CN102949752A - Construction method for tissue engineering skin containing complete skin appendage - Google Patents
Construction method for tissue engineering skin containing complete skin appendage Download PDFInfo
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- CN102949752A CN102949752A CN2011102494252A CN201110249425A CN102949752A CN 102949752 A CN102949752 A CN 102949752A CN 2011102494252 A CN2011102494252 A CN 2011102494252A CN 201110249425 A CN201110249425 A CN 201110249425A CN 102949752 A CN102949752 A CN 102949752A
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Abstract
The invention relates to the field of tissue engineering skin research of molecular biology, in particular to a method for constructing full-layer three-dimensional skin containing a complete skin appendage in vitro by applying the tissue engineering method.
Description
Technical field
The invention belongs to the tissue engineering technique field of molecular biology skin model, relate to a kind of a new generation more near the organization engineering skin that contains complete skin appendages and the construction method thereof of human skin structure.
Background technology
Skin easily is subject to the infringement of the factors such as wound, burn, inflammation, ulcer, tumor post-operation and congenital disease as the vital tissue organ that covers and protect body surface.The wound repair area research that appears as of organization engineering skin provides a kind of new treatment approach, at present, has had the various skin product to come out, and has been used for clinical treatment, has obtained certain curative effect.Yet the real desirable permanent Graftskin of organization engineering skin distance has remained certain gap.Especially present technical method can not construct the appendages structure of skin in organization engineering skin, such as hair follicle, sweat gland, sebaceous gland etc.And these structures play vital effect in skin trauma repair and reconstruction process.Therefore, how setting up what contain skin appendages is one of problem demanding prompt solution near the threedimensional model of natural skin more.
The patent of the people such as Ri Wujinjin application in February 6 calendar year 2001 (application number: 01107099.4) and the patent of JIUYUE in 2002 people such as banket application on the 2nd (application number: 02139398.2) all relate to the preparation of organization engineering skin, above invention comprises that various solution preparations, collagen gel preparation and cell three-dimensional cultivate.Constructed organization engineering skin is structurally similar with human body skin, has both had epidermal area and skin corium, rebuilds but can not reach real skin owing to lacking skin appendages, and all biological that therefore not yet has normal skin is learned function.And the equal various cell proportions during undeclared preparation organization engineering skin of above invention, can not clearly interaction between iuntercellular, cell and extracellular matrix etc. in the organization engineering skin model.Extracellular matrix components is as the functional activity zone, with the tight related also differentiation direction of regulating cell of the differentiation of cell; Simultaneously, growing multiplication of cell etc. also depends on the reconstruction of microenvironment.Yet invention does not in the past relate to the effect of extracellular matrix and microenvironment to the impact of cell.We are at the patent (application number: 200910253998.5) related to the construction method of the organization engineering skin that contains sweat gland of application in 2009, the organization engineering skin that does not relate to other skin appendages makes up, yet the symbiosis of various skin appendages and mutual synergism are the keys of organization engineering skin performance function.Therefore, make up a kind of organization engineering skin substitute that contains complete skin appendages, can promote the development of imitation biochemistry functional organization engineering skin.
Summary of the invention
One of purpose of the present invention is the deficiency for existing organization engineering skin, provides a kind of more near the organization engineering skin of new generation that contains complete skin appendages of natural skin.Two of purpose of the present invention provides the preparation method of above-mentioned skin model.May further comprise the steps:
A) microsphere as cell microcarrier and growth factor release carrier prepares, employing has good biocompatibility and contains the gelatin of various kinds of cell epimatrix composition as the main natural substrates of carrier construction, composite growth factor was to keep skin appendages growth and proliferation of cell state after preparation was finished, and perhaps external evoked adult stem cell generates skin appendages cell (0.25-0.5 μ g somatomedin/g microsphere);
B) separation and cultivation hair follicle cell, sweat gland cells, sebaceous gland cell or the external evoked adult stem cell that can generate the skin appendages cell of process, described various cells microsphere supported and that cultivate are carried out compound the cultivation altogether 5-7 days, and the density of inoculating the cell of microsphere is 5 * 10
3-1 * 10
4/ cm
2
The hair follicle cell that c) will obtain through common cultivation, sweat gland cells, sebaceous gland cell microcarrier complex or generated the adult stem cell of various skin appendages cells and inoculated epiderm skin cell and fibroblastic collagen gel and mix (skin appendages cell: epidermis cell: fibroblastic ratio is 1: 1: 2) through external evoked, and cultivated 15-20 days according to the mode of three-dimensional structure;
D) constructed organization engineering skin is made morphological observation, carry out HE dyeing, the skin appendages such as the skin texture that observation post makes up and hair follicle cell, sweat gland cells, sebaceous gland generate situation; Detect the expression of skin appendages mark in the constructed organization engineering skin with immunohistochemical method.
Description of drawings
Fig. 1 is the organization engineering skin model HE dyeing photo (a) of the compound complete skin appendages of dimensional culture, wherein anti--CK5 (b), CK14 (c) immunofluorescence dyeing positive expression.
The specific embodiment
Following the present invention specifically sets forth invention in the mode of specific embodiment.It will be appreciated by persons skilled in the art that following embodiment is in order to set forth invention, and non-limiting invention scope of the present invention.
Embodiment
Embodiment 1 microsphere supported and various skin appendages cell carries out compound altogether cultivation
Getting that sterilization seals up for safekeeping microsphere supported soak 24h with DMEM after, be tiled in 12 orifice plates, add the DMEM culture fluid that contains 10% hyclone, with the hair follicle cell of former culture, sweat gland cells, sebaceous gland cell respectively with 5 * 10
3-1 * 10
4/ cm
2Density is inoculated in particulate carrier and puts 37 ℃, 5%CO
2In the incubator compound cultivation 5-7 days.
The three-dimensional skin that embodiment 2 contains complete skin appendages makes up
Get the good cell of growth conditions, with epidermis cell and dermal fibroblast with 1 * 10
7The density of/ml is mixed (collagen concentration 8mg/ml with extraction from the type i collagen 3ml of calf-skin, add the DMEM that contains 10% hyclone before mixing, abundant mixing, regulate pH to 7.4), to import collagen gel (skin appendages cell summation: epidermis cell: fibroblastic ratio was as 1: 1: 2) take injection system with hair follicle cell, sweat gland cells, sebaceous gland cell carrier complex (ratio is 1: 1: 1) simultaneously, and hatch for 37 ℃ and solidify rear adding culture fluid cultivation 3 days; The collagen gel of compound cells is moved to the gas-liquid face carry out the organization engineering skin that the organ culture namely obtains to contain complete skin appendages 1～2 week.
Claims (10)
1. a structure contains the method for the three-dimensional skin model of holostrome of whole skin appendages, it is characterized in that described method is as means take stem cell and regenerative medicine and engineered technical method, external the skin holostrome is comprised that all types cell of skin appendages (hair follicle, sweat gland, sebaceous gland) and compound the constructing of biologic bracket material have and the mutually dermatoid method of normal skin configuration, may further comprise the steps:
A) microsphere as cell microcarrier and growth factor release carrier prepares;
B) separation and cultivation epiderm skin, corium and skin appendages cell are perhaps through external evoked all adult stem cells (comprise and coming from from body, allogeneic etc.) that can generate the skin appendages cell;
C) described microsphere supported different cells with cultivating are carried out respectively compound altogether cultivation;
D) will mix with the gel that contains matrix components of inoculation epidermis cell and hypodermal cell by your different proportion through the various cell microcarrier complexs that common cultivation obtains, and continue to cultivate according to tridimensional model;
E) constructed organization engineering skin is made morphological observation, carry out HE dyeing, the skin texture that observation post makes up and the formational situation of various skin appendages cells; Detect the expression of skin appendages cell sign thing in the constructed organization engineering skin with immunofluorescence staining.
2. method according to claim 1 is characterized in that described microsphere supported preparation method is to be prepared from by the acceptable Biodegradable material of body.
3. method according to claim 2 is characterized in that described Biodegradable material comprises any or several combinations of gelatin, collagen, hyaluronic acid, chitosan, alginate, polylactic acid, polyglycolic acid, polyglycolic-lactic acid.
4. method according to claim 2 is characterized in that described microsphere supported particle diameter is 100-500 μ m.
5. method according to claim 1 is characterized in that the compound somatomedin of described microsphere comprises epithelical cell growth factor (EGF), fibroblast growth factor (acidity: aFGF; Alkalescence: bFGF), any or several combinations of the signal in platelet-derived endothelial cell growth factor (PD-ECGF/PDGF), VEGF (VEGF), transforming growth factor β (TFG-β) and cytokine and extracellular matrix source etc.
6. method according to claim 1, it is characterized in that described cell is the one-tenth somatic cell from body/allogeneic source, comprise all skin-derived cells and adult stem cell etc., it is characterized in that described cell is 5 * 103-1 * 104/cm2 at the inoculum density on microsphere supported.
7. method according to claim 1, the gel that contains matrix components comprises the prepared gels of natural macromolecular material such as gelatin, collagen, hyaluronic acid, chitosan, alginate.
8. each described method according to claim 1-7, it is characterized in that described skin appendages cell: epidermis cell: fibroblastic ratio is 1: 1: 2.
9. method according to claim 1 is characterized in that described various skin appendages cell (or through external evoked adult stem cell that can generate the skin appendages cell) and microsphere supported compound incubation time are 5-7 days.
10. method according to claim 1 is characterized in that the time that described skin appendages cell microcarrier complex and epidermis cell and fibroblast carry out compound cultivation according to the dimensional culture mode is 15-20 days.
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| CN2011102494252A CN102949752A (en) | 2011-08-22 | 2011-08-22 | Construction method for tissue engineering skin containing complete skin appendage |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104491931A (en) * | 2014-12-02 | 2015-04-08 | 清华大学深圳研究生院 | Sebaceous gland-containing skin tissue as well as formation method and application thereof |
| CN106421916A (en) * | 2016-10-24 | 2017-02-22 | 广州润虹医药科技有限公司 | Tissue engineering skin and preparation method thereof |
| CN107217028A (en) * | 2017-05-27 | 2017-09-29 | 广州润虹医药科技有限公司 | A kind of organization engineering skin containing appendicle and preparation method thereof |
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| CN101773688A (en) * | 2010-02-05 | 2010-07-14 | 中国人民解放军第四军医大学 | Preparation method of tissue engineering skin containing appendant organs |
| CN102091352A (en) * | 2009-12-09 | 2011-06-15 | 中国人民解放军总医院第一附属医院 | Method for constructing tissue engineering skin model containing sweat gland |
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| CN1415384A (en) * | 2002-09-02 | 2003-05-07 | 中国人民解放军第四军医大学口腔医学院 | Method for preparing all layers of skin used by tissue engineering |
| CN101147810A (en) * | 2007-11-01 | 2008-03-26 | 南昌大学第一附属医院 | Cell-biodegradable material compound and its preparation method and application |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104491931A (en) * | 2014-12-02 | 2015-04-08 | 清华大学深圳研究生院 | Sebaceous gland-containing skin tissue as well as formation method and application thereof |
| WO2016086527A1 (en) * | 2014-12-02 | 2016-06-09 | 清华大学深圳研究生院 | Sebaceous gland-containing skin tissue, formation method and usage thereof |
| CN106421916A (en) * | 2016-10-24 | 2017-02-22 | 广州润虹医药科技有限公司 | Tissue engineering skin and preparation method thereof |
| CN107217028A (en) * | 2017-05-27 | 2017-09-29 | 广州润虹医药科技有限公司 | A kind of organization engineering skin containing appendicle and preparation method thereof |
| CN107217028B (en) * | 2017-05-27 | 2020-05-08 | 广州润虹医药科技股份有限公司 | Tissue engineering skin containing accessory and preparation method thereof |
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Application publication date: 20130306 |