CN102166375A - Reconstruction method of tissue engineering human corneal epithelium - Google Patents
Reconstruction method of tissue engineering human corneal epithelium Download PDFInfo
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- CN102166375A CN102166375A CN2011100390992A CN201110039099A CN102166375A CN 102166375 A CN102166375 A CN 102166375A CN 2011100390992 A CN2011100390992 A CN 2011100390992A CN 201110039099 A CN201110039099 A CN 201110039099A CN 102166375 A CN102166375 A CN 102166375A
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Abstract
本发明涉及一种组织工程人角膜上皮的重建方法，是利用含20％小牛血清的DMEM/F12培养液将人角膜上皮细胞体外培养至对数生长期，采用胰蛋白酶和胰蛋白酶-EDTA消化法获得完全去除上皮层的消化羊膜载体支架，平铺在插入式培养皿中牢固干贴后，将悬浮于含有IV型胶原蛋白和10％小牛血清的DMEM/F12培养液的对数生长期人角膜上皮细胞，接种至平铺有去上皮层消化羊膜载体支架的插入式培养皿中采用气-液界面培养法进行组织工程人角膜上皮的体外重建。本发明工艺科学合理，所重建的组织工程人角膜上皮可用于批量生产，满足广大角膜上皮病盲患者临床角膜移植治疗对组织工程人角膜上皮的大量需求，且组织工程人角膜上皮体外重建和临床治疗的成本低。The present invention relates to a reconstruction method of tissue engineered human corneal epithelium. Human corneal epithelial cells are cultured in vitro to the logarithmic growth phase with DMEM/F12 culture solution containing 20% calf serum, and digested with trypsin and trypsin-EDTA. The digested amniotic membrane carrier scaffold that completely removed the epithelial layer was obtained by the method, and after it was flatly spread in the insert culture dish and dried firmly, it was suspended in the logarithmic growth phase of DMEM/F12 medium containing type IV collagen and 10% calf serum. Human corneal epithelial cells were inoculated into an insert culture dish covered with de-epithelialized digested amnion carrier scaffolds, and the tissue-engineered human corneal epithelium was reconstructed in vitro by air-liquid interface culture method. The process of the present invention is scientific and reasonable, and the reconstructed tissue-engineered human corneal epithelium can be used in batch production to meet the large demand for tissue-engineered human corneal epithelium in the clinical corneal transplantation treatment of blind patients with corneal epithelial diseases, and the tissue-engineered human corneal epithelium can be reconstructed in vitro and clinically. The cost of treatment is low.
Description
Technical field
The invention belongs to corneal epithelium constructing technology field, be specifically related to a kind of method for reconstructing of organizational project people's corneal epithelium, promptly utilize people's corneal epithelial cell and take off the method that epithelial layer digestion amniotic membrane is rebuild organizational project people corneal epithelium.
Background technology
People's corneal epithelium is made up of the multilamellar epithelial cell, is the first road barrier of cornea, can prevent scattering and disappearing and the invasion of extraneous pathogen of cornea moisture, and stops that liquid and electrolyte enter hypothallus in the tear, makes cornea be in pellucidity; In addition, the microvillus on corneal epithelial cell surface and tear membrane interaction are for the preceding refractive surface of transparency cornea provides optical interface (Xie Lixin, 2000).Because corneal epithelium directly contact with extraneous, so be vulnerable to damage and infection and cause corneal epithelial wound or pathological changes, cause visual deterioration, severe patient can cause corneal epithelium blind (Fan Tingjun etc., 2010).Keratopathy is to be only second to cataractous second largest diseases causing blindness, and wherein majority is because corneal epithelium 26S Proteasome Structure and Function caused unusually (Whitcher etc., 2001).Cornea epithelial transplantation is the unique channel of the sick blind healing of present corneal epithelium, but because the donor's cornea quantity wretched insufficiency of contributing causes the blind patient of most corneal epitheliums to see light again by cornea epithelial transplantation.In recent years, the rise of cornea histoengineering makes reconstruction in vitro go out the normal tissue engineering comea epithelium of morphosis becomes possibility, be to solve the insufficient a kind of new feasible way of donor's cornea material at present, for the sick blind patient's of corneal epithelium clinical treatment has brought new hope, become the research focus but how to utilize corneal epithelial cell and suitable carrier bracket to go out organizational project people corneal epithelium at reconstruction in vitro.
Utilize tissue engineering technique that the reconstruction in vitro research of people's corneal epithelium is started from 1993, many scholars successively utilize limbal stem cell, epidermal stem cells, mesenchymal stem cells MSCs, oral mucosa epithelial cell and the amniotic epithelial cells etc. of inducing differentiation as seed cell, with composite collagen, amniotic membrane, cell-eliminating coanea matrix, chitosan, fibrin and PNIPAM gel etc. is reconstruction in vitro research (Minami etc., 1993 that carrier bracket has been carried out organizational project people's corneal epithelium; Koizumi etc., 2001; Nakamura etc., 2003; Jin etc., 2004; Ma etc., 2006; Fan Tingjun etc., 2010), and can make corneal wound rabbit or corneal injury patient recover certain vision after transplanting.These results of study are that the reconstruction in vitro of organizational project people corneal epithelium has been opened up road; but because used seed cell is the cell of inducing differentiation; because of seed cell is originated limited; or carry on as before and have the characteristics of original kind cell; so can't carry out the scale reconstruction in vitro of organizational project people corneal epithelium; or organizational project people's corneal epithelium of reconstruction in vitro because of structure function is unstable can't clinical practice, still be only limited to experimentation at present.
2009; Fan Tingjun etc. have successfully set up people's corneal epithelial cell system of non-transfection, no oncogenicity; and therefrom filter out the normal and normal corneal epithelial cell strain of structure function of caryogram, successfully solved the source problem that comes that required a large amount of seed cells are rebuild in the scale of people's tissue engineering comea epithelium.Therefore; set up a kind of people's of utilization corneal epithelial cell monoclonal cell strain as early as possible and go the method for epithelial layer digestion amniotic membrane at reconstruction in vitro organizational project people corneal epithelium; having become various countries scholar's main goal of attack, also is the key that the sick blind patient of whole world corneal epithelium is benefited in large-scale production of tissue engineering comea epithelium and clinical practice.
Summary of the invention
The method for reconstructing that the purpose of this invention is to provide a kind of organizational project people's corneal epithelium, utilize the monoclonal cell strain of people's corneal epithelial cell and go epithelial layer digestion amniotic membrane carrier bracket to come reconstruction in vitro people corneal epithelium, thereby satisfy the transplanting demand of patients with corneal epithelium, to remedy the deficiencies in the prior art.
Method for reconstructing of the present invention comprises the steps:
1) preparation of people's corneal epithelium seed cell suspension:
Get the cell of people's corneal epithelial cell system, with the DMEM/F12 culture fluid that contains 20% calf serum 37 ℃ of amplification cultivation to exponential phase, through the digestion of 0.25% trypsin solution and 1000～1500 rev/mins of centrifugal 10～15 minutes acquisition people corneal epithelial cell precipitations, reuse contains the DMEM/F12 culture fluid of 10% calf serum and 0.005%～0.01%IV collagen type with this cell precipitation suspension adult corneal epithelial cell suspension, behind cell counting, adjust cell concentration to 8 * 10
6～3 * 10
7Individual cells/ml;
Wherein the construction method of people's corneal epithelial cell system is: cornea is taken out the back with containing antibiotic disinfectant solution sterilization, after again cornea being carried out digestion process, take off the corneal epithelium that has bowman's lamina, after being cut into little corneal epithelial tissue's piece, corneal epithelial tissue's piece is affixed on the bottom of culture hole downwards, makes bowman's lamina upwards; Add culture fluid then and carry out pasting board cultivate in CO2 gas incubator, under 37 ℃, change culture fluid between culture period, cell carries out successive transfer culture with trypsin digestion after growing up to monolayer, is passaged to the structure of having finished people's corneal epithelial cell system more than 60 generations; Wherein, culture fluid is to contain 20% hyclone, 0.02 ‰～0.04 ‰ human epidermal growth factor, human alkaline fibroblast growth factor, 0.4 ‰～1.0 ‰ carboxymethyl chitosan oligosaccharides of 0.01 ‰～0.02 ‰ and the DMEM/F12 culture fluid of 0.25 ‰～1 ‰ chondroitin sulfate.
It is above-mentioned that to contain antibiotic disinfectant solution be 20%～70% gentamycin solution than concentration for the quality of preparing with 0.9% normal saline.
It is to be the digestion of 0.2～0.5% trypsin solution with concentration that above-mentioned cornea carries out digestion process, and digestion time is 1～2min.
2) preparation of carrier bracket
Utilize 0.25% trypsin-0.1%EDTA solution that the epithelial surface of lyophilizing amniotic membrane is inverted digestion, to be removed the amniotic membrane of epithelial layer fully, after D-hanks solution rinsing 2 times, to go epithelial layer digestion amniotic membrane to break into the go epithelial layer identical with card punch and digest the amniotic membrane disk with 6 well culture plate plug-in type culture dish apertures, make its epithelial surface be tiled in the bottom of plug-in type culture dish up, be positioned over 5%CO
2It is standby to make carrier bracket in the incubator behind firm dry doubling under 37 ℃ of conditions;
3) structure of organizational project people corneal epithelium
At last, in the plug-in type culture dish that posts epithelial layer digestion amniotic membrane carrier bracket, add 0.1～0.2 milliliter of above-mentioned ready people's corneal epithelial cell suspension gently, cell suspension is dispersed in the plug-in type culture dish, do not add culture fluid in the 6 well culture plate holes, put 5%CO
2After 37 ℃ of cultivations treated that cell attached on the carrier bracket fully in 20～24 hours in the incubator, the old culture fluid in the plug-in type culture dish is removed in suction, the plug-in type culture dish put into gently added 0.8 milliliter of 6 well culture plate that contain the DMEM culture fluid of 10% calf serum, set up the liquid-vapor interface culture environment, at 5%CO
2Cultivate the reconstruction in vitro that starts organizational project people corneal epithelium for 37 ℃ in the incubator, shook 6 well culture plates gently every 12 hours, changed liquid once every 24～36 hours, treat that people's corneal epithelial cell is growing up to the organizational project people's corneal epithelium that is reconstruction after 6～8 layers on the carrier bracket.
The present invention utilizes people's corneal epithelial cell of no oncogenicity, non-transfection, can be the seed cell that the scale reconstruction in vitro of organizational project people corneal epithelium provides capacity by continuous passage; Utilize the prepared epithelial layer that goes of commercialization lyophilizing amniotic membrane to digest amniotic membrane, can satisfy organizational project people corneal epithelium and rebuild needed a large amount of carrier bracket material in batches; And can directly apply to batch process and clinical corneal transplantation; And the cost of its production and clinical treatment is low.
The specific embodiment
Below in conjunction with specific embodiment method of the present invention is described in detail
One, the foundation of people's corneal epithelial cell system
1, the startup of corneal film pasting board cultivation: take out 2 complete people's corneas from eye bank, put into 100 milliliters of glass beakers, adding 10～30 ml concns are 0.9% normal saline, clean 5～9 minutes; Behind the sucking-off normal saline, the concentration that adds 10～30 milliliters is 20%～70% gentamycin, soaks 15～35 minutes, carries out disinfection in superclean bench, and below operation is all carried out in superclean bench, and all articles for use all are aseptic; From gentamycin liquid, take out cornea with the ophthalmology tweezer, the concave surface of cornea is lain against in the glass culture dish up, add 5～10 milliliters of digestion of 0.2～0.5% trypsin 1～2 minute in the glass culture dish; Remove trypsin solution, with the ophthalmology tweezer tear the band bowman's lamina corneal epithelium, along CC point the corneal epithelium that has bowman's lamina on average is cut into 8 with eye scissors, with the ophthalmology tweezer corneal epithelium is faced down at the bottom of the smooth hole of going into 24 well culture plates, in going into the culture hole of corneal epithelial sheet, subsides add 0.1～0.3 milliliter of culture fluid, the volume of culture fluid guarantees that promptly corneal epithelial sheet can be good at being attached at the bottom of the culture hole, guarantees that again tissue can be not dry.Culture plate is put into 5% CO2 gas incubator, cultivate for 37 ℃;
2, tissue block method carries out people's corneal epithelial cell and cultivates: the corneal epithelial sheet pasting board was cultivated after 12-24 hour, added corneal epithelial cell special culture solution to 1 milliliter in each culture hole, in 5% CO2 gas incubator, 37 ℃ of cultivations; The corneal epithelial cell special culture solution is more substituted once in every interval 3～5 days.
3, the preparation of people's corneal epithelial cell special culture solution: get 3.0 milliliters of the conventional DMEM/F12 culture fluid of preparing, add 1～4 milligram of chondroitin sulfate then successively, 0.08～0.4 milligram of 0.2～0.6 milligram of carboxymethyl chitosan oligosaccharide and IV Collagen Type VI, dissolving back is with 0.22 micron filtering with microporous membrane degerming fully, add 0.8 milliliter of hyclone, 0.01 ‰～0.02 ‰ human epidermal growth factor and 0.01 ‰～0.02 ‰ basic fibroblast growth factor have been added again, add the DMEM/F12 culture fluid to 4.0 milliliter of conventional preparation, be people's corneal epithelial cell special culture solution of the present invention.
4, the successive transfer culture of people's corneal epithelial cell: after treating that people's corneal epithelial cell grows up to monolayer, the culture fluid in the sucking-off culture hole, adding 0.5 ml concn is 0.25% trypsin solution in each culture hole, leaves standstill digestion 1～2 minute; Trypsin solution is removed in suction, adds 1 milliliter above-mentioned special culture solution in every culture hole, with making people's corneal epithelial cell suspension at the bottom of the dropper piping and druming culture hole in 3～5 minutes; Take out 0.5 milliliter of corneal epithelial cell suspension respectively from every culture hole, join respectively in the new culture hole, each culture hole is added 0.5 milliliter of above-mentioned special culture solution, makes it final volume to 1 milliliter; After treating that people's corneal epithelial cell grows up to monolayer once more, still carry out successive transfer culture with above-mentioned same procedure.
Embodiment 1
From eye bank, take out 2 complete people's corneas, put into 100 milliliters of glass beakers, add 10 ml concns and be 0.9% normal saline, cleaned 5 minutes; Behind the sucking-off normal saline, the concentration that adds 10 milliliters is 20% gentamycin, soaks 35 minutes, carries out disinfection in superclean bench; Below operation is all carried out in superclean bench, and all articles for use all are aseptic; From gentamycin liquid, take out cornea with the ophthalmology tweezer, lie against the concave surface of cornea in the glass culture dish up, add 5 milliliters of digestion of 0.2% trypsin in the culture dish after 2 minutes, remove trypsin solution, tear with the ophthalmology tweezer and to have the corneal epithelium of bowman's lamina, along CC point the corneal epithelium that has bowman's lamina on average is cut into 8 with eye scissors, with the ophthalmology tweezer corneal epithelium is faced down at the bottom of the smooth hole of going into 24 well culture plates, each culture hole is added 0.1 milliliter of the DMEM/F12 culture fluid that contains 20% hyclone; Culture plate is put into 5% CO2 gas incubator, cultivate for 37 ℃.Get 3.0 milliliters of the conventional DMEM/F12 culture fluid of preparing, dissolving back is with 0.22 micron filtering with microporous membrane degerming fully, add 0.8 milliliter of hyclone, add 0.01 ‰～0.02 ‰ human epidermal growth factor and 0.01 ‰～0.02 ‰ basic fibroblast growth factor again, adding the DMEM/F12 culture fluid to 4.0 milliliter of conventional preparation, promptly is people's corneal epithelial cell special culture solution.Pasting board was cultivated after 12 hours, added above-mentioned special culture solution to 1 milliliter in each culture hole, in 5% CO2 gas incubator, 37 ℃ of cultivations; The corneal epithelial cell special culture solution is more substituted once in every interval 3 days.After treating that people's corneal epithelial cell grows up to monolayer, the culture fluid in the sucking-off culture hole, adding 0.5 ml concn is 0.25% trypsin solution in each culture hole, leaves standstill digestion 1 minute; Trypsin solution is removed in suction, adds 1 milliliter above-mentioned special culture solution in every culture hole, with making people's corneal epithelial cell suspension at the bottom of the dropper piping and druming culture hole in 3 minutes; Take out 0.5 milliliter of corneal epithelial cell suspension respectively from each culture hole, join respectively in the new culture hole, every culture hole is added 0.5 milliliter of above-mentioned special culture solution, makes it final volume to 1 milliliter; After treating that people's corneal epithelial cell grows up to monolayer once more, still carry out successive transfer culture with above-mentioned same procedure.
Two, the reconstruction of organizational project people corneal epithelium
1, corneal epithelium is rebuild the preparation of special culture solution: get 80 milliliters of the conventional DMEM/F12 culture fluid of preparing, add 4～8 milligrams of IV collagen types, dissolving back is with 0.22 micron filtering with microporous membrane degerming fully, add 10 milliliters of calf serums, add the DMEM/F12 culture fluid to 100 milliliter of conventional preparation, be corneal epithelium of the present invention and rebuild special culture solution.
2, the preparation of people's corneal epithelium seed cell: get people's corneal epithelial cell, be suspended in 20% calf serum-DMEM/F12 culture fluid, be seeded to floor space and be in 75 square centimeters the culture bottle, put 37 ℃ and carry out amplification cultivation, cell proliferation 72～80 hours is to the logarithmic growth after date, with glass dropper sucking-off culture fluid, add 0.25% trypsin solution digestion 1～2 minute, the culture fluid that adds sucking-off before this stops digestion, 1000～1500 rev/mins centrifugal 10～15 minutes, cell precipitation suspends with 3 milliliters of above-mentioned special culture solution and evenly makes people's corneal epithelium seed cell suspension, after utilizing Casy cell counter or blood counting chamber to carry out cell counting, rebuild special culture solution with corneal epithelium and adjust seed cell concentration to 8 * 10
6～3 * 10
7Individual cells/ml.
3, go the preparation of epithelial layer digestion amniotic membrane carrier bracket: utilize 0.25% trypsin-0.1%EDTA solution (1: 1) that the epithelial surface of lyophilizing amniotic membrane is inverted digestion 15～30 minutes, to be removed the amniotic membrane of epithelial layer fully, after D-hanks solution rinsing 2 times, the epithelial layer that goes that will go epithelial layer digestion amniotic membrane to break into 2.5 centimetres of diameters with card punch digests the amniotic membrane disk.
4, the reconstruction in vitro of organizational project people corneal epithelium: go epithelial layer digestion amniotic membrane disk to be tiled in the bottom of 6 well culture plate plug-in type culture dishs, be positioned in 37 ℃ of incubators dry doubling and handle and make epithelial layer digestion amniotic membrane carrier bracket after 20～24 hours according to the supine direction of epithelium.In the plug-in type culture dish of carrier bracket, add 0.1～0.2 milliliter of above-mentioned ready seed cell suspension gently, piping and druming is dispersed in the plug-in type culture dish cell suspension gently, puts 5%CO
2After 37 ℃ of cultivations treated that cell attached on the carrier bracket fully in 20～26 hours in the incubator, the old culture fluid in the plug-in type culture dish is removed in suction, the plug-in type culture dish is put into 6 well culture plates that added 0.8 milliliter of 10% calf serum-DMEM/F12 culture fluid gently, at 5%CO
237 ℃ of reconstruction in vitro that carry out organizational project people corneal epithelium in the incubator, shook 6 well culture plates gently every 12 hours, changed liquid once every 24～36 hours, treat that people's corneal epithelial cell is growing up to the organizational project people's corneal epithelium that is reconstruction after 6～8 layers on the carrier bracket.
Embodiment 2
Get people's corneal epithelial cell, be suspended in 20% calf serum-DMEM/F12 culture fluid, be seeded to floor space and be in 75 square centimeters the culture bottle, put 37 ℃ and carried out amplification cultivation 80 hours.Get 80 milliliters of the conventional DMEM/F12 culture fluid of preparing, add 8 milligrams of IV collagen types, dissolving back is with 0.22 micron filtering with microporous membrane degerming fully, add 10 milliliters of calf serums, add the DMEM/F12 culture fluid to 100 milliliter of conventional preparation, be corneal epithelium of the present invention and rebuild special culture solution.To the culture bottle after the amplification cultivation, with glass dropper sucking-off culture fluid, add 0.25% trypsin solution digestion 1.5 minutes, the old culture fluid that adds sucking-off before this stops digestion, 1500 rev/mins centrifugal 10 minutes, obtain cell precipitation, suspend with 3 milliliters of above-mentioned special culture solution and evenly make people's corneal epithelial cell suspension; Utilize Casy cell counter or blood counting chamber to carry out cell counting, adjust cell concentration to 1.5 * 10 with above-mentioned special culture solution
7Individual cells/ml.
Utilize 0.25% trypsin-0.1%EDTA solution (1: 1) that the epithelial surface of lyophilizing amniotic membrane is inverted digestion 20 minutes, to be removed the amniotic membrane of epithelial layer fully, after D-hanks solution rinsing 2 times, the epithelial layer that goes that will go epithelial layer digestion amniotic membrane to break into 2.5 centimetres of diameters with card punch digests the amniotic membrane disk, be tiled in the bottom of 6 well culture plate plug-in type culture dishs according to the supine direction of epithelium, be positioned in 37 ℃ of incubators dry doubling and handle and make epithelial layer digestion amniotic membrane carrier bracket after 24 hours.In the plug-in type culture dish of carrier bracket, add 0.15 milliliter of above-mentioned ready seed cell suspension gently, piping and druming is dispersed in the plug-in type culture dish cell suspension gently, puts 5%CO
2After 37 ℃ of cultivations treated that cell attached on the carrier bracket fully in 24 hours in the incubator, the old culture fluid in the plug-in type culture dish hole is removed in suction, the plug-in type culture dish is put into gently 6 well culture plates that added 0.8 milliliter of 10% calf serum-DMEM culture fluid, set up the liquid-vapor interface culture environment, at 5%CO
237 ℃ of reconstruction in vitro that carry out organizational project people corneal epithelium in the incubator, shook 6 well culture plates gently every 12 hours, changed liquid once every 30 hours, treat that people's corneal epithelial cell is growing up to the organizational project people's corneal epithelium that is reconstruction after 6～8 layers on the carrier bracket.
Embodiment 3
Get people's corneal epithelium monoclonal cell strain cell, be suspended in 20% calf serum-DMEM/F12 culture fluid, be seeded to floor space and be in 75 square centimeters the culture bottle, put 37 ℃ and carried out amplification cultivation 72 hours.Get 80 milliliters of the conventional DMEM/F12 culture fluid of preparing, add 6 milligrams of IV collagen types, dissolving back is with 0.22 micron filtering with microporous membrane degerming fully, add 10 milliliters of calf serums, add the DMEM/F12 culture fluid to 100 milliliter of conventional preparation, be corneal epithelium of the present invention and rebuild special culture solution.To the culture bottle after the amplification cultivation, with glass dropper sucking-off culture fluid, add 0.25% trypsin solution digestion 1 minute, the old culture fluid that adds sucking-off before this stops digestion, 1000 rev/mins centrifugal 15 minutes, obtain cell precipitation, suspend with 3 milliliters of above-mentioned special culture solution and evenly make people's corneal epithelial cell suspension; Utilize Casy cell counter or blood counting chamber to carry out cell counting, adjust cell concentration to 3 * 10 with above-mentioned special culture solution
7Individual cells/ml.
Utilize 0.25% trypsin-0.1%EDTA solution (1: 1) that the epithelial surface of lyophilizing amniotic membrane is inverted digestion 15 minutes, to be removed the amniotic membrane of epithelial layer fully, after D-hanks solution rinsing 2 times, the epithelial layer that goes that will go epithelial layer digestion amniotic membrane to break into 2.5 centimetres of diameters with card punch digests the amniotic membrane disk, be tiled in the bottom of 6 well culture plate plug-in type culture dishs according to the supine direction of epithelium, be positioned in 37 ℃ of incubators dry doubling and handle and make epithelial layer digestion amniotic membrane carrier bracket after 20 hours.In the plug-in type culture dish of carrier bracket, add 0.1 milliliter of above-mentioned ready seed cell suspension gently, piping and druming is dispersed in the plug-in type culture dish cell suspension gently, puts 5%CO
2After 37 ℃ of cultivations treated that cell attached on the carrier bracket fully in 26 hours in the incubator, the old culture fluid in the plug-in type culture dish is removed in suction, the plug-in type culture dish is put into 6 well culture plates that added 0.8 milliliter of 10% calf serum-DMEM/F12 culture fluid gently, at 5%CO
237 ℃ of reconstruction in vitro that carry out organizational project people corneal epithelium in the incubator, shook 6 well culture plates gently every 12 hours, changed liquid once every 24 hours, treat that people's corneal epithelial cell is growing up to the organizational project people's corneal epithelium that is reconstruction after 6～8 layers on the carrier bracket.
Embodiment 4
Get people's corneal epithelium monoclonal cell strain cell, be suspended in 20% calf serum-DMEM/F12 culture fluid, be seeded to floor space and be in 75 square centimeters the culture bottle, put 37 ℃ and carried out amplification cultivation 75 hours.Get 80 milliliters of the conventional DMEM/F12 culture fluid of preparing, add 4 milligrams of IV collagen types, dissolving back is with 0.22 micron filtering with microporous membrane degerming fully, add 10 milliliters of calf serums, add the DMEM/F12 culture fluid to 100 milliliter of conventional preparation, be corneal epithelium of the present invention and rebuild special culture solution.To the culture bottle after the amplification cultivation, with glass dropper sucking-off culture fluid, add 0.25% trypsin solution digestion 2 minutes, the old culture fluid that adds sucking-off before this stops digestion, 1200 rev/mins centrifugal 10 minutes, obtain cell precipitation, suspend with 3 milliliters of above-mentioned special culture solution and evenly make people's corneal epithelial cell suspension; Utilize Casy cell counter or blood counting chamber to carry out cell counting, adjust cell concentration to 8 * 10 with above-mentioned special culture solution
6Individual cells/ml.
Utilize 0.25% trypsin-0.1%EDTA solution (1: 1) solution that the epithelial surface of lyophilizing amniotic membrane is inverted digestion 30 minutes, to be removed the amniotic membrane of epithelial layer fully, after D-hanks solution rinsing 2 times, the epithelial layer that goes that will go epithelial layer digestion amniotic membrane to break into 2.5 centimetres of diameters with card punch digests the amniotic membrane disk, be tiled in the bottom of 6 well culture plate plug-in type culture dishs according to the supine direction of epithelium, be positioned in 37 ℃ of incubators dry doubling and handle and make epithelial layer digestion amniotic membrane carrier bracket after 22 hours.In the plug-in type culture dish of carrier bracket, add 0.2 milliliter of above-mentioned ready seed cell suspension gently, piping and druming is dispersed in the plug-in type culture dish cell suspension gently, puts 5%CO
2After 37 ℃ of cultivations treated that cell attached on the carrier bracket fully in 20 hours in the incubator, the old culture fluid in the plug-in type culture dish is removed in suction, the plug-in type culture dish is put into 6 well culture plates that added 0.8 milliliter of 10% calf serum-DMEM/F12 culture fluid gently, at 5%CO
237 ℃ of reconstruction in vitro that carry out organizational project people corneal epithelium in the incubator, shook 6 well culture plates gently every 12 hours, changed liquid once every 36 hours, treat that people's corneal epithelial cell is growing up to the organizational project people's corneal epithelium that is reconstruction after 6～8 layers on the carrier bracket.
Organizational project people's corneal epithelium that method of the present invention reconstructed is similar to the normal cornea epithelium and have a function of normal cornea epithelium on morphosis and transparency.For better checking organizational project people's corneal epithelium that the inventive method obtained has biologically function, with organizational project people's cornea epithelial transplantation that the present invention rebuild to removing limbal stem cell and striking off in New Zealand's lagophthalmos of corneal epithelium, experimental result shows, organizational project people's corneal epithelium of transplanting can make rabbit cornea recover transparent reaching more than 180 days, and the epithelium of transplanting can defense against bacterial infects.Three reproducible results show that all organizational project people's corneal epithelium of using the inventive method structure has the prospect that substitutes spontaneous cornea.
Claims (6)
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Cited By (11)
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| CN102787095A (en) * | 2012-07-06 | 2012-11-21 | 广州医学院第一附属医院 | Method for cultivating rat primary airway epithelial cells |
| RU2481087C1 (en) * | 2012-02-15 | 2013-05-10 | Федеральное государственное бюджетное учреждение "Московский научно-исследовательский институт глазных болезней имени Гельмгольца" Министерства здравоохранения и социального развития Российской Федерации | Method of treating inflammatory and trophic injuries of cornea |
| CN104877958A (en) * | 2014-02-28 | 2015-09-02 | 上海尚瑞生物医药科技有限公司 | Cell strain for maintaining transparency of corneal epithelial cell layer |
| CN106591216A (en) * | 2016-12-12 | 2017-04-26 | 深圳市眼科医院 | Human normal corneal epithelium cells and application thereof |
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| CN107164309A (en) * | 2017-06-18 | 2017-09-15 | 广东博溪生物科技有限公司 | The 3D models and its construction method of a kind of utilization serum-containing medium external structure |
| CN107326003A (en) * | 2017-06-18 | 2017-11-07 | 广东博溪生物科技有限公司 | The 3D models and its construction method of a kind of utilization serum-free medium external structure |
| CN108277204A (en) * | 2018-01-10 | 2018-07-13 | 山东麦德克斯生物科技有限公司 | A kind of method that bioengineering cultivates eye Full-thickness corneal |
| CN109517051A (en) * | 2018-12-03 | 2019-03-26 | 洛阳轩智生物科技有限公司 | Human epidermal stem cell induction is the method for corneal epithelial cell |
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| RU2481087C1 (en) * | 2012-02-15 | 2013-05-10 | Федеральное государственное бюджетное учреждение "Московский научно-исследовательский институт глазных болезней имени Гельмгольца" Министерства здравоохранения и социального развития Российской Федерации | Method of treating inflammatory and trophic injuries of cornea |
| CN102787095A (en) * | 2012-07-06 | 2012-11-21 | 广州医学院第一附属医院 | Method for cultivating rat primary airway epithelial cells |
| CN104877958A (en) * | 2014-02-28 | 2015-09-02 | 上海尚瑞生物医药科技有限公司 | Cell strain for maintaining transparency of corneal epithelial cell layer |
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| CN106955372B (en) * | 2017-04-12 | 2020-08-14 | 山东省眼科研究所 | Method for constructing tissue engineering corneal endothelium |
| CN107164309A (en) * | 2017-06-18 | 2017-09-15 | 广东博溪生物科技有限公司 | The 3D models and its construction method of a kind of utilization serum-containing medium external structure |
| CN107326003A (en) * | 2017-06-18 | 2017-11-07 | 广东博溪生物科技有限公司 | The 3D models and its construction method of a kind of utilization serum-free medium external structure |
| CN107129966A (en) * | 2017-06-18 | 2017-09-05 | 广东博溪生物科技有限公司 | A kind of corneal epithelial cell nutrient solution containing serum |
| CN107326003B (en) * | 2017-06-18 | 2020-09-25 | 广东博溪生物科技有限公司 | 3D model constructed in vitro by using serum-free culture solution and construction method thereof |
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| CN108277204A (en) * | 2018-01-10 | 2018-07-13 | 山东麦德克斯生物科技有限公司 | A kind of method that bioengineering cultivates eye Full-thickness corneal |
| CN109517051A (en) * | 2018-12-03 | 2019-03-26 | 洛阳轩智生物科技有限公司 | Human epidermal stem cell induction is the method for corneal epithelial cell |
| CN109517051B (en) * | 2018-12-03 | 2020-10-16 | 拉菲尔(深圳)投资咨询有限公司 | Method for inducing human epidermal stem cells into corneal epithelial cells |
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