CN102166375B - Reconstruction method of tissue engineering human corneal epithelium - Google Patents
Reconstruction method of tissue engineering human corneal epithelium Download PDFInfo
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- CN102166375B CN102166375B CN 201110039099 CN201110039099A CN102166375B CN 102166375 B CN102166375 B CN 102166375B CN 201110039099 CN201110039099 CN 201110039099 CN 201110039099 A CN201110039099 A CN 201110039099A CN 102166375 B CN102166375 B CN 102166375B
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Abstract
The invention relates to a reconstruction method of tissue engineering human corneal epithelium. The method comprises the following steps of: adopting a DMEM/F12 culture medium containing 20% calf serum to carry out the in vitro culture on human corneal epithelium cells to a logarithmic growth phase, and adopting trypsin and a trypsin-EDTA digestion method to obtain a digestive amniotic carrier bracket of which the epithelium is completely removed; and after the digestive amniotic carrier bracket of which the epithelium is removed is flatly laid in a plug-in Petri dish and is firmly and pasted in a drying way, inoculating the human corneal epithelium cells at the logarithmic growth phase suspended in the DMEM/F12 culture medium containing IV type collagen and 20% calf serum to the plug-in Petri dish flatly laid with the digestive amniotic carrier bracket of which the epithelium is removed, and carrying out the in vitro reconstruction on the tissue engineering human corneal epithelium by a gas-liquid interface culture method. The invention has scientific and reasonable process, the reconstructed tissue engineering human corneal epithelium can be used for mass production, a lot of demands of vast blind patients with corneal epithelium diseases for the tissue engineering human corneal epithelium in clinical corneal transplantation treatment can be met, and the costs of the in vitro reconstruction and clinical treatment of the tissue engineering human corneal epithelium are low.
Description
Technical field
The invention belongs to corneal epithelium constructing technology field, be specifically related to a kind of method for reconstructing of tissue engineering human corneal epithelium, namely utilize people's corneal epithelial cell and take off the method that epithelial layer digestion amniotic membrane is rebuild tissue engineering human corneal epithelium.
Background technology
People's corneal epithelium is comprised of the multilamellar epithelial cell, is the first 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 front 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 unusually caused (Whitcher etc., 2001).Cornea epithelial transplantation is the unique channel of the blind healing of present corneal epithelial diseases, 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, it is a kind of new feasible way that solves at present donor's cornea material deficiency, for the blind patient's of corneal epithelial diseases clinical treatment has brought new hope, become study hotspot but how to utilize corneal epithelial cell and suitable carrier bracket to go out tissue engineering human 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, reconstruction in vitro research (Minami etc., 1993 of tissue engineering human corneal epithelium have been carried out take composite collagen, amniotic membrane, cell-eliminating coanea matrix, chitosan, fibrin and PNIPAM gel etc. as carrier bracket; 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 tissue engineering human corneal epithelium has been opened up road; but because used seed cell is the cell of inducing differentiation; because 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 tissue engineering human corneal epithelium; or the tissue engineering human corneal epithelium of reconstruction in vitro because of structure function is unstable can't clinical practice, still be only limited at present experimentation.
2009; Fan Tingjun etc. have successfully set up non-transfection, without people's Characteristic Analysis of Corneal Epithelial Cell Line of 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 as early as possible a kind of people's of utilization corneal epithelial cell monoclonal cell strain and go epithelial layer digestion amniotic membrane in the method for reconstruction in vitro tissue engineering human corneal epithelium; having become various countries scholar's main goal of attack, also is the key that the blind patient of whole world corneal epithelial diseases is benefited in the 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 tissue engineering human corneal epithelium, utilize the monoclonal cell strain of people's corneal epithelial cell and go epithelial layer digestion amnion carrying support 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 Characteristic Analysis of Corneal Epithelial Cell Line, 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, use again the DMEM/F12 culture fluid that contains 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 Characteristic Analysis of Corneal Epithelial Cell Line is: sterilize with containing antibiotic disinfectant solution after cornea is taken out, after again cornea being carried out digestion process, take off the corneal epithelium with 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; Then add culture fluid 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 Characteristic Analysis of Corneal Epithelial Cell Line more than 60 generations; Wherein, culture fluid is the DMEM/F12 culture fluid that contains 20% hyclone, 0.02 ‰～0.04 ‰ human epidermal growth factor, 0.01 ‰～0.02 ‰ human alkaline fibroblast growth factor, 0.4 ‰～1.0 ‰ carboxymethyl chitosan oligosaccharides and 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 6 well culture plate culture plate insert apertures with card punch and digest the amniotic membrane disk, make its epithelial surface be tiled in the bottom of culture plate insert up, be positioned over 5%CO
2It is for subsequent use to make carrier bracket in the incubator behind firm dry doubling under 37 ℃ of conditions;
3) structure of tissue engineering human corneal epithelium
At last, in the culture plate insert that posts epithelial layer digestion amnion carrying support, add gently 0.1～0.2 milliliter of above-mentioned ready people's corneal epithelial cell suspension, cell suspension is dispersed in the culture plate insert, do not add culture fluid in the 6 well culture plate holes, put 5%CO
2Cultivate 20～24 hours after cell attaches on the carrier bracket fully for 37 ℃ in the incubator, suck the old culture fluid in the culture plate insert, culture plate insert 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 tissue engineering human corneal epithelium for 37 ℃ in the incubator, shook gently 6 well culture plates every 12 hours, change liquid once every 24～36 hours, grow up to the tissue engineering human corneal epithelium that is reconstruction after 6～8 layers at carrier bracket until people's corneal epithelial cell.
The present invention utilizes the people's corneal epithelial cell without oncogenicity, non-transfection, can be the seed cell that the scale reconstruction in vitro of tissue engineering human 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 tissue engineering human corneal epithelium and rebuild in batches needed a large amount of carrier bracket material; And can directly apply to batch production 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 Characteristic Analysis of Corneal Epithelial Cell Line
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 ophthalmic tweezers, 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, tear with the corneal epithelium of bowman's lamina with ophthalmic tweezers, along CC point the corneal epithelium with bowman's lamina on average is cut into 8 with eye scissors, with ophthalmic tweezers corneal epithelium is faced down at the bottom of the smooth hole that enters 24 well culture plates, in the culture hole that is pasted into corneal epithelial sheet, add 0.1～0.3 milliliter of culture fluid, the volume of culture fluid guarantees that namely 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: 3.0 milliliters of DMEM/F12 culture fluid getting normal compound, then add successively 1～4 milligram of chondroitin sulfate, 0.08～0.4 milligram of 0.2～0.6 milligram of carboxymethyl chitosan oligosaccharide and IV Collagen Type VI, fully after the dissolving with 0.22 micron filtering with microporous membrane degerming, 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 normal compound, 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 people's corneal epithelial cell grew up to monolayer, the culture fluid in the sucking-off culture hole added 0.5 ml concn in each culture hole and is 0.25% trypsin solution, leaves standstill digestion 1～2 minute; Suck trypsin solution, add 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 respectively 0.5 milliliter of corneal epithelial cell suspension 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 people's corneal epithelial cell grows up to monolayer again, 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 ophthalmic tweezers, 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 corneal epithelium of bowman's lamina with ophthalmic tweezers, along CC point the corneal epithelium with bowman's lamina on average is cut into 8 with eye scissors, with ophthalmic tweezers corneal epithelium is faced down at the bottom of the smooth hole that enters 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 DMEM/F12 culture fluid of normal compound, fully after the dissolving with 0.22 micron filtering with microporous membrane degerming, add 0.8 milliliter of hyclone, add again 0.01 ‰～0.02 ‰ human epidermal growth factor and 0.01 ‰～0.02 ‰ basic fibroblast growth factor, adding the DMEM/F12 culture fluid to 4.0 milliliter of normal compound, namely 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 people's corneal epithelial cell grew up to monolayer, the culture fluid in the sucking-off culture hole added 0.5 ml concn in each culture hole and is 0.25% trypsin solution, leaves standstill digestion 1 minute; Suck trypsin solution, add 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 respectively 0.5 milliliter of corneal epithelial cell suspension 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 people's corneal epithelial cell grows up to monolayer again, still carry out successive transfer culture with above-mentioned same procedure.
Two, the reconstruction of tissue engineering human corneal epithelium
1, the preparation of corneal epithelial reconstruction special culture solution: 80 milliliters of DMEM/F12 culture fluid getting normal compound, add 4～8 milligrams of IV collagen types, fully after the dissolving with 0.22 micron filtering with microporous membrane degerming, add 10 milliliters of calf serums, add the DMEM/F12 culture fluid to 100 milliliter of normal compound, be corneal epithelial reconstruction special culture solution of the present invention.
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 before this sucking-off 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, adjust seed cell concentration to 8 * 10 with the corneal epithelial reconstruction special culture solution
6～3 * 10
7Individual cells/ml.
3, go the preparation of epithelial layer digestion amnion carrying support: 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 tissue engineering human corneal epithelium: go epithelial layer digestion amniotic membrane disk to be tiled in the bottom of 6 well culture plate culture plate inserts according to the supine direction of epithelium, be positioned in 37 ℃ of incubators dry doubling and process and make epithelial layer digestion amnion carrying support after 20～24 hours.In the culture plate insert of carrier bracket, add gently 0.1～0.2 milliliter of above-mentioned ready seed cell suspension, piping and druming is dispersed in the culture plate insert cell suspension gently, puts 5%CO
2Cultivate 20～26 hours after cell attaches on the carrier bracket fully for 37 ℃ in the incubator, suck the old culture fluid in the culture plate insert, culture plate insert 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 tissue engineering human corneal epithelium in the incubator, shook gently 6 well culture plates every 12 hours, change liquid once every 24～36 hours, grow up to the tissue engineering human corneal epithelium that is reconstruction after 6～8 layers at carrier bracket until people's corneal epithelial cell.
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 DMEM/F12 culture fluid of normal compound, add 8 milligrams of IV collagen types, fully after the dissolving with 0.22 micron filtering with microporous membrane degerming, add 10 milliliters of calf serums, add the DMEM/F12 culture fluid to 100 milliliter of normal compound, be corneal epithelial reconstruction special culture solution of the present invention.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 before this sucking-off 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 culture plate inserts according to the supine direction of epithelium, be positioned in 37 ℃ of incubators dry doubling and process and make epithelial layer digestion amnion carrying support after 24 hours.In the culture plate insert of carrier bracket, add gently 0.15 milliliter of above-mentioned ready seed cell suspension, piping and druming is dispersed in the culture plate insert cell suspension gently, puts 5%CO
2Cultivate 24 hours after cell attaches on the carrier bracket fully for 37 ℃ in the incubator, suck the old culture fluid in the culture plate insert hole, culture plate insert 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 tissue engineering human corneal epithelium in the incubator, shook gently 6 well culture plates every 12 hours, change liquid once every 30 hours, grow up to the tissue engineering human corneal epithelium that is reconstruction after 6～8 layers at carrier bracket until people's corneal epithelial cell.
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 DMEM/F12 culture fluid of normal compound, add 6 milligrams of IV collagen types, fully after the dissolving with 0.22 micron filtering with microporous membrane degerming, add 10 milliliters of calf serums, add the DMEM/F12 culture fluid to 100 milliliter of normal compound, be corneal epithelial reconstruction special culture solution of the present invention.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 before this sucking-off 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 culture plate inserts according to the supine direction of epithelium, be positioned in 37 ℃ of incubators dry doubling and process and make epithelial layer digestion amnion carrying support after 20 hours.In the culture plate insert of carrier bracket, add gently 0.1 milliliter of above-mentioned ready seed cell suspension, piping and druming is dispersed in the culture plate insert cell suspension gently, puts 5%CO
2Cultivate 26 hours after cell attaches on the carrier bracket fully for 37 ℃ in the incubator, suck the old culture fluid in the culture plate insert, culture plate insert 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 tissue engineering human corneal epithelium in the incubator, shook gently 6 well culture plates every 12 hours, change liquid once every 24 hours, grow up to the tissue engineering human corneal epithelium that is reconstruction after 6～8 layers at carrier bracket until people's corneal epithelial cell.
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 DMEM/F12 culture fluid of normal compound, add 4 milligrams of IV collagen types, fully after the dissolving with 0.22 micron filtering with microporous membrane degerming, add 10 milliliters of calf serums, add the DMEM/F12 culture fluid to 100 milliliter of normal compound, be corneal epithelial reconstruction special culture solution of the present invention.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 before this sucking-off 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 culture plate inserts according to the supine direction of epithelium, be positioned in 37 ℃ of incubators dry doubling and process and make epithelial layer digestion amnion carrying support after 22 hours.In the culture plate insert of carrier bracket, add gently 0.2 milliliter of above-mentioned ready seed cell suspension, piping and druming is dispersed in the culture plate insert cell suspension gently, puts 5%CO
2Cultivate 20 hours after cell attaches on the carrier bracket fully for 37 ℃ in the incubator, suck the old culture fluid in the culture plate insert, culture plate insert 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 tissue engineering human corneal epithelium in the incubator, shook gently 6 well culture plates every 12 hours, change liquid once every 36 hours, grow up to the tissue engineering human corneal epithelium that is reconstruction after 6～8 layers at carrier bracket until people's corneal epithelial cell.
The tissue engineering human corneal epithelium that method of the present invention reconstructs is similar to the normal cornea epithelium and have a function of normal cornea epithelium on morphosis and transparency.The tissue engineering human corneal epithelium that obtains for better checking the inventive method has function biologically, the tissue engineering human corneal epithelium that the present invention is rebuild is transplanted to and is removed limbal stem cell and strike off in New Zealand's lagophthalmos of corneal epithelium, experimental result shows, the tissue engineering human 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 the tissue engineering human corneal epithelium of using the inventive method structure has the prospect that substitutes spontaneous cornea.
Claims (6)
1. the method for reconstructing of a tissue engineering human corneal epithelium, comprise 1) preparation of people's corneal epithelium seed cell suspension, 2) preparation of amnion carrying support and the 3) incubation step of tissue engineering human corneal epithelium, it is characterized in that, above-mentioned steps 1) be that the cell of employment Characteristic Analysis of Corneal Epithelial Cell Line prepares people's corneal epithelium seed cell suspension, wherein the construction method of people's Characteristic Analysis of Corneal Epithelial Cell Line is: sterilize with containing antibiotic disinfectant solution after cornea is taken out, after again cornea being carried out digestion process, take off the corneal epithelium with 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; Then add culture fluid 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 Characteristic Analysis of Corneal Epithelial Cell Line more than 60 generations; Described culture fluid is the DMEM/F12 culture fluid that contains 20% hyclone, 0.02 ‰～0.04 ‰ human epidermal growth factor, 0.01 ‰～0.02 ‰ human alkaline fibroblast growth factor, 0.4 ‰～1.0 ‰ carboxymethyl chitosan oligosaccharides and 0.25 ‰～1 ‰ chondroitin sulfate.
2. method for reconstructing as claimed in claim 1 is characterized in that above-mentioned to contain antibiotic disinfectant solution be 20%～70% gentamycin solution than concentration for the quality of preparing with 0.9% normal saline.
3. method for reconstructing as claimed in claim 1 is characterized in that it is to be 0.2～0.5% trypsin solution digestion with concentration that above-mentioned cornea carries out digestion process, and digestion time is 1～2min.
4. method for reconstructing as claimed in claim 1, it is characterized in that above-mentioned step 1) preparation people corneal epithelium seed cell suspension is to get people's corneal epithelial cell, 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, use the DMEM/F12 culture fluid that contains 10% calf serum and 0.005%～0.01%IV collagen type with this cell precipitation suspension adult corneal epithelial cell suspension, wherein cell concentration is 8 * 10 again
6～3 * 10
7Individual/mL.
5. method for reconstructing as claimed in claim 1, it is characterized in that above-mentioned step 2) preparation of amnion carrying support is to mix with 0.25% trypsin-0.1%EDTA solution equal-volume the epithelial surface of lyophilizing amniotic membrane is inverted digestion, with thorough removal amniotic epithelial cells, after D-hanks solution rinsing 2 times, to go epithelial layer digestion amniotic membrane to break into the go epithelial layer identical with 6 well culture plate culture plate insert apertures with card punch and digest the amniotic membrane disk, make its epithelial surface be tiled in the bottom of 6 well culture plate culture plate inserts up, be positioned over 5%CO
2Firmly making carrier bracket behind the dry doubling under 37 ℃ of conditions in the incubator.
6. method for reconstructing as claimed in claim 1, it is characterized in that above-mentioned step 3) cultivation of tissue engineering human corneal epithelium is with in the culture plate insert that posts epithelial layer digestion amnion carrying support, add gently 0.1～0.2 milliliter of above-mentioned ready people's corneal epithelial cell suspension, cell suspension is dispersed in the culture plate insert, do not add culture fluid in the 6 well culture plate holes, put 5% CO
2Cultivate 20～24 hours after cell attaches on the carrier bracket fully for 37 ℃ in the incubator, suck the old culture fluid in the culture plate insert, culture plate insert 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 tissue engineering human corneal epithelium for 37 ℃ in the incubator, shook gently 6 well culture plates every 12 hours, change liquid once every 24～36 hours, grow up to the tissue engineering human corneal epithelium that is reconstruction after 6～8 layers at carrier bracket until people's 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 |
| CN104877958B (en) * | 2014-02-28 | 2018-01-02 | 上海尚瑞生物医药科技有限公司 | A kind of cell line for keeping corneal epithelium shape cellular layer transparency |
| CN106591216B (en) * | 2016-12-12 | 2021-07-27 | 深圳市眼科医院 | Human normal corneal epithelial cell and application thereof |
| CN106955372B (en) * | 2017-04-12 | 2020-08-14 | 山东省眼科研究所 | Method for constructing tissue engineering corneal endothelium |
| CN107129966B (en) * | 2017-06-18 | 2023-03-03 | 广东博溪生物科技有限公司 | Serum-containing corneal epithelial cell culture solution |
| 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 |
| CN107164309B (en) * | 2017-06-18 | 2023-06-16 | 广东博溪生物科技有限公司 | 3D model constructed outside serum-containing culture liquid and construction method thereof |
| 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 |
| 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 |
| CN112941014A (en) * | 2021-02-22 | 2021-06-11 | 深圳艾尼尔角膜工程有限公司 | Method for improving adhesion of tissue engineering anterior lamellar corneal epithelial cells |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1426289A (en) * | 2000-04-27 | 2003-06-25 | 雷·瑞芳·蔡 | Method for expansion of epithelial stem cells |
| CN1749393A (en) * | 2005-07-27 | 2006-03-22 | 中国海洋大学 | Constructing method for human corneal endothelium cell system |
| CN101508971A (en) * | 2009-03-23 | 2009-08-19 | 中国海洋大学 | Tissue engineering reconstruction method for human corneal endothelium |
-
2011
- 2011-02-16 CN CN 201110039099 patent/CN102166375B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1426289A (en) * | 2000-04-27 | 2003-06-25 | 雷·瑞芳·蔡 | Method for expansion of epithelial stem cells |
| CN1749393A (en) * | 2005-07-27 | 2006-03-22 | 中国海洋大学 | Constructing method for human corneal endothelium cell system |
| CN101508971A (en) * | 2009-03-23 | 2009-08-19 | 中国海洋大学 | Tissue engineering reconstruction method for human corneal endothelium |
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