CN1635115A - Human corneal limbal stem cell tissue engineering product nourished by fibroblast and preparing process thereof - Google Patents
Human corneal limbal stem cell tissue engineering product nourished by fibroblast and preparing process thereof Download PDFInfo
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- CN1635115A CN1635115A CNA2004100193424A CN200410019342A CN1635115A CN 1635115 A CN1635115 A CN 1635115A CN A2004100193424 A CNA2004100193424 A CN A2004100193424A CN 200410019342 A CN200410019342 A CN 200410019342A CN 1635115 A CN1635115 A CN 1635115A
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Abstract
A human corneal limbal stem cell-amnion tissue engineering complex using human corneal limbal fibroblasts as the trophoblast is characterized in that: it is close to the human corneal epithelium form in histology form, and is analogous to the human corneal epithelium in immunology phenotype, its ultrastructure shows that the connection between the cells grows mature and the connection between the epithelia and the amnion carrier is a close conglutination. The culture method includes the following steps: (1) preparing the amnion for culturing the human corneal limbal stem cell in vitro; (2) preparing the human corneal limbal fibroblast trophoblast; (3) preparing the human corneal epithelia suspension; (4) culturing the human corneal limbal stem cell-amnion complex. The human corneal limbal stem cell-amnion complex has analogous phenotype to the human corneal epithelium, and provides a safe and effective biology tissue engineering product to treat corneal limbal stem cell defective eye diseases.
Description
Affiliated field:
The present invention relates to be used for the treatment of product of eye surface diseases and preparation method thereof in a kind of bioengineering field, be particularly related to a kind of can the damage by the repairing corneal limbal stem cell, be used for human limbal stem cell-amnion tissue engineering complex body of eye table reconstruction and preparation method thereof.
Background technology:
In the treatment of eye surface diseases, most important breakthrough is the function and the localized understanding of corneal limbal stem cell in recent years.The experimental results shows that the part cell of corneal limbus stratum basale has kept not differentiation characteristic, " stem cell " for corneal epithelial cell, these cells have following feature: (1) has multiplication potentiality, (2) can self, (3) can produce the functional cell that a large amount of ends eventually break up, (4) can the repair tissue damage.The eye surface diseases that with the limbal stem cell deficiency is feature is because the limbal stem cell damage causes the corneal epithelium rectification of defects, finally cause the corneal stroma scarring, its extent of disease only relates to an eye table epithelium, but grievous injury visual function often is serious blinding illness in eye.For this type of patient, its cornea rear portion matrix and endothelium and eyeball other organize all very healthyly, only needs to solve because the eye table that stem cell defect causes is rebuild an obstacle and can improve patient's visual function.Yet traditional operation method is as: tarsorrhaphy, all have many limitation from the transplanting of body conjunctiva, oral mucosa transplanting, amnion transplantation, limbal stem cell transplantation.New treatment technology and treatment product are demanded in the healing of serious limbal stem cell deficiency eye surface diseases urgently.
At present, along with developing rapidly of tissue engineering technique, using for reference on the basis of chrotoplast culture system traditionally, introducing the carrier of amnion as the limbal stem cell vitro culture, the limbal stem cell transplantation of cultivation has obtained initial success in clinical application.In this technology, reasonably the vitro culture system is to obtain the most important prerequisite of good result, wherein the most important thing is to guarantee in culture system, to contain the stem cell of some amount, and used culture system can be kept the initiating cell characteristic of limbal stem cell preferably.In addition, need to add the 3T3 inoblast in the epithelial cell culture system and make trophoderm, but, contain mouse source property heterogenous gene and reverse transcription virus gene, for clinical application has brought potential threat because the 3T3 inoblast is the immortal cell line of mouse source property.Therefore set up the vitro culture system of rational human limbal stem cell-amnion complex body, make it to have validity and biological safety, then have the clinical meaning of particularly important.
Summary of the invention:
Purpose of the present invention just is to overcome above-mentioned the deficiencies in the prior art, and the human limbal stem cell tissue engineering product and the preparation method that provide a kind of human fibroblasts to nourish, this amnion complex body has validity and biological safety.
Technical scheme of the present invention is: the human limbal stem cell tissue engineering product that a kind of human fibroblasts nourishes, it is characterized in that: on the histology form near people's corneal epithelium form, similar with people's limbal epithelium on immunological phenotype, being connected between its ultrastructure shows cell and the cell reached maturity, and is adhesion closely between epithelial cell and the amnion carrier.
The histology form of above-mentioned human limbal stem cell-amnion tissue engineering complex body is: the multiple layer people corneal limbal epithelial cell of growing on the amnion carrier is divided into the multilayer structure of 4～6 confluent monolayer cells, and basal cell is column.
The phenotype of above-mentioned human limbal stem cell-amnion tissue engineering complex body is: multiple layer corneal limbal epithelial cell cells of superficial layer Cytokeratin (CK3) positive of growing on amnion, the basal layer cell feminine gender, most of cells of superficial layer Δ Np63 positive, Np63 is negative for the basal layer cell Δ; Basal layer cell is seldom expressed the slit and is connected albumen (Cx43), only at cells of superficial layer a small amount of expression is arranged.
The ultrastructure of above-mentioned human limbal stem cell-amnion tissue engineering complex body is: corneal limbal epithelial cell grows fine, and easily sees epithelium mitotic division phase, and nucleus is big; There is abundant desmosome to connect between the cell; The basis pontis endochylema of basis pontis cell has a large amount of projections to go deep into substrate plate and reticular lamina, does not have the space between cell and the basilar membrane, and more complete by the substrate plate growth that emiocytosis produces, reticular lamina thickens; And merge with collegen filament.
The preparation method of the human limbal stem cell tissue engineering product that above-mentioned human fibroblasts nourishes, it is characterized in that: finish: the 1. preparation of the vitro culture carrier-amnion of human limbal stem cell: separate and the preservation amnion according to the following step, take out frozen amnion during use, 0.1% EDTA and 0.1% trypsinase successively act on the removal amniotic epithelial cells; 2. the trophoblastic preparation of people's corneal limbus inoblast: isolating fetal corneal limbus stroma, cultivator corneal limbus inoblast is with 4 μ g/ml ametycin handler corneal limbus inoblasts; 3. the preparation of people's corneal limbal epithelial cell suspension: may further comprise the steps: separation of human corneal limbus tissue, with the Dispase II separation of human corneal limbal epithelial cell of 1.2U/ml, trypsinase with 0.1% and machinery suction preparation single cell suspension; 4. the cultivation of human limbal stem cell-amnion complex body: above-mentioned treated amnion put into is covered with the fibroblastic culture plate of people's corneal limbus that ametycin was handled, and with people's corneal limbal epithelial cell suspension with 5 * 10
3/ cm
2Density be inoculated on the amnion, through adopting the air-lifting technology behind cultivation 14-21 days of nutrient solution, cell was placed gas-liquid interface 7 days.
Above-mentioned amnion is from the caesarean delivered fetal membrane of healthy pregnant women.
Above-mentioned corneal limbus stroma is from the eyeball of fetus.
Above-mentioned people's corneal limbal epithelial cell is from the healthy eyeball of fresh cadaver eye, patient self eyeball or relatives.
Above-mentioned nutrient solution adopts 60%DMEM and 30%Ham ' s F12 nutrient solution, in add 10% foetal calf serum, 5 μ g/ml Regular Insulin, 0.18mM VITAMIN B4,0.1nM Toxins,exo-, cholera, 0.4 μ g/ml hydrocortisone, 2nM triiodothyronine, 4mM glutamine, 50IU/ml hydrogen Streptomycin sulphate and 10ng/ml Urogastron.
Advantage of the present invention is: 1, the present invention introduces the vitro culture carrier of amnion as limbal stem cell in the preparation method of human limbal stem cell tissue engineering product, make product have biocompatibility, and can bring into play the superiority that amnion is used for the eye surface diseases treatment.Adopt the cell suspension method to cultivate, can guarantee in culture system, to contain the stem cell of some amount.Adopt the air-lifeing technology to promote cytodifferentiation, and utilize people's corneal limbus inoblast to be trophoderm, can bring into play trophoblastic effect effectively, help to keep the feature of limbal stem cell initiating cell, can remove the potential threat of the heterology gene that mouse source property 3T3 inoblast may bring again, make product have security.2, the product human limbal stem cell tissue engineering product among the present invention has the phenotype similar with people's limbal epithelium, connect between the ultrastructure shows cell and reach maturity, set up tight adhesion between cell and the amnion, thereby made product in clinical application, have validity.
The present invention has the purposes of particularly important:
1. according to technology of preparing provided by the invention, can provide suitable carrier for the vitro culture of human limbal stem cell.
2. according to the trophoblastic technology of preparing of people's corneal limbus inoblast provided by the invention, can set up people's corneal limbus fibroblast, be used for trophoderm as the human limbal stem cell vitro culture.
3. according to the technology of preparing of people's corneal limbal epithelial cell suspension provided by the invention, can obtain to comprise human limbal stem cell at interior pure epithelial cell suspension.
4. according to the technology of preparing of human limbal stem cell tissue engineering product provided by the invention, can be so that limbal stem cell amplification in a large number on amnion, and be divided into the stratified epithelium spline structure, be used to prepare the human limbal stem cell tissue engineering product.
5. can be used for the treatment of with the limbal stem cell deficiency according to the human limbal stem cell-amnion tissue engineering complex body of technology of preparing of the present invention preparation is the eye surface diseases of feature.
Description of drawings:
Fig. 1 is the histological structure figure of human limbal stem cell tissue engineering product, wherein a: the monolayer cell that forms on amnion is comparatively flat; B: the multiple layer people corneal limbal epithelial cell of growing on amnion is divided into the multilayer structure of 4～6 confluent monolayer cells, and basal cell is column.
Fig. 2 is the phenotypic map of normal cornea and corneal limbal epithelial cell, wherein a: the limbal epithelium cells of superficial layer is expressed CK3, and basal layer cell is not expressed; B: the corneal epithelium holostrome is expressed CK3; C: corneal limbus basal layer cell Δ Np63 expresses positive; D: between peripheral cornea basal layer cell or the expression of Δ Np63 arranged; E: the central cornea epithelium is not expressed; F: Cx43 is less for corneal limbus basis pontis cell expressing; G: the basis pontis cell great expression Cx43 of corneal epithelium.
Fig. 3 is the phenotypic map of human limbal stem cell tissue engineering product, wherein a: the individual layer corneal limbal epithelial cell CK3 positive rate 14.3% of growing on amnion; B: the individual layer corneal limbal epithelial cell Δ Np63 positive rate of growing on amnion is 87.5%; C: the individual layer corneal limbal epithelial cell of growing on amnion only has the expression of Cx43 by chance; D: the multiple layer corneal limbal epithelial cell cells of superficial layer CK3 positive of on amnion, growing, basal layer cell feminine gender; E: the multiple layer corneal limbal epithelial cell basal layer cell Δ Np63 positive of on amnion, growing, the cells of superficial layer major part is negative; F: the multiple layer corneal limbal epithelial cell basal layer cell of growing on amnion is seldom expressed Cx43, only at cells of superficial layer a small amount of expression is arranged.
Fig. 4 is the ultra micro Electronic Speculum structure iron (cell grows to the form of individual layer on amnion) of human limbal stem cell tissue engineering product, and wherein: a: corneal limbal epithelial cell nuclear is big, and chromatin is abundant, and big kernel is arranged; B: iuntercellular can be seen desmosome structure (arrow) and closely be connected (arrowhead); C: the endochylema projection of basis pontis stretches into the substrate plate (arrow) of interruption, accidental anchor shape fiber (arrowhead); D: there is the protein calmness (arrow) that concentrates part endochylema prominence, and the epithelial cell secretion forms the substrate plate (arrowhead) that is interrupted, reticular lamina imperfect (star), the amnion stroma that it is formed for collegen filament down.
Fig. 5 is the ultra micro Electronic Speculum structure iron (cell grows to the form of multiple layer on amnion) of human limbal stem cell tissue engineering product, and wherein: a: corneal limbal epithelial cell grows fine, and easily sees epithelium mitotic division phase (arrowhead), and nucleus is big; B: having abundant desmosome to connect (arrowhead), is many far beyond monolayer cell; C: the nipple that the basis pontis endochylema of basis pontis cell gos deep into substrate plate and reticular lamina is many and big, does not have tangible space between cell and the basilar membrane substantially, and substrate plate is grown more complete (arrow), and reticular lamina thickens (star); D: substrate plate is grown complete, and reticular lamina thickens, and merges (star) with collegen filament.
Embodiment:
A kind of cultural method that is used for the treatment of the human limbal stem cell-amnion tissue engineering complex body of limbal stem cell deficiency eye surface diseases is finished according to the following step: 1, the preparation of amnion: get healthy pregnant women and cut open palace product fetal membrane, separate amnion and chorion along the physiology gap, be tiled in the amnion epithelial surface on the nitrocellulose membrane up, put into by 50% DMEM (Gibco, USA) and in the preservation liquid formed of 50% glycerine, put-80 ℃ and preserve more than three months.Take out frozen amnion from refrigerator during use, put in 37 ℃ of water baths and melt.EDTA effect with 0.1% 30 minutes was used 0.1% trypsin acting 30 seconds again, scraped off amniotic epithelial cells gently with the cell sleaker.With the amnion epithelial surface be tiled in up culture insert (Millipore, standby in USA).2, the trophoblastic preparation of people's corneal limbus inoblast: microscopically is got fetus cornea edge tissue, Dispase II (Millipore with 1.2U/ml, USA) epithelium is removed in the effect back, to go up subcutaneous reticular tissue inserts in the 25cm culturing bottle, adding DMEM (10% foetal calf serum, 100U/ml mycillin) nutrient solution cultivates.Treat people's corneal limbus inoblast be paved with about 1/2 bottle at the bottom of after go down to posterity.With the nearly warm people's corneal limbus inoblast of the ametycin effect of 4 μ g/ml growth 2 hours, the cell cryopreservation after the effect was standby.Time spent is with 2 * 10
4/ cm
2Density inoculate six orifice plates, standby after 24 hours.3, the preparation of people's corneal limbal epithelial cell suspension: fetch the fresh cadaver eyeball from eye bank, microscopically is got the lamellar cornea edge tissue of thick about 100 μ m, uses the Dispase II of 1.2U/ml to act on 2 hours for 37 ℃.Microscopically isolated cornea edge epithelium was inserted epithelium in 0.1% the trypsin solution effect 4 minutes.Make single cell suspension with No. 4 syringe needle suction of cells.4, the cultivation of human limbal stem cell-amnion complex body: the cultivation of human limbal stem cell-amnion complex body: the culture insert that will be covered with amnion puts into and is covered with fibroblastic 6 orifice plates of people's corneal limbus that ametycin was handled, and people's corneal limbal epithelial cell suspension is with 5 * 10
3/ cm
2Density be inoculated on the amnion, nutrient solution adopts DMEM and Ham ' s F12 (Gibco, USA) nutrient solution (mixing at 2: 1), in add foetal calf serum (10%) (Hyclone, USA), Regular Insulin (5 μ g/ml) (Sigma, USA), VITAMIN B4 (0.18mM) (Sigma, USA), Toxins,exo-, cholera (0.1nM) (Sigma, USA), hydrocortisone (0.4 μ g/ml) (Sigma, USA), triiodothyronine (2nM) (Sigma, USA), glutamine (4mM), hydrogen Streptomycin sulphate (50IU/ml), put 37 ℃, 5% CO
2Cultivate in the incubator.Inoculate after 3 days, begin in the nutrient solution to add Urogastron (10ng/ml) (PeproTech, UAS).Changed liquid once in per afterwards two days.Cultivate after 21 days, adopt the air-lifting technology, cell was placed gas-liquid interface 7 days.
Adopt the experiment of the human limbal stem cell-amnion complex body of aforesaid method cultivation to detect:
1, histological observation: human limbal stem cell-amnion complex body of cultivating 14 days and cultivating 28 days is made frozen section, HE dyeing.
2, the expression of Δ Np63 and CK3 detects in immunohistochemistry technology: after 14 days cells of cultivation form individual layer, directly be laid on human limbal stem cell-amnion complex body on the slide glass, normal people's cornea tissue and cultivation were divided into the human limbal stem cell-amnion complex body of stratified epithelium in 28 days and make frozen section, adopt the detection of row Δ Np63 of immunohistochemistry technology and CK3, respectively with mouse-anti CK3 monoclonal antibody (ICN, USA) and mouse-anti Δ Np63 monoclonal antibody (BD, USA) be one anti-, adopt SAB test kit (middle mountain company), the DAB colour developing, Hematorylin is redyed.
3, immunofluorescence technique detects the expression of Cx43: individual layer human limbal stem cell-amnion complex body directly is laid on the slide glass, normal people's cornea tissue and multiple layer human limbal stem cell-amnion complex body are made frozen section, adopt immunofluorescence technique to detect the expression of Cx43.(CHEMICON is one anti-USA), and (Sigma is two anti-USA) to the sheep anti-mouse igg of FITC mark, and (Sigma USA) redyes nuclear, fluorescence microscope with the Hoechst fluorescence dye with mouse-anti Cx43 monoclonal antibody.
4, transmission electron microscope observing: get corneal limbal epithelial cell-amnion complex body of cultivating 14 days and 28 days, fix, fix behind 1% the perosmic anhydride with glutaraldehyde/Paraformaldehyde 96 of 2.5%, after the rising gradient alcohol dehydration, the embedding of usefulness Epon812 Resins, epoxy.Behind LKB-V ultramicrotome semithin section (1 μ) location, carry out ultrathin section(ing).Acetic acid uranium, lead citrate dyeing, the JEOL-100CX transmission electron microscope observing.
Detected result is as follows:
1) the histology form of human limbal stem cell-amnion complex body
Histological section shows that the monolayer cell that forms is comparatively flat, and is tight with amniotic adhesion on amnion.After 28 days, epithelial cell is divided into the multilayer structure of 4～6 confluent monolayer cells, and basal layer cell is near column.(accompanying drawing 1)
2) phenotype of human limbal stem cell-amnion complex body
Normal people's limbal epithelium cells of superficial layer is expressed CK3, and basal layer cell is not expressed, the corneal epithelium holostrome CK3 positive; Corneal limbus basal layer cell Δ Np63 expresses strong positive, and between peripheral cornea basal layer cell or the expression of Δ Np63 is arranged, the central cornea epithelium is not expressed Δ Np63; Cx43 is less for corneal limbus basis pontis cell expressing, and the basis pontis cell great expression Cx43 of corneal epithelium.(accompanying drawing 2)
When people's corneal limbal epithelial cell of cultivating grew up to individual layer, Δ Np63 The positive expression rate was 87.5%, and the cell of express cell keratoprotein 3 has only 14.3%, and immunofluorescence shows an only a few cell expressing Cx43.The basal layer cell Δ Np63 that is divided into people's corneal limbal epithelial cell of multiple layer expresses strong positive, does not express CK3 and Cx43, the above cell CK3 of the stratum basale positive.Above result shows that in our culture system the initiating cell characteristic of limbal stem cell can be kept in whole culturing process.(accompanying drawing 3)
3) human limbal stem cell-amnion complex body ultrastructure
Cultivate after 14 days, the individual layer corneal limbal epithelial cell nuclear of as seen growing on amnion under the Electronic Speculum is big, and chromatin is abundant, and big kernel is arranged, and iuntercellular has complete desmosome and closely is connected to form.The corneal limbal epithelial cell basis pontis has the endochylema projection to stretch into the substrate plate and the reticular lamina of interruption, and the endochylema of basis pontis and substrate plate leave the space, many places, in part endochylema prominence the protein calmness that concentrates is arranged, accidental oblique or slightly vertical anchor shape fiber.The epithelial cell secretion forms the substrate plate that is interrupted, and reticular lamina is not too complete.(accompanying drawing 4)
Cultivate after 28 days, corneal limbal epithelial cell grows fine, and easily sees epithelium mitotic division phase, and nucleus is big, and surface epithelial cell microvillus hyperplasia is complicated interdigitate.Having abundant tight connection to be connected with desmosome, is many far beyond monolayer cell.The nipple that the basis pontis endochylema of basis pontis cell gos deep into substrate plate and reticular lamina is many and big, does not have tangible space between cell and the basilar membrane substantially; Substrate plate is grown ripe, more complete more; Reticular lamina thickens, and is more complete, and local collegen filament with amnion merge.(accompanying drawing 5)
Claims (9)
1, a kind of human limbal stem cell tissue engineering product of human fibroblasts's nourishing, it is characterized in that: on the histology form near people's corneal epithelium form, similar with people's limbal epithelium on immunological phenotype, being connected between its ultrastructure shows cell and the cell reached maturity, and is adhesion closely between epithelial cell and the amnion carrier.
2, the human limbal stem cell tissue engineering product of human fibroblasts's nourishing according to claim 1, it is characterized in that: the histology form of above-mentioned human limbal stem cell tissue engineering product is: the multiple layer people corneal limbal epithelial cell of growing on the amnion carrier is divided into the multilayer structure of 4～6 confluent monolayer cells, and basal cell is column.
3, the human limbal stem cell tissue engineering product of human fibroblasts's nourishing according to claim 1, it is characterized in that: the phenotype of above-mentioned human limbal stem cell tissue products is: multiple layer corneal limbal epithelial cell cells of superficial layer Cytokeratin (CK3) positive of growing on amnion, the basal layer cell feminine gender, most of cells of superficial layer Δ Np63 positive, Np63 is negative for the basal layer cell Δ; Basal layer cell is seldom expressed the slit and is connected albumen (Cx43), only at cells of superficial layer a small amount of expression is arranged.
4, the human limbal stem cell tissue engineering product of human fibroblasts's nourishing according to claim 1, it is characterized in that: the ultrastructure of above-mentioned human limbal stem cell tissue engineering product is: corneal limbal epithelial cell grows fine, easily see epithelium mitotic division phase, nucleus is big; There is abundant desmosome to connect between the cell; The basis pontis endochylema of basis pontis cell has a large amount of projections to go deep into substrate plate and reticular lamina, does not have the space between cell and the basilar membrane, and more complete by the substrate plate growth that emiocytosis produces, reticular lamina thickens; And merge with collegen filament.
5, a kind of preparation method of human limbal stem cell tissue engineering product of human fibroblasts's nourishing according to claim 1, it is characterized in that: finish: the 1. preparation of the vitro culture carrier-amnion of human limbal stem cell: separate and the preservation amnion according to the following step, take out frozen amnion during use, 0.1% EDTA and 0.1% trypsinase successively act on the removal amniotic epithelial cells; 2. the trophoblastic preparation of people's corneal limbus inoblast: isolating fetal corneal limbus stroma, cultivator corneal limbus inoblast is with 4 μ g/ml ametycin handler corneal limbus inoblasts; 3. the preparation of people's corneal limbal epithelial cell suspension: may further comprise the steps: separation of human corneal limbus tissue, with the DispaseII separation of human corneal limbal epithelial cell of 1.2U/ml, trypsinase with 0.1% and machinery suction preparation single cell suspension; 4. the cultivation of human limbal stem cell-amnion complex body: above-mentioned treated amnion put into is covered with the fibroblastic culture plate of people's corneal limbus that ametycin was handled, and with people's corneal limbal epithelial cell suspension with 5 * 10
3/ cm
2Density be inoculated on the amnion, through adopting the air-lifting technology behind cultivation 14-21 days of nutrient solution, cell was placed gas-liquid interface 7 days.
6, the preparation method of the human limbal stem cell tissue engineering product of human fibroblasts's nourishing according to claim 5, it is characterized in that: above-mentioned amnion is from the caesarean delivered fetal membrane of healthy pregnant women.
7, the preparation method of the human limbal stem cell tissue engineering product of human fibroblasts's nourishing according to claim 5, it is characterized in that: above-mentioned corneal limbus stroma is from the eyeball of fetus.
8, the preparation method of the human limbal stem cell tissue engineering product of human fibroblasts's nourishing according to claim 5 is characterized in that: above-mentioned people's corneal limbal epithelial cell is from the healthy eyeball of fresh cadaver eye, patient self eyeball or relatives.
9, the preparation method of the human limbal stem cell tissue engineering product of human fibroblasts's nourishing according to claim 5, it is characterized in that: above-mentioned nutrient solution adopts 60%DMEM and 30%Ham ' s F12 nutrient solution, in add 10% foetal calf serum, 5 μ g/ml Regular Insulin, 0.18mM VITAMIN B4,0.1nM Toxins,exo-, cholera, 0.4 μ g/ml hydrocortisone, 2nM triiodothyronine, 4mM glutamine, 50IU/ml hydrogen Streptomycin sulphate and 10ng/ml Urogastron.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100487113C (en) * | 2006-12-11 | 2009-05-13 | 中国农业科学院北京畜牧兽医研究所 | Liancheng white duck embryo fibroblast and culture method thereof |
| CN1916166B (en) * | 2006-09-08 | 2010-08-11 | 北京赛尔泰和生物医药科技有限公司 | Method for preparing epithelium of autologous cornea |
| CN100998526B (en) * | 2006-01-10 | 2011-10-05 | 上海组织工程研究与开发中心 | Corneal graft |
| CN103816571A (en) * | 2014-02-24 | 2014-05-28 | 钟春燕 | Preparation method of bacterial cellulose reinforced amnion composite material used for corneal reconstruction |
| CN103893825A (en) * | 2014-02-24 | 2014-07-02 | 钟春燕 | Method for preparing bacterial cellulose compounded amnion extracellular matrix material containing collagen |
| 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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| CN106916787A (en) * | 2017-02-15 | 2017-07-04 | 中山大学中山眼科中心 | A kind of limbal stem cell culture medium and its cultural method |
| CN107118552A (en) * | 2017-05-02 | 2017-09-01 | 中山大学中山眼科中心 | A kind of composite membrane based on gelatin and amino acid and the method that limbal stem cell is cultivated on film |
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| CN100998526B (en) * | 2006-01-10 | 2011-10-05 | 上海组织工程研究与开发中心 | Corneal graft |
| CN1916166B (en) * | 2006-09-08 | 2010-08-11 | 北京赛尔泰和生物医药科技有限公司 | Method for preparing epithelium of autologous cornea |
| CN100487113C (en) * | 2006-12-11 | 2009-05-13 | 中国农业科学院北京畜牧兽医研究所 | Liancheng white duck embryo fibroblast and culture method thereof |
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| CN103893825A (en) * | 2014-02-24 | 2014-07-02 | 钟春燕 | Method for preparing bacterial cellulose compounded amnion extracellular matrix material containing collagen |
| 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 |
| CN105833738A (en) * | 2016-05-05 | 2016-08-10 | 北京科技大学 | Nanocellulose/soybean protein composite filter material and preparation method and purposes thereof |
| CN106916787A (en) * | 2017-02-15 | 2017-07-04 | 中山大学中山眼科中心 | A kind of limbal stem cell culture medium and its cultural method |
| CN107118552A (en) * | 2017-05-02 | 2017-09-01 | 中山大学中山眼科中心 | A kind of composite membrane based on gelatin and amino acid and the method that limbal stem cell is cultivated on film |
| CN109321527A (en) * | 2018-10-10 | 2019-02-12 | 中国海洋大学 | The extracorporeal culturing method of limbal stem cell stability |
| CN112126626A (en) * | 2020-09-30 | 2020-12-25 | 广东康盾生物工程技术有限公司 | Limbal stem cell culture medium and culture method |
| CN112126626B (en) * | 2020-09-30 | 2023-04-07 | 广东康盾创新产业集团股份公司 | Limbal stem cell culture medium and culture method |
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