CN101590292A - The preparation method of acellular conjunctiva matrix and application - Google Patents
The preparation method of acellular conjunctiva matrix and application Download PDFInfo
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- CN101590292A CN101590292A CNA2009100163436A CN200910016343A CN101590292A CN 101590292 A CN101590292 A CN 101590292A CN A2009100163436 A CNA2009100163436 A CN A2009100163436A CN 200910016343 A CN200910016343 A CN 200910016343A CN 101590292 A CN101590292 A CN 101590292A
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Abstract
The present invention relates to a kind of preparation method and application of the acellular conjunctiva matrix as the tissue engineering comea timbering material.The present invention removes the cell composition in the bulbar conjunctiva first, prepare acellular conjunctiva matrix, and with it as the tissue engineering comea support, rabbit corneal epithelium or endotheliocyte can form the good cell monolayer on support, construct tissue engineering comea epithelium or endothelium, and successfully transplant on rabbit animal model eye.The constructed tissue engineering comea degradation time of the present invention is longer, and lagophthalmos does not have obvious immunological rejection to it, and the corneal limbal stem cell lacks or the endothelial cell function to lose compensatory therapeutic effect all remarkable.Preparation method of the present invention is simple; Good biocompatibility; Antigenicity is very low; Seed cell is grown thereon easily and is bred; It is slower to degrade; In incubation and after transplanting, remain its transparency; And the bulbar conjunctiva wide material sources have wide application and development prospect.
Description
Technical field
The present invention relates to a kind of preparation method and application of the acellular conjunctiva matrix as the tissue engineering comea timbering material.
Background technology
Corneal blindness is the second largest blinding oculopathy of China, and corneal transplantation is unique effective treatment means of recovering lost eyesight, but the scarcity of cornea donor material has a strong impact on carrying out of corneal transplantation.In recent years, along with the development of tissue engineering technique, tissue engineering comea will be opened up new approach for the treatment of keratopathy.Three big key elements of organizational project comprise seed cell, timbering material and microenvironment.Timbering material is the place of cells survival and functionating, can grow by cell guiding simultaneously, is determining the form of constructed tissue.Have only and selected good timbering material, seed cell could be grown better thereon, be bred.Ideal timbering material should meet the following conditions: (1) good biocompatibility, and can keep three dimensions and not disturb other physiological process with promoting normal cell growth and differentiation; (2) there is not bad tissue reaction; (3) material is simple, and can reinvent by different shapes and size; (4) implant after, material can be degraded or absorb.Because the particularity of cornea tissue itself, the tissue engineering comea timbering material also should have certain transparency and anti-tractive ability simultaneously.Do not find also that up to now a kind of timbering material possesses above-mentioned these features fully.
Collagen in existing tissue engineering comea timbering material such as the natural macromolecular material, gelatin etc., good biocompatibility, but mechanics poor performance, degraded shortcoming such as too fast are all arranged; The macromolecular material of synthetic such as polyglycolic acid, polylactic-co-glycolic acid etc., acid degradation product then easily are deposited in the part, are unfavorable for the cell growth; Bio-derived material is as " removing epithelium " amniotic membrane or bioamnion etc., and degradation in vivo is very fast, can not satisfy the needs of tissue engineering comea.Ideal timbering material is the research focus of tissue engineering comea always, also is the bottleneck problem of restriction tissue engineering comea development, is very important so seek a kind of ideal timbering material.
Summary of the invention
The preparation method that the purpose of this invention is to provide a kind of acellular conjunctiva matrix, and be applied to make up tissue engineering comea epithelium or endothelium as the tissue engineering comea timbering material, to remedy the deficiency of existing tissue engineering comea timbering material.
The present invention is a material with the bulbar conjunctiva of people in the bulbar conjunctiva of animal (mainly being the pig in slaughterhouse) or the eye bank, by volume fraction is that 0.2% glutaraldehyde solution is crosslinked, volume fraction is 1%tritonX-100, the quality volume fraction is that 0.25% pancreatin and 0.02%EDTA take off cell, processes such as disinfection by ultraviolet light prepare acellular conjunctiva matrix, and be that with the acellular conjunctiva matrix tissue engineering comea timbering material cultivates people's corneal epithelium or endotheliocyte, people's corneal epithelium or endotheliocyte form close-connected cell monolayer on acellular conjunctiva matrix, construct tissue engineering comea epithelium or endothelium, and can be used for successfully that human limbal stem cell lacks or treatment that the endothelial cell function is lost keratopathy such as compensatory.
The preparation method of acellular conjunctiva matrix of the present invention:
1. get the fresh pig eyeball in slaughterhouse, after washing repeatedly with PBS, cut off ball week tissue, cut bulbar conjunctiva again, place the PBS that contains tobramycin 1000U/ml to soak 30 minutes; If take the bulbar conjunctiva of people in the eye bank, directly place the PBS that contains tobramycin 1000U/ml to soak 30 minutes;
2. in super-clean bench, the Tenon capsule of above-mentioned bulbar conjunctiva is wiped out totally, and with PBS flushing as far as possible, be soaked in volume fraction and be in 0.2% glutaraldehyde solution 5 minutes, to carry out crosslinked protection in the optical stereo microscopically;
3. reuse PBS flushing is to remove residual glutaraldehyde solution;
4. then bulbar conjunctiva being taken off cell and handle, is 1% tritonX-100 processing 24 hours with volume fraction on 37 ℃ of constant temperature shaking tables, then at 37 ℃, and co
2Concentration is in 5% the incubator, the quality volume fraction is the EDTA effect 2 hours of 0.25% pancreatin and 0.02%, with PBS concussion flushing 10 minutes, is placed on aeration-drying in the super-clean bench, bulbar conjunctiva two sides ultraviolet disinfection each 30 minutes promptly obtains acellular conjunctiva matrix.
Acellular conjunctiva matrix of the present invention is applied to make up tissue engineering comea epithelium or endothelium as the tissue engineering comea timbering material.
Acellular conjunctiva matrix is as the application process of tissue engineering comea timbering material:
1. will wash with PBS before use as the acellular conjunctiva matrix of tissue engineering comea timbering material, be put in then in 24 orifice plates, in aseptic super-clean bench, dry, it firmly is attached on 24 orifice plates;
2. will make cell suspension as the people's corneal epithelium or the endotheliocyte of seed cell again, the DMEM/F12 culture fluid that use contains 10%FBS is seeded in above-mentioned 24 orifice plates that post acellular conjunctiva matrix, changed culture fluid in 3 days, close-connected corneal epithelium to be formed or endothelial cell monolayer promptly are built into tissue engineering comea epithelium or endothelium;
3. at last above-mentioned tissue engineering comea epithelium or endothelium are transplanted to respectively on the human eye that limbal stem cell lacks or the mistake of endothelial cell function is compensatory.
With the tissue engineering comea timbering material of acellular conjunctiva matrix as a kind of practicality, its preparation method is simple first in the present invention, and good biocompatibility is transparent and have certain refractive power and certain elasticity and an anti-tractive ability; Seed cell is growth and propagation on acellular conjunctiva matrix easily; Antigenicity is very low; It is slower to degrade, and helps seed cell propagation and brings into play its function; In incubation and after transplanting, remain its transparency; And the bulbar conjunctiva wide material sources have wide application and development prospect.
The specific embodiment
The present invention is with the bulbar conjunctiva of animal (mainly being the pig in slaughterhouse), or people's bulbar conjunctiva is a raw material in the eye bank, prepare acellular conjunctiva matrix, and with acellular conjunctiva matrix as the tissue engineering comea timbering material, construct the tissue engineering comea epithelium or the endothelium that can be used for transplanting.
The preparation method of acellular conjunctiva matrix:
1. get the fresh pig eyeball in slaughterhouse, after washing repeatedly with PBS, cut off ball week tissue, cut bulbar conjunctiva again, place the PBS that contains tobramycin 1000U/ml to soak 30 minutes;
2. in super-clean bench, the Tenon capsule of above-mentioned bulbar conjunctiva is wiped out totally, and with PBS flushing as far as possible, be put in volume fraction and be in 0.2% glutaraldehyde solution and to carry out crosslinked protection in 5 minutes in optics body condition microscopically;
3. reuse PBS flushing is to remove residual glutaraldehyde solution;
4. then bulbar conjunctiva being taken off cell and handle, is 1%tritonX-100 effect 24 hours with volume fraction on 37 ℃ of constant temperature shaking tables, then at 37 ℃, and co
2Concentration is in 5% the incubator, and the quality volume fraction is 0.25% pancreatin and 0.02%EDTA effect 2 hours, with PBS concussion flushing 10 minutes, is placed on aeration-drying in the super-clean bench, and bulbar conjunctiva two sides ultraviolet disinfection each 30 minutes obtains acellular conjunctiva matrix.
The bulbar conjunctiva of other animal preparation in the also available slaughterhouse of the inventive method, method is the same.
If take the bulbar conjunctiva of people in the eye bank, in step 1, directly place the PBS that contains tobramycin 1000U/ml to soak 30 minutes, step is identical thereafter.
People's tissue engineering comea epithelium or endothelium be applied to clinical before, done a large amount of zooperies, it is clinical to prove that it can be applied to fully.
People's tissue engineering comea epithelium that acellular conjunctiva matrix constructs as the tissue engineering comea timbering material or endothelium are applied to clinical, are transplanted on the human eye, and concrete application process is as follows:
(1) with the PBS flushing, is put in then in 24 orifice plates before acellular conjunctiva matrix uses, in aseptic super-clean bench, dries, it firmly is attached on 24 orifice plates;
(2) again the people's corneal epithelium or the endotheliocyte of In vitro culture are made cell suspension, the DMEM/F12 culture fluid that use contains 10%FBS is seeded in 24 orifice plates that post acellular conjunctiva matrix, changed culture fluid in 3 days, form close-connected corneal epithelium or endothelial cell monolayer in 7 days, promptly construct tissue engineering comea epithelium or endothelium;
(3) above-mentioned tissue engineering comea epithelium or endothelium are transplanted to respectively on the human eye that limbal stem cell lacks or the mistake of endothelial cell function is compensatory, the result shows that human eye does not have immunological rejection to tissue engineering comea epithelium or endothelium, tissue engineering comea epithelium or endothelium are in transplanting degraded not yet in back 30 days, and the corneal limbal stem cell lacks or the endothelial cell function is lost the compensatory significant curative effect that all has.
In order to guarantee that constructed people's tissue engineering comea epithelium or endothelium can effectively use, and will extract it usually and carry out following detection:
Adopt trypan blue-alizarin red dyeing, the showed cell sharpness of border with the homomorphosis of normal person's corneal epithelium or endotheliocyte, and has only only a few cell indigo plant to dye as a result, and prompting has activity at the people's corneal epithelium or the endotheliocyte of acellular conjunctiva matrix growth;
Adopt the cellular immunofluorescence method can detect the label K3/12 of people's corneal epithelial cell or the label NSE of endotheliocyte, prompting has function at the people's corneal epithelium or the endotheliocyte of acellular conjunctiva matrix growth.
Acellular conjunctiva matrix can adopt following several method to preserve:
(1) be placed in the DMEM/F12 culture fluid that contains 10%FBS, 4 ℃ of sealings are preserved standby;
(2) directly be sealed in the aseptic plastic packaging bag, room temperature is preserved; Use be prepended in the DMEM/F12 culture fluid that contains 10%FBS soak standby more than 12 hours;
(3) place pure glycerin-20 ℃ preservation; After washing repeatedly with PBS before using, be put in again soak in the DMEM/F12 culture fluid that contains 10%FBS standby more than 12 hours;
(4) place DMEM and ℃ preservation of pure glycerin equal-volume mixed liquor-20; Method is with (3) before using
(5) place FBS and ℃ preservation of pure glycerin equal-volume mixed liquor-20; Wash repeatedly with PBS before using.
With following a plurality of embodiment, describe the present invention in detail.
Embodiment 1
With the Oculus sus domestica bulbar conjunctiva is the material preparation acellular conjunctiva matrix:
The fresh pig eyeball is fetched by the slaughterhouse, after washing repeatedly with PBS, cuts off ball week tissue, cuts bulbar conjunctiva again, places the PBS that contains tobramycin 1000U/ml to soak 30 minutes; In super-clean bench, the Tenon capsule of above-mentioned bulbar conjunctiva is wiped out totally, and with PBS flushing as far as possible, be put in volume fraction and be in 0.2% glutaraldehyde solution and to carry out crosslinked protection in 5 minutes in the optical stereo microscopically; Reuse PBS flushing is to remove residual glutaraldehyde solution; Then bulbar conjunctiva being taken off cell and handle, is 1%tritonX-100 effect 24 hours with volume fraction on 37 ℃ of constant temperature shaking tables., then at 37 ℃, co
2Concentration is in 5% the incubator, and the quality volume fraction is 0.25% pancreatin and 0.02%EDTA effect 2 hours, with PBS concussion flushing 10 minutes, is placed on aeration-drying in the super-clean bench, and bulbar conjunctiva two sides ultraviolet disinfection each 30 minutes obtains acellular conjunctiva matrix.Be placed in the DMEM/F12 culture fluid that contains 10%FBS, 4 ℃ of sealings are preserved standby or directly are sealed in the aseptic plastic packaging bag, preserve at normal temperatures.
Embodiment 2
Bulbar conjunctiva with the people is the material preparation acellular conjunctiva matrix:
Get the bulbar conjunctiva of people in the eye bank, place the PBS that contains tobramycin 1000U/ml to soak 30 minutes; In super-clean bench, the Tenon capsule of above-mentioned bulbar conjunctiva is wiped out totally, and with PBS flushing as far as possible, be put in volume fraction and be in 0.2% glutaraldehyde solution and to carry out crosslinked protection in 5 minutes in the optical stereo microscopically; Reuse PBS flushing is to remove residual glutaraldehyde solution; Then bulbar conjunctiva being taken off cell and handle, is 1%tritonX-100 effect 24 hours with volume fraction on 37 ℃ of constant temperature shaking tables, then at 37 ℃, and co
2Concentration is in 5% the incubator, and the quality volume fraction is 0.25% pancreatin and 0.02%EDTA effect 2 hours, with PBS concussion flushing 10 minutes, is placed on aeration-drying in the super-clean bench, and bulbar conjunctiva two sides ultraviolet disinfection each 30 minutes obtains acellular conjunctiva matrix.Place pure glycerin,, or place DMEM and pure glycerin equal-volume mixed liquor ,-20 ℃ of preservations, or place FBS and pure glycerin equal-volume mixed liquor ,-20 ℃ of preservations-20 ℃ of preservations.
Embodiment 3
Make up rabbit tissue engineering comea epithelium or endothelium with the acellular conjunctiva matrix for the tissue engineering comea timbering material:
Take out the acellular conjunctiva matrix as the tissue engineering comea timbering material of embodiment 1 preparation, PBS flushing back is stretched and is put in 24 orifice plates, dries in aseptic super-clean bench, makes it firmly be attached at the ground of 24 orifice plates.The rabbit corneal epithelium or the endotheliocyte of In vitro culture are digested to single cell suspension, the DMEM/F12 culture fluid that use contains 15%FBS is inoculated in 24 orifice plates that post acellular conjunctiva matrix, changed culture fluid in 3 days, 5 days corneal epitheliums or endotheliocyte can form close-connected cell monolayer on acellular conjunctiva matrix, promptly be built into rabbit tissue engineering comea epithelium or endothelium.
Embodiment 4
Make up people's tissue engineering comea epithelium or endothelium with the acellular conjunctiva matrix for the tissue engineering comea timbering material:
Take out the acellular conjunctiva matrix as the tissue engineering comea timbering material of embodiment 2 preparations, PBS flushing back is stretched and is put in 24 orifice plates, dries in aseptic super-clean bench, makes it firmly be attached at the ground of 24 orifice plates.The people's corneal epithelium or the endotheliocyte of In vitro culture are digested to single cell suspension, the DMEM/F12 culture fluid that use contains 10%FBS is inoculated in 24 orifice plates that post acellular conjunctiva matrix, changed culture fluid in 3 days, 7 days corneal epitheliums or endotheliocyte can form close-connected cell monolayer on acellular conjunctiva matrix, promptly make up adult's tissue engineering comea epithelium or endothelium.
Embodiment 5
Make the rabbit animal model that limbal stem cell lacks (as alkali burn), the rabbit tissue engineering comea epithelial transplantation of getting embodiment 3 structures is to rabbit animal model eye.The result shows that lagophthalmos does not have immunological rejection to the tissue engineering comea epithelium, and transplants the degraded not yet in 25 days of back tissue engineering comea epithelium, and can reduce cornea rebirth blood vessel and leucomatous formation, proves its good effect.
Embodiment 6
Make the endothelial cell function and lose the rabbit animal model of compensatory (as removing entocornea), get the rabbit tissue engineering comea endothelium of embodiment 3 structures and be transplanted on the rabbit animal model eye.The result shows that lagophthalmos does not have immunological rejection to the tissue engineering comea endothelium, and transplants the degraded not yet in 25 days of back tissue engineering comea endothelium, and can recover the transparency of cornea and avoid the formation of cornea bulla, its good effect.
Embodiment 7
People's tissue engineering comea epithelial transplantation of getting embodiment 4 structures is to the human eye of suffering from the limbal stem cell shortage.The result shows that human eye does not have immunological rejection to the tissue engineering comea epithelium, and transplants the degraded not yet in 30 days of back tissue engineering comea epithelium, and can reduce cornea rebirth blood vessel and leucomatous formation, its good effect.
Embodiment 8
Getting people's tissue engineering comea endothelium that embodiment 4 makes up is transplanted to and suffers from the endothelial cell function and lose on the compensatory human eye.The result shows that human eye does not have immunological rejection to the tissue engineering comea endothelium, and transplants the degraded not yet in 30 days of back tissue engineering comea endothelium, and can recover the transparency of cornea and avoid the formation of cornea bulla, its good effect.
Embodiment 9
Make the rabbit animal model of corneal epithelial defect (as mechanical curettage), the amniotic membrane directly transplanting that respectively acellular conjunctiva matrix of embodiment 2 preparations and existing tissue engineering comea timbering material " removed epithelium " is on rabbit animal model eye.The result shows, though lagophthalmos does not all have immunological rejection to acellular matrix and " removing epithelium " amniotic membrane, " removing epithelium " amniotic membrane is in transplanting degraded in back 10 days, and acellular conjunctiva matrix is in transplanting degraded not yet in back 25 days.
Embodiment 10
Make the rabbit animal model that limbal stem cell lacks (as alkali burn), will with " removing epithelium " amniotic membrane be the rabbit tissue engineering comea epithelial transplantation that makes up of tissue engineering comea timbering material to rabbit animal model eye, observe its curative effect.The result shows that though lagophthalmos does not have immunological rejection to the tissue engineering comea epithelium, " removing epithelium " amniotic membrane was degraded in transplanting in back 10 days, and can not play the effect that suppresses cornea rebirth blood vessel and corneal leukoma formation.
Embodiment 11
Making the endothelial cell function and lose the rabbit animal model of compensatory (as removing entocornea), will be that the rabbit tissue engineering comea endothelium that the tissue engineering comea timbering material makes up is transplanted on the rabbit animal model eye with " removing epithelium " amniotic membrane, observe its curative effect.The result shows that though lagophthalmos does not have immunological rejection to the tissue engineering comea endothelium, " removing epithelium " amniotic membrane was degraded in transplanting in back 10 days, and can not play the transparency of recovering cornea and suppress the effect that the cornea bulla forms.
Claims (4)
1, the preparation method of acellular conjunctiva matrix:
(1) gets the fresh pig eyeball in slaughterhouse, after washing repeatedly with PBS, cut off ball week tissue, cut bulbar conjunctiva again; Or people's bulbar conjunctiva in the eye bank, place the PBS that contains tobramycin 1000U/ml to soak 30 minutes;
(2) in super-clean bench, the Tenon capsule of above-mentioned bulbar conjunctiva is wiped out totally, and with PBS flushing as far as possible, be put in volume fraction and be in 0.2% glutaraldehyde solution 5 minutes to carry out crosslinked protection in the optical stereo microscopically;
(3) reuse PBS flushing is to remove residual glutaraldehyde solution;
(4) then bulbar conjunctiva being taken off cell and handle, is 1%tritonX-100 effect 24 hours with volume fraction on 37 ℃ of constant temperature shaking tables, at 37 ℃, and co
2Concentration is in 5% the incubator, and the quality volume fraction is 0.25% pancreatin and 0.02%EDTA effect 2 hours, with PBS concussion flushing 10 minutes, is placed on aeration-drying in the super-clean bench, and two sides ultraviolet disinfection each 30 minutes obtains acellular conjunctiva matrix.
2, acellular conjunctiva matrix is applied to make up tissue engineering comea epithelium or endothelium as the tissue engineering comea timbering material.
3, acellular conjunctiva matrix is as the application process of tissue engineering comea timbering material:
(1) will wash with PBS before use as the acellular conjunctiva matrix of tissue engineering comea timbering material, be put in then in 24 orifice plates, in aseptic super-clean bench, dry, it firmly is attached on 24 orifice plates:
(2) again the people's corneal epithelium or the endotheliocyte of In vitro culture are made cell suspension, the DMEM/F12 culture fluid that use contains 10%FBS is seeded in above-mentioned 24 orifice plates that post acellular conjunctiva matrix, changed culture fluid in 3 days, form close-connected corneal epithelium or endothelial cell monolayer in 7 days, promptly be built into tissue engineering comea epithelium or endothelium;
(3) at last above-mentioned tissue engineering comea epithelium or endothelium are transplanted to respectively on the human eye that limbal stem cell lacks or the mistake of endothelial cell function is compensatory.
The store method of the acellular conjunctiva matrix that 4, prepares:
(1) is placed in DMEM/F 12 culture fluid that contain 10%FBS, preserves 4 ℃ of sealings; Or
(2) directly be sealed in the aseptic plastic packaging bag, preserve at normal temperatures; Or
(3) place pure glycerin ,-20 ℃ of preservations; Or
(4) place DMEM and pure glycerin equal-volume mixed liquor ,-20 ℃ of preservations; Or
(5) place FBS and pure glycerin equal-volume mixed liquor ,-20 ℃ of preservations.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101985051A (en) * | 2010-10-21 | 2011-03-16 | 暨南大学 | Acellular cornea or acellular corneal stroma, preparation method and application thereof |
| CN102462561A (en) * | 2010-11-19 | 2012-05-23 | 北京迈迪顶峰医疗科技有限公司 | Small intestinal submucosa (SIS) soft tissue repair patch and preparation method thereof |
| CN103550826A (en) * | 2013-10-31 | 2014-02-05 | 广州优得清生物科技有限公司 | Method for preparing heterogenic cornea material |
| CN105770994A (en) * | 2016-05-09 | 2016-07-20 | 拜欧迪赛尔（北京）生物科技有限公司 | Preparation method and application of bioengineering decellularized eye conjunctiva |
| CN106172369A (en) * | 2016-07-04 | 2016-12-07 | 拜欧迪赛尔（北京）生物科技有限公司 | The antioxidation of a kind of optimization takes off Cell protective solutions |
| CN106421908A (en) * | 2016-07-01 | 2017-02-22 | 山东省眼科研究所 | Method for preparing decellularized cornea |
| CN110101916A (en) * | 2019-05-17 | 2019-08-09 | 张勇 | Synthesize cornea and its preparation method and application |
| CN112426566A (en) * | 2020-11-30 | 2021-03-02 | 山东大学 | Regenerative cornea material and preparation method and application thereof |
| CN115105637A (en) * | 2022-07-28 | 2022-09-27 | 上海交通大学医学院附属第九人民医院 | Application of subconjunctival fibroblast acellular matrix in conjunctival reconstruction |
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Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101985051A (en) * | 2010-10-21 | 2011-03-16 | 暨南大学 | Acellular cornea or acellular corneal stroma, preparation method and application thereof |
| CN102462561A (en) * | 2010-11-19 | 2012-05-23 | 北京迈迪顶峰医疗科技有限公司 | Small intestinal submucosa (SIS) soft tissue repair patch and preparation method thereof |
| CN103550826A (en) * | 2013-10-31 | 2014-02-05 | 广州优得清生物科技有限公司 | Method for preparing heterogenic cornea material |
| WO2015062177A1 (en) * | 2013-10-31 | 2015-05-07 | 广州优得清生物科技有限公司 | Method for preparing heterogenous corneal material |
| CN103550826B (en) * | 2013-10-31 | 2015-07-08 | 广州优得清生物科技有限公司 | Method for preparing heterogenic cornea material |
| US10072244B2 (en) | 2013-10-31 | 2018-09-11 | Youvision Biotech Co., Ltd. | Method for preparing heterogenetic corneal material |
| CN105770994A (en) * | 2016-05-09 | 2016-07-20 | 拜欧迪赛尔（北京）生物科技有限公司 | Preparation method and application of bioengineering decellularized eye conjunctiva |
| CN106421908B (en) * | 2016-07-01 | 2018-01-16 | 深圳艾尼尔角膜工程有限公司 | A kind of preparation method of de- cell cornea |
| CN106421908A (en) * | 2016-07-01 | 2017-02-22 | 山东省眼科研究所 | Method for preparing decellularized cornea |
| CN106172369B (en) * | 2016-07-04 | 2018-01-12 | 拜欧迪赛尔（北京）生物科技有限公司 | A kind of anti-oxidant de- Cell protective solutions of optimization |
| CN106172369A (en) * | 2016-07-04 | 2016-12-07 | 拜欧迪赛尔（北京）生物科技有限公司 | The antioxidation of a kind of optimization takes off Cell protective solutions |
| CN110101916A (en) * | 2019-05-17 | 2019-08-09 | 张勇 | Synthesize cornea and its preparation method and application |
| CN110101916B (en) * | 2019-05-17 | 2019-12-31 | 张勇 | Synthetic cornea and preparation method and application thereof |
| CN112426566A (en) * | 2020-11-30 | 2021-03-02 | 山东大学 | Regenerative cornea material and preparation method and application thereof |
| CN115105637A (en) * | 2022-07-28 | 2022-09-27 | 上海交通大学医学院附属第九人民医院 | Application of subconjunctival fibroblast acellular matrix in conjunctival reconstruction |
| CN115105637B (en) * | 2022-07-28 | 2023-11-10 | 上海交通大学医学院附属第九人民医院 | Application of subconjunctival fibroblast acellular matrix in conjunctival reconstruction |
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Application publication date: 20091202 |