer maturation of the organization engineering skin that makes up and the differentiation not exclusively to a great extent.Because horny layer is ripe and differentiation not exclusively, cause that toxigenous data are not inconsistent in toxicity that detection compound produces competent cell and itself body, cause False Rate higher, can not meet the requirements of prediction accuracy.
At present, Mechanism Study ripe about horny layer and differentiation is more, and the differentiation of the verification table chrotoplast that the research of vivo and vitro is all strong is relevant with epithelial-mesenchymal interaction, and is promptly closely related with dermal fibroblast.So the organization engineering skin that adds dermal fibroblast is stopping that chemical compound has remarkable effect aspect competent cell toxicity.But, be survival rate at present with MTT method meter chrotoplast to the detection of toxicity of compound, like this, be mixed with epidermis cell and fibroblastic double-layer tissue engineering skin just can't obtain accurate result.Thus, promptly allow to form the intact horny layer of differentiation, contain epidermis cell and fibroblastic organization engineering skin detection compound toxicity is inapplicable at present.By retrieval, promote that about utilizing fibroblast the horny layer differentiation is intact, product does not contain fibroblastic organization engineering skin construction method again simultaneously, and not seeing as yet so far has technical literature openly.
Summary of the invention
The object of the present invention is to provide the differentiation of a kind of horny layer of epidermis structure intact, product does not contain the construction method of fibroblastic organization engineering skin again and with the organization engineering skin of its preparation simultaneously.
The technical solution used in the present invention is as follows:
A kind of organization engineering skin construction method may further comprise the steps:
(1) preparation tissue engineering skin culture liquid: with commercial serum-free epidermis cell culture fluid is basal liquid, adds insulin 20～50ng, hydrocortisone 100～300 μ g, vitamin C 20～40mg, calcium chloride 1～2mM by every 500mL basal liquid standard again;
(2) preparation human body fibroblast feeder layer: the fibroblast in human body source after suppressing propagation and handling, is added volume ratio 2～20% serum with conventional cell culture fluid and cultivates;
(3) preparation gel biological material: with type i collagen albumen, chitosan, hyaluronic acid and chondroitin sulfate according to 8～12: 1～3: 1～2: 1～2 proportion by weight mix homogeneously, regulate PH to 7.2～7.4 with the 1M sodium hydroxide solution then;
(4) structure and cultured tissue engineering skin: with 1～10 * 10 of described step (2) preparation
5The gel biological material of individual fibroblast and the described step of 1～3mL (3) preparation fully mixes, complex, described complex is at 37 ℃, 5%CO
2Cultivate 2h in the incubator, add 1～10 * 10 in the above then
5/ mL people source epidermal cell suspension 1mL, after continuing to cultivate 1h, interpolation is by the tissue engineering skin culture liquid 3～5mL of described step (1) preparation, continue to be cultured to the 5th day, the organization engineering skin of gained is carried out the cultivation of gas-liquid face, finished in the 20th day to cultivate, obtain to contain the organization engineering skin of intact differentiation and sophisticated epidermal structure.
Optimum in the organization engineering skin construction method of the present invention, described step (1) for adding Insulin 3 5ng, hydrocortisone 200 μ g, vitamin C 30mg, calcium chloride 1.5mM by every 500mL basal liquid standard.
Organization engineering skin construction method of the present invention, the inhibition propagation in the described step (2) is treated to 5～100Gy roentgenization to be handled 0.5～20h or utilizes compound treatment 0.5～20h.Described ray can be γ, χ ray etc.; Described chemical compound can be ametycin, bleomycin etc.
Optimum in the organization engineering skin construction method of the present invention, described step (2) with the cultivation of conventional cell culture fluid interpolation volume ratio 10% serum.
Organization engineering skin construction method of the present invention, in the described step (3) optimum with type i collagen albumen, chitosan, hyaluronic acid and chondroitin sulfate according to 10: 2: 1: 1 proportion by weight mix homogeneously.
Utilize the organization engineering skin and the application in the vitro detection toxicity of compound thereof of organization engineering skin construction method preparation of the present invention.
In the organization engineering skin construction method of the present invention, used raw and auxiliary material people source epidermis cell, people source fibroblast, chitosan, type i collagen albumen, hyaluronic acid, chondroitin sulfate, calcium chloride etc. all should meet aseptic requirement.
Organization engineering skin construction method of the present invention, the fibroblast that system adopts interpolation to handle through inhibition propagation is finished the interaction with epidermis cell, thereby improve the maturation and the differentiation degree of epidermis cell, the degree of accuracy that improves the detection of chemical compound in vitro toxicity there is important effect, thereby make the organization engineering skin of its preparation can more effectively stop the toxic action of chemical compound to competent cell, improve the prediction accuracy of organization engineering skin, the final alternative detection method of more effective animals that produces based on organization engineering skin to toxicity of compound.
The present invention has the following advantages: the one, and method is easy, and all material is all with liquid form, and the synthetic and scale dirt of being convenient to product produces; The 2nd, product has the intact horny layer of differentiation, is beneficial to stop toxicity of compound; The 3rd, when product was used for analyzed in vitro, not mixing had fibroblast, improved accuracy of analysis; The 4th, the product that obtains is convenient to quality control, variation between dwindling batch.
The specific embodiment
For further specifying the present invention, specifically set forth with the following Examples:
Embodiment 1:
Be basal liquid at first, press 500mL basal liquid standard again and add Insulin 3 5ng, hydrocortisone 200 μ g, vitamin C 30mg, calcium chloride 1.5mM, preparation tissue engineering skin culture liquid with the serum-free epidermis cell culture fluid K-SFM of Gibco company; With type i collagen albumen, chitosan, hyaluronic acid and chondroitin sulfate according to 10: 2: 1: 1 proportion by weight mix homogeneously, regulate PH to 7.2 with the 1M sodium hydroxide then, preparation gel biological material; After the fibroblast in human body source handled 2.5h with ametycin, add 10% serum with conventional cell culture fluid DMEM and cultivate preparation human body fibroblast feeder layer; With the preparation human body fibroblast feeder layer cells with 5 * 10
5/ mL fully mixes with gel biological material 2mL, complex, complex is at 37 ℃, 5%CO
2Cultivate 2h in the incubator, add 5 * 10 in the above then
5/ mL epidermal cell suspension 1mL continues to cultivate 1h, adds tissue engineering skin culture liquid 4mL.The 5th day, organization engineering skin is carried out the gas-liquid face cultivate, the 20th day finishes to cultivate, obtains to contain the organization engineering skin of intact differentiation and sophisticated horny layer of epidermis structure.
Embodiment 2:
Be basal liquid at first, press 500mL basal liquid standard again and add insulin 20ng, hydrocortisone 300 μ g, vitamin C 20mg, calcium chloride 2mM, preparation tissue engineering skin culture liquid with the serum-free epidermis cell culture fluid K-SFM of Gibco company; With type i collagen albumen, chitosan, hyaluronic acid and chondroitin sulfate according to 12: 2: 1: 1 proportion by weight mix homogeneously, regulate PH to 7.2 with the 1M sodium hydroxide then, preparation gel biological material; After the fibroblast in human body source handled 18h with the 10Gy radiation gamma, add 2% serum with conventional cell culture fluid DMEM and cultivate preparation human body fibroblast feeder layer; With the preparation human body fibroblast feeder layer cells with 10 * 10
5/ mL fully mixes with gel biological material 3mL, complex, complex is at 37 ℃, 5%CO
2Cultivate 2h in the incubator, add 10 * 10 in the above then
5/ mL epidermal cell suspension 1mL continues to cultivate 1h, adds tissue engineering skin culture liquid 5mL.The 5th day, organization engineering skin is carried out the gas-liquid face cultivate, the 20th day finishes to cultivate, obtains to contain the organization engineering skin of intact differentiation and sophisticated horny layer of epidermis structure.
Embodiment 3:
Be basal liquid at first, press 500mL basal liquid standard again and add insulin 50ng, hydrocortisone 100 μ g, vitamin C 40mg, calcium chloride 1mM, preparation tissue engineering skin culture liquid with the serum-free epidermis cell culture fluid K-SFM of Gibco company; With type i collagen albumen, chitosan, hyaluronic acid and chondroitin sulfate according to 9: 3: 1: 1 proportion by weight mix homogeneously, regulate PH to 7.3 with the 1M sodium hydroxide then, preparation gel biological material; After the fibroblast in human body source handled 5h with bleomycin, add 8% serum with conventional cell culture fluid DMEM and cultivate preparation human body fibroblast feeder layer; With the preparation human body fibroblast feeder layer cells with 1 * 10
5/ mL fully mixes with gel biological material 3mL, complex, complex is at 37 ℃, 5%CO
2Cultivate 2h in the incubator, add 1 * 10 in the above then
5/ mL epidermal cell suspension 1mL continues to cultivate 1h, adds tissue engineering skin culture liquid 3mL.The 5th day, organization engineering skin is carried out the gas-liquid face cultivate, the 20th day finishes to cultivate, obtains to contain the organization engineering skin of intact differentiation and sophisticated horny layer of epidermis structure.
Embodiment 4:
Be basal liquid at first, press 500mL basal liquid standard again and add insulin 40ng, hydrocortisone 150 μ g, vitamin C 25mg, calcium chloride 1mM, preparation tissue engineering skin culture liquid with the serum-free epidermis cell culture fluid K-SFM of Gibco company; With type i collagen albumen, chitosan, hyaluronic acid and chondroitin sulfate according to 8: 2: 2: 2 proportion by weight mix homogeneously, regulate PH to 7.4 with the 1M sodium hydroxide then, preparation gel biological material; After the fibroblast in human body source handled 3h with 60Gy χ ray, add 15% serum with conventional cell culture fluid DMEM and cultivate preparation human body fibroblast feeder layer; With the preparation human body fibroblast feeder layer cells with 8 * 10
5/ mL fully mixes with gel biological material 1mL, complex, complex is at 37 ℃, 5%CO
2Cultivate 2h in the incubator, add 8 * 10 in the above then
5/ mL epidermal cell suspension 1mL continues to cultivate 1h, adds tissue engineering skin culture liquid 4mL.The 5th day, organization engineering skin is carried out the gas-liquid face cultivate, the 20th day finishes to cultivate, obtains to contain the organization engineering skin of intact differentiation and sophisticated horny layer of epidermis structure.
Embodiment 5:
Be basal liquid at first, press 500mL basal liquid standard again and add Insulin 3 0ng, hydrocortisone 250 μ g, vitamin C 35mg, calcium chloride 1mM, preparation tissue engineering skin culture liquid with the serum-free epidermis cell culture fluid K-SFM of Gibco company; With type i collagen albumen, chitosan, hyaluronic acid and chondroitin sulfate according to 11: 3: 2: 1 proportion by weight mix homogeneously, regulate PH to 7.4 with the 1M sodium hydroxide then, preparation gel biological material; After the fibroblast in human body source handled 15h with ametycin, add 15% serum with conventional cell culture fluid DMEM and cultivate preparation human body fibroblast feeder layer; With the preparation human body fibroblast feeder layer cells with 3 * 10
5/ mL fully mixes with gel biological material 2mL, complex, complex is at 37 ℃, 5%CO
2Cultivate 2h in the incubator, add 3 * 10 in the above then
5/ mL epidermal cell suspension 1mL continues to cultivate 1h, adds tissue engineering skin culture liquid 3mL.The 5th day, organization engineering skin is carried out the gas-liquid face cultivate, the 20th day finishes to cultivate, obtains to contain the organization engineering skin of intact differentiation and sophisticated horny layer of epidermis structure.
The test example:
The organization engineering skin that contains intact differentiation and sophisticated horny layer of epidermis structure that will make up according to the method for embodiment 1 places fixedly 24h of neutral formalin solution, routine preparation Histological section, and carry out HE dyeing.By the painted result of HE, find that the organization engineering skin epidermal area cutin differentiation of adopting this method to make up is intact, do not see that fibroblast mixes.
Annotate: the terminological interpretation in this description:
The MTT method:
Based on living cells metabolite Reducing agent MTT tetrazolium bromide, act on the respiratory chain in the living cells mitochondrion, generate blue formazan crystallization under the effect of succinate dehydrogenase and cytochrome C, the crystalline growing amount of formazan only is directly proportional with the living cells number.The formazan crystallization that generates can be dissolved in specific lysate, utilizes microplate reader to measure optical density value, thereby detects cell number.
HE dyeing:
The dyeing that promptly utilizes h and E that section is carried out, after HE dyeing, nucleus is shown as bluish violet, the shown in red or pale red of Cytoplasm.
The % that relates among the present invention if no special instructions outside, all refer to percentage by weight.
Above-described embodiment is described preferred implementation of the present invention; be not that scope of the present invention is limited; design under the prerequisite of spirit not breaking away from the present invention; various distortion and improvement that the common engineers and technicians in this area make technical scheme of the present invention all should fall in the definite protection domain of claims of the present invention.
Claims (6)
1. an organization engineering skin construction method is characterized in that, may further comprise the steps:
(1) preparation tissue engineering skin culture liquid: with commercial serum-free epidermis cell culture fluid is basal liquid, adds insulin 20～50ng, hydrocortisone 100～300 μ g, vitamin C 20～40mg, calcium chloride 1～2mM by every 500mL basal liquid standard again;
(2) preparation human body fibroblast feeder layer: the fibroblast in human body source after suppressing propagation and handling, is added volume ratio 2～20% serum with conventional cell culture fluid and cultivates; Described inhibition propagation is treated to 5～100Gy γ or χ roentgenization and handles 0.5～20h or utilize ametycin or bleomycin processing 0.5～20h;
(3) preparation gel biological material: with type i collagen albumen, chitosan, hyaluronic acid and chondroitin sulfate according to 8～12: 1～3: 1～2: 1～2 proportion by weight mix homogeneously, regulate PH to 7.2～7.4 with the 1M sodium hydroxide solution then;
(4) structure and cultured tissue engineering skin: with 1～10 * 10 of described step (2) preparation
5The gel biological material of individual fibroblast and the described step of 1～3mL (3) preparation fully mixes, complex, described complex is at 37 ℃, 5%CO
2Cultivate 2h in the incubator, add 1～10 * 10 in the above then
5/ mL people source epidermal cell suspension 1mL, after continuing to cultivate 1h, interpolation is by the tissue engineering skin culture liquid 3～5mL of described step (1) preparation, continue to be cultured to the 5th day, the organization engineering skin of gained is carried out the cultivation of gas-liquid face, finished in the 20th day to cultivate, obtain to contain the organization engineering skin of intact differentiation and sophisticated epidermal structure.
2. organization engineering skin construction method according to claim 1 is characterized in that: add Insulin 3 5ng, hydrocortisone 200 μ g, vitamin C 30mg, calcium chloride 1.5mM by every 500mL basal liquid standard in the described step (1).
3. organization engineering skin construction method according to claim 1 is characterized in that: add volume ratio 10% serum with conventional cell culture fluid in the described step (2) and cultivate.
4. organization engineering skin construction method according to claim 1 is characterized in that: in the described step (3) with type i collagen albumen, chitosan, hyaluronic acid and chondroitin sulfate according to 10: 2: 1: 1 proportion by weight mix homogeneously.
5. utilize the organization engineering skin of each described organization engineering skin construction method preparation of claim 1～4.
6. the application of the described organization engineering skin of claim 5 in the vitro detection toxicity of compound.
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| CN2008101161081A CN101297981B (en) | 2008-07-03 | 2008-07-03 | Tissue engineering skin construction method and tissue engineering skin produced by the same |
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Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101532950B (en) * | 2008-12-31 | 2011-09-07 | 程树军 | Method for analyzing toxicity and effect of skin whitening agent through human being skin melanocyte |
| CN101485905B (en) * | 2009-02-26 | 2012-12-19 | 中国检验检疫科学研究院 | Method for constructing tissue engineering skin |
| CN101879329A (en) * | 2010-06-21 | 2010-11-10 | 蒋明军 | Artificial skin containing basilar membrane and construction method thereof |
| CN107287150B (en) * | 2017-06-18 | 2020-08-04 | 广东博溪生物科技有限公司 | Construction method of 3D (three-dimensional) epidermis model |
| CN109529123B (en) * | 2018-11-08 | 2021-02-19 | 中国人民解放军第四军医大学 | Vascularized full-layer tissue engineering skin formed by assembling hydrogel, nanofiber scaffold and skin cells layer by layer and preparation method thereof |
| CN109504651A (en) * | 2018-11-05 | 2019-03-22 | 合肥中科干细胞再生医学有限公司 | A kind of method for building up of external epidermis threedimensional model |
| CN110669726A (en) * | 2019-09-19 | 2020-01-10 | 上海长征医院 | Cell strain capable of being passaged for long time after gamma ray irradiation of human skin fibroblasts and construction method thereof |
| CN110607273A (en) * | 2019-09-19 | 2019-12-24 | 上海长征医院 | Cell strain capable of being passaged for long time after gamma ray irradiation of mouse embryo fibroblast and construction method thereof |
| CN111337494A (en) * | 2020-03-27 | 2020-06-26 | 济南磐升生物技术有限公司 | Preparation method and application of artificial epidermis cosmetic detection kit |
| CN113755554A (en) * | 2021-09-15 | 2021-12-07 | 中国医学科学院皮肤病医院(中国医学科学院皮肤病研究所) | Method for testing drug sensitivity by using tissue engineering skin |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1310027A (en) * | 2001-02-06 | 2001-08-29 | 中国人民解放军第三军医大学第三附属医院 | Preparation of compound artificial skin |
| CN1569258A (en) * | 2003-07-25 | 2005-01-26 | 北京以岭生物工程有限公司 | Tissue engineering autologous complex skin and its preparation method |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1310027A (en) * | 2001-02-06 | 2001-08-29 | 中国人民解放军第三军医大学第三附属医院 | Preparation of compound artificial skin |
| CN1569258A (en) * | 2003-07-25 | 2005-01-26 | 北京以岭生物工程有限公司 | Tissue engineering autologous complex skin and its preparation method |
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