CN107287150A - A kind of construction method of 3D epidermises model - Google Patents
A kind of construction method of 3D epidermises model Download PDFInfo
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- CN107287150A CN107287150A CN201710460494.5A CN201710460494A CN107287150A CN 107287150 A CN107287150 A CN 107287150A CN 201710460494 A CN201710460494 A CN 201710460494A CN 107287150 A CN107287150 A CN 107287150A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0629—Keratinocytes; Whole skin
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
- C12N2500/14—Calcium; Ca chelators; Calcitonin
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
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- C12N2500/84—Undefined extracts from animals from mammals
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/33—Insulin
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- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
- C12N2502/1323—Adult fibroblasts
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Abstract
The invention belongs to tissue engineering technique field, and in particular to a kind of construction method of 3D epidermises model.This method mitomycin C（Mitomycin C, MC）The fibroblast of processing is inoculated with keratinocyte thereon as trophoderm, is needed using the supply cutin formation cell growth such as various factors and albumen of fibroblasts to secrete, while coordinating low concentration serum and EGF, insulin and CaCl2, the culture through two stages of under liquid and gas-liquid face, structure form 3D epidermis models.3D epidermises model constructed by the present invention is consistent with human epidermal structure height, and reduces production cost, meets economic, effective and efficient requirement needed for large-scale production 3D epidermis models.
Description
Technical field
The invention belongs to tissue engineering technique field, and in particular to a kind of construction method of 3D epidermises model.
Background technology
Traditional cosmetic material screening and product development checking is mainly completed by zoopery, due to species variation
And individual difference causes the result of zoopery can not its security of accurate evaluation and both effectiveness, and result poor reproducibility.Animal
Welfare protective tissue is for resisting the cry also more and more higher of zoopery.In view of the foregoing, European Union, the U.S. and other countries
Legislation forbids cosmetics or raw material through zoopery in its market sale in succession, in order to meet cosmetics exploitation demand, has
Effect assesses its security and both effectiveness, promotes cosmetic industry upgrading, and animal substitutes detection and arisen at the historic moment, and with human body skin knot
The highly consistent skin model of structure turns into the core tool substituted in detection method, and epidermis model is even more to have as one of which
Very important status, therefore exploitation one kind meets detection requirement, and economical and practical epidermis modeling sense is great.
The keratinocyte for building epidermis model is the main cell in epidermal cell, and it can form epidermis mould
The peculiar basic structure of type, i.e. basalis, spinous layer, stratum granulosum and cuticula.Being selected general keratinocyte culture without blood more
Clear cultivating system, by adding the various exogenous factors in basic culture solution, it is ensured that keratinocyte is in stable condition, prolong
Delay aging.But the addition factor in serum-free medium is most all costly such as bovine brain pituitary extract（Bovine
Pituitary Extract, BPE）, have a strong impact on keratinocyte and expand the production of the scale of product, the cell exists
Easily occur problem of aging again in traditional system containing serum free culture system and cause model construction to fail, how to solve epidermal cell and exist
Propagation, Differentiation Problems in system containing serum free culture system turn into the key of technology.
Because keratinocyte has stem cell properties, therefore provided using trophocyte to break through the technical barrier
May.Trophocyte is stem cell screening, culture and induction type versatile stem cell（iPSCs）A kind of conventional skill of culture
Art, the primary or initial-stage culture stage, which typically all needs to depend on to secrete them and survive in vitro, breeds necessary growth factor
Feeder cells.The growth factor of different types of feeder cells secretion is slightly different, but requires the taste in incubation
Foster confluent monolayer cells, which do not divide, does not breed and still keeps metabolic activity.Common can have fibroblast, 3T3 as trophoblastic cell
Cell, mesenchymal cell etc., conventional mitotic block agent are mitomycin C（Mitomycin C, MC）, it is with DNA shape
Into crosslinking, suppress DNA replication dna, also have inhibitory action to RNA, so as to reach above-mentioned purpose.
CN104459145A discloses a kind of cosmetics detection, by coating IV type glue on commercially available polycarbonate membrane
Original, then passes through serum-free medium（Epilife）Epidermis model is built, invention publicity content is first according to, only in nutrient solution
Middle addition lipid mixture and Ca2+ can successfully construct epidermis model and lack theories integration.Secondly the lipid addition in nutrient solution
Thing is expensive, and because most of sub- need to unavoidably to introduce solubilizing agents such as dimethyl in non-aqueous material processing procedure
Peak（DMSO）, excessively it is introduced into or extended residual can brings uncertain influence in nutrient solution on the growth of cell.
Chinese patent 201310425798.X discloses a kind of construction method of tissue engineering epidermis model, first to silkworm
Silk carries out degumming, dissolving, dialysis and concentration and prepares silk fibroin protein solution, then with the silk fibroin protein solution infiltration thermoplasticity prepared
Non-woven fabrics, after by vacuum drying and Ethanol Treatment, be prepared into inoculation supporter, then above inoculation human epidermal cell strain
HaCat is built into epidermis model.Whole supporter processing procedure is cumbersome, complexity, and introduces the thermoplastic nonwoven of non-skin structure
Cloth, is either transmitted to the liquid nutritional built in culture, or to the Histological section after late detection and interpretation of result all
Strong influence can be produced.
The content of the invention
The technical problems to be solved by the invention are that there is provided a kind of 3D epidermises model for above-mentioned the deficiencies in the prior art
Construction method.The fibroblast that this method is handled with mitomycin C utilizes each of fibroblasts to secrete as trophoderm
Planting the supply cutin formation cell growth such as the factor and albumen needs, while coordinate low concentration serum and EGF, insulin and CaCl2,
Growth and the differentiation demand of keratinocyte can be met to greatest extent, reduce the production cost of 3D epidermis models.
In order to solve the above technical problems, the technical solution adopted by the present invention is：A kind of construction method of 3D epidermises model, its
It is characterised by, comprises the following steps：
Counted Step 1: being resuspended after the fibroblast recovered is digested, according to the close of 3 × 104/cm2~6 × 104/cm2
Fibroblast after recovery is inoculated in the nutrient solution containing serum by degree, is discarded after cultivating 18h~24h in CO2 incubators
Nutrient solution, then adds the nutrient solution containing mitomycin C, and nutrient solution is discarded after handling 2h~6h in CO2gas incubator,
Rinsed 3~5 times with PBS cushioning liquid, obtain fibroblast trophoderm；
Counted Step 2: being resuspended after the keratinocyte recovered is digested, cell concentration needed for being calculated according to inoculum density, so
Afterwards according to 1 × 105/cm2~5 × 105/cm2 density by the keratinocyte after resuspension be seeded to described in step one into
Fibrocyte nourishes layer surface, then is placed under liquid culture under progress liquid in nutrient solution, changes be transferred to after cultivating 48h under liquid, liquid daily
Gas-liquid face culture is carried out in the nutrient solution of gas-liquid face, liquid is changed daily, 3D epidermis models are obtained；Nutrient solution is with low-sugar type under the liquid
Liquid based on DMEM nutrient solutions, α-MEM nutrient solutions, F12 nutrient solutions or DF12 nutrient solutions, addition hyclone, calcium chloride, EGF
The volumn concentration of hyclone is 1%~5%, the concentration of calcium chloride in nutrient solution under EGF and insulin, liquid
For 0.1mM~0.5mM, the concentration of EGF EGFs is the μ g/mL of 0.01 μ g/mL~0.1, and the concentration of insulin is
0.05mM~2mM；Gas-liquid face nutrient solution is trained with low-sugar type DMEM nutrient solutions, α-MEM nutrient solutions, F12 nutrient solutions or DF12
Tire ox blood in liquid based on nutrient solution, addition hyclone, calcium chloride, EGF EGFs and insulin, gas-liquid face nutrient solution
Clear volumn concentration is 1%~5%, and the concentration of calcium chloride is 1.0mM~1.5mM, and the concentration of EGF EGFs is
The μ g/mL of 0.01 μ g/mL~0.1, the concentration of insulin is 0.05mM~2mM.
A kind of construction method of above-mentioned 3D epidermises model, it is characterised in that the nutrient solution containing serum described in step one
The liquid based on low-sugar type DMEM nutrient solutions, α-MEM nutrient solutions, F12 nutrient solutions or DF12 nutrient solutions, adds hyclone, institute
The volumn concentration for stating hyclone in the nutrient solution containing serum is 5%~15%.
A kind of construction method of above-mentioned 3D epidermises model, it is characterised in that the training containing mitomycin C described in step one
Nutrient solution liquid based on low-sugar type DMEM nutrient solutions, α-MEM nutrient solutions, F12 nutrient solutions or DF12 nutrient solutions, adds hyclone
And mitomycin C, the volumn concentration of hyclone is 5%~15%, mitomycin in the nutrient solution containing mitomycin C
C concentration is the μ g/mL of 10 μ g/mL~40.
A kind of construction method of above-mentioned 3D epidermises model, it is characterised in that gas-liquid face described in step 2 culture when
Between be 8~10 days.
The present invention has advantages below compared with prior art：
1st, the identical tissue-derived fibroblast that the present invention is handled using mitomycin C can keep away as trophocyte
Exempt from because the uncertain influence of external source that species variation is introduced, while reciprocation that can to greatest extent between analogue body inner cell,
Ensure cell or tissue normal growth.The fibroblast handled by mitomycin C does not breed but keeps metabolic activity, it is only necessary to
The purpose for reducing exterior addition factor is just can reach using less nutrition, it secret out of into fiber production factor（FGF）, especially
It is Basic Fibroblast Growth Factor（β-FGF）Endothelioid cells can be promoted to breed, preferably be repaiied while having to the cell of damage
Multiple effect；The TGF secreted out of（TGF）It is a multifunctional protein for cell, the life of various kinds of cell can be influenceed
The functions, transforming growth factor-β therein such as long, differentiation, Apoptosis and immunological regulation（TGF-β）SMAD signals can be passed through
Path and/or DAXX signal paths play its biological function, promote cell propagation.Because fibroblast can secrete natural
Collagen, is more beneficial for the attaching and growth of keratinocyte.
2nd, the basic ingredient in nutrient solution of the invention can meet keratinocyte basic physical needs, add low concentration
Serum can partly substitute in keratinocyte nutrient solution add the factor effect, provide nutrition for keratinocyte,
Such as part replacement bovine brain pituitary extract, keratinocyte is set to be unlikely in the serum of high concentration（Routine serum is dense
Spend for 10%）And occur aging.The other kinds of factor and albumen common guarantee cutin shape of fibroblasts to secrete are utilized simultaneously
Into cell normal growth demand, the EGF EGFs of addition are for promoting keratinocyte growth and angling to have important
Effect, insulin Human Keratinocytes have obvious proliferation.
3rd, the present invention cultivates the Ca2+ concentration difference of the nutrient solution used in gentle liquid level incubation, low concentration under liquid
Ca2+（0.1mM~0.5mM）It can promote the propagation of keratinocyte, and the Ca2+ of high concentration（1.0mM~1.5mM）Then
It can be promoted to break up, different cultivation stages, the CaCl2 of various concentrations is added in serum-containing medium can be effectively ensured angle
Matter formation cell is broken up according to predetermined direction, maturation.
4th, the fibroblast that the present invention is handled with mitomycin C utilizes each of fibroblasts to secrete as trophoderm
Kind of the factor and albumen such as FGF, MMP and TGF etc. supply cutin formation cell growth need, while cooperation low concentration serum and EGF,
Insulin and CaCl2, growth and the differentiation demand of keratinocyte can be met to greatest extent, reduce the life of 3D epidermis models
Produce cost.Meet required economic, effective and efficient requirement of large-scale production 3D epidermis models.
With reference to the accompanying drawings and examples, technical scheme is described in further detail.
Brief description of the drawings
Fig. 1 is the HE slice maps for the 3D epidermis models that the embodiment of the present invention 1 is built.
Fig. 2 is the HE slice maps for the 3D epidermis models that the embodiment of the present invention 2 is built.
Fig. 3 is the HE slice maps for the 3D epidermis models that the embodiment of the present invention 3 is built.
Fig. 4 is the HE slice maps for the 3D epidermis models that the embodiment of the present invention 4 is built.
Fig. 5 is the HE slice maps for the 3D epidermis models that the embodiment of the present invention 5 is built.
Fig. 6 is the HE slice maps for the 3D epidermis models that the embodiment of the present invention 6 is built.
Fig. 7 is the HE slice maps for the 3D epidermis models that the embodiment of the present invention 7 is built.
Fig. 8 is the HE slice maps for the 3D epidermis models that the embodiment of the present invention 8 is built.
Fig. 9 is the HE slice maps for the 3D epidermis models that the embodiment of the present invention 9 is built.
Figure 10 is the HE slice maps for the 3D epidermis models that the embodiment of the present invention 10 is built.
Figure 11 is the HE slice maps for the 3D epidermis models that the embodiment of the present invention 11 is built.
Figure 12 is the HE slice maps for the 3D epidermis models that the embodiment of the present invention 12 is built.
Embodiment
Embodiment 1
The construction method of the 3D epidermis models of the present embodiment comprises the following steps：
Step 1: recovery P2 carries out cellar culture to the fibroblast of recovery, treats that plating cells rate reaches for human fibroblasts
Had digestive transfer culture is carried out to 85% or so, is inoculated in after cell count according to 6 × 104/cm2 density in the nutrient solution containing serum, in
37 DEG C, discard nutrient solution after culture 24h in 5%CO2 incubators, the nutrient solution containing mitomycin C is then added, in carbon dioxide
Nutrient solution is discarded after handling 2h in incubator, cell is softly rinsed 3 times with the PBS cushioning liquid of 37 DEG C of preheatings, obtains into fiber
Cytotrophoblast；The nutrient solution containing serum liquid based on low-sugar type DMEM nutrient solutions, adds hyclone, described to contain blood
The volumn concentration of hyclone is 10% in clear nutrient solution；The nutrient solution containing mitomycin C is trained with low-sugar type DMEM
The volumn concentration of hyclone in nutrient solution, addition hyclone and mitomycin C, the nutrient solution containing mitomycin C
For 10%, the concentration of mitomycin C is 40 μ g/mL；
Counted Step 2: being resuspended after the keratinocyte recovered is digested, cell concentration needed for being calculated according to inoculum density, so
The keratinocyte after resuspension is seeded into fibroblast described in step one according to 5 × 105/cm2 density afterwards to nourish
Layer surface, then culture under progress liquid in nutrient solution is placed under liquid, change daily after cultivating 48h under liquid, liquid and be transferred to gas-liquid face nutrient solution
Middle progress gas-liquid face culture, the time of the gas-liquid face culture is 10 days, and liquid is changed daily, 3D epidermis models are obtained；Under the liquid
Trained under nutrient solution liquid based on DF12 nutrient solutions, addition hyclone, calcium chloride, EGF EGFs and insulin, liquid
The volumn concentration of hyclone is 5% in nutrient solution, and the concentration of calcium chloride is 0.5mM, and the concentration of EGF EGFs is
0.01 μ g/mL, the concentration of insulin is 2mM；Gas-liquid face nutrient solution liquid based on DF12 nutrient solutions, adds tire ox blood
Clearly, the volumn concentration of hyclone is 5%, chlorine in calcium chloride, EGF EGFs and insulin, gas-liquid face nutrient solution
The concentration for changing calcium is 1.5mM, and the concentration of EGF EGFs is 0.01 μ g/mL, and the concentration of insulin is 2mM.
The 3D epidermises model and people's epidermal structure that the present embodiment is built are highly consistent（Such as Fig. 1）.Can be obvious in HE slice maps
Find out that the 3D epidermises model being built into using this method has consistent with human epidermal, distinctive four-layer structure, from the bottom up
It is followed successively by basalis, spinous layer, stratum granulosum and cuticula.
Embodiment 2
The construction method of the 3D epidermis models of the present embodiment comprises the following steps：
Step 1: recovery P2 carries out cellar culture to the fibroblast of recovery, treats that plating cells rate reaches for human fibroblasts
Had digestive transfer culture is carried out to 85% or so, is inoculated in after cell count according to 3 × 104/cm2 density in the nutrient solution containing serum, in
37 DEG C, discard nutrient solution after culture 18h in 5%CO2 incubators, the nutrient solution containing mitomycin C is then added, in carbon dioxide
Nutrient solution is discarded after handling 3h in incubator, cell is softly rinsed 5 times with the PBS cushioning liquid of 37 DEG C of preheatings, obtains into fiber
Cytotrophoblast；The nutrient solution containing serum liquid based on α-MEM nutrient solutions, adds hyclone, described containing serum
The volumn concentration of hyclone is 5% in nutrient solution；The nutrient solution containing mitomycin C is based on α-MEM nutrient solutions
The volumn concentration of hyclone is in liquid, addition hyclone and mitomycin C, the nutrient solution containing mitomycin C
5%, the concentration of mitomycin C is 20 μ g/mL；
Counted Step 2: being resuspended after the keratinocyte recovered is digested, cell concentration needed for being calculated according to inoculum density, so
The keratinocyte after resuspension is seeded into fibroblast described in step one according to 1 × 105/cm2 density afterwards to nourish
Layer surface, then culture under progress liquid in nutrient solution is placed under liquid, change daily after cultivating 48h under liquid, liquid and be transferred to gas-liquid face nutrient solution
Middle progress gas-liquid face culture, the time of the gas-liquid face culture is 8 days, and liquid is changed daily, 3D epidermis models are obtained；Trained under the liquid
Trained under nutrient solution liquid based on α-MEM nutrient solutions, addition hyclone, calcium chloride, EGF EGFs and insulin, liquid
The volumn concentration of hyclone is 1% in nutrient solution, and the concentration of calcium chloride is 0.1mM, and the concentration of EGF EGFs is
0.1 μ g/mL, the concentration of insulin is 0.05mM；Gas-liquid face nutrient solution liquid based on α-MEM nutrient solutions, adds tire ox
The volumn concentration of hyclone is 1% in serum, calcium chloride, EGF EGFs and insulin, gas-liquid face nutrient solution,
The concentration of calcium chloride is 1.0mM, and the concentration of EGF EGFs is 0.1 μ g/mL, and the concentration of insulin is 0.05mM.
The 3D epidermises model and people's epidermal structure that the present embodiment is built are highly consistent（Such as Fig. 2）.
Embodiment 3
The construction method of the 3D epidermis models of the present embodiment comprises the following steps：
Step 1: recovery P2 carries out cellar culture to the fibroblast of recovery, treats that plating cells rate reaches for human fibroblasts
Had digestive transfer culture is carried out to 85% or so, is inoculated in after cell count according to 5 × 104/cm2 density in the nutrient solution containing serum, in
37 DEG C, discard nutrient solution after culture 20h in 5%CO2 incubators, the nutrient solution containing mitomycin C is then added, in carbon dioxide
Nutrient solution is discarded after handling 6h in incubator, cell is softly rinsed 4 times with the PBS cushioning liquid of 37 DEG C of preheatings, obtains into fiber
Cytotrophoblast；The nutrient solution containing serum liquid based on DF12 nutrient solutions, adds hyclone, the training containing serum
The volumn concentration of hyclone is 15% in nutrient solution；The nutrient solution containing mitomycin C is based on DF12 nutrient solutions
The volumn concentration of hyclone is in liquid, addition hyclone and mitomycin C, the nutrient solution containing mitomycin C
15%, the concentration of mitomycin C is 10 μ g/mL；
Counted Step 2: being resuspended after the keratinocyte recovered is digested, cell concentration needed for being calculated according to inoculum density, so
The keratinocyte after resuspension is seeded into fibroblast described in step one according to 3 × 105/cm2 density afterwards to nourish
Layer surface, then culture under progress liquid in nutrient solution is placed under liquid, change daily after cultivating 48h under liquid, liquid and be transferred to gas-liquid face nutrient solution
Middle progress gas-liquid face culture, the time of the gas-liquid face culture is 9 days, and liquid is changed daily, 3D epidermis models are obtained；Trained under the liquid
Cultivated under nutrient solution liquid based on F12 nutrient solutions, addition hyclone, calcium chloride, EGF EGFs and insulin, liquid
The volumn concentration of hyclone is 3% in liquid, and the concentration of calcium chloride is 0.2mM, and the concentration of EGF EGFs is
0.05 μ g/mL, the concentration of insulin is 1mM；Gas-liquid face nutrient solution is with F12 nutrient solution basal liquids, addition hyclone, chlorine
The volumn concentration for changing hyclone in calcium, EGF EGFs and insulin, gas-liquid face nutrient solution is 3%, calcium chloride
Concentration be 1.2mM, the concentration of EGF EGFs is 0.05 μ g/mL, and the concentration of insulin is 1mM.
The 3D epidermises model and people's epidermal structure that the present embodiment is built are highly consistent（Such as Fig. 3）.
Embodiment 4
The construction method of the 3D epidermis models of the present embodiment comprises the following steps：
Step 1: recovery P2 carries out cellar culture to the fibroblast of recovery, treats that plating cells rate reaches for human fibroblasts
Had digestive transfer culture is carried out to 85% or so, is inoculated in after cell count according to 3 × 104/cm2 density in the nutrient solution containing serum, in
37 DEG C, discard nutrient solution after culture 24h in 5%CO2 incubators, the nutrient solution containing mitomycin C is then added, in carbon dioxide
Nutrient solution is discarded after handling 5h in incubator, cell is softly rinsed 5 times with the PBS cushioning liquid of 37 DEG C of preheatings, obtains into fiber
Cytotrophoblast；The nutrient solution containing serum liquid based on F12 nutrient solutions, adds hyclone, the training containing serum
The volumn concentration of hyclone is 10% in nutrient solution；The nutrient solution containing mitomycin C liquid based on F12 nutrient solutions,
The volumn concentration for adding hyclone in hyclone and mitomycin C, the nutrient solution containing mitomycin C is 10%,
The concentration of mitomycin C is 30 μ g/mL；
Counted Step 2: being resuspended after the keratinocyte recovered is digested, cell concentration needed for being calculated according to inoculum density, so
The keratinocyte after resuspension is seeded into fibroblast described in step one according to 5 × 105/cm2 density afterwards to nourish
Layer surface, then culture under progress liquid in nutrient solution is placed under liquid, change daily after cultivating 48h under liquid, liquid and be transferred to gas-liquid face nutrient solution
Middle progress gas-liquid face culture, the time of the gas-liquid face culture is 10 days, and liquid is changed daily, 3D epidermis models are obtained；Under the liquid
Nutrient solution liquid based on low-sugar type DMEM nutrient solutions, addition hyclone, calcium chloride, EGF EGFs and insulin,
The volumn concentration of hyclone is 4% in nutrient solution under liquid, and the concentration of calcium chloride is 0.3mM, EGF EGFs
Concentration is 0.1 μ g/mL, and the concentration of insulin is 0.5mM；Gas-liquid face nutrient solution is based on low-sugar type DMEM nutrient solutions
The volume hundred of hyclone in liquid, addition hyclone, calcium chloride, EGF EGFs and insulin, gas-liquid face nutrient solution
It is 4% to divide content, and the concentration of calcium chloride is 1.2mM, and the concentration of EGF EGFs is 0.1 μ g/mL, and the concentration of insulin is
0.5mM。
The 3D epidermises model and people's epidermal structure that the present embodiment is built are highly consistent（Such as Fig. 4）.
Embodiment 5
The construction method of the 3D epidermis models of the present embodiment comprises the following steps：
Step 1: recovery P2 carries out cellar culture to the fibroblast of recovery, treats that plating cells rate reaches for human fibroblasts
Had digestive transfer culture is carried out to 85% or so, is inoculated in after cell count according to 6 × 104/cm2 density in the nutrient solution containing serum, in
37 DEG C, discard nutrient solution after culture 18h in 5%CO2 incubators, the nutrient solution containing mitomycin C is then added, in carbon dioxide
Nutrient solution is discarded after handling 2h in incubator, cell is softly rinsed 5 times with the PBS cushioning liquid of 37 DEG C of preheatings, obtains into fiber
Cytotrophoblast；The nutrient solution containing serum liquid based on low-sugar type DMEM nutrient solutions, adds hyclone, described to contain blood
The volumn concentration of hyclone is 5% in clear nutrient solution；The nutrient solution containing mitomycin C is trained with low-sugar type DMEM
The volume of hyclone in liquid based on nutrient solution, addition hyclone and mitomycin C, the nutrient solution containing mitomycin C
Percentage composition is 5%, and the concentration of mitomycin C is 10 μ g/mL；
Counted Step 2: being resuspended after the keratinocyte recovered is digested, cell concentration needed for being calculated according to inoculum density, so
The keratinocyte after resuspension is seeded into fibroblast described in step one according to 3 × 105/cm2 density afterwards to nourish
Layer surface, then culture under progress liquid in nutrient solution is placed under liquid, change daily after cultivating 48h under liquid, liquid and be transferred to gas-liquid face nutrient solution
Middle progress gas-liquid face culture, the time of the gas-liquid face culture is 8 days, and liquid is changed daily, 3D epidermis models are obtained；Trained under the liquid
Nutrient solution liquid based on low-sugar type DMEM nutrient solutions, addition hyclone, calcium chloride, EGF EGFs and insulin, liquid
The volumn concentration of hyclone is 5% in lower nutrient solution, and the concentration of calcium chloride is 0.1mM, EGF EGFs it is dense
Spend for 0.01 μ g/mL, the concentration of insulin is 2mM；Gas-liquid face nutrient solution liquid based on low-sugar type DMEM nutrient solutions, adds
Plus hyclone, calcium chloride, EGF EGFs and insulin, the volume basis of hyclone contains in the nutrient solution of gas-liquid face
Measure as 5%, the concentration of calcium chloride is 1.0mM, and the concentration of EGF EGFs is 0.01 μ g/mL, and the concentration of insulin is
2mM。
The 3D epidermises model and people's epidermal structure that the present embodiment is built are highly consistent（Such as Fig. 5）.
Embodiment 6
The construction method of the 3D epidermis models of the present embodiment comprises the following steps：
Step 1: recovery P2 carries out cellar culture to the fibroblast of recovery, treats that plating cells rate reaches for human fibroblasts
Had digestive transfer culture is carried out to 85% or so, is inoculated in after cell count according to 6 × 104/cm2 density in the nutrient solution containing serum, in
37 DEG C, discard nutrient solution after culture 24h in 5%CO2 incubators, the nutrient solution containing mitomycin C is then added, in carbon dioxide
Nutrient solution is discarded after handling 2h in incubator, cell is softly rinsed 3 times with the PBS cushioning liquid of 37 DEG C of preheatings, obtains into fiber
Cytotrophoblast；The nutrient solution containing serum liquid based on DF12 nutrient solutions, adds hyclone, the training containing serum
The volumn concentration of hyclone is 10% in nutrient solution；The nutrient solution containing mitomycin C adds tire with DF12 nutrient solutions
The volumn concentration of hyclone is 10% in cow's serum and mitomycin C, the nutrient solution containing mitomycin C, and mitogen is mould
Plain C concentration is 40 μ g/mL；
Counted Step 2: being resuspended after the keratinocyte recovered is digested, cell concentration needed for being calculated according to inoculum density, so
The keratinocyte after resuspension is seeded into fibroblast described in step one according to 5 × 105/cm2 density afterwards to nourish
Layer surface, then culture under progress liquid in nutrient solution is placed under liquid, change daily after cultivating 48h under liquid, liquid and be transferred to gas-liquid face nutrient solution
Middle progress gas-liquid face culture, the time of the gas-liquid face culture is 10 days, and liquid is changed daily, 3D epidermis models are obtained；Under the liquid
Under nutrient solution liquid based on α-MEM nutrient solutions, addition hyclone, calcium chloride, EGF EGFs and insulin, liquid
The volumn concentration of hyclone is 5% in nutrient solution, and the concentration of calcium chloride is 0.5mM, the concentration of EGF EGFs
For 0.01 μ g/mL, the concentration of insulin is 2mM；Gas-liquid face nutrient solution liquid based on α-MEM nutrient solutions, adds tire ox
The volumn concentration of hyclone is 5% in serum, calcium chloride, EGF EGFs and insulin, gas-liquid face nutrient solution,
The concentration of calcium chloride is 1.5mM, and the concentration of EGF EGFs is 0.01 μ g/mL, and the concentration of insulin is 2mM.
The 3D epidermises model and people's epidermal structure that the present embodiment is built are highly consistent（Such as Fig. 6）.
Embodiment 7
The construction method of the 3D epidermis models of the present embodiment comprises the following steps：
Step 1: recovery P2 carries out cellar culture to the fibroblast of recovery, treats that plating cells rate reaches for human fibroblasts
Had digestive transfer culture is carried out to 85% or so, is inoculated in after cell count according to 3 × 104/cm2 density in the nutrient solution containing serum, in
37 DEG C, discard nutrient solution after culture 18h in 5%CO2 incubators, the nutrient solution containing mitomycin C is then added, in carbon dioxide
Nutrient solution is discarded after handling 3h in incubator, cell is softly rinsed 5 times with the PBS cushioning liquid of 37 DEG C of preheatings, obtains into fiber
Cytotrophoblast；The nutrient solution containing serum liquid based on F12 nutrient solutions, adds hyclone, the training containing serum
The volumn concentration of hyclone is 5% in nutrient solution；The nutrient solution containing mitomycin C liquid based on F12 nutrient solutions,
The volumn concentration for adding hyclone in hyclone and mitomycin C, the nutrient solution containing mitomycin C is 5%,
The concentration of mitomycin C is 40 μ g/mL；
Counted Step 2: being resuspended after the keratinocyte recovered is digested, cell concentration needed for being calculated according to inoculum density, so
The keratinocyte after resuspension is seeded into fibroblast described in step one according to 1 × 105/cm2 density afterwards to nourish
Layer surface, then culture under progress liquid in nutrient solution is placed under liquid, change daily after cultivating 48h under liquid, liquid and be transferred to gas-liquid face nutrient solution
Middle progress gas-liquid face culture, the time of the gas-liquid face culture is 8 days, and liquid is changed daily, 3D epidermis models are obtained；Trained under the liquid
Cultivated under nutrient solution liquid based on F12 nutrient solutions, addition hyclone, calcium chloride, EGF EGFs and insulin, liquid
The volumn concentration of hyclone is 1% in liquid, and the concentration of calcium chloride is 0.1mM, and the concentration of EGF EGFs is 0.1
μ g/mL, the concentration of insulin is 0.05mM；Gas-liquid face nutrient solution liquid based on F12 nutrient solutions, addition hyclone,
The volumn concentration of hyclone is 1%, chlorination in calcium chloride, EGF EGFs and insulin, gas-liquid face nutrient solution
The concentration of calcium is 1.0mM, and the concentration of EGF EGFs is 0.1 μ g/mL, and the concentration of insulin is 0.05mM.
The 3D epidermises model and people's epidermal structure that the present embodiment is built are highly consistent（Such as Fig. 7）.
Embodiment 8
The construction method of the 3D epidermis models of the present embodiment comprises the following steps：
Step 1: recovery P2 carries out cellar culture to the fibroblast of recovery, treats that plating cells rate reaches for human fibroblasts
Had digestive transfer culture is carried out to 85% or so, is inoculated in after cell count according to 5 × 104/cm2 density in the nutrient solution containing serum, in
37 DEG C, discard nutrient solution after culture 20h in 5%CO2 incubators, the nutrient solution containing mitomycin C is then added, in carbon dioxide
Nutrient solution is discarded after handling 6h in incubator, cell is softly rinsed 4 times with the PBS cushioning liquid of 37 DEG C of preheatings, obtains into fiber
Cytotrophoblast；The nutrient solution containing serum liquid based on F12 nutrient solutions, adds hyclone, the training containing serum
The volumn concentration of hyclone is 15% in nutrient solution；The nutrient solution containing mitomycin C liquid based on F12 nutrient solutions,
The volumn concentration for adding hyclone in hyclone and mitomycin C, the nutrient solution containing mitomycin C is 15%,
The concentration of mitomycin C is 10 μ g/mL；
Counted Step 2: being resuspended after the keratinocyte recovered is digested, cell concentration needed for being calculated according to inoculum density, so
The keratinocyte after resuspension is seeded into fibroblast described in step one according to 3 × 105/cm2 density afterwards to nourish
Layer surface, then culture under progress liquid in nutrient solution is placed under liquid, change daily after cultivating 48h under liquid, liquid and be transferred to gas-liquid face nutrient solution
Middle progress gas-liquid face culture, the time of the gas-liquid face culture is 9 days, and liquid is changed daily, 3D epidermis models are obtained；Trained under the liquid
Cultivated under nutrient solution liquid based on F12 nutrient solutions, addition hyclone, calcium chloride, EGF EGFs and insulin, liquid
The volumn concentration of hyclone is 5% in liquid, and the concentration of calcium chloride is 0.5mM, and the concentration of EGF EGFs is
0.01 μ g/mL, the concentration of insulin is 2mM；Gas-liquid face nutrient solution is with F12 nutrient solution basal liquids, addition hyclone, chlorine
The volumn concentration for changing hyclone in calcium, EGF EGFs and insulin, gas-liquid face nutrient solution is 3%, calcium chloride
Concentration be 1.5mM, the concentration of EGF EGFs is 0.01 μ g/mL, and the concentration of insulin is 2mM.
The 3D epidermises model and people's epidermal structure that the present embodiment is built are highly consistent（Such as Fig. 8）.
Embodiment 9
The construction method of the 3D epidermis models of the present embodiment comprises the following steps：
Step 1: recovery P2 carries out cellar culture to the fibroblast of recovery, treats that plating cells rate reaches for human fibroblasts
Had digestive transfer culture is carried out to 85% or so, is inoculated in after cell count according to 3 × 104/cm2 density in the nutrient solution containing serum, in
37 DEG C, discard nutrient solution after culture 20h in 5%CO2 incubators, the nutrient solution containing mitomycin C is then added, in carbon dioxide
Nutrient solution is discarded after handling 3h in incubator, cell is softly rinsed 3 times with the PBS cushioning liquid of 37 DEG C of preheatings, obtains into fiber
Cytotrophoblast；The nutrient solution containing serum liquid based on α-MEM nutrient solutions, adds hyclone, described containing serum
The volumn concentration of hyclone is 10% in nutrient solution；The nutrient solution containing mitomycin C is using α-MEM nutrient solutions as base
The volumn concentration of hyclone in plinth liquid, addition hyclone and mitomycin C, the nutrient solution containing mitomycin C
For 10%, the concentration of mitomycin C is 10 μ g/mL；
Counted Step 2: being resuspended after the keratinocyte recovered is digested, cell concentration needed for being calculated according to inoculum density, so
The keratinocyte after resuspension is seeded into fibroblast described in step one according to 5 × 105/cm2 density afterwards to nourish
Layer surface, then culture under progress liquid in nutrient solution is placed under liquid, change daily after cultivating 48h under liquid, liquid and be transferred to gas-liquid face nutrient solution
Middle progress gas-liquid face culture, the time of the gas-liquid face culture is 10 days, and liquid is changed daily, 3D epidermis models are obtained；Under the liquid
Under nutrient solution liquid based on α-MEM nutrient solutions, addition hyclone, calcium chloride, EGF EGFs and insulin, liquid
The volumn concentration of hyclone is 4% in nutrient solution, and the concentration of calcium chloride is 0.3mM, the concentration of EGF EGFs
For 0.05 μ g/mL, the concentration of insulin is 1mM；Gas-liquid face nutrient solution liquid based on α-MEM nutrient solutions, adds tire ox
The volumn concentration of hyclone is 4% in serum, calcium chloride, EGF EGFs and insulin, gas-liquid face nutrient solution,
The concentration of calcium chloride is 1.2mM, and the concentration of EGF EGFs is 0.05 μ g/mL, and the concentration of insulin is 1mM.
The 3D epidermises model and people's epidermal structure that the present embodiment is built are highly consistent（Such as Fig. 9）.
Embodiment 10
The construction method of the 3D epidermis models of the present embodiment comprises the following steps：
Step 1: recovery P2 carries out cellar culture to the fibroblast of recovery, treats that plating cells rate reaches for human fibroblasts
Had digestive transfer culture is carried out to 85% or so, is inoculated in after cell count according to 5 × 104/cm2 density in the nutrient solution containing serum, in
37 DEG C, discard nutrient solution after culture 20h in 5%CO2 incubators, the nutrient solution containing mitomycin C is then added, in carbon dioxide
Nutrient solution is discarded after handling 6h in incubator, cell is softly rinsed 4 times with the PBS cushioning liquid of 37 DEG C of preheatings, obtains into fiber
Cytotrophoblast；The nutrient solution containing serum liquid based on DF12 nutrient solutions, adds hyclone, the training containing serum
The volumn concentration of hyclone is 5% in nutrient solution；The nutrient solution containing mitomycin C liquid based on DF12 nutrient solutions,
The volumn concentration for adding hyclone in hyclone and mitomycin C, the nutrient solution containing mitomycin C is 5%,
The concentration of mitomycin C is 20 μ g/mL；
Counted Step 2: being resuspended after the keratinocyte recovered is digested, cell concentration needed for being calculated according to inoculum density, so
The keratinocyte after resuspension is seeded into fibroblast described in step one according to 3 × 105/cm2 density afterwards to nourish
Layer surface, then culture under progress liquid in nutrient solution is placed under liquid, change daily after cultivating 48h under liquid, liquid and be transferred to gas-liquid face nutrient solution
Middle progress gas-liquid face culture, the time of the gas-liquid face culture is 9 days, and liquid is changed daily, 3D epidermis models are obtained；Trained under the liquid
Cultivated under nutrient solution liquid based on DF12 nutrient solutions, addition hyclone, calcium chloride, EGF EGFs and insulin, liquid
The volumn concentration of hyclone is 3% in liquid, and the concentration of calcium chloride is 0.2mM, and the concentration of EGF EGFs is
0.05 μ g/mL, the concentration of insulin is 1mM；Gas-liquid face nutrient solution with DF12 nutrient solution basal liquids, addition hyclone,
The volumn concentration of hyclone is 3%, chlorination in calcium chloride, EGF EGFs and insulin, gas-liquid face nutrient solution
The concentration of calcium is 1.2mM, and the concentration of EGF EGFs is 0.05 μ g/mL, and the concentration of insulin is 1mM.
The 3D epidermises model and people's epidermal structure that the present embodiment is built are highly consistent（Such as Figure 10）.
Embodiment 11
The construction method of the 3D epidermis models of the present embodiment comprises the following steps：
Step 1: recovery P2 carries out cellar culture to the fibroblast of recovery, treats that plating cells rate reaches for human fibroblasts
Had digestive transfer culture is carried out to 85% or so, is inoculated in after cell count according to 6 × 104/cm2 density in the nutrient solution containing serum, in
37 DEG C, discard nutrient solution after culture 24h in 5%CO2 incubators, the nutrient solution containing mitomycin C is then added, in carbon dioxide
Nutrient solution is discarded after handling 2h in incubator, cell is softly rinsed 3 times with the PBS cushioning liquid of 37 DEG C of preheatings, obtains into fiber
Cytotrophoblast；The nutrient solution containing serum liquid based on low-sugar type DMEM nutrient solutions, adds hyclone, described to contain blood
The volumn concentration of hyclone is 15% in clear nutrient solution；The nutrient solution containing mitomycin C is trained with low-sugar type DMEM
The volumn concentration of hyclone in nutrient solution, addition hyclone and mitomycin C, the nutrient solution containing mitomycin C
For 15%, the concentration of mitomycin C is 30 μ g/mL；
Counted Step 2: being resuspended after the keratinocyte recovered is digested, cell concentration needed for being calculated according to inoculum density, so
The keratinocyte after resuspension is seeded into fibroblast described in step one according to 5 × 105/cm2 density afterwards to nourish
Layer surface, then culture under progress liquid in nutrient solution is placed under liquid, change daily after cultivating 48h under liquid, liquid and be transferred to gas-liquid face nutrient solution
Middle progress gas-liquid face culture, the time of the gas-liquid face culture is 10 days, and liquid is changed daily, 3D epidermis models are obtained；Under the liquid
Trained under nutrient solution liquid based on DF12 nutrient solutions, addition hyclone, calcium chloride, EGF EGFs and insulin, liquid
The volumn concentration of hyclone is 1% in nutrient solution, and the concentration of calcium chloride is 0.1mM, and the concentration of EGF EGFs is
0.1 μ g/mL, the concentration of insulin is 0.05mM；Gas-liquid face nutrient solution liquid based on DF12 nutrient solutions, adds tire ox blood
Clearly, the volumn concentration of hyclone is 1%, chlorine in calcium chloride, EGF EGFs and insulin, gas-liquid face nutrient solution
The concentration for changing calcium is 1.0mM, and the concentration of EGF EGFs is 0.1 μ g/mL, and the concentration of insulin is 0.05mM.
The 3D epidermises model and people's epidermal structure that the present embodiment is built are highly consistent（Such as Figure 11）.
Embodiment 12
The construction method of the 3D epidermis models of the present embodiment comprises the following steps：
Step 1: recovery P2 carries out cellar culture to the fibroblast of recovery, treats that plating cells rate reaches for human fibroblasts
Had digestive transfer culture is carried out to 85% or so, is inoculated in after cell count according to 3 × 104/cm2 density in the nutrient solution containing serum, in
37 DEG C, discard nutrient solution after culture 18h in 5%CO2 incubators, the nutrient solution containing mitomycin C is then added, in carbon dioxide
Nutrient solution is discarded after handling 3h in incubator, cell is softly rinsed 5 times with the PBS cushioning liquid of 37 DEG C of preheatings, obtains into fiber
Cytotrophoblast；The nutrient solution containing serum liquid based on α-MEM nutrient solutions, adds hyclone, described containing serum
The volumn concentration of hyclone is 15% in nutrient solution；The nutrient solution containing mitomycin C is using α-MEM nutrient solutions as base
The volumn concentration of hyclone in plinth liquid, addition hyclone and mitomycin C, the nutrient solution containing mitomycin C
For 15%, the concentration of mitomycin C is 40 μ g/mL；
Counted Step 2: being resuspended after the keratinocyte recovered is digested, cell concentration needed for being calculated according to inoculum density, so
The keratinocyte after resuspension is seeded into fibroblast described in step one according to 1 × 105/cm2 density afterwards to nourish
Layer surface, then culture under progress liquid in nutrient solution is placed under liquid, change daily after cultivating 48h under liquid, liquid and be transferred to gas-liquid face nutrient solution
Middle progress gas-liquid face culture, the time of the gas-liquid face culture is 8 days, and liquid is changed daily, 3D epidermis models are obtained；Trained under the liquid
Nutrient solution liquid based on low-sugar type DMEM nutrient solutions, addition hyclone, calcium chloride, EGF EGFs and insulin, liquid
The volumn concentration of hyclone is 1% in lower nutrient solution, and the concentration of calcium chloride is 0.5mM, EGF EGFs it is dense
Spend for 0.05 μ g/mL, the concentration of insulin is 0.05mM；Gas-liquid face nutrient solution is based on low-sugar type DMEM nutrient solutions
The volume hundred of hyclone in liquid, addition hyclone, calcium chloride, EGF EGFs and insulin, gas-liquid face nutrient solution
It is 1% to divide content, and the concentration of calcium chloride is 1.5mM, and the concentration of EGF EGFs is 0.05 μ g/mL, the concentration of insulin
For 0.05mM.
The 3D epidermises model and people's epidermal structure that the present embodiment is built are highly consistent（Such as Figure 12）.
It is described above, only it is presently preferred embodiments of the present invention, any limitation is not done to the present invention, it is every according to invention skill
Any simple modification, change and equivalent structure change that art is substantially made to above example, still fall within the technology of the present invention
In the protection domain of scheme.
Claims (4)
1. a kind of construction method of 3D epidermises model, it is characterised in that comprise the following steps：
Counted Step 1: being resuspended after the fibroblast recovered is digested, according to the close of 3 × 104/cm2~6 × 104/cm2
Fibroblast after recovery is inoculated in the nutrient solution containing serum by degree, is discarded after cultivating 18h~24h in CO2 incubators
Nutrient solution, then adds the nutrient solution containing mitomycin C, and nutrient solution is discarded after handling 2h~6h in CO2gas incubator,
Rinsed 3~5 times with PBS cushioning liquid, obtain fibroblast trophoderm；
Counted Step 2: being resuspended after the keratinocyte recovered is digested, cell concentration needed for being calculated according to inoculum density, so
Afterwards according to 1 × 105/cm2~5 × 105/cm2 density by the keratinocyte after resuspension be seeded to described in step one into
Fibrocyte nourishes layer surface, then is placed under liquid culture under progress liquid in nutrient solution, changes be transferred to after cultivating 48h under liquid, liquid daily
Gas-liquid face culture is carried out in the nutrient solution of gas-liquid face, liquid is changed daily, 3D epidermis models are obtained；Nutrient solution is with low-sugar type under the liquid
Liquid based on DMEM nutrient solutions, α-MEM nutrient solutions, F12 nutrient solutions or DF12 nutrient solutions, addition hyclone, calcium chloride, EGF
The volumn concentration of hyclone is 1%~5%, the concentration of calcium chloride in nutrient solution under EGF and insulin, liquid
For 0.1mM~0.5mM, the concentration of EGF EGFs is the μ g/mL of 0.01 μ g/mL~0.1, and the concentration of insulin is
0.05mM~2mM；Gas-liquid face nutrient solution is trained with low-sugar type DMEM nutrient solutions, α-MEM nutrient solutions, F12 nutrient solutions or DF12
Tire ox blood in liquid based on nutrient solution, addition hyclone, calcium chloride, EGF EGFs and insulin, gas-liquid face nutrient solution
Clear volumn concentration is 1%~5%, and the concentration of calcium chloride is 1.0mM~1.5mM, and the concentration of EGF EGFs is
The μ g/mL of 0.01 μ g/mL~0.1, the concentration of insulin is 0.05mM~2mM.
2. the construction method of a kind of 3D epidermises model according to claim 1, it is characterised in that contain blood described in step one
Clear nutrient solution liquid based on low-sugar type DMEM nutrient solutions, α-MEM nutrient solutions, F12 nutrient solutions or DF12 nutrient solutions, adds tire
The volumn concentration of hyclone is 5%~15% in cow's serum, the nutrient solution containing serum.
3. the construction method of a kind of 3D epidermises model according to claim 1, it is characterised in that contain silk described in step one
Rimocidin C nutrient solution liquid based on low-sugar type DMEM nutrient solutions, α-MEM nutrient solutions, F12 nutrient solutions or DF12 nutrient solutions,
Add the volumn concentration of hyclone in hyclone and mitomycin C, the nutrient solution containing mitomycin C for 5%~
15%, the concentration of mitomycin C is the μ g/mL of 10 μ g/mL~40.
4. a kind of construction method of 3D epidermises model according to claim 1, it is characterised in that gas-liquid described in step 2
The time of face culture is 8~10 days.
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