CN1562392A - Method for fabricating activated artificial skin tissue in bilayer by using bioreactor - Google Patents
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Abstract
A process for preparing artificial active dual-layer skin tissue by use of bioreactor includes putting the composite collagen sponge as scaffold on the porous supporter in bioreactor, adding fibroblast suspension to the bioreactor for configuring the dermis, inoculating horny cells, and culturing for configuring epiderm.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the method that a kind of preparation has corium and the double-deck active artificial skin tissue of epidermis, particularly a kind of method that adopts bioreactor to prepare the active artificial skin tissue.
Background technology
Skin is the organ of human body maximum, plays a part the protection organismic internal environment and keeps that it is stable, because it is in the body surface of human body, is subject to various damages and produces disease.Treat various acute and existing history in several thousand of chronic dermatosis with skin transplantation, traditional therapy is to adopt from body or heteroplastic transplantation, but because autotransplantation skin source is limited, heterogenous skin transplant have rejection, spread disease, problem such as preservation difficulty, be difficult to satisfy the patient and transplant needs.In recent years, skin transplantation has begun to develop into the transplanting of tissue engineering skin substitute from auto-skin grafting and allograft, and seeking ideal permanent Graftskin becomes the target that people dream of.
Tissue engineering skin is divided into: epiderm substitute, dermal substitute and double-layer active skin.Rheinwald in 1975 and Green have set up horn cell In vitro culture and amplification technique (RheinwaldJG, Green H.Serial cultivation of human epidermal keratinocytes:the formationof keratining colonies from single cell.Cell, 1975,6:331-336), subsequently O ' Connor and Gallico reported respectively the epiderm substitute of In vitro culture be used for the treatment of the fire victim and succeed (O ' Connor NE, Mulliken JB, Banks-Schlegel S, et al.Graftingof burns with cultured epithelium prepared form autologous epidermalcell.Lancet, 1981,1:75.Gallico GG, O ' connor NE, Comton CC, etal.Permanent coverage of large burn wounds with autologous cultured humanepithelium.N Engl J Med, 1984,311:448-451).This class substitute still has application when the treatment large-area burns at present, but there is following shortcoming in it: cultivation cycle is long, and anti-infection ability is poor, lacks the corium composition, shrink greatly, not wear-resisting, easy ulceration, elasticity is not good enough, and transplanting succeed rate is low, thereby is subjected to certain limitation in clinical practice.In order to overcome these problems, software engineering researchers invent dermal substitute, it can improve the epiderm substitute transplanting succeed rate and improve healing quality, and existing multiple commercial dermal substitute has been applied to clinical.Integra is collagen-aminopolysaccharide dermal substitute (the Phillips TJ that is produced by American I ntegra lifesciences company, Arch Dermatol, 1998,110 (6): 344-349.), its shortcoming is to cost an arm and a leg, can not extensive use, implant the back and be difficult for observing wound surface early infection situation, especially need the second operation could wound closure.Hansbrough etc. are seeded to the polylactic acid net with fibroblast and make up dermal substitute, and called after Dermagraft, successfully are used for the treatment of diabetic ulcer clinically.Simple dermal substitute is owing to lack the protection of epidermis, infection rate height.
Ideal Graftskin should be the corium and the epidermal area that are lacked can be repaired simultaneously, the Apligraf that U.S. Organogenesis company produces is the Graftskin of first kind of double-layer active, its skin corium is by fibroblast, the cattle type i collagen, the gel that culture medium etc. are formed, the surface seeding horn cell, In vitro culture constitutes the bilayer skin tissue about 20 days, clinical proof has certain curative effect to various intractables and chronic skin ulcer, but because of there are major defect in this product and preparation technology thereof, cause clinical practice to be obstructed, seriously shrink as collagen gel, the anticollagenase degradation capability is poor, and because artificial skin makes up is to carry out under the quiescent conditions in culture dish, cell culture environment is uncontrollable, the transmission capacity of nutrient and by-product is limited, is unfavorable for collagen gel interior detail intracellular growth, in addition manual operations, cause the production process complexity, organize homogeneity and quality stability to can not get ensureing.
United States Patent (USP) RE35,399 and Chinese invention patent (application number: 91109937.9) set forth a kind of preparation method of bilayer skin substitute, this Graftskin is made up of three parts: the horn cell of cultivation constitutes epidermis, the centre is the atresia collagen layer, cultivates fibroblast and constitute skin corium on the collagen sponge substrate of porous, crosslinkedization.At first angle of departure cell plastid and fibroblast from skin samples, behind In vitro culture, fibroblast is inoculated on the collagen sponge, the another side of sponge is made atresia collagen, horn cell " is dripped and falls " to atresia collagen, cultivate the back and form Graftskin.Because this Graftskin is produced in culture dish, cause the fibroblast major part of inoculation to be trapped in the collagen sponge surface on the one hand, and skewness in collagen sponge, make being organized on the anatomical structure of structure very big difference be arranged with natural tissues, static as mentioned above on the other hand incubation is unfavorable for cell growth and substrate secretion, moreover between each culture dish, batch and criticize between differ greatly.
Halberstadt (Halberstadt CR, Hardin R, Bezverkov K, et al.The in vitrogrowth of a three-dimensional human dermal replacement using a single-passperfusion system, Biotechnol Bioeng, 1994,43:740-746.) wait the people with 2 * 10
5Cells/cm
2Human fibroblasts be inoculated on the holey support that the PGA/PLA material makes, unidirectional perfusion cultures system by a kind of sealing, can construct dermal substitute through the cultivation in 2-～3 weeks with biologic activity, and duplication of production dermal tissue substitute effectively.Its advantage is to compare with static cultivation and can shortens the time that corium makes up; The degradation speed of PGA/PLA timbering material is controlled; The artificial dermis that makes up can direct freezing preservation; But this system is only limited to the structure artificial dermis.
Prenosil and Villeneuve (Prenosil JE, Villeneuve PE.Automated productionof cultured epidermal autograpts and sub-confluent epiderlmal autografts in acomputer controlled bioreactor.Biotechnol Bioeng, 1998,59:679-683.) with people's epidermis cell direct inoculation to fep film, and fep film is placed in the Merlon growth room, a plurality of growth rooms can pile up, on the peristaltic pump that is connected a multitube road, can carry out flow-control automatically.Its advantage is can monitor the formation situation of epidermis because the gripper shoe that makrolon material is done is transparent.Equally, this reactor assembly is only limited to the structure epiderm substitute, can't realize the external structure of double-layer active skin.
Summary of the invention
The technical issues that need to address of the present invention are to disclose a kind of method that adopts bioreactor to prepare double-layer active artificial skin tissue, to overcome the prior art above shortcomings, satisfy the needs in relevant field.
Method of the present invention comprises the steps:
(1) is timbering material with the composite collagen sponge, places on the porous support of filling type bioreactor that add the DMEM culture medium that contains 5～15% new-born calf serum, soaked overnight is abandoned culture medium, by 1 * 10
5Cells/cm
2～5 * 10
6Cells/cm
2Cell density adds the fibroblast suspension in the reactor, pour into inoculation by peristaltic pump, to the culture fluid residual cell density be lower than inoculum density 5～20% after, carry out perfusion cultures, simultaneously concentration of glucose in the culture fluid of detection reaction device exit.Regulating the culture medium rate of flooding is 0.5～4d-1, and control exit concentration of glucose is not less than 1.0～2.0g/L.In this reactor,, finish corium and make up through the perfusion cultures of 10～20d;
(2) with the DMEM culture medium of horn cell serum-free medium replacement new-born calf serum, on above-mentioned artificial dermis, press density 1 * 10
5Cells/cm
2～5 * 10
6Cells/cm
2The inoculation horn cell, behind static cultivation 1～2d, with horn cell culture medium perfusion cultures 2～5d, irrigation rate is 0.5～2d
-1Then remove the horn cell serum-free medium, adopt the DMEM culture medium that contains 2～10% new-born calf serum to carry out gas-liquid interface and cultivate, concentration of glucose in the while detection reaction device exit culture fluid, the control irrigation rate is 0.5～4d
-1, make concentration of glucose be not less than 1.0～2.0g/L.After cultivating 8～14d, finish epidermis and make up.
Said composite collagen sponge is made up of aminopolysaccharide class material and collagen solution, and aminopolysaccharide class content of material is 5～25% (g/g) of collagen dry weight; Said aminopolysaccharide class material comprises in receiving one or more of chitosan, 6-chondroitin sulfate, hyaluronic acid or heparin.
Wherein: said collagen solution is that the tendon powder that tendon Bovis seu Bubali goes fascia, lipid to make is soaked in the collagen-HAc solution that obtains in the acetum, its concentration is generally 5.0～10.0mg/mL, this collagen-HAc solution is preparation like this: tendon Bovis seu Bubali goes fascia, lipid to make the tendon powder, be soaked in the acetum, making final concentration is collagen-HAc solution of 8.0mg/mL.Perhaps directly buy commercial collagen, being mixed with final concentration is collagen-HAc solution of 5.0～10.0mg/mL.
Said composite collagen sponge is like this preparation: chitosan, 6-chondroitin sulfate, hyaluronic acid and heparin are received one or more add collagen-HAc solution,-20 ℃ of refrigerator freeze overnight, vacuum lyophilization 24hr, adopt 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (Sigma) that collagen sponge is carried out chemical crosslinking, clean, lyophilizing, sterilization promptly obtains said composite collagen sponge.This composite collagen sponge is through 1-ethyl-3-(3-dimethyl aminopropyl) after carbodiimide is crosslinked, can overcome the simple collagen gel or shortcomings such as the collagen sponge mechanical strength is not enough, shrinkage factor is high, degradation speed is too fast, glutaraldehyde cross-linking tool cytotoxicity, and can improve the biocompatibility of material, promote the attaching and the propagation of cell on three-dimensional stent material;
Said horn cell is an isolating horn cell from epidermal tissue, separation method is a kind of method of routine, at Johnson EW, Meunier SF, Roy CJ, Parenteau NL.Serialcultivation of normal human keratinocytes:a defined system for studying theregulation of growth and differentiation.In Vitro Cell Dev Biol, 1992, detailed report has been arranged in the 28A:429-435. document;
Said fibroblast is an isolating fibroblast from dermal tissue, separation method is a kind of method of routine, at Halberstadt CR, Hardin R, Bezverkov K, et al.The in vitrogrowth of a three-dimensional human dermal replacement using a single-passperfusion system, Biotechnol Bioeng, 1994, detailed report has been arranged in the 43:740-746. document;
Said filling type bioreactor has disclosed description in Chinese patent application numbers 200410016651.6.
Said horn cell serum-free medium has detailed description in Chinese patent application numbers 200410016367.9, it is characterized in that this serum-free medium is a kind of aqueous solution, comprises basal medium and following component:
Basal medium 12-18g/L
Histidine 0.1-1.0mmol/L
Isoleucine 0.1-0.6mmol/L
Tryptophan 0.05-0.3mmol/L
Phenylalanine 0.3-0.6mmol/L
Lysine 0.7-1.2mmol/L
Methionine 0.05-0.3mmol/L
Tyrosine 0.3-0.6mmol/L
Keratin cell growth factor 2 0-200ng/L and/or epidermal growth factor 5-20
ng/L
Pantothenate 0.02-0.04mmol/L
Choline chloride 0.1-0.5mmol/L
Folic acid 0.01-0.03mmol/L
Inose 0.1-0.4mmol/L
Nicotiamide 0.05-0.5mmol/L
Vitamin B6 0.02-0.06mmol/L
Riboflavin 0.001-0.01mmol/L
Thiamine 0.01-0.03mmol/L
Insulin 1-20 μ g/L
Transferrins 1-20 μ g/L
Ethanolamine 0.01-0.05mmol/L
Phosphoethanolamine 0.01-0.05mmol/L
Strontium chloride 1-5mmol/L
Bovine serum albumin 5-20 μ g/L
Hydrocortisone 0.1-1 μ g/L
Triiodo thyroid amine 10-40pmol/L
Calcium chloride 0.03-0.5mmol/L
Magnesium chloride 5-10mmol/L
Antioxidant is an amount of
Penicillin 50-200U/L
Streptomycin 50-200U/L
Said basal medium comprises MCDB153, no calcium DMEM or does not have calcium DMEM and Ham ' s F12 is 1: 0.3～3 mixture with volume ratio.
Said antioxidant comprises mercaptoethanol 0.01-0.05mmol/L and/or catalase 50-150U/L and/or superoxide dismutase 50-150U/L and/or sodium selenite 0.01-0.05mmol/L.
Adopt novel bioreactor system, can in same reactor, make up double-layer active artificial skin with skin corium and epidermal area, avoided bioreactor system in the past to make up the limitation of artificial dermis or artificial epidermis only, stack by reactor simultaneously, make a preparation process can make up the polylith artificial skin simultaneously, compare with manual operations, preparation process is more effective and can repeat.
Adopt bioreactor system to prepare tool corium and the double-deck active artificial skin of epidermis, make the certain mechanical strength of artificial skin tool made, delay degradation speed, cell and extracellular matrix and in material, be evenly distributed, manufacturing cycle is short, production can repeat, characteristics such as difference is little between criticizing and criticizing.
Compare with preparation method in the past, adopt perfusion inoculation and training method, the transfer mode of material becomes active transfer by passive diffusion on the one hand, make cell being more evenly distributed in collagen sponge, the secretion of the formation that helps organizing, cell growth and cell extracellular matrix, in this dynamic culture environment, can alleviate the diffusion-restricted of nutrient and by-product to a certain extent, reduced at the inner nutrient of three-dimensional material and exhausted and by-product toxicity pair cell normal growth and metabolic influence, thus structure, composition and the function of further betterment works tissue; Adopt the perfusion cultures mode on the other hand, import and export the variation of concentration of glucose according to reactor, regulate the perfusion strategy of culture medium, keep culture environment a metastable level, make it more near cells in vivo and organize stable interior environment, promote cell proliferation and functional expression, shorten incubation time, make being organized in of structure more approach natural tissues on the anatomical structure.
Description of drawings
Fig. 1 is the pouring type bioreactor system structure chart.
Fig. 2 is the reactor result schematic diagram.
The specific embodiment
Referring to Fig. 1 and Fig. 2, the present invention is used for the pouring type bioreactor system that double-layer active skin is organized external structure, comprising: liquid-adding device, filling type bioreactor, receipts liquid device, liquid reflux device and feeder;
Said filling type bioreactor comprises: the closed container 2 that is provided with loam cake 1; Be arranged on the porous support plate 5 that in the closed container 2 closed container 2 is divided into last chamber 3 and following chamber 4, be arranged on the fit sealing spare 6 of porous support plate 5 tops near closed container 2 inwalls; The timbering material or the cell material complex 7 that are used for the skin structure lie in porous support plate 5, and fixing by fit sealing spare 6, and porous support plate 5 can provide gas-liquid interface when epidermal area made up; Last chamber 3 is provided with liquid inlet 8 and liquid outlet 9, following chamber is provided with liquid inlet 8 and liquid outlet 9, loam cake 1 is provided with liquid inlet 8, gas access 10 and gas outlet 11, and loam cake 1 below is provided with liquid distribution trough 101, is used to the culture medium that distributes and enter from the liquid inlet 8 of loam cake 1.
Said liquid-adding device is connected with the liquid inlet 8 of filling type bioreactor, said receipts liquid device is connected with the liquid outlet 9 of filling type bioreactor, said liquid reflux device is connected with liquid-adding device and receipts liquid device respectively by pipeline, and said feeder is connected with the gas access 10 of filling type bioreactor by pipeline;
Said liquid-adding device comprises liquid feeding peristaltic pump 12 and coupled liquid feeding liquid containing bottle 13, liquid feeding liquid containing bottle 13 is connected with the liquid inlet 8 of filling type bioreactor by pipeline, because said bioreactor has three liquid inlets 8, select the liquid inlet 8 of loam cake 1 during inoculation for use, perfusion cultures may be selected the liquid inlet 8 of avris, therefore be preferably between liquid feeding peristaltic pump 12 and the pipeline that each liquid inlet 8 of filling type bioreactor links to each other by-pass valve control 26 is set, entering the mouth from which with controlling liquid 8 enters;
Said receipts liquid device comprises: receive liquid peristaltic pump 14, receive liquid liquid containing bottle 15, receive hydraulic control valve 16 and receive liquid bottle 17, the import of receiving liquid peristaltic pump 14 is connected with the liquid outlet 9 of filling type bioreactor, the outlet of receiving liquid peristaltic pump 14 is connected with the inlet of receiving liquid liquid containing bottle 15, the outlet of receiving liquid liquid containing bottle 15 is connected with receipts liquid bottle 17 by liquid storage peristaltic pump 25, receiving hydraulic control valve 16 is arranged on the pipeline of receiving between liquid liquid containing bottle 15 and the liquid storage peristaltic pump 25, be used for control and receive liquid measure, be provided with by-pass valve control 26 between each outlet of bioreactor and the receipts liquid peristaltic pump 14.
Said liquid reflux device comprises by reflux pipeline 19 and is arranged on the recycle control valve of receiving between liquid liquid containing bottle 15 and the liquid feeding liquid containing bottle 13 18;
Said feeder comprises interconnective successively air pump 20, mass air flow sensor 21 and air filter 22, and air filter 22 is connected with filling type bioreactor;
Said checkout gear comprises and is inserted in liquid feeding liquid containing bottle 13, receives the dissolved oxygen electrode 23 in the liquid liquid containing bottle 15 and receives pH electrode 24 in the liquid liquid containing bottle 15, dissolved oxygen electrode 23 is connected with the pH analyzer with dissolved-oxygen content analyser respectively with pH electrode 24, to detect and to control the oxyty and the pH of culture medium in liquid feeding liquid containing bottle 13, the receipts liquid liquid containing bottle 15.
As seen from Figure 1, a plurality of said filling type bioreactors can be connected in parallel, to satisfy the needs that enlarge volume of production.
Get child's foreskin, be cut into 1cm
2About skin chunk, clean 3 times with the PBS that contains the 200U/mL gentamycin, 75% alcohol wash 2 times, reuse PBS cleans 2 times.Skin histology is joined in the neutral protease (Sigma) of 200U/mL, 4 ℃ digest 18hr down, corium are separated with epidermis.From epidermal tissue, obtain horn cell, from dermal tissue, obtain fibroblast.
Epidermis is shredded, and the pancreatin with 0.25% (Sigma)/0.06%EDTA is 6mL (volume of pancreatin/EDTA Digestive system is approximately 3 times by the digestion tissue volume) effect 8min altogether, and with in the calf serum of equivalent with the pancreatin activity.
150 eye mesh screens filter, and remove the epidermis fragment, obtain the horn cell suspension.The centrifugal 5min of 2000r/min removes supernatant, and PBS cleans 2 times, adds the horn cell serum-free medium (Gibco) that contains the 200U/mL gentamycin, and the cell inoculation amount is 2 * 10
4Cells/cm
2, each 25cm
2Culture bottle (Nunc) in add culture medium 5mL, at 37 ℃, 5%CO
2Incubator (Revco) in cultivate, changed liquid once in per three days, obtain former generation cultured cells.When cell cover with the square vase floor space 75% the time, the cultivation of going down to posterity, remove supernatant, the pancreatin that adds 1.5mL digests, and when cell rounding, the back that comes off adds equivalent calf serum termination pancreatin activity, cell suspension removes supernatant after with the centrifugal 5min of the speed of 2000r/min, PBS cleans twice, add culture medium, be seeded to square vase or bioreactor system and go down to posterity and cultivate and amplification, obtain to make up the required horn cell of epidermal area.
Corium partly shreds, and adds 0.05% collagenase (Gibco) solution, enzymolysis 40min under 37 ℃ of conditions; Remove by filter residual pieces, cell is with PBS washing and centrifugal collection, and the fibroblast suspension is according to inoculum concentration 5 * 10
4/ cm
2Be seeded in square vase Central Plains and be commissioned to train fosterly, be culture medium, changed liquid once in per 3 days with the DMEM (Gibco) that contains 10% calf serum.It is about about 80% to treat that fibroblast is paved with square vase bottom, with the digestion of 0.25% trypsin solution, presses 2 * 10
4/ cm
2Inoculum concentration in square vase or bioreactor, go down to posterity and cultivate and amplification, obtain to make up the required fibroblast of skin histology skin corium.
Tendon Bovis seu Bubali goes fascia, lipid to make the packing of tendon powder, and is standby in-20 ℃ of storages.The tendon end is soaked in the HAc solution of 0.1mol/L, making final concentration is collagen-HAc solution of 8.0mg/mL.Chitosan solution, heparin are received solution, chondroitin sulfate cellulose solution to be added in the collagen solution, make final concentration be respectively 10% (g/g), 7% (g/g) and 3% (g/g) of collagen dry weight,-20 ℃ of refrigerator freeze overnight promptly get collagen sponge behind the vacuum lyophilization 24hr.Adopt 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (Sigma) that collagen sponge is carried out chemical crosslinking, fully clean and lyophilizing with distilled water, oxirane disinfection is made the composite collagen sea host material that can be used for preparing the double-layer active skin tissue.
Adopt the identical method of embodiment 2, but in the composite collagen sponge, aminopolysaccharide class material is a hyaluronic acid, final concentration is 20% (g/g) of collagen dry weight.
Embodiment 4
Collagen sponge is placed on the porous stainless steel plate washer support of filling type bioreactor of double-layer active skin external structure, add the DMEM culture medium that contains 10% new-born calf serum, soaked overnight.Abandon culture medium, by 5 * 10
5Cells/cm
2Cell density adds the fibroblast suspension in the reactor, pours into inoculation by peristaltic pump, to the culture fluid residual cell density be lower than inoculum density 10% after, carry out perfusion cultures.
The control rate of flooding is 1d
-1, concentration of glucose in the culture fluid of detection reaction device exit when concentration of glucose is lower than 2g/L, improves irrigation rate to 2.5d simultaneously
-1In this reactor,, finish corium and make up through the perfusion cultures of 16d.
Replace the DMEM culture medium of 10% new-born calf serum with the horn cell serum-free medium, on above-mentioned artificial dermis by density 5 * 10
5Cells/cm
2The inoculation horn cell, behind the static cultivation 1d, with horn cell culture medium perfusion cultures 2d, irrigation rate is 1d
-1Then remove the horn cell serum-free medium, adopt the DMEM culture medium that contains 10% new-born calf serum to carry out gas-liquid interface and cultivate, liquid level position is controlled by upper outlet, and culture medium is imported and exported by the lower end and poured into, and the control irrigation rate is 1d
-1, concentration of glucose in the culture fluid of detection reaction device exit when concentration of glucose is lower than 2g/L, improves irrigation rate to 3d simultaneously
-1After cultivating 14d, finish epidermis and make up.
Claims (6)
1. a composite collagen sponge that is used for external structure double-layer active artificial skin tissue is characterized in that being made up of aminopolysaccharide class material and collagen solution, and aminopolysaccharide class content of material is 5～25% (g/g) of collagen dry weight; Said aminopolysaccharide class material comprises in receiving one or more of chitosan, 6-chondroitin sulfate, hyaluronic acid or heparin, said collagen solution is that the tendon powder that tendon Bovis seu Bubali goes fascia, lipid to make is soaked in the collagen-HAc solution that obtains in the acetum, or by the collagen-HAc solution of business-like collagen product preparation.
2. a method that adopts bioreactor to prepare double-layer active artificial skin tissue is characterized in that, comprises the steps:
(1) be timbering material with the composite collagen sponge, place on the porous support of filling type bioreactor, adding the DMEM culture medium that contains 5～15% new-born calf serum soaks, abandon culture medium, perfusion inoculation fibroblast, to the culture fluid residual cell density be lower than inoculum density 5～20% after, carry out perfusion cultures, in reactor,, finish corium and make up through the perfusion cultures of 10～20d;
(2) inoculate horn cell on above-mentioned artificial dermis, replace containing the DMEM culture medium of new-born calf serum with the horn cell serum-free medium, behind static cultivation 1～2d, with horn cell culture medium perfusion cultures 2～5d, irrigation rate is 0.5～2d
-1, then remove the horn cell serum-free medium, adopt the DMEM culture medium that contains 2～10% new-born calf serum to carry out gas-liquid interface and cultivate, and adjust irrigation rate (0.5～4d as required
-1), finish epidermis behind 8～14d and make up.
Said horn cell is an isolating horn cell from epidermal tissue, and said fibroblast is an isolating fibroblast from dermal tissue.
3. method according to claim 2 is characterized in that, with 1 * 10
5Cells/cm
2～5 * 10
6Cells/cm
2Cell density adds the fibroblast suspension in the reactor.
4. method according to claim 2 is characterized in that, the irrigation rate of step (1) is 0.5～4d
-1, make that concentration of glucose is not less than 1.0～2.0g/L in the reactor exit culture fluid.
5. method according to claim 2 is characterized in that, presses density 1 * 10 on artificial dermis
5Cells/cm
2～5 * 10
6Cells/cm
2The inoculation horn cell.
6. method according to claim 2 is characterized in that, step (2) is carried out perfusion cultures with the DMEM that contains serum, and the determining of irrigation rate makes the concentration of glucose in the reactor exit culture fluid be not less than 1.0～2.0g/L.
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| WO2009003375A1 (en) * | 2007-06-29 | 2009-01-08 | Shanghai Tissue Engineering Life Science Co. Ltd. | Tissue engineering tendon and construction methods in vitro thereof |
| CN101532950B (en) * | 2008-12-31 | 2011-09-07 | 程树军 | Method for analyzing toxicity and effect of skin whitening agent through human being skin melanocyte |
| CN101352586B (en) * | 2008-08-26 | 2012-03-07 | 程树军 | Method for preparing full-thickness skin for toxicity test by stem cell raft type cultivation |
| CN105219697A (en) * | 2014-07-04 | 2016-01-06 | 赫柏慧康生物科技无锡有限公司 | A kind of people's primary keratinocyte is separated the method for preparation |
| US9259445B2 (en) | 2006-06-07 | 2016-02-16 | Universidad Tecnica Federico Santa Maria | Integrated implant system (IIS) biocompatible, biodegradable and bioactive, comprising a biocompatible sterile porous polymeric matrix and a gel, integrating in situ the tridimensional matrix structure |
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| EP1263459A2 (en) * | 1999-12-02 | 2002-12-11 | Ibex Technologies, Inc. | Attenuation of fibroblast proliferation |
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| WO2009003375A1 (en) * | 2007-06-29 | 2009-01-08 | Shanghai Tissue Engineering Life Science Co. Ltd. | Tissue engineering tendon and construction methods in vitro thereof |
| CN101352586B (en) * | 2008-08-26 | 2012-03-07 | 程树军 | Method for preparing full-thickness skin for toxicity test by stem cell raft type cultivation |
| CN101532950B (en) * | 2008-12-31 | 2011-09-07 | 程树军 | Method for analyzing toxicity and effect of skin whitening agent through human being skin melanocyte |
| CN105219697A (en) * | 2014-07-04 | 2016-01-06 | 赫柏慧康生物科技无锡有限公司 | A kind of people's primary keratinocyte is separated the method for preparation |
| CN106421931A (en) * | 2016-11-10 | 2017-02-22 | 广东泰宝医疗科技股份有限公司 | Skin repair material with biological activity and method for preparing skin repair material |
| CN109055303A (en) * | 2018-09-12 | 2018-12-21 | 山东麦德克斯生物科技有限公司 | A kind of construction method of skin histology |
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