CN106552295A - A kind of bilayer skin containing blood capillary tube chamber and preparation method thereof - Google Patents
A kind of bilayer skin containing blood capillary tube chamber and preparation method thereof Download PDFInfo
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Abstract
One kind tube chamber containing blood capillary bilayer skin prepared by the present invention, with two-layer skin texture, respectively epidermal area from top to bottom, the skin corium containing blood capillary tube chamber；Epidermal area is made up of epidermis cell hair growth promoting cladding, skin corium containing blood capillary tube chamber is built-up by fibroblast, umbilical cord mesenchymal stem cells, huve cell, which passes through to mix fibroblast, umbilical cord mesenchymal stem cells and huve cell with certain proportion, using multiple inocalation method, using cell own extracellular matrix secretion capacity and paracrine system, on the one hand form the histological structure of dermis of skin, on the other hand promote the propagation of endotheliocyte, migration, segment dislocation and stable.This simulation normal human skin is assembled, by cell-cell interaction and influencing each other reach skin corium constitute and the micro- segment dislocation of intradermal tissue construction method, it is to avoid before artificial addition extraneous scaffold and blood vessel the brought 26S Proteasome Structure and Function of the newborn factor unstable and operate it is loaded down with trivial details.
Description
Technical field
The invention belongs to tissue engineering technique field in biomedical engineering, relates generally to a kind of bilayer skin containing blood capillary tube chamber and preparation method thereof.
Background technology
Normal skin cells to the demand of oxygen and nutrition, the removing of metabolic waste be by cell near blood diffusion and translocation realize, and the bilayer skin of fibroblast compound support frame material and epidermis cell structure is currently based on due to a lack of blood vessel structure, long-term surviving and functional requirement cannot be met, cause fibroblastic functional disorder, the transfer ability of keratinocyte and multiplication capacity are reduced, and ultimately cause the function limitation of skin histology；The bilayer skin for building is transplanted on wound surface, as the sepage that the acquisition of living cells nutritional labeling in bilayer skin and the excretion of waste rely primarily on wound surface substrate is completed, therefore the mass exchange speed of cell is slow, has a strong impact on the survival of living cells in bilayer skin and its realization of function.In addition to improving skin time-to-live and function, this bilayer skin with differentiation structure can be also used for the basic scientific research of vascular mechanism to skin containing blood vessel, and screening has the medicine of suppression or facilitation to angiogenesis.
Therefore, how to construct the bilayer skin containing blood vessel and accelerate artificial skin substitute vascularization to cause the concern of vast researcher.Existing vascularization engineering skin majority is all with extracellular matrix（ECM）Substitute is built for support, such as in May, 2004, and banket et al. has applied for relevant patent（Chinese Patent Application No. 03134540.9）, the invention constructs organization engineering skin containing blood vessel structure as supporting structure by the use of NTx.Extracellular matrix substitute is although the outside microenvironment of cell growth can be constituted, but while also bringing many exogenous materials, such as bovine collagen, allogeneic dermis layer, man-made polymer etc., all there is certain defect in these, the immunogenicity of i.e. different exogenous materials, degradation rate, the toxicity of catabolite, host immune response, the tissue fibering that scaffold degradation causes, and with the mechanical mismatch of surrounding tissue, being all likely to become affects the key factor of the chronobiological activity and primary biological function of building skin texture.It is and extracellular matrix substitute is secreted by cell itself, ectogenic to add the conduction for being unfavorable for intercellular signal, and then affect to build the structure and Function of skin.
In addition, build at present mainly add endotheliocyte in the skin corium based on fibroblast containing microvascular bilayer skin model, while the newborn factor is promoting the angiogenesis in skin before adding blood vessel.Now the conventional addition factor has：VEGF (VEGF), basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) etc., the method is frequently necessary to repeat to implement or strict controlled release durg delivery system, the concentration of such as the added factor, proportionings between the different addition factors and each factor action time etc., these inevitably cause the complexity and unstability of whole building process, limit its application to a certain extent.
The content of the invention
In view of the shortcomings of the prior art, the purpose of the present invention is to propose to a kind of bilayer skin containing blood capillary tube chamber and preparation method thereof, by fibroblast, umbilical cord mesenchymal stem cells and huve cell are mixed with certain proportion, using multiple inocalation method, using cell own extracellular matrix secretion capacity and paracrine system, the histological structure of dermis of skin is on the one hand formed, on the other hand promotes the propagation of endotheliocyte, migration, segment dislocation and stable.This simulation normal human skin is assembled, by cell-cell interaction and influencing each other reach skin corium constitute and the micro- segment dislocation of intradermal tissue construction method, it is to avoid before artificial addition extraneous scaffold and blood vessel the brought 26S Proteasome Structure and Function of the newborn factor unstable and operate it is loaded down with trivial details.
The bilayer skin of tube chamber containing blood capillary prepared by the present invention, with two-layer skin texture, respectively epidermal area from top to bottom, the skin corium containing blood capillary tube chamber；The epidermal area is made up of epidermis cell hair growth promoting cladding；The skin corium containing blood capillary tube chamber is built-up by fibroblast, umbilical cord mesenchymal stem cells, huve cell.
The preparation method of the bilayer skin of tube chamber containing blood capillary proposed by the invention, comprises the following steps：
The extraction of step one, fibroblast and epidermis cell：
Skin histology block is immersed in 75% ethanol solution, then is embathed in proceeding to PBS solution to without blood stains；Piece of tissue is added into Dispase Digestive systems, 16 ~ 17 h under the conditions of inserting 4 DEG C, are placed；Hatch 1 ~ 3 h or so under the conditions of adding 37 DEG C of collagenase, after terminating digesting under 1000rpm/min, 8 ~ 10min is centrifuged, adds Fibroblast culture solution (Gibico) 37 DEG C of 5%, CO2CMC model；Epidermis under piece of tissue is peeled off is put into the 0.25% pancreatin/EDTA Digestive systems (Gibico) of 37 DEG C of preheatings, digests 8 ~ 10 min, terminate after the completion of digestion under the conditions of proceeding to 37 DEG C, the epidermis that filtration is obtained, and 800
R/min is centrifuged 5 ~ 6 min；Centrifugation pours out supernatant after terminating, add PBS, 800 r/min that 5 ~ 6 min are centrifuged；Supernatant is poured out after centrifugation, cultured epidermal cell liquid (Gibico) is added, is carried out inoculated and cultured；
Step 2, primary extraction umbilical cord mesenchymal stem cells and huve cell
By 0.8 ~ 1.2cm3The stroma of the placental lobules of size is rinsed with PBS, after rejecting blood vessel, connective tissue, is placed in 300 ~ 800 in centrifuge tube
Rpm/min, is centrifuged 5 ~ 8 min, and after abandoning supernatant, recentrifuge is to supernatant redfree；Piece of tissue is inoculated with according to interval 0.5cm, 10～20min is aseptically placed, when piece of tissue dry tack free, (0.1 ~ 0.6mmol/L) containing non essential amino acid is added, L-glutaminate (1 ~ 10mmol/L), 1 × 105The DMEM in high glucose culture medium of U/L penicillins, streptomycin, beta -mercaptoethanol (0.1 ~ 1.0mmol/L) and 5 ~ 10% hyclones, in 37 DEG C, 5%CO2Under conditions of cultivate, liquid 1 time is changed per 3～5d；After cell 80% merges, piece of tissue is removed, add 0.25% pancreatin/EDTA had digestive transfer culture cultures, often digest 2 ~ 3min with 0.25% pancreatin/EDTA for after the fusion of cell 80% afterwards, 500 ~ 1000r/min is passed on after 5 ~ 10min is centrifuged.
Aseptically, two sections of 18 ~ 20 cm length, section neat umbilical cords are rinsed to without bloodstain with PBS respectively, the tremulous pulse two ends of umbilical cord is ligatured, with anti-pollution；Fill which in the umbilical vein that 0.1 ~ 0.5% collagenase II is injected section of umbilical cord again, another is injected 0.1 ~ 0.5% Collagenase I and 0.25% pancreatin/EDTA grade ratio mixture slaking liquid in the same way and fill which；10 ~ 20 min are incubated under the conditions of umbilical cord is placed in 37 DEG C；Collect the Digestive system in two sections of umbilical veines, RPMI-1640 culture fluid of the injection containing 10% hyclone rinses tube chamber again, Digestive system is collected in the lump with flushing liquor, 500 ~ 1000 r/min, 5 ~ 8min of centrifugation, abandons supernatant, adds RPMI-1640 complete culture solutions, piping and druming is uniformly inoculated with, and is placed in 5%CO2,
Culture fluid is changed after 8 ~ 24 h are cultivated under the conditions of 37 DEG C, is removed not adherent cell, then liquid is changed every 18 ~ 24 h sxemiquantitative and be paved with bottom of bottle to cell growth 1 time.
The RPMI-1640 complete culture solutions refer to, 5 ~ 20% hyclones, 6 ~ 20 ng/mL are added in RPMI-1640 culture fluid
VEGF, L-glutaminate 1 ~ 10
MmoL/L, 100 U/mL of penicillin, streptomycin 100
mg/mL；
Step 3, the corium layer building containing blood capillary luminal structure：
When the fibroblast of the third generation, umbilical cord mesenchymal stem cells and endotheliocyte are fused to 70% ~ 80%, by three kinds of cells with 1.0 × 105~1.5×105The density of individual/ml is resuspended in basal medium is built respectively and 5 ~ 8min is centrifuged in 500 ~ 1000 rpm/min；
Consisting of for basal medium is built wherein, in volume ratio 1:Add 1 ~ 35 ug/ml of insulin, 10 ~ 50ug/ml of adenine, 0.1 ~ 4.5ug/ml of hydrocortisone, VC in 1 α-MEM and RPMI-1640 mixed-culture medium
20 ~ 80ug/ml, 5 ~ 10% hyclones.
Incubation is as follows：The cell culture cell of 1 ~ 5% gelatin is being coated with advance within first day（Nunc, hereinafter abbreviation cell）It is middle to be inoculated with 1:1:Three kinds of cells of 1 ratio mixing, inoculum density are 0.15 × 106~0.5 ×106Individual/cell, discards structure basal medium after 18 ~ 24h, continue thereafter with paving with 1 ~ 5% gelatin, is incubated 45 ~ 60min, abandons gelatin, be inoculated with 1 again under the conditions of 37 DEG C:1:Three kinds of cells of 1 ratio mixing, inoculum density are 0.15 × 106~0.5
×106Individual/cell, after 70 ~ 72h of culture, spreads with 1 ~ 5% gelatin, continues 45 ~ 60min of incubation, abandon gelatin, according to volume ratio 2:1 inoculation fibroblast and the cell suspension of umbilical cord mesenchymal stem cells, inoculum density is 0.15 × 106~0.5 ×106Individual/cell, continues 2 ~ 3d of culture, changes liquid every other day.
This step is the committed step for building.By fibroblast, umbilical cord mesenchymal stem cells and huve cell are co-cultured in three-dimensional environment in varing proportions, to set up the dermal tissue structure containing blood capillary tube chamber.Mescenchymal stem cell not only has good film-formation result but also with important endocrine function, conventional mescenchymal stem cell has drawn from bone marrow, it is invasive because drawing materials, and increase its cell viability with donor age and quantity declines equivalent risk therewith and its application is gradually restricted.The research in later stage shows that umbilical cord mesenchymal stem cells have most of biological property of mesenchymal stem cells MSCs, such as：Cytokine spectrum, phenotypic features etc..And umbilical cord is puerperal garbage, and wide material sources, yield height disclosure satisfy that later stage industrialized requirement.
Skin corium introduce umbilical cord extract mescenchymal stem cell advantage have it is following some：1）Secretion extracellular matrix ability is strong, the effect for playing secretion skin corium framework ingredient can be cooperateed with fibroblast, by increasing capacitance it is possible to increase skin corium thickness；2）The hypertrophy of blood vessel is promoted by paracrine action.Umbilical cord extracts the active factorses that mescenchymal stem cell can secrete the angiogenesis promotings such as VEFG, bFGF.VEGF is the rush endothelial cell mitogen factor of high special, and the propagation of stimulating endothelial cell, differentiation, induction of vascular are generated；BFGF can stimulate the rise of vegf receptor, produce synergism with VEGF, promote blood capillary segment dislocation.In addition, umbilical cord extracts the generation that mescenchymal stem cell can also secrete the extracellular matrix components such as participation such as integrin, the cadherinses blood vessel required for angiogenesiss；3）Umbilical cord mesenchymal stem cells have differentiation capability, which is co-cultured by the present invention with Human umbilical vein endothelial cells, the microenvironment formed by endotheliocyte can affect the differentiation direction of umbilical cord mesenchymal stem cells so as to break up to blood vessel endothelium induction, further form blood capillary tube-like structures.
Skin corium extracellular matrix secretion amount need to reach.Therefore, in the incubation of skin corium, three inocalation methods be present invention employs.Due to the function of the tuberosity formula growth characteristics and a large amount of extracellular matrixs of secretion of umbilical cord mesenchymal stem cells itself, easily there is pattern contract balling-up, causes the failure of corium layer building in long-term cultivation skin corium.Therefore the mode that coating method and continuous three skin coriums are inoculated with advance before the present invention uses the inoculation of 1 ~ 5% gelatin, to alleviate the contraction of skin corium, increase skin corium thickness, shorten skin corium cultivation cycle.When the inoculation of third time skin corium is carried out, with fibroblast：Umbilical cord mesenchymal stem cells=2:1 ratio carries out culture structure, it is therefore an objective to avoid impact of the umbilical cord mesenchymal stem cells paracrine factor to epidermal area hair growth promoting.
Step 4, the bilayer skin containing blood capillary luminal structure build：
Fusion rate is built culture fluid SKc1 re-suspended cells and is centrifuged up to 60% or so forth generation epidermis cell, with 0.1 ~ 0.8 × 106The cell density of individual/cell is inoculated with.The 1st day of SKc1 cultures is training method under liquid, and the 2nd cultivated under SKc1 liquid day proceeds by the culture of solution-air face.SKc2 culture fluid solution-airs face is changed after 2 ~ 3d is cultivated under continuous liquid and cultivates 2 ~ 3d, then changed SKc3 culture fluid solution-airs face and cultivate 2 ~ 3d, finally changed SKc4 culture fluid solution-airs face and cultivate 3 ~ 4d, building process is completed.
The composition of above-mentioned culture fluid is as follows：
The composition of basal medium：It is 1 in volume ratio:Add 1 ~ 35 ug/ml of insulin, 10 ~ 50ug/ml of adenine, 0.1 ~ 4.5ug/ml of hydrocortisone, 5 ~ 10% hyclones in 1 α-MEM and F12 mixed liquor；
Culture fluid SKc1 be in basal medium add 10 ~ 50 μ g/ml BPE, the EGF of 0.1 ~ 0.5ng/ml, 0.01 ~ 0.09nM of ethanolamine-phosphate, 2 ~ 10nM of Progesterone, 60 ~ 90 μ g/ml of commercial heparin；
Culture fluid SKc2 is the BPE, the EGF of 0.1 ~ 0.5ng/ml, the CaCl of 0.5 ~ 2.5 mM for adding 10 ~ 50 μ g/ml in basal medium2, the Vc of 20 ~ 80 μ g/ml, 0.01 ~ 0.09nM of ethanolamine-phosphate, 2 ~ 10nM of Progesterone；
Culture fluid SKc3 is the BPE, the EGF of 0.1 ~ 0.5ng/ml, the CaCl of 2.6 ~ 3.5 mM for adding 10 ~ 50 μ g/ml in basal medium2, the Vc of 20 ~ 80 μ g/ml, 0.01 ~ 0.09nM of ethanolamine-phosphate, 2 ~ 10nM of Progesterone；
Culture fluid SKc4 is the BPE, the EGF of 0.1 ~ 0.5ng/ml, the CaCl of 3.6 ~ 4.5mM for adding 10 ~ 50 μ g/ml in basal medium2, the Vc of 20 ~ 80 μ g/ml, 0.01 ~ 0.09nM of ethanolamine-phosphate, 2 ~ 10nM of Progesterone, 60 ~ 90 μ g/ml of commercial heparin；
Hair growth promoting process of this step by simulation normal human skin epidermal area, adds each somatomedin in the epidermis difference hair growth promoting stage, by the change of training method, makes the normal hair growth promoting of epidermal area be basal layer, spinous layer, granular layer, horny layer.The factor of addition not only supports the hair growth promoting of epidermal area but also is able to maintain that the growth of skin corium, the formation of blood capillary luminal structure.
The present invention is in the bilayer skin model containing blood capillary tube chamber is built with fibroblast, umbilical cord mesenchymal stem cells and huve cell as seed cell, using the multiple inocalation method of skin corium, and building the hair growth promoting of different phase addition trophic factors guarantee corium and epidermal area.The umbilical cord mesenchymal stem cells wide material sources of introducing, easily obtain, and yield is high, disclosure satisfy that later stage industrialized requirement.Participate in constituting skin corium skeleton and vessel lumen using the extracellular matrix secretion ability and paracrine ability of umbilical cord mesenchymal stem cells.Using multiple vaccinization, increase skin corium thickness, shorten incubation time, it is to avoid because of extracellular matrix secretion when skin corium long-time is cultivated, cell is subject to the contraction problem brought by mechanical stretch, the inoculation for next step epidermal area to provide advantage.Whole building process does not introduce the newborn factor before exogenous support and blood vessel, using cell own extracellular matrix secretory function and paracrine system assembling skin corium tissue scaffold, constitute blood capillary luminal structure, the normal hair growth promoting process of simulation human body skin, reaches the high similarity of histology and human body skin structure.The present invention can construct the bilayer skin containing blood capillary tube chamber in a short time, and operating process is easy, repeatable strong, and can realize industrialization.
Description of the drawings
The photo of Fig. 1, normal Morphology of Human Umbilical Vein Endothelial；
Fig. 2, human fibroblasts are carried out in 24 orifice plates, the 2D of three kinds of cells of human umbilical cord mesenchymal stem cells and Human umbilical vein endothelial cells is co-cultured, the photo after 6d.It can be seen that without external source sexual stimuluses angiogenesis factor in figure, by the co-cultivation of three kinds of cells, Human umbilical vein endothelial cells can join end to end, tube-like structures are constituted.
Fig. 3, human fibroblasts are carried out for the present embodiment, after the co-cultivation of three kinds of cells of human umbilical cord mesenchymal stem cells and Human umbilical vein endothelial cells, the tube-like structures for being formed express the immunohistochemical staining result of endothelial cell marker thing CD31.
Specific embodiment
Fibroblast culture solution, cultured epidermal cell liquid, pancreatin/EDTA Digestive systems used in embodiment is purchased from Gibico companies, and the cell culture cell for being used is purchased from Nunc companies.
Embodiment 1 ：A kind of preparation method of the bilayer skin of tube chamber containing blood capillary
The extraction of step one, fibroblast and epidermis cell：
Skin histology is immersed in 75% ethanol solution, then is embathed in proceeding to PBS solution to without blood stains；Piece of tissue is added into Dispase Digestive systems, 16 h under the conditions of inserting 4 DEG C, are placed；Add 37 DEG C of incubators of collagenase and hatch 1 h or so, terminate 1000rpm/min after digestion, 8min is centrifuged, add Fibroblast culture solution to be cultivated, the epidermis under peeling off is put to 8 ml
In the 0.25% pancreatin/EDTA Digestive systems of 37 DEG C of preheatings, in proceeding to 37 DEG C of incubators, 8 min are digested, terminated after the completion of digestion, filter the epidermis for obtaining, 800 r/min are centrifuged 5 min；Centrifugation adds PBS, 800 r/min that 5 min are centrifuged after terminating；Supernatant is poured out, cultured epidermal cell liquid is added, is carried out inoculated and cultured；
The primary extraction of step 2, umbilical cord mesenchymal stem cells and huve cell
By 0.8 ~ 1.2cm3The stroma of the placental lobules of size is rinsed with PBS, after rejecting blood vessel, connective tissue, is placed in 300rpm/min in 50ml centrifuge tubes, 5min is centrifuged, supernatant is abandoned, and recentrifuge is to supernatant redfree；Piece of tissue is inoculated in into 10 cm2In culture dish, it is spaced in 0.5 cm, super-clean bench and places 10min, when piece of tissue dry tack free, addition 4ml (0.1mmol/L) containing non essential amino acid, L-glutaminate (2mmol/L), 1 × 105The DMEM in high glucose culture medium of U/L penicillins, streptomycin, beta -mercaptoethanol (0.2mmol/L) and 10% hyclone, in 37 DEG C, 5%CO2Incubator in culture, liquid 1 time is changed per 3d；After cell 80% merges, piece of tissue is removed, add 0.25% pancreatin/EDTA had digestive transfer culture cultures.Often pass on after 1000r/min centrifugation 5min for 0.25% pancreatin after the fusion of cell 80%/EDTA digestion 3min afterwards.
On super-clean bench, umbilical cord is put in PBS culture dishs, is extruded the blood in umbilical cord, neat two section is repaiied with shears, it is divided into one section of 20 cm, find out syringe of the umbilical vein with plain head and umbilical vein is inserted from one end, vascular forcepss are fixed, then rinsed more than 3 times with the PBS of preheating, bloodstain is rinsed well completely, blood to prevent umbilical artery residual is mixed into, and tremulous pulse can be separated a little sterilization knot and be pricked, with the mosquito forcepss clamp other end.0.1% collagenase II is injected from the syringe needle for rinsing fills which, and another is injected the ratio mixture slaking liquid such as 0.1% Collagenase I and 0.25% pancreatin in the same way；Syringe needle is taken out, injection end is clamped with mosquito forcepss, move in sterile beaker, be placed in 37 DEG C of incubators and be incubated 15 min；Take out, the vascular forcepss of injection end are unclamped, the Digestive system in umbilical vein is collected, is then injected into the RPMI-1640 culture fluid containing 10% hyclone and rinses tube chamber again, Digestive system and flushing liquor are collected in into centrifuge tube in the lump, 1000 r/min, are centrifuged 8min, abandon supernatant, add 1640 complete culture solutions of RPMI-, gently blown and beaten uniformly with pasteur pipet, be inoculated in culture bottle, be placed in 5%CO2, cultivate in 37 DEG C of incubators, 24
The not adherent cell of broth out is changed after h, then liquid is changed every 24 h sxemiquantitative and is paved with bottom of bottle to cell growth 1 time.
The RPMI-1640 complete culture solutions refer to, 5 ~ 20% hyclones, 6 ~ 20 ng/mL are added in RPMI-1640 culture fluid
VEGF, L-glutaminate 1 ~ 10
MmoL/L, 100 U/mL of penicillin, streptomycin 100
mg/mL。
Step 3, the corium layer building containing blood capillary luminal structure：
Cell reaches the third generation, works as fibroblast, and umbilical cord mesenchymal stem cells and endotheliocyte are fused to 70% ~ 80%, and three kinds of cells are with 1.5 × 105The density of individual/ml is resuspended in basal medium is built respectively and 5min is centrifuged in 800rpm/min；
Consisting of for basal medium is built wherein：α-MEM, RPMI-1640（Volume ratio α-MEM：RPMI-1640=1:1）, insulin 15ug/ml, adenine 25ug/ml, hydrocortisone 3ug/ml, Vc50ug/ml, 10% hyclone.
Incubation is as follows：It is inoculated with 1 in the cell culture cell for being coated with 1% gelatin in advance when first day:1:Three kinds of cells of 1 ratio mixing, inoculum density are 0.2 × 106Individual/cell, discards cell culture fluid, and continues paving with 1% gelatin, be incubated 45min, abandon gelatin, be inoculated with according to 1 in incubator after 18h:1:Three kinds of cells of 1 ratio mixing, inoculum density are 0.2 × 106Individual/cell, after 72h, spreads with 1% gelatin, 45min is incubated in incubator, abandon gelatin, be inoculated with fibroblast：Umbilical cord mesenchymal stem cells=2:1 cell suspension, inoculum density are 0.2 × 106Individual/cell, continues culture 3d, changes liquid every other day.
Step 4, the bilayer skin containing blood capillary luminal structure build：
Epidermis cell is passaged to forth generation, when cell culture to fusion rate is up to 60% or so, with building culture fluid SKc1 re-suspended cells and being centrifuged, with 0.5 × 106The cell density of/cell is seeded in cell culture cell.The 1st day of SKc1 cultures is training method under liquid, and the 2nd cultivated under SKc1 liquid day proceeds by the culture of solution-air face.Culture 2d in SKc2 culture fluid solution-airs face is changed after 2d is cultivated under continuous liquid, SKc3 culture fluid solution-airs face culture 2d is then changed, SKc4 culture fluid solution-airs face culture 3d is finally changed, building process is completed.
The composition of above-mentioned culture fluid is as follows：
Build basal medium：α-MEM, F12（Volume ratio α-MEM：F12=1:1）, insulin 1ug/ml, adenine 22ug/ml, hydrocortisone 0.22ug/ml, 10% hyclone.Culture fluid SKc1 is to add the BPE of 15 μ g/ml, the EGF of 0.1ng/ml, ethanolamine-phosphate 0.03nM, Progesterone 3nM, 60 μ g/ml of commercial heparin in basal medium is built；
Culture fluid SKc1 is to add the BPE of 15 μ g/ml, the EGF of 0.5ng/ml, ethanolamine-phosphate 0.04nM, Progesterone 5nM, 75 μ g/ml of commercial heparin in basal medium is built；
Culture fluid SKc2 is the BPE, the EGF of 0.1ng/ml, ethanolamine-phosphate 0.03nM, Progesterone 3nM, the CaCl of 2.5 mM for adding 15 μ g/ml in basal medium is built2, the Vc of 50 μ g/ml；
Culture fluid SKc3 is the BPE, the EGF of 0.1ng/ml, ethanolamine-phosphate 0.03nM, Progesterone 3nM, the CaCl of 3.5 mM for adding 15 μ g/ml in basal medium is built2, the Vc of 50 μ g/ml；
Culture fluid SKc4 is the BPE, the EGF of 0.1ng/ml, ethanolamine-phosphate 0.03nM, the CaCl of 4.5 mM for adding 15 μ g/ml in basal medium is built2, Progesterone 3nM
, the Vc of 50 μ g/ml, 60 μ g/ml of commercial heparin；
Embodiment 2 ：A kind of preparation method of the bilayer skin of tube chamber containing blood capillary
The extraction of step one, fibroblast and epidermis cell：
Skin histology is immersed in 75% ethanol solution, blood stains is removed, then is embathed in proceeding to PBS solution to without blood stains；Piece of tissue is added into Dispase Digestive systems, 17 h under the conditions of inserting 4 DEG C, are placed；Add 37 DEG C of incubators of collagenase and hatch 2 h or so, terminate 1000rpm/min after digestion, 10min is centrifuged, add Fibroblast culture solution to be cultivated；Epidermis under peeling off is put into the 0.25% pancreatin/EDTA Digestive systems of 37 DEG C of preheatings, digests 10 min in proceeding to 37 DEG C of incubators, and the epidermis of acquisition is filtered after termination, and 800 rpm/min are centrifuged 5 min；
Centrifugation pours out supernatant after terminating, add 10 mL PBSs, 800 rpm/min that 5 min are centrifuged；Supernatant is poured out after centrifugation, cultured epidermal cell liquid is added, is carried out inoculated and cultured；
The primary extraction of step 2, umbilical cord mesenchymal stem cells and huve cell
By 0.8 ~ 1.2cm3The stroma of the placental lobules of size is rinsed with PBS, after rejecting blood vessel, connective tissue, is placed in 300rpm/min in 50ml centrifuge tubes, 5min is centrifuged, supernatant is abandoned, and recentrifuge is to supernatant redfree；Piece of tissue is inoculated in culture dish, interval 0.5
Cm, places 10min in super-clean bench, when piece of tissue dry tack free, is carefully added into containing non essential amino acid (0.1mmol/L), L-glutaminate (2mmol/L), 1 × 105The DMEM in high glucose culture medium of U/L penicillins, streptomycin, beta -mercaptoethanol (0.2mmol/L) and 10% hyclone, in 37 DEG C, 5%CO2Incubator in culture, liquid 1 time is changed per 3～5d；After cell 80% merges, piece of tissue is removed, add 0.25% pancreatin/EDTA had digestive transfer culture cultures；Afterwards often for 0.25% pancreatin after the fusion of cell 80%/EDTA digestion 2min, 1000r/min centrifugation 5min, 1：3 pass on.
Umbilical cord is put under aseptic condition and fill in preheating PBS culture dishs in advance, cut off the part of clamp trace and hematoma, extrude the blood in umbilical cord, neat two section is repaiied with shears, be divided into one section of 20 cm.Umbilical vein is inserted from one end with the syringe with plain head, vascular forcepss are fixed, then rinsed to without bloodstain with the PBS of preheating；0.1% collagenase II is injected from the syringe needle for rinsing fills which, and another is injected the ratio mixture slaking liquid such as 0.1% Collagenase I and 0.25% pancreatin in the same way；Syringe needle is taken out, injection end is clamped with mosquito forcepss, move in sterile beaker, be placed in 37 DEG C of incubators and be incubated 15 min.Take out, unclamp the vascular forcepss of injection end, collect the Digestive system in umbilical vein, it is then injected into the RPMI-1640 culture fluid containing 10% hyclone and rinses tube chamber again, Digestive system and flushing liquor is collected in into centrifuge tube, 1000 r/min in the lump, centrifugation 8min, supernatant is abandoned, is added RPMI-1640 complete culture solutions to be inoculated in culture bottle, is placed in 5%CO2, cultivate in 37 DEG C of incubators, 24
The not adherent cell of broth out is changed after h, then every 24 h
Sxemiquantitative is changed liquid and is paved with bottom of bottle to cell growth 1 time.
The RPMI-1640 complete culture solutions refer to, 5 ~ 20% hyclones, 6 ~ 20 ng/mL are added in RPMI-1640 culture fluid
VEGF, L-glutaminate 1 ~ 10
MmoL/L, 100 U/mL of penicillin, streptomycin 100
mg/mL
Step 3, the corium layer building containing blood capillary luminal structure：
Cell reaches the third generation, works as fibroblast, and umbilical vein mescenchymal stem cell and endotheliocyte are fused to 70% ~ 80%, and three kinds of cells are with 1.0 × 105The density of individual/ml is resuspended in basal medium is built respectively and 5min is centrifuged in 800rpm/min.
Consisting of for basal medium is built wherein：α-MEM, RPMI-1640（Volume ratio α-MEM：RPMI-1640=1:1）, insulin 10ug/ml, adenine 25ug/ml, hydrocortisone 1ug/ml, Vc50ug/ml, 10% hyclone.
Incubation is as follows：It is inoculated with 1 in the cell culture cell for being coated with 2% gelatin in advance when first day:1:Three kinds of cells of 1 ratio mixing, inoculum density is 0.2 x106Individual/cell, discards cell culture fluid, and continues paving with 2% gelatin, 45min is incubated in incubator, abandon gelatin, inoculation 1 after 24h:1:Three kinds of cells of 1 ratio mixing, inoculum density are 0.2 x106/ cell, after 72h, abandons gelatin, is inoculated with fibroblast：Umbilical cord mesenchymal stem cells=2:1 cell suspension, inoculum density are 0.15 x106Individual/cell, continues culture 3d, changes liquid every other day.
Step 4, the bilayer skin containing blood capillary luminal structure build：
Epidermis cell is passaged to forth generation, when cell culture to fusion rate is up to 60% or so, with building culture fluid SKc1 re-suspended cells and being centrifuged, with 0.5 × 106The cell density of individual/cell is seeded in cell culture cell.The 1st day of SKc1 cultures is training method under liquid, and the 2nd cultivated under SKc1 liquid day proceeds by the culture of solution-air face.Culture 2d in SKc2 culture fluid solution-airs face is changed after 2d is cultivated under continuous liquid, SKc3 culture fluid solution-airs face culture 2d is then changed, SKc4 culture fluid solution-airs face culture 4d is finally changed, building process is completed.
The composition of above-mentioned culture fluid is as follows：
Build basal medium：α-MEM, F12（Volume ratio α-MEM：F12=1:1）, insulin 10ug/ml, adenine 25ug/ml, hydrocortisone 1ug/ml, 10% hyclone.
Culture fluid SKc1 is to add the BPE of 15 μ g/ml, the EGF of 0.5ng/ml, ethanolamine-phosphate 0.04nM, Progesterone 5nM, 75 μ g/ml of commercial heparin in basal medium is built；
Culture fluid SKc2 is the BPE, the EGF of 0.5ng/ml, ethanolamine-phosphate 0.04nM, Progesterone 5nM, the CaCl of 2.0 mM for adding 15 μ g/ml in basal medium is built2, the Vc of 35 μ g/ml；
Culture fluid SKc3 is the BPE, the EGF of 0.5ng/ml, ethanolamine-phosphate 0.04nM, Progesterone 5nM, the CaCl of 3.0 mM for adding 15 μ g/ml in basal medium is built2, the Vc of 35 μ g/ml；
Culture fluid SKc4 is to add BPE, the EGF of 0.5ng/ml of 15 μ g/ml, 0.04 nM of ethanolamine-phosphate, Progesterone 5nM, the CaCl of 4.0 mM in basal medium is built2, the Vc of 35 μ g/ml, 75 μ g/ml of commercial heparin
Embodiment 3 ：A kind of preparation method of the bilayer skin of tube chamber containing blood capillary
The extraction of step one, fibroblast and epidermis cell：
Skin histology is immersed in 75% ethanol solution, blood stains is removed, then is embathed in proceeding to PBS solution；Piece of tissue is added into Dispase Digestive systems, 16 h under the conditions of inserting 4 DEG C, are placed；Add 37 DEG C of incubators of collagenase and hatch 2 h or so, terminate 1000rpm/min after digestion, 10min is centrifuged, add Fibroblast culture solution to be cultivated；Epidermis under peeling off is put into the 0.25% pancreatin/EDTA Digestive systems of 37 DEG C of preheatings, digests 10 min in proceeding to 37 DEG C of incubators, and the epidermis of acquisition is filtered after termination, and 800 rpm/min are centrifuged 5 min；
Centrifugation pours out supernatant after terminating, add 10 mL PBSs, 800 rpm/min that 5 min are centrifuged；Supernatant is poured out after centrifugation, cultured epidermal cell liquid is added, is carried out inoculated and cultured；
The primary extraction of step 2, umbilical cord mesenchymal stem cells and huve cell
The stroma of placental lobules is rinsed with PBS, after rejecting blood vessel, connective tissue, 300rpm/min in 50ml centrifuge tubes is placed in, 5min is centrifuged, supernatant is abandoned, recentrifuge is to supernatant redfree；Piece of tissue is inoculated in culture dish, interval 0.5
Cm, places 10min in super-clean bench, when piece of tissue dry tack free, is carefully added into containing non essential amino acid (0.1mmol/L), L-glutaminate (2mmol/L), 1 × 105The DMEM in high glucose culture medium of U/L penicillins, streptomycin, beta -mercaptoethanol (0.2mmol/L) and 10% hyclone, in 37 DEG C, 5%CO2Incubator in culture, liquid 1 time is changed per 3～5d；After cell 80% merges, piece of tissue is removed, add 0.25% pancreatin/EDTA had digestive transfer culture cultures；Afterwards often for 0.25% pancreatin after the fusion of cell 80%/EDTA digestion 2min, 1000r/min centrifugation 5min, 1：3 pass on.
Umbilical cord is put under aseptic condition and fill in preheating PBS culture dishs in advance, cut off the part of clamp trace and hematoma, extrude the blood in umbilical cord, neat two section is repaiied with shears, be divided into one section of 20 cm.Umbilical vein is inserted from one end with the syringe with plain head, vascular forcepss are fixed, then rinsed to without bloodstain with the PBS of preheating；0.1% collagenase II is injected from the syringe needle for rinsing fills which, and another is injected the ratio mixture slaking liquid such as 0.1% Collagenase I and 0.25% pancreatin in the same way；Syringe needle is taken out, injection end is clamped with mosquito forcepss, move in sterile beaker, be placed in 37 DEG C of incubators and be incubated 15 min.Take out, unclamp the vascular forcepss of injection end, collect the Digestive system in umbilical vein, it is then injected into the RPMI-1640 culture fluid containing 10% hyclone and rinses tube chamber again, Digestive system and flushing liquor is collected in into centrifuge tube, 1000 r/min in the lump, centrifugation 8min, supernatant is abandoned, is added RPMI-1640 complete culture solutions to be inoculated in culture bottle, is placed in 5%CO2, cultivate in 37 DEG C of incubators, 24
The not adherent cell of broth out is changed after h, then every 24 h
Sxemiquantitative is changed liquid and is paved with bottom of bottle to cell growth 1 time.
The RPMI-1640 complete culture solutions refer to, 5 ~ 20% hyclones, 6 ~ 20 ng/mL are added in RPMI-1640 culture fluid
VEGF, L-glutaminate 1 ~ 10
MmoL/L, 100 U/mL of penicillin, streptomycin 100
mg/mL
Step 3, the corium layer building containing blood capillary luminal structure：
Cell reaches the third generation, works as fibroblast, and umbilical vein mescenchymal stem cell and endotheliocyte are fused to 70% ~ 80%, and three kinds of cells are with 1.0 × 105The density of individual/ml is resuspended in basal medium is built respectively and 5min is centrifuged in 800rpm/min.
Consisting of for basal medium is built wherein：α-MEM, RPMI-1640（Volume ratio α-MEM：RPMI-1640=1:1）, insulin 10ug/ml, adenine 25ug/ml, hydrocortisone 1ug/ml, Vc50ug/ml, 10% hyclone.
Incubation is as follows：It is inoculated with 1 in the cell culture cell for being coated with 2% gelatin in advance when first day:1:Three kinds of cells of 1 ratio mixing, inoculum density is 0.2 x106Individual/cell, discards cell culture fluid, and continues paving with 2% gelatin, 45min is incubated in incubator, abandon gelatin, inoculation 1 after 24h:1:Three kinds of cells of 1 ratio mixing, inoculum density are 0.2 x106/ cell, after 72h, abandons gelatin, is inoculated with fibroblast：Umbilical cord mesenchymal stem cells=2:1 cell suspension, inoculum density are 0.15 x106Individual/cell, continues culture 3d, changes liquid every other day.
Step 4, the bilayer skin containing blood capillary luminal structure build：
Epidermis cell is passaged to forth generation, when cell culture to fusion rate is up to 60% or so, with building culture fluid SKc1 re-suspended cells and being centrifuged, with 0.5 × 106The cell density of individual/cell is seeded in cell culture cell.The 1st day of SKc1 cultures is training method under liquid, and the 2nd cultivated under SKc1 liquid day proceeds by the culture of solution-air face.Culture 2d in SKc2 culture fluid solution-airs face is changed after 2d is cultivated under continuous liquid, SKc3 culture fluid solution-airs face culture 2d is then changed, SKc4 culture fluid solution-airs face culture 4d is finally changed, building process is completed.
The composition of above-mentioned culture fluid is as follows：
Build basal medium：α-MEM, F12（Volume ratio α-MEM：F12=1:1）, insulin 10ug/ml, adenine 25ug/ml, hydrocortisone 1ug/ml, 10% hyclone.
Culture fluid SKc1 is to add the BPE of 15 μ g/ml, the EGF of 0.5ng/ml, ethanolamine-phosphate 0.04nM, Progesterone 5nM, 75 μ g/ml of commercial heparin in basal medium is built；
Culture fluid SKc2 is the BPE, the EGF of 0.5ng/ml, ethanolamine-phosphate 0.04nM, Progesterone 5nM, the CaCl of 0.5 mM for adding 15 μ g/ml in basal medium is built2, the Vc of 35 μ g/ml；
Culture fluid SKc3 is the BPE, the EGF of 0.5ng/ml, ethanolamine-phosphate 0.04nM, Progesterone 5nM, the CaCl of 2.6 mM for adding 15 μ g/ml in basal medium is built2, the Vc of 35 μ g/ml；
Culture fluid SKc4 is to add BPE, the EGF of 0.5ng/ml of 15 μ g/ml, 0.04 nM of ethanolamine-phosphate, Progesterone 5nM, the CaCl of 3.6 mM in basal medium is built2, the Vc of 35 μ g/ml, 75 μ g/ml of commercial heparin
Fig. 1 is normal Morphology of Human Umbilical Vein Endothelial, it can be seen that endotheliocyte is dispersed in and is uniformly distributed；Fig. 2 is that the present embodiment carries out human fibroblasts in 24 orifice plates, the 2D of three kinds of cells of human umbilical cord mesenchymal stem cells and Human umbilical vein endothelial cells is co-cultured, visible after 6d to have obvious tube-like structures to be formed；After Fig. 3 carries out human fibroblasts, the co-cultivation of three kinds of cells of human umbilical cord mesenchymal stem cells and Human umbilical vein endothelial cells for the present embodiment, the tube-like structures for being formed express the immunohistochemical staining result of endothelial cell marker thing CD31.
Claims (2)
1. a kind of bilayer skin of tube chamber containing blood capillary, it is characterised in that:With two-layer skin texture, respectively epidermal area from top to bottom, the skin corium containing blood capillary tube chamber；
The epidermal area is made up of epidermis cell hair growth promoting cladding；
The skin corium containing blood capillary tube chamber is made up of fibroblast, umbilical cord mesenchymal stem cells, huve cell.
2. the preparation method of the bilayer skin of tube chamber containing blood capillary described in a kind of claim 1, it is characterised in that comprise the following steps：
The extraction of step one, fibroblast and epidermis cell：
Skin histology block is immersed in 75% ethanol solution, then is embathed in proceeding to PBS solution to without blood stains；Piece of tissue is added into Dispase Digestive systems, 16 ~ 17 h under the conditions of inserting 4 DEG C, are placed；Hatch 1 ~ 3 h or so under the conditions of adding 37 DEG C of collagenase, after terminating digesting under 1000rpm/min, 8 ~ 10min is centrifuged, adds 37 DEG C of Fibroblast culture solution, 5%CO2CMC model；Epidermis under piece of tissue is peeled off is put into the 0.25% pancreatin/EDTA Digestive systems of 37 DEG C of preheatings, digests 8 ~ 10 min, terminate after the completion of digestion under the conditions of proceeding to 37 DEG C, filters the epidermis for obtaining, and 800 r/min are centrifuged 5 ~ 6 min；Centrifugation pours out supernatant after terminating, add PBS, 800 r/min that 5 ~ 6 min are centrifuged；Supernatant is poured out after centrifugation, cultured epidermal cell liquid is added, is carried out inoculated and cultured；
Step 2, primary extraction umbilical cord mesenchymal stem cells and huve cell
By 0.8 ~ 1.2cm3The stroma of the placental lobules of size is rinsed with PBS, after rejecting blood vessel, connective tissue, is placed in 300 ~ 800 in centrifuge tube
Rpm/min, is centrifuged 5 ~ 8 min, and after abandoning supernatant, recentrifuge is to supernatant redfree；Piece of tissue is inoculated with according to interval 0.5cm, 10～20min is aseptically placed, when piece of tissue dry tack free, 0.1 ~ 0.6mmol/L containing non essential amino acid, 1 ~ 10mmol/L of L-glutaminate, 1 × 10 is added5The DMEM in high glucose culture medium of U/L penicillins, streptomycin, 0.1 ~ 1.0mmol/L of beta -mercaptoethanol and 5 ~ 10% hyclones, in 37 DEG C, 5%CO2Under conditions of cultivate, liquid 1 time is changed per 3～5d；After cell 80% merges, piece of tissue is removed, add 0.25% pancreatin/EDTA had digestive transfer culture cultures, often digest 2 ~ 3min with 0.25% pancreatin/EDTA for after the fusion of cell 80% afterwards, 500 ~ 1000r/min is passed on after 5 ~ 10min is centrifuged；
Aseptically, by 18 ~ 20
Two sections of neat umbilical cords of cm length, section are rinsed to without bloodstain with PBS respectively, the tremulous pulse two ends of umbilical cord are ligatured, with anti-pollution；Fill which in the umbilical vein that 0.1 ~ 0.5% collagenase II is injected section of umbilical cord again, another is injected 0.1 ~ 0.5% Collagenase I and 0.25% pancreatin/EDTA grade ratio mixture slaking liquid in the same way and fill which, and 10 ~ 20 min are incubated under the conditions of being placed in 37 DEG C；Collect the Digestive system in two sections of umbilical veines, RPMI-1640 culture fluid of the injection containing 10% hyclone rinses tube chamber again, Digestive system is collected in the lump with flushing liquor, 500 ~ 1000 r/min, 5 ~ 8min of centrifugation, abandons supernatant, adds RPMI-1640 complete culture solutions, piping and druming is uniformly inoculated with, and is placed in 5%CO2, change culture fluid, remove not adherent cell, then change liquid every 18 ~ 24 h sxemiquantitative and be paved with bottom of bottle to cell growth 1 time after 8 ~ 24 h being cultivated under the conditions of 37 DEG C；
The RPMI-1640 complete culture solutions refer to, 5 ~ 20% hyclones, 6 ~ 20 ng/mL VEGF, 1 ~ 10 mmoL/L of L-glutaminate, 100 U/mL of penicillin, 100 mg/mL of streptomycin are added in RPMI-1640 culture fluid；
Step 3, the corium layer building containing blood capillary luminal structure：
When the fibroblast of the third generation, umbilical cord mesenchymal stem cells and endotheliocyte are fused to 70% ~ 80%, by three kinds of cells with 1.0 × 105~1.5×105The density of individual/ml is resuspended in basal medium is built respectively and 5 ~ 8min is centrifuged in 500 ~ 1000 rpm/min；
Consisting of for basal medium is built wherein, in volume ratio 1:Add 1 ~ 35 ug/ml of insulin, 10 ~ 50ug/ml of adenine, 0.1 ~ 4.5ug/ml of hydrocortisone, 20 ~ 80ug/ml of VC, 5 ~ 10% hyclones in 1 α-MEM and RPMI-1640 mixed-culture medium；
Incubation is as follows：It is inoculated with the cell culture cell for being coated with 1 ~ 5% gelatin in advance with 1 within first day:1:Three kinds of cells of 1 ratio mixing, inoculum density are 0.15 × 106~0.5 ×106Individual/cell, discards structure basal medium after 18 ~ 24h, continue thereafter with paving with 1 ~ 5% gelatin, is incubated 45 ~ 60min, abandons gelatin, be inoculated with 1 again under the conditions of 37 DEG C:1:Three kinds of cells of 1 ratio mixing, inoculum density are 0.15 × 106~0.5 ×106Individual/cell, after 70 ~ 72h of culture, spreads with 1 ~ 5% gelatin, continues 45 ~ 60min of incubation, abandon gelatin, according to volume ratio 2:1 inoculation fibroblast and the cell suspension of umbilical cord mesenchymal stem cells, inoculum density is 0.15 × 106~0.5 ×106Individual/cell, continues 2 ~ 3d of culture, changes liquid every other day；
Step 4, the bilayer skin containing blood capillary luminal structure build：
Fusion rate is built culture fluid SKc1 re-suspended cells and is centrifuged up to 60% or so forth generation epidermis cell, with 0.1 ~ 0.8 × 106The cell density of individual/cell is seeded in cell culture cell；The 1st day of SKc1 cultures is training method under liquid, and the 2nd cultivated under SKc1 liquid day proceeds by the culture of solution-air face；
SKc2 culture fluid solution-airs face is changed after 2 ~ 3d is cultivated under continuous liquid and cultivates 2 ~ 3d, then changed SKc3 culture fluid solution-airs face and cultivate 2 ~ 3d, finally changed SKc4 culture fluid solution-airs face and cultivate 3 ~ 4d, building process is completed；
The composition of above-mentioned culture fluid is as follows：
The composition of basal medium：It is 1 in volume ratio:Add insulin 1 ~ 35 in 1 α-MEM and F12 mixed liquor
Ug/ml, 10 ~ 50ug/ml of adenine, 0.1 ~ 4.5ug/ml of hydrocortisone, 5 ~ 10% hyclones；
Culture fluid SKc1 be in basal medium add 10 ~ 50 μ g/ml BPE, the EGF of 0.1 ~ 0.5ng/ml, 0.01 ~ 0.09nM of ethanolamine-phosphate, 2 ~ 10nM of Progesterone, 60 ~ 90 μ g/ml of commercial heparin；
Culture fluid SKc2 is the BPE, the EGF of 0.1 ~ 0.5ng/ml, the CaCl of 1.5 ~ 2.5 mM for adding 10 ~ 50 μ g/ml in basal medium2, the Vc of 20 ~ 80 μ g/ml, 0.01 ~ 0.09nM of ethanolamine-phosphate, 2 ~ 10nM of Progesterone；
Culture fluid SKc3 is the BPE, the EGF of 0.1 ~ 0.5ng/ml, the CaCl of 1.5 ~ 3.0 mM for adding 10 ~ 50 μ g/ml in basal medium2, the Vc of 20 ~ 80 μ g/ml, 0.01 ~ 0.09nM of ethanolamine-phosphate, 2 ~ 10nM of Progesterone；
Culture fluid SKc4 is the BPE, the EGF of 0.1 ~ 0.5ng/ml, the CaCl of 2.5 ~ 3.5mM for adding 10 ~ 50 μ g/ml in basal medium2, the Vc of 20 ~ 80 μ g/ml, 0.01 ~ 0.09nM of ethanolamine-phosphate, 2 ~ 10nM of Progesterone, 60 ~ 90 μ g/ml of commercial heparin；
Culture fluid SKc3 is the BPE, the EGF of 0.1 ~ 0.5ng/ml, the CaCl of 1.5 ~ 3.0 mM for adding 10 ~ 50 μ g/ml in basal medium2, the Vc of 20 ~ 80 μ g/ml, 0.01 ~ 0.09nM of ethanolamine-phosphate, 2 ~ 10nM of Progesterone；
Culture fluid SKc4 is the BPE, the EGF of 0.1 ~ 0.5ng/ml, the CaCl of 2.5 ~ 3.5mM for adding 10 ~ 50 μ g/ml in basal medium2, the Vc of 20 ~ 80 μ g/ml, 0.01 ~ 0.09nM of ethanolamine-phosphate, 2 ~ 10nM of Progesterone, 60 ~ 90 μ g/ml of commercial heparin.
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| CN202110442485.XA CN113201482A (en) | 2015-09-25 | 2015-09-25 | Culture solution for double-layer skin culture containing microvascular lumens |
| CN202110442492.XA CN113249300A (en) | 2015-09-25 | 2015-09-25 | Double-layer skin containing microvascular lumen and preparation method thereof |
| CN202110442085.9A CN113249299A (en) | 2015-09-25 | 2015-09-25 | Preparation method of double-layer skin containing microvascular lumen |
| CN201510619910.2A CN106552295B (en) | 2015-09-25 | 2015-09-25 | Double-layer skin containing microvascular lumen and preparation method thereof |
| CN202110442075.5A CN113355274A (en) | 2015-09-25 | 2015-09-25 | Double-layer skin containing microvascular lumen and preparation method thereof |
| CN202110442493.4A CN113106055A (en) | 2015-09-25 | 2015-09-25 | Double-layer skin containing microvascular lumen and preparation method thereof |
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| CN202110442485.XA Division CN113201482A (en) | 2015-09-25 | 2015-09-25 | Culture solution for double-layer skin culture containing microvascular lumens |
| CN202110442493.4A Division CN113106055A (en) | 2015-09-25 | 2015-09-25 | Double-layer skin containing microvascular lumen and preparation method thereof |
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| CN202110442085.9A Withdrawn CN113249299A (en) | 2015-09-25 | 2015-09-25 | Preparation method of double-layer skin containing microvascular lumen |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109136185A (en) * | 2017-06-28 | 2019-01-04 | 中国科学院生物物理研究所 | The preparation method and application of one type brain organ device |
| CN109529123A (en) * | 2018-11-08 | 2019-03-29 | 中国人民解放军第四军医大学 | The vascularization holostrome organization engineering skin and preparation method thereof that hydrogel, nano fiber scaffold and Skin Cell assemble layer by layer |
| CN109550080A (en) * | 2019-01-24 | 2019-04-02 | 中国人民解放军陆军特色医学中心 | A kind of artificial bilayer's skin and preparation method thereof |
| CN110201232A (en) * | 2019-06-06 | 2019-09-06 | 中国人民解放军第四军医大学 | A kind of preparation method of the Prevascularized stem cell diaphragm of self assembly |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111494423B (en) * | 2020-04-22 | 2022-01-25 | 上海市东方医院（同济大学附属东方医院） | Stem cell composition, application thereof and stem cell dropping liquid |
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| CN109529123A (en) * | 2018-11-08 | 2019-03-29 | 中国人民解放军第四军医大学 | The vascularization holostrome organization engineering skin and preparation method thereof that hydrogel, nano fiber scaffold and Skin Cell assemble layer by layer |
| CN109529123B (en) * | 2018-11-08 | 2021-02-19 | 中国人民解放军第四军医大学 | Vascularized full-layer tissue engineering skin formed by assembling hydrogel, nanofiber scaffold and skin cells layer by layer and preparation method thereof |
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| CN110201232A (en) * | 2019-06-06 | 2019-09-06 | 中国人民解放军第四军医大学 | A kind of preparation method of the Prevascularized stem cell diaphragm of self assembly |
| CN110201232B (en) * | 2019-06-06 | 2020-10-23 | 中国人民解放军第四军医大学 | Preparation method of self-assembled prevascularized stem cell membrane |
Also Published As
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| CN113355274A (en) | 2021-09-07 |
| CN113201482A (en) | 2021-08-03 |
| CN113249299A (en) | 2021-08-13 |
| CN106552295B (en) | 2021-05-25 |
| CN113106055A (en) | 2021-07-13 |
| CN113249300A (en) | 2021-08-13 |
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