CN101063109A - Construction method and application for complexion adjustable organization engineering skin - Google Patents
Construction method and application for complexion adjustable organization engineering skin Download PDFInfo
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- CN101063109A CN101063109A CNA2006100763658A CN200610076365A CN101063109A CN 101063109 A CN101063109 A CN 101063109A CN A2006100763658 A CNA2006100763658 A CN A2006100763658A CN 200610076365 A CN200610076365 A CN 200610076365A CN 101063109 A CN101063109 A CN 101063109A
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Abstract
The invention discloses a method to construct engineering skin to adjust flesh in biological medicine domain, which comprises the following steps: orient-evoking and differentiating stem cell of diverse tissue to sheath and dermal cell; combining the cell group and culturing melanin cell with biological material at a finite proportion; preparing substitution good to restore and heal clinical burn, ulcer, would, deformed, defection after operation and scar. This invention can meet diverse needs of individual flesh according to add diverse melanin cell, which possesses giant latent capacity and wide application prospect.
Description
Technical field
The present invention relates to biomedical sector, specifically relate to the stem cell from different tissues is broken up to epidermis with to the dermal cell directional induction respectively external, and will induce the cell mass of differentiation and the melanocyte of separation and Culture to combine with biomaterial in certain proportion, the method that common structure can be regulated the tissue engineering skin of the colour of skin utilizes this method can prepare having specified shape, thickness, can regulating the skin histology surrogate of the colour of skin of skin tissue recovering such as being used for clinical burn, ulcer, wound, deformity, operative defect, scar and healing.
Background technology
Skin has the function of barrier, protection, regulate body temperature and sensation as the largest organ of human body.Because the skin injury that factors such as inflammation, ulcer, scald, burn cause, but serious threat to life.Clinical common employing is at present treated the damaged surface of a wound from body flap or dermatoplastic method, but faces the new wound defective of skin donor site, and the deficiency in skin donor site source.There is the difference on color, quality, the function in the skin that closes on of graft and transplantation site in addition.
Organizational engineering is the principle and the method for application project and life science, make up biological surrogate, to recover, to keep or promote a science (the Nerem RM.Tissue engineeringin the USA.Med Biol Eng Comput of impaired organ or tissue function, 1992,30 (4): CE8).The foundation of organizational engineering and develop into skin injury treatment and started brand-new approach.
Be applied to clinical tissue engineering product at present and mainly contain the dressing of synthetic property, artificial skin graft, artificial dermis surrogate and artificial compound holostrome skin.Integra
TMIt is artificial dermis by the double-deck matrix of American I ntegra Life Sciences company production.Its corium is to make the support with certain hole size by the collagen protein of ox tendon and chondroitin sulfate, be beneficial to vascular endothelial cell by growing into fibrocyte, epidermis is made up of pellosil, can remove after the skin corium healing, carries out the transplanting of artificial epidermis again.U.S. FDA was checked and approved Integra in 1996
TMBe used for the treatment of burn and scald.Yet, Integra
TMOwing to lack the cellular constituent of living, may delay the reconstruction of corium.
Also having a class is the skin that contains single cell, as artificial epidermis and artificial dermis.Epicel
TMBe the artificial epidermis of producing by U.S. Genzyme tissue repair company, be mainly used in treatment big area burn and scald patient.It is with patient from the keratinocyte of body in external amplification culture, be transplanted to ulcer, hemorrhoid, epidermolysis bullosa position.Though they stick finely in residual dermal sites, they do not stick fat, chronic wound or infective wound surface; And transplantation site peels off easily, perhaps forms blister, and is extremely sensitive to physical abuse; Lack the corium composition; Transplanting succeed rate is main relevant with surface of a wound bacterial contamination degree; The expense costliness.Dermagraft
TMIt is the artificial dermis of producing by Advanced Tissue Sciences company.It is online to be that the inoblast that will obtain in newborn infant's foreskin is inoculated in biodegradable acid fiber by polylactic, transplanted back 14 days, inoblast breeds extracellular matrix and cytokines such as justacrine collagen in a large number, 3～4 all acid fiber by polylactic nets disappear because of biological degradation, are used for the treatment of the foot ulcers that diabetes cause.But produce this synthetic nethike embrane dermal substitute and need a large amount of inoblasts; Be difficult to change the thickness of nethike embrane; The commercially available prod only reaches corium and rebuilds.
Apligraft
TMBe first kind of business-like tissue engineering skin that had not only contained epidermal area but also contained skin corium, produce by U.S. Organogenesis company.It is that the inoblast that will obtain in newborn infant's foreskin is inoculated in the I collagen protein of ox, and emiocytosis extracellular matrix after 6 days forms the tissue of similar skin corium, and the keratinocyte of newborn infant's foreskin is gone up in plantation again.Keratinocyte is attached to beginning to be differentiated to form epidermal area on the corium.Again whole mixture is placed liquid-vapo(u)r interface to continue to cultivate, impel the keratinocyte maturation.Though Apligraft
TMRealized the purpose that surgical operation is rebuild corium and epidermis simultaneously, but owing to adopt homogeneous variant cell to originate as seed cell, thereby exist inevitable immunological rejection, and the risk of potential pathogeny infected by microbes; And used seed cell is the mature cell of end differentiation eventually, the ability that does not have self duplication to break up.
Adult stem cell is the cell that a class has self and multidirectional differentiation potential, existing both at home and abroad isolated experiment research report of inducing differentiation in a large number about adult stem cell to bone, cartilage, fat, liver and nerve etc., and when it is transplanted in the body, has ability (the Mackenzie TC that moves and be divided into the needed corresponding cell of body to diseased region, Flake AW.Blood Cells Mol Dis, 2001,27 (3): 601.), utilize adult stem cell as seed cell, will open up new approach for the treatment of skin injury.
Be applied to clinical tissue engineering skin at present and still can not carry out corresponding dermatoplasty reparation according to the difference of patient's colour of skin.
Summary of the invention
The invention provides a kind of stem cell directional induction in vitro and be divided into epidermis and dermal cell, again the method that makes up the tissue engineering skin that to regulate the colour of skin with the melanocyte and the biomaterial of separation and Culture.It is characterized in that the melanocyte of epidermic cell group, dermal cell group and the separation and Culture of external evoked one-tenth skin is mixed in proportion with Method of Tissue Engineering, make up the artificial skin that contains melanocyte by the additional proportion of regulating melanocyte.Skin histology surrogate specified shape, thickness, that can regulate the colour of skin that has that can prepare skin tissue recovering such as being used for clinical burn, ulcer, wound, deformity, operative defect, scar and healing by this method.
The present invention is achieved by the following technical solutions:
(1) external evoked stem cell breaks up to epidermic cell
Tissue-derived MSC or other adult stem cells such as marrow, fat, embryo, Cord blood, peripheral blood are planted in be covered with gelatin (Sigma company in advance, article No. G-1890), collagen, fibrin gel, Matrigel glue (BD company, article No. 354240) or on the orifice plate of other biological activity glue, add and contain 10% foetal calf serum (Biochrom company, article No. S0115) low sugar DMEM substratum (Sigma company, article No. D-5523) or the corresponding minimum medium of other adult stem cell, in 37 ℃, 5%CO
2Be cultured to cell in the incubator and reach 30%～50% fusion, then use epidermis inductive condition nutrient solution: low sugar DMEM/DF12 (1: 1) substratum (DF12 substratum instead, Sigma company, article No. D-0547) contains following ingredients in: 10～20ng/ml EGF (R﹠amp; D company, article No. 236-EG), 10～20ng/ml bFGF (R﹠amp; D company, article No. 234-FSE), 5～20ng/ml PDGF (R﹠amp; D company, article No. 222-AB), 1%ITS (Sigma company, article No. I-3146), 5～15mmol/L dexamethasone (Sigma company, article No. D-1756), 100IU/ml penicillin, the composition of two or more in the 100IU/ml Streptomycin sulphate carries out inducing culture, can obtain epidermic cell after 10～14 days.
(2) external evoked stem cell breaks up to dermal cell
The MSC that marrow, fat, embryo, Cord blood, peripheral blood etc. are tissue-derived or other adult stem cell are cultivated and are being contained 10% foetal calf serum (Biochrom company, article No. S0115) low sugar DMEM substratum (Sigma company, article No. D-5523) or in the corresponding minimum medium of other adult stem cell, at 37 ℃, 5%CO
2Be cultured to cell in the incubator and reach 30%～50% fusion, then use corium inductive condition nutrient solution instead: high sugared DMEM (Sigma company, article No. D-5648) contains following ingredients in: 2～10% foetal calf serums (Biochrom company, article No. S0115), 5～15ng/mlTGF-β
1(R﹠amp; D company, article No. 240-B), 10～20ng/ml bFGF (R﹠amp; D company, article No. 234-FSE), 1%ITS (Sigma company, article No. I-3146), 5～15mmol/L dexamethasone (Sigma company, article No. D-1756), 100IU/ml penicillin, the composition of two or more in the 100IU/ml Streptomycin sulphate carries out inducing culture, can obtain dermal cell after 10～14 days.
(3) separation and Culture of melanocyte:
Get the part skin histology, remove subcutis and sarcolemma, place the 75% ethanol 1min that sterilizes then, physiological saline rinsing number time changes in the culture dish, with eye scissors tissue is cut to 2mm * 10mm fine strip shape, 2.5g/L Dispase enzyme (Gibco company, article No. 17105-041) digestion spend the night.Gently epidermis is separated with corium with pincet, collect epidermis, with 0.25% trysinization 10min, piping and druming obtains single cell suspension repeatedly, add an amount of serum and stop digestion, 400 eye mesh screens filter, cell suspension is the centrifugal 5min of 1000 commentaries on classics/min in centrifuge tube, abandon supernatant, add melanocyte nutrient solution (melanocyte growth medium M2, the c-24110 that contains 100 μ g/ml Geneticins (Gibco company, article No. 11811-023) in the precipitation, PromoCell company) in 37 ℃, 5%CO
2Cultivate in the incubator.Change liquid behind the 36h, remove not adherent cell, the next day change a not good liquor.When primary cell 70%～80% merged, conventional trysinization was gone down to posterity.
(4) preparation of collagen solution preparation and support material
Fresh oxtail is dipped in 75% alcohol, peels off the ox tendon under the aseptic condition, and the physiological saline washing shreds, and adds 4 ℃ of jolting dissolvings repeatedly of 0.03～0.05M Glacial acetic acid, the preparation collagen solution.10 * low sugar the DMEM (Sigma company, article No. D-5523) that adds 1/10 volume, an amount of foetal calf serum (Biochrom company, article No. S0115) is with NaOH adjusting pH value to 7.2～7.4 of 1N.Before using required collagen solution is injected culture dish, place on ice, ultraviolet lamp is irradiation 30min～60min down.
Carry out cobalt 60 radiation sterilizations with the skin acellular matrix in various Mammalss such as pig, ox, sheep or other source or other commercial acellular matrix or such as degradable, loose porous biology or polymer materialss such as collagen sponge, fibroin, PLA, PGA, PLGA, wash 3 times with low sugar DMEM substratum, place 4 ℃ to deposit afterwards, stand-by.
(5) preparation of organizational project holostrome skin
Acellular matrix or other biologic bracket material or polymer materials (purify as domestic silkworm silk or tussah silk the dissolving silk gum, fibroin and the chitin that obtain, Mierocrystalline cellulose, polyamino acid, PLGA, PGA, PLA, polymeric amide, polyester, polystyrene, polypropylene, polyacrylic ester, polyvinyl chloride, polycarbonate, tetrafluoroethylene, nitrocotton etc.) are placed in the culture plate (or other culture dish) that siliconizing crosses.To induce 10～14 days cell mass digestion to dermal cell, (Sigma company, article No. D-5523) is resuspended with the low sugar DMEM substratum that contains 10% foetal calf serum (Biochrom company, article No. S0115), and counting and collagen solution mix, by 1 * 10
3～5 * 10
4Individual/cm
2The density kind advance in the support material.Add the low sugar DMEM substratum that contains 10% foetal calf serum in right amount, left standstill 10 minutes, put into 37 ℃ subsequently, 5%CO
2Cultivate in the incubator, behind the 4h substratum suction is gone, change the dermal cell nutrient solution into and cultivate.To induce 10～14 days cell mass to digest respectively to epidermic cell and melanocyte after 4 days, resuspended with the low sugar DMEM substratum that contains 10% foetal calf serum, counting, in 3～30: 1 ratio, with 2 * 10
3～2 * 10
5Individual/cm
2Density evenly plant in the material surface that contains dermal cell.Behind the 2h substratum inhaled and go, change the epidermic cell nutrient solution into and cultivate, change liquid-vapo(u)r interface after 3 days into and cultivate, continue to cultivate that tissue engineering skin promptly can be used for transplanting after 7 days.
The present invention is that the stem cell with different tissue sources breaks up to epidermis with to the dermal cell directional induction respectively external, mix with certain proportion with the melanocyte of separation and Culture then, again with pig, ox, the skin acellular matrix in various Mammalss such as sheep or other source or other commercial acellular matrix or such as collagen sponge, fibroin, PLA, PGA, degradables such as PLGA, loose porous biology or polymer materials combine, common formation can be regulated the tissue engineering skin of the colour of skin, can prepare and be used for clinical burn, ulcer, wound, deformity, operative defect, skin tissue recovering such as scar and healing have a specified shape, thickness, can regulate the skin histology surrogate of the colour of skin.Because stem cell has multidirectional differentiation potential, can under inductive condition, be divided into epidermic cell and dermal cell, the cell by inducing differentiation and the acting in conjunction of the excretory soluble cell factor and extracellular matrix isoreactivity composition thereof, can quicken the healing of the damaged surface of a wound, can also satisfy the needs of the Different Individual colour of skin simultaneously according to the difference that adds the melanocyte ratio, therefore, marketable value has a high potential, and has broad application prospects.
Description of drawings
Fig. 1 stem cell is induced differentiation to epidermic cell
A: induce back 5 days of differentiation, cell is epidermic cell typical " paving stone shape " (* 200)
B: induce back 10 days transmission electron microscope observings of differentiation, occur a large amount of tonofilaments and melanosome (* 60000) in the endochylema
C: identified by immunofluorescence, induce the cell expressing CK19 (* 100) of differentiation
D: immunohistochemistry is identified, is induced the parts of fine cellular expression CK10 (* 200) of differentiation
Fig. 2 stem cell is induced differentiation to dermal cell
A: induce back 10 days transmission electron microscope observings of differentiation, a small amount of collagen microfibril (* 60000) appears in the extracellular
B: immunohistochemistry is identified, induces differentiation parts of fine cellular expression type i collagen
Immunofluorescence detects in Fig. 3 tissue engineering skin nude mice transplant experiment and the body
A: the nude mice transplant experiment model that covers tissue engineering skin
B: identified by immunofluorescence, tissue engineering skin were transplanted back 3 days, nude mice skin injury surface of a wound cell expressing CK10
C: identified by immunofluorescence, tissue engineering skin were transplanted back 7 days, and cell moves to the skin injury surface of a wound
D: tissue engineering skin was transplanted back 7 days, and tissue engineering skin wound healing rate reaches 50%
E: tissue engineering skin nude mice transplant experiment is after 21 days, and the damaged surface of a wound heals fully
Embodiment
Embodiment 1, be example with marrow MSC, the stem cell of in-vitro separation different tissue sources
Aseptic condition directly extracts patient's marrow down, add the dilution of equivalent physiological saline, 200 eye mesh screens filter, add 6%HES (B.Braun company) sedimented red cell, draw the upper strata cell suspension, the centrifugal supernatant that goes, add physiological saline and make cell suspension, separate (1000g * 20min), careful sucking-off intermediate layer cell with the Percoll parting liquid (AmershamBiosciences company, article No. 17-0891-01) of proportion 1.073g/ml, with containing 10% foetal calf serum (Biochrom company, article No. S0115) (Sigma company, article No. D-5523) is resuspended for low sugar DMEM substratum, is inoculated in the T-25 culturing bottle.Put CO
2(temperature is set at 37 ℃ to incubator, CO
2Concentration is 5%) the interior cultivation; Change liquid after cultivating 72h, after this according to the cell speed of growth culture system is changed liquid, treat that the culturing bottle inner cell grows to when merging more than 80%, 37 ℃ of peptic cells of the trypsinase with 0.25% are respectively by 5.5～7.0 * 10
3Individual/cm
2Be inoculated in the culturing bottle, put CO
2Incubator is cultivated.
Embodiment 2, external evoked MSC break up to epidermic cell
The MSC kind of derived from bone marrow planted be covered with Matrigel glue (BD company in advance, article No. 354240) on the orifice plate, add and contain 10% foetal calf serum (Biochrom company, article No. S0115) low sugar DMEM substratum (Sigma company, article No. D-5523) is in 37 ℃, be cultured to cell in the 5%CO2 incubator and reach 50% fusion, then use epidermis inductive condition nutrient solution: low sugar DMEM/DF12 (1: 1) substratum (Sigma company, article No. D-0547) instead, 15ng/ml EGF (R﹠amp; D company, article No. 236-EG), 15ng/ml bFGF (R﹠amp; D company, article No. 234-FSE), 5ng/ml PDGF (R﹠amp; D company, article No. 222-AB), 1%ITS (Sigma company, article No. I-3146), 10mmol/L dexamethasone (Sigma company, article No. D-1756), 100IU/ml penicillin, the 100IU/ml Streptomycin sulphate carries out inducing culture, can obtain epidermic cell after 10～14 days.The result shows that stem cell is induced back 5 days of differentiation to epidermic cell, and cell is epidermic cell typical " paving stone shape ".Induce back 10 days of differentiation, occur a large amount of tonofilaments and melanosome in the endochylema, immunofluorescence detects cell expressing CK19, immunohistochemical methods test section cell expressing CK10 (Fig. 1).
Embodiment 3, external evoked MSC break up to dermal cell
The MSC kind of derived from bone marrow is planted on the orifice plate that is covered with Matrigel glue in advance, add the low sugar DMEM substratum (Sigma company, article No. D-5523) that contains 10% foetal calf serum (Biochrom company, article No. S0115), in 37 ℃, 5%CO
2Be cultured to cell in the incubator and reach 50% fusion, use corium inductive condition nutrient solution then instead: high sugared DMEM (Sigma company, article No. D-5648), 5% foetal calf serum (Biochrom company, article No. S0115), 15ng/mlTGF-β 1 (R﹠amp; D company, article No. 240-B), 15g/ml bFGF (R﹠amp; D company, article No. 234-FSE), 1%ITS (Sigma company, article No. I-3146), 10mmol/L dexamethasone (Sigma company, article No. D-1756), 100IU/ml penicillin, the 100IU/ml Streptomycin sulphate carries out inducing culture, can obtain dermal cell after 10～14 days.The result shows that stem cell is induced back 10 days of differentiation to dermal cell, and a small amount of collagen microfibril appears in the extracellular, and immunohistochemical methods detects, parts of fine cellular expression type i collagen (Fig. 2).
The separation of embodiment 4, melanocyte and vitro culture
Get the part skin histology, remove subcutis and sarcolemma, place the 75% ethanol 1min that sterilizes then, physiological saline rinsing number time changes in the culture dish, with eye scissors tissue is cut to 2mm * 10mm fine strip shape, 2.5g/L Dispase enzyme (Gibco company, article No. 17105-041) digestion spend the night.Gently epidermis is separated with corium with pincet, collect epidermis, with 0.25% trysinization 10min, piping and druming obtains single cell suspension repeatedly, add an amount of serum and stop digestion, 400 eye mesh screens filter, cell suspension is the centrifugal 5min of 1000 commentaries on classics/min in centrifuge tube, abandon supernatant, add melanocyte nutrient solution (melanocyte growth medium M2, the c-24110 that contains 100 μ g/ml Geneticins (Gibco company, article No. 11811-023) in the precipitation, PromoCell company), cultivates in the 5%CO2 incubator in 37 ℃.Change liquid behind the 36h, remove not adherent cell, the next day change a not good liquor.When primary cell 70%～80% merged, conventional trysinization was gone down to posterity.
Embodiment 5, collagen solution are prepared
Fresh oxtail is dipped in 75% alcohol, peels off the ox tendon under the aseptic condition, and the physiological saline washing shreds, and adds 4 ℃ of jolting dissolvings repeatedly of 0.05M Glacial acetic acid, the preparation collagen solution.10 * low sugar the DMEM that adds 1/10 volume, an amount of foetal calf serum is with NaOH adjusting pH value to 7.2～7.4 of 1N.Before using required collagen solution is injected culture dish, place on ice, ultraviolet lamp is irradiation 40min down.
Embodiment 6, be example with the skin acellular matrix, preparation can be regulated the tissue engineering skin of the colour of skin
The skin acellular matrix of Niu Laiyuan is carried out cobalt 60 radiation sterilizations, wash 3 times, the culture plate (or other culture dish) that places siliconizing to cross afterwards with low sugar DMEM substratum.To induce 10～14 days cell mass digestion to dermal cell, resuspended with the low sugar DMEM substratum that contains 10% foetal calf serum, counting and collagen solution mix, by 5 * 10
4Individual/cm
2The density kind advance in the support material.Add the low sugar DMEM substratum that contains 10% foetal calf serum in right amount, left standstill 10 minutes, put into 37 ℃ subsequently, cultivate in the 5%CO2 incubator, behind the 4h substratum suction is gone, change the dermal cell nutrient solution into and cultivate.To induce 10～14 days cell mass to digest respectively to epidermic cell and melanocyte after 4 days, resuspended with the low sugar DMEM substratum that contains 10% foetal calf serum, counting be in 30: 1 ratio, with 2 * 10
5Individual/cm
2Density evenly plant in the material surface that contains dermal cell.Behind the 2h substratum inhaled and go, change the epidermic cell nutrient solution into and cultivate, change liquid-vapo(u)r interface after 3 days into and cultivate, continue to cultivate that tissue engineering skin promptly can be used for transplanting after 7 days.
Embodiment 7, organization engineering skin are repaired the experiment of the nude mice surface of a wound
Behind 3.5% chloral hydrate anesthesia nude mice, cut the round defect surface of a wound that holostrome skin is made diameter 1.5cm in back tail side, transplanted tissue's engineering Graftskin; Postoperative was removed wrapping dressing in 3 days, and the skin and the surface of a wound are fitted tighter, survive, and the 7th day, the wound healing rate reached 50%, the 21 day surface of a wound and heals fully, wound repair good (Fig. 3).
Claims (10)
1, a kind of construction process that can regulate the tissue engineering skin of the colour of skin is characterized in that: by inducing the cell mass of differentiation, the melanocyte and common structure of biomaterial of separation and Culture to form to epidermic cell with to dermal cell.
2, the construction process that can regulate the tissue engineering skin of the colour of skin according to claim 1 is characterized in that described to epidermic cell or induce the cell of differentiation to comprise from the stem cell of separate tissue such as Cord blood, peripheral blood, marrow, fat, placenta, umbilical cord to dermal cell to obtain through external evoked differentiation.
3, the construction process that can regulate the tissue engineering skin of the colour of skin according to claim 1 is characterized in that described biomaterial comprises that degradable biomaterial comprises silk gum, fibroin and collagen, chitin, Mierocrystalline cellulose, polyamino acid, PLGA, PGA, PLA that domestic silkworm silk or tussah silk dissolving are purified and obtained; Also comprise acellular dermal matrix; Non-degraded type biomaterial such as polymeric amide, polyester, polystyrene, polypropylene, polyacrylic ester, polyvinyl chloride, polycarbonate, tetrafluoroethylene, nitrocotton.
4, the construction process that can regulate the tissue engineering skin of the colour of skin according to claim 1 is characterized in that described melanocyte separates from skin, obtains through cultured and amplified in vitro.
5, the construction process that can regulate the tissue engineering skin of the colour of skin according to claim 1, include the melanocyte of the separation of stem cell, the external evoked differentiation of stem cell, the cell of inducing differentiation and separation and Culture and the common cultivation of biomaterial, it is characterized in that the inventive method may further comprise the steps:
(1) separation and Culture of different tissue sources stem cell;
(2) separation and Culture of melanocyte;
(3) the different tissue sources stem cell is induced differentiation to epidermic cell;
(4) the different tissue sources stem cell is induced differentiation to dermal cell;
(5) preparation of collagen solution preparation and support material;
(6) contain the structure of tissue engineering skin of the regulated colour of skin of the epidermic cell of inducing differentiation and dermal cell and melanocyte:
1) acellular matrix or other biology or polymer materials are placed in the culture plate (or other culture dish) that siliconizing crosses, to induce 10～14 days cell mass digestion to dermal cell, resuspended with the low sugar DMEM substratum that contains 10% foetal calf serum, counting, mix with collagen solution, by 1 * 10
3～5 * 10
4Individual/cm
2The density kind advance in the support material, add the low sugar DMEM substratum contain 10% foetal calf serum in right amount, left standstill 10 minutes, put into 37 ℃ subsequently, 5%CO
2Cultivate in the incubator, behind the 4h substratum suction is gone, change the dermal cell nutrient solution into and cultivate;
2) will induce 10～14 days the cell mass and the melanocyte of cultivation to digest respectively to epidermic cell after 4 days, resuspended with the low sugar DMEM substratum that contains 10% foetal calf serum, counting, in 3～30: 1 ratio, with 2 * 10
3～2 * 10
5Individual/cm
2Density evenly plant in the material surface that contains dermal cell;
3) behind the 2h substratum inhaled and go, change the epidermic cell nutrient solution into and cultivate, change liquid-vapo(u)r interface after 3 days into and cultivate, continue to cultivate that tissue engineering skin promptly can be used for transplanting after 7 days.
6, the construction process of regulating the tissue engineering skin of the colour of skin according to claim 1, it is characterized in that being used for inducing the different tissue sources stem cell is to contain following ingredients at low sugar DMEM/DF12 (1: 1) substratum to the substratum of epidermic cell differentiation: EGF, bFGF, PDGF, ITS, dexamethasone, penicillin, the composition of two or more in the Streptomycin sulphate.
7, the construction process of regulating the tissue engineering skin of the colour of skin according to claim 1, it is characterized in that being used for induced dry-cell is to contain following ingredients at the sugared DMEM substratum of height to the substratum of dermal cell differentiation: foetal calf serum, TGF-β
1, bFGF, ITS, dexamethasone, penicillin, the composition of two or more in the Streptomycin sulphate.
8, the construction process of regulating the tissue engineering skin of the colour of skin according to claim 1, it is characterized in that being used for stem cell to a kind of induction scheme of epidermic cell differentiation can be: low sugar DMEM/DF12 (1: 1) substratum, 15ng/ml EGF, 15ng/ml bFGF, 5ng/ml PDGF, 1%ITS, 10mmol/L dexamethasone, 100IU/ml penicillin, the 100IU/ml Streptomycin sulphate.
9, the construction process of regulating the tissue engineering skin of the colour of skin according to claim 1 is characterized in that being used for stem cell and to a kind of induction scheme of dermal cell differentiation can be: high sugared DMEM, 5% foetal calf serum, 15ng/mlTGF-β
1, 15g/ml bFGF, 1%ITS, 10mmol/L dexamethasone, 100IU/ml penicillin, 100IU/ml Streptomycin sulphate.
10, can regulate the application of the tissue engineering skin of the colour of skin, it is characterized in that the tissue engineering skin of the regulated colour of skin that makes up according to the described method of claim 1 can prepare the skin histology surrogate of skin tissue recovering such as being applied to clinical burn, ulcer, wound, deformity, operative defect, scar and healing.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101856517A (en) * | 2010-06-18 | 2010-10-13 | 杭州市第三人民医院 | Tissue engineering material-based culture method and applications of melanophore |
| CN103194423A (en) * | 2013-03-07 | 2013-07-10 | 中国人民解放军军事医学科学院野战输血研究所 | Medium and purpose thereof |
| CN105238739A (en) * | 2015-11-11 | 2016-01-13 | 朱宁文 | Selective culture method for large-scale preparation of human extracellular matrix through melanocytes for clinic treatment level cell therapy |
| CN105255813A (en) * | 2015-08-14 | 2016-01-20 | 上海交通大学医学院附属第九人民医院 | Epithelial tissue and epithelial tissue in-vitro construction method |
| CN107802891A (en) * | 2017-11-09 | 2018-03-16 | 清华大学深圳研究生院 | Organization engineering skin and preparation method thereof |
| CN112074596A (en) * | 2018-02-09 | 2020-12-11 | 亥姆霍兹慕尼黑-德国健康与环境研究中心(有限公司) | Device and method for monitoring scar formation |
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2006
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101856517A (en) * | 2010-06-18 | 2010-10-13 | 杭州市第三人民医院 | Tissue engineering material-based culture method and applications of melanophore |
| CN101856517B (en) * | 2010-06-18 | 2013-04-03 | 杭州市第三人民医院 | Tissue engineering material-based culture method and applications of melanophore |
| CN103194423A (en) * | 2013-03-07 | 2013-07-10 | 中国人民解放军军事医学科学院野战输血研究所 | Medium and purpose thereof |
| CN103194423B (en) * | 2013-03-07 | 2015-02-25 | 中国人民解放军军事医学科学院野战输血研究所 | Medium and purpose thereof |
| CN105255813A (en) * | 2015-08-14 | 2016-01-20 | 上海交通大学医学院附属第九人民医院 | Epithelial tissue and epithelial tissue in-vitro construction method |
| CN105255813B (en) * | 2015-08-14 | 2018-09-25 | 上海交通大学医学院附属第九人民医院 | A kind of method of epithelial tissue and external structure epithelial tissue |
| CN105238739A (en) * | 2015-11-11 | 2016-01-13 | 朱宁文 | Selective culture method for large-scale preparation of human extracellular matrix through melanocytes for clinic treatment level cell therapy |
| CN107802891A (en) * | 2017-11-09 | 2018-03-16 | 清华大学深圳研究生院 | Organization engineering skin and preparation method thereof |
| CN112074596A (en) * | 2018-02-09 | 2020-12-11 | 亥姆霍兹慕尼黑-德国健康与环境研究中心(有限公司) | Device and method for monitoring scar formation |
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