CN102952779A - Cultural method for inducing human embryonic stem cell to directionally differentiate into corneal limbal stem cell - Google Patents
Cultural method for inducing human embryonic stem cell to directionally differentiate into corneal limbal stem cell Download PDFInfo
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Abstract
The invention discloses a cultural method for inducing a human embryonic stem cell to directionally differentiate into a corneal limbal stem cell, which comprises the following steps that firstly, a DMEM (Dulbecco's Modified Eagle Medium)/F12 conditioned medium is adopted to culture a human primary corneal limbal stem cell to prepare a corneal limbal stem cell conditioned medium, and then the corneal limbal stem cell conditioned medium is utilized and combined with IV type collagen culture in vitro to induce the human embryonic stem cell to directionally differentiate into the corneal limbal stem cell. According to the corneal limbal stem cell obtained by utilizing the cultural method, through light microscope observation in vitro, electron microscope observation, real-time quantitative polymerase chain reaction, immunofluorescence, flow cytometry, cloning efficiency determination and the like, the induced cell has a similar shape and phenotype with a normal corneal limbal stem cell, has good differentiation and proliferation capacity in vitro, can be transferred in vitro for more than four generations and can be used as a seed cell for preparing a corneal graft.
Description
Technical field
The present invention relates to the cultural method that a kind of inducing human embryo stem cell for directional is divided into limbal stem cell, and the limbal stem cell that utilizes this cultural method to obtain unites and takes off the method that cell amnion support makes up organizational project Allograft thing, belongs to organizational project and field of ophthalmology.
Background technology
Limbal stem cell is the basic foundation of corneal epithelial cell self, reparation and regeneration, also is barrier structure important between the conjunctiva of angle, has extremely important effect for the performance of keeping Corneal transparency, stability and normal physiological function.Serious eye surface diseases, such as eye table chemical injury, inflammation, eye table immunological disease etc., can cause the disappearance of limbal stem cell and the destruction of barrier structure, thereby cause the reaction of a series of eye table, corneal epithelium repeatedly damaged, rotten to the corn, ulcer, new vessel is grown into and the false triangular mass of mucous membrane growing from the inner corner of the eye forms, finally cause losing one's sight.The serious scarcity of limbal stem cell transplantation donor and the difficult situation of cultured and amplified in vitro cause lifelong blind so that numerous patient has lost the treatment machine meeting in the wait of misery at present.Therefore, seek to be fit to derived cell substitute limbal stem cell, improve corneal limbal stem cell total deficiency for damage after the reparation of corneal epithelium and the reconstruction of stable eye table have important clinical practice meaning.
From Thomson in 1998 etc. since the first time, separation and Culture went out human embryo stem cell from the embryo, the concern that embryonic stem cell enjoys scientific circles because of its powerful self-renewal capacity that has and totipotency, it is as seed cell, can repair damaged part, recover body function, for the treatment of some refractory diseases provides new approach.Therefore select human embryo stem cell to have advantageous Superiority of Scientific Research as the source of inducing cell.
The outer embryo stem cell for directional of Current Domestic is induced in the research aspect the eye table and is paid close attention to eventually last corneal epithelial cell more.But studies show that of Ahmad S in 2007 etc. adopts corneal limbus conditioned mediums of fibroblast inducing human embryo stem cell to be divided into to express the cell of the special cell opposite sex of corneal epithelium marker CK3/12, and the expression of the cornea limbal stem cell marker such as p63 has the peak to occur in 21 days by a definite date atomization.Homma etc. then find to utilize the IV Collagen Type VI that mouse embryo stem cell is carried out that contact induces can be external acquisition individual layer class epithelioid cell, this cell CK12 is positive, and squamous cell marker CK14 is negative, and inducing cell is transplanted in corneal injury mouse model upper 24 hour of visible transplanted cells close attachment in recipient cornea matrix also fully covering damage top layer.But the success ratio of this kind induction method only has less than 20%.In the research at home, Wang Zhichong etc. by the contact induction method, with Embryonic Stem Cell and surface cornea edge matrix co-cultivation, make the former vitro differentiation become single form than maxicell.Inducing cell can form the multiple layer of cell transplanting behind nude mice by subcutaneous, can be observed the part morphological feature of limbal stem cell under the electron microscope.Above-mentioned external and experimentation on animals confirmation, embryonic stem cell has to the potential of corneal epithelial cell directed differentiation in in-vitro simulated corneal epithelium microenvironment.But existing induced differentiation of embryonic stem cells scheme is induced inefficiency, induce the gained cell to be eventually end differentiation corneal epithelial cell, without the phenotype of normal cornea limbal stem cell and vitro differentiation amplification ability, so can't satisfy needs because of eye surface diseases clinical treatment due to the limbal stem cell deficiency.There is not yet at present the bibliographical information about the effective induction method of human embryo stem cell source limbal stem cell both at home and abroad.
In sum, originate in the urgent need to the vicarious species daughter cell with normal cornea limbal stem cell function in this area, and the engineered ocular surface graft that is suitable for transplanting use that utilizes seed cell to make up.
Summary of the invention
For above-mentioned prior art, in order to solve the serious deficient present situation of limbal stem cell transplantation donor, the present invention has set up the induction method that a kind of efficient feasible human embryo stem cell for directional is divided into limbal stem cell, provides seed cell for the tissue engineering comea epithelium makes up.Inducing cell has the form similar to the normal cornea limbal stem cell and phenotype, and good vitro differentiation multiplication capacity, but subculture in vitro separately is more than 4 generations; Its with take off cell amnion timbering material and have good biocompatibility; Lose in the compensatory animal model at limbal stem cell, the inducing cell graft can effectively be repaired the damage corneal epithelium, suppresses new vessel and generates, and reduces inflammatory reaction and scar and generates.
The present invention is achieved by the following technical solutions:
A kind of inducing human embryo stem cell for directional is divided into the cultural method of limbal stem cell, and step is as follows: at first, adopt DMEM/F12 conditioned medium cultivator limbal stem cell of former generation, preparation limbal stem cell conditioned medium; Then, utilize this limbal stem cell conditioned medium associating IV Collagen Type VI vitro culture inducing human embryo stem cell for directional to be divided into limbal stem cell.
The concrete grammar of described " adopting DMEM/F12 conditioned medium cultivator limbal stem cell of former generation, preparation limbal stem cell conditioned medium " is as follows: adopt DMEM/F12 conditioned medium cultivator limbal stem cell of former generation, change liquid every day; When adjacent limbal stem cell cloning cluster begins to merge, filter with liquid-transfering gun absorption substratum supernatant and by 0.22 μ m filter, be the limbal stem cell conditioned medium.
Described DMEM/F12 conditioned medium is to add following material at common DMEM/F12 medium base to make: foetal calf serum, human epidermal growth factor, hydrocortisone, Regular Insulin, Transferrins,iron complexes and Niu Chuiti extract; Wherein, after the interpolation, foetal calf serum accounts for the 10%(of total amount of liquid in present patent application, if no special instructions, solution per-cent all represents the volume fraction of additive in whole solution), the Niu Chuiti extract accounts for 0.2% of total amount of liquid, human epidermal growth factor's concentration is 5ng/ml, the concentration of hydrocortisone is 0.18 μ g/ml, and the concentration of Regular Insulin is 5 μ g/ml, and the concentration of Transferrins,iron complexes is 5 μ g/ml.
Described people limbal stem cell of former generation prepares by the following method: get the corneal ring tissue, (end level of penicillin and Streptomycin sulphate respectively is 100U/ml with the PBS balanced salt solution that contains penicillin-Streptomycin sulphate, together lower) irrigation and disinfection, remove corneal endothelium and descemet's membrane, the residual angle membrane tissue is placed in the 2.4U/ml DispaseII, in 37 ℃ of 5%CO
2Hatched in the incubator 90 minutes; With micro-tweezers separation of corneal epithelium tissue; Collect and separate the corneal epithelial tissue that obtains, in 37 ℃ of digestion of 0.25% pancreatin (the 0.25g pancreatin is dissolved in the PBS balanced salt solution, is settled to 100ml and is prepared from) 10 minutes, namely get the single cell suspension of limbal stem cell, with 1X10
3/ cm
2Density be inoculated on the 3T3 cell that mitomycin C processed, namely get people's limbal stem cell of former generation.
The concrete grammar of described " utilizing this limbal stem cell conditioned medium associating IV Collagen Type VI vitro culture inducing human embryo stem cell for directional to be divided into limbal stem cell " is as follows: utilize the in-vitro simulated corneal limbus microenvironment of limbal stem cell conditioned medium associating IV Collagen Type VI, adopt inducing culture cultivator embryonic stem cell 6～12 days (preferred 9 days), inducing human embryo stem cell for directional is divided into limbal stem cell; Wherein, 1) use-pattern of IV Collagen Type VI is: add 1mlIV Collagen Type VI (final concentration 0.5mg/ml) in every diameter 35mm culture dish, coated culture dish was absorbed coated liquid more than 2 hours before the inoculation human embryo stem cell, carry out cell cultures; 2) inducing culture is comprised of the DMEM/F12 conditioned medium (containing the additives such as foetal calf serum) of 75% limbal stem cell conditioned medium and 25%.
The limbal stem cell that the cultural method that utilizes inducing human embryo stem cell for directional of the present invention to be divided into limbal stem cell obtains, have the form similar to the normal cornea limbal stem cell and phenotype through confirmation inducing cells such as external observation by light microscope, electron microscope observation, real-time quantitative polymerase chain reaction, immunofluorescence, flow cytometry and cloning efficiency mensuration, and show good vitro differentiation multiplication capacity, but subculture in vitro separately is more than 4 generations.
The limbal stem cell that the present invention obtains can be used as seed cell for the preparation of corneal graft, and when specifically using, mode is as follows:
(1) the Freshman amnion is used PBS balanced salt solution (end level of penicillin and Streptomycin sulphate respectively the is 100U/ml) irrigation and disinfection that contains penicillin-Streptomycin sulphate, with 37 ℃ of digestion of 0.25% pancreatin 30 minutes, remove amnion cell, obtain taking off cell amnion timbering material, under dissecting microscope, will take off the disk that cell amnion timbering material is cut to diameter 3cm with eye scissors;
(2) with 0.125% pancreatin (the 0.125g pancreatin is dissolved in the PBS balanced salt solution, is settled to 100ml and is prepared from) in 37 ℃ of digestion induced dry-cells (limbal stem cell) 5 minutes, be prepared into single cell suspension, then press 1.5X10
4Individual cell/cm
2It is inoculated in taking off of cutting, and (the timbering material vaccination area is about 7cm on the cell amnion timbering material
2, the inoculation total amount is about 10.5X10
5Individual cell), in 2 weeks of vitro culture, wherein, submerged culture 7 days, liquid-gas interface was cultivated 7 days, can form the inducing cell graft with stratified epithelium spline structure.
Adopted 1mol/L soda ash burned rabbits cornea 40 seconds, the preparation Rabbit Corneal Limbal Stem Cells loses compensatory model, burn is planted sheet (the resulting inducing cell graft with stratified epithelium spline structure of the present invention) with inducing cell after January and is transplanted in the harmed eye table, transplanting after March inducing cell plants sheet and can effectively repair the harmed eye table, suppress new vessel and generate, reduce inflammatory reaction and scar and generate.
The present invention has set up one and has overlapped the induction scheme that efficient feasible human embryo stem cell for directional is divided into limbal stem cell, provides seed cell for the tissue engineering comea epithelium makes up; Set up a kind of method that adopts inducing cell to unite to take off cell amnion support to make up organizational project eye table, for the structure of engineered eye table has been established solid basis.
Description of drawings
Fig. 1 former generation limbal stem cell and the opticmicroscope picture of inducing cell of behaving, wherein, a: people's limbal stem cell opticmicroscope of former generation picture; B: inducing cell opticmicroscope picture, as seen, inducing cell is arranged closely, is the paving stone sample, with Corneal limbal stem cell plesiomorphism among the figure.
Fig. 2 is that inducing cell is united and taken off cell amnion timbering material external structure organizational project Allograft thing HE dyeing picture, as seen, has formed the stratified epithelium spline structure among the figure.
Fig. 3 is before the inducing cell transplantation and the slit lamp picture in postoperative March, wherein, a: slit lamp picture before the transplantation, among the figure as seen, corneal epithelial defect, in a large number new vesseles open into; B: post-transplantation slit lamp in March picture, among the figure as seen, damage corneal epithelium reparation, without obvious new vessel open into.
Fig. 4 is before the inducing cell transplantation and the Laser Scanning Confocal Microscope picture in postoperative March, wherein, a: Laser Scanning Confocal Microscope picture before the transplantation, as seen, the normal cornea epithelial structure is destroyed among the figure, new vessel open into and the reflective scar tissue of height; B: post-transplantation slit lamp in March picture, as seen, transplanted cells is arranged closely, is the paving stone sample among the figure, and scar tissue scope limitation has no new vessel.
Fig. 5 is that inducing cell is united and taken off cell porcine cornea timbering material external structure organizational project Allograft thing HE dyeing picture, as seen, has formed the stratified epithelium spline structure among the figure.
Embodiment
The present invention is further illustrated below in conjunction with embodiment.
Embodiment 1 inducing human embryo stem cell for directional is divided into limbal stem cell, and the preparation corneal graft
With the Freshman amnion with after containing the PBS balanced salt solution irrigation and disinfection of 100U/ml penicillin-Streptomycin sulphate, digested 30 minutes in 37 ℃ with 0.25% pancreatin, amnion cell is removed in liquid-transfering gun piping and druming, obtain taking off cell amnion timbering material, under dissecting microscope, will take off the disk that cell amnion timbering material is cut to diameter 3cm with eye scissors.Adopt inducing culture cultivator embryonic stem cell 9 days, and made it be divided into limbal stem cell like cell (shown in Fig. 1 b), in 37 ℃ of digestion inducing cells 5 minutes, prepare single cell suspension with 0.125% pancreatin, press 1.5X10
4Individual cell/cm
2It is inoculated in taking off of cutting, and (the timbering material vaccination area is about 7cm on the cell amnion timbering material
2, the inoculation total amount is about 10.5X10
5Individual cell), in 2 weeks of vitro culture, wherein, submerged culture 7 days, liquid-gas interface was cultivated 7 days, can form the cell corneal graft with stratified epithelium spline structure, as shown in Figure 2.
Described " adopted inducing culture cultivator embryonic stem cell 9 days, and made it be divided into the limbal stem cell like cell " concrete steps are as follows:
Utilize the in-vitro simulated corneal limbus microenvironment of limbal stem cell conditioned medium associating IV Collagen Type VI, adopted inducing culture cultivator embryonic stem cell 9 days, inducing human embryo stem cell for directional is divided into limbal stem cell; Wherein, 1) use-pattern of IV Collagen Type VI is: add the 1mlIV Collagen Type VI in every diameter 35mm culture dish, final concentration 0.5mg/ml, coated culture dish absorbed coated liquid more than 2 hours before the inoculation human embryo stem cell, carry out cell cultures; 2) inducing culture is comprised of the DMEM/F12 conditioned medium of 75% limbal stem cell conditioned medium and 25%.
Described limbal stem cell conditioned medium prepares by the following method: adopt DMEM/F12 conditioned medium cultivator limbal stem cell of former generation, change liquid every day; When adjacent limbal stem cell cloning cluster begins to merge, get the substratum supernatant, be the limbal stem cell conditioned medium.
Described DMEM/F12 conditioned medium is to add following material at common DMEM/F12 medium base to make: foetal calf serum, human epidermal growth factor, hydrocortisone, Regular Insulin, Transferrins,iron complexes and Niu Chuiti extract; Wherein, after the interpolation, foetal calf serum accounts for 10% of total amount of liquid, ox hypophysis extract accounts for 0.2% of total amount of liquid, and human epidermal growth factor's concentration is 5ng/ml, and the concentration of hydrocortisone is 0.18 μ g/ml, the concentration of Regular Insulin is 5 μ g/ml, and the concentration of Transferrins,iron complexes is 5 μ g/ml.
Described people limbal stem cell of former generation prepares by the following method: get the corneal ring tissue, with the PBS balanced salt solution irrigation and disinfection that contains penicillin-Streptomycin sulphate, remove corneal endothelium and descemet's membrane, the residual angle membrane tissue is placed in the 2.4U/mlDispaseII, in 37 ℃ of 5%CO
2Hatched in the incubator 90 minutes; With micro-tweezers separation of corneal epithelium tissue; Collect and separate the corneal epithelial tissue that obtains, in 37 ℃ of digestion of 0.25% pancreatin 10 minutes, namely get the single cell suspension of limbal stem cell, with 1X10
3/ cm
2Density be inoculated on the 3T3 cell that mitomycin C processed, namely get people's limbal stem cell of former generation (as shown in Figure 1a).
The engineered Ocular surface healing chemical damage corneal epithelium that embodiment 2 makes up
The annular filter scraps of paper were soaked 20 seconds in 1mol/L soda ash, suck unnecessary liquid with sterile gauze, place the lagophthalmos table to burn 40 seconds after, give a large amount of stroke-physiological saline solution flushing eye table, the preparation Rabbit Corneal Limbal Stem Cells loses compensatory model.Burn is after January, the Rabbit Corneal Limbal Stem Cells that selection successfully constructs loses compensatory model, and microscopically is carefully removed the new vessel tunica fibrosa, the cleaning plant bed, inducing cell is planted sheet (embodiment 1 preparation) place on the ready plant bed, sew up fixing with the 10-0 nylon wire planting the sheet edge.Postoperative was observed under slit lamp and is planted the sheet situation in per 3 days, and per January is the observation of cell growing state under Laser Scanning Confocal Microscope.Put to death laboratory animal row histology after transplanting March, as shown in Figure 3.
The engineered Ocular surface healing physical abuse corneal epithelium that embodiment 3 makes up
Utilize the flaggy cutter in microscopically circular resection corneal tissue, the preparation Rabbit Corneal Limbal Stem Cells loses compensatory model.Surgical injury is after January, the Rabbit Corneal Limbal Stem Cells that selection successfully constructs loses compensatory model, and microscopically is carefully removed the new vessel tunica fibrosa, the cleaning plant bed, inducing cell is planted sheet (embodiment 1 preparation) place on the ready plant bed, sew up fixing with the 10-0 nylon wire planting the sheet edge.Postoperative was observed under slit lamp and is planted the sheet situation in per 3 days, and per January is the observation of cell growing state under Laser Scanning Confocal Microscope.Put to death laboratory animal row histology after transplanting March, as shown in Figure 4.
Embodiment 4 is external to be united and takes off cell porcine cornea support and make up engineered front flaggy
Collect the fresh pig angle, with the balanced salt solution irrigation and disinfection that contains penicillin-Streptomycin sulphate, microscopically is treated to porcine cornea the tissue of 10 millimeters of diameters.Place 0.5%SDS solution in 4 ℃ of stirrings 24 hours the tissue of processing, re-use aseptic balanced salt solution rinsing tissue 8 times, each time is 2 hours, amounts to 16 hours, can obtain to take off cell porcine cornea timbering material.Use 0.125% trysinization inducing cell, the preparation single cell suspension is pressed 1.5X10
4Individual cell/cm
2It is inoculated in takes off that (the timbering material vaccination area is about 0.8cm on the cell porcine cornea timbering material
2, the inoculation total amount is about 1.2X10
4Individual cell) external liquid-gas interface cultivated for 2 weeks, and wherein submerged culture 7 days, liquid-gas interface was cultivated 7 days, can form the inducing cell graft with stratified epithelium spline structure, as shown in Figure 5.
In embodiment 2, inducing cell is planted sheet can effectively repair the harmed eye table, suppresses new vessel and generates, and reduces inflammatory reaction and scar and generates.In embodiment 4, cultivated for 2 weeks after, inducing cell can form the inducing cell graft with stratified epithelium spline structure taking off cell porcine cornea support.Above embodiment shows that the method for inducing differentiation that the present invention makes up effectively inducing human embryo stem cell for directional is divided into limbal stem cell, and induces graft to play good repair the transplanting animal, has wide DEVELOPMENT PROSPECT.
Claims (7)
1. an inducing human embryo stem cell for directional is divided into the cultural method of limbal stem cell, and it is characterized in that: step is as follows: at first, adopt DMEM/F12 conditioned medium cultivator limbal stem cell of former generation, preparation limbal stem cell conditioned medium; Then, utilize this limbal stem cell conditioned medium associating IV Collagen Type VI vitro culture inducing human embryo stem cell for directional to be divided into limbal stem cell;
The concrete grammar of described " adopting DMEM/F12 conditioned medium cultivator limbal stem cell of former generation, preparation limbal stem cell conditioned medium " is as follows: adopt DMEM/F12 conditioned medium cultivator limbal stem cell of former generation, change liquid every day; When adjacent limbal stem cell cloning cluster begins to merge, get the substratum supernatant, be the limbal stem cell conditioned medium;
The concrete grammar of described " utilizing this limbal stem cell conditioned medium associating IV Collagen Type VI vitro culture inducing human embryo stem cell for directional to be divided into limbal stem cell " is as follows: utilize the in-vitro simulated corneal limbus microenvironment of limbal stem cell conditioned medium associating IV Collagen Type VI, adopted inducing culture cultivator embryonic stem cell 6～12 days, inducing human embryo stem cell for directional is divided into limbal stem cell; Wherein, 1) use-pattern of IV Collagen Type VI is: add the 1mlIV Collagen Type VI in every diameter 35mm culture dish, final concentration 0.5mg/ml, coated culture dish absorbed coated liquid more than 2 hours before the inoculation human embryo stem cell, carry out cell cultures; 2) inducing culture is comprised of the DMEM/F12 conditioned medium of 75% limbal stem cell conditioned medium and 25%.
2. inducing human embryo stem cell for directional according to claim 1 is divided into the cultural method of limbal stem cell, it is characterized in that: described DMEM/F12 conditioned medium is to add following material at common DMEM/F12 medium base to make: foetal calf serum, the human epidermal growth factor, hydrocortisone, Regular Insulin, Transferrins,iron complexes and Niu Chuiti extract; Wherein, after the interpolation, foetal calf serum accounts for 10% of total amount of liquid, ox hypophysis extract accounts for 0.2% of total amount of liquid, and human epidermal growth factor's concentration is 5ng/ml, and the concentration of hydrocortisone is 0.18 μ g/ml, the concentration of Regular Insulin is 5 μ g/ml, and the concentration of Transferrins,iron complexes is 5 μ g/ml.
3. inducing human embryo stem cell for directional according to claim 1 is divided into the cultural method of limbal stem cell, it is characterized in that: described people limbal stem cell of former generation prepares by the following method: get the corneal ring tissue, with the PBS balanced salt solution irrigation and disinfection that contains penicillin-Streptomycin sulphate, remove corneal endothelium and descemet's membrane, the residual angle membrane tissue is placed in the 2.4U/mlDispaseII, in 37 ℃ of 5%CO
2Hatched in the incubator 90 minutes; With micro-tweezers separation of corneal epithelium tissue; Collect and separate the corneal epithelial tissue that obtains, in 37 ℃ of digestion of 0.25% pancreatin 10 minutes, namely get the single cell suspension of limbal stem cell, with 1X10
3/ cm
2Density be inoculated on the 3T3 cell that mitomycin C processed, namely get people's limbal stem cell of former generation.
4. the limbal stem cell that utilizes each described inducing human embryo stem cell for directional is divided into limbal stem cell in the claim 1～3 cultural method to obtain.
5. limbal stem cell claimed in claim 4 is as the purposes of seed cell for the preparation of corneal graft.
6. described purposes according to claim 5, it is characterized in that: when specifically using, mode is as follows:
(1) with the Freshman amnion PBS balanced salt solution irrigation and disinfection that contains penicillin-Streptomycin sulphate, with 37 ℃ of digestion of 0.25% pancreatin 30 minutes, remove amnion cell, obtain taking off cell amnion timbering material;
(2) digested limbal stem cells 5 minutes with 0.125% pancreatin in 37 ℃, be prepared into single cell suspension, then it is inoculated in and takes off on the cell amnion timbering material, 2 weeks of vitro culture, wherein, submerged culture 7 days, liquid-gas interface was cultivated 7 days, can form the inducing cell graft with stratified epithelium spline structure.
7. corneal graft is characterized in that: prepare by the following method:
(1) with the Freshman amnion PBS balanced salt solution irrigation and disinfection that contains penicillin-Streptomycin sulphate, with 37 ℃ of digestion of 0.25% pancreatin 30 minutes, remove amnion cell, obtain taking off cell amnion timbering material;
(2) with the limbal stem cell 5 minute of 0.125% pancreatin in 37 ℃ of digestion claims 4, be prepared into single cell suspension, then it is inoculated in and takes off on the cell amnion timbering material, 2 weeks of vitro culture, wherein, submerged culture 7 days, liquid-gas interface was cultivated 7 days, can form the inducing cell graft with stratified epithelium spline structure, be corneal graft.
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| CN106591235A (en) * | 2017-01-24 | 2017-04-26 | 吴欣怡 | Method for promoting function and characteristic of corneal endothelial cells |
| CN107075469A (en) * | 2014-06-27 | 2017-08-18 | 加利福尼亚大学董事会 | Mammal limbal stem cell, its production method and its purposes of culture |
| CN107164309A (en) * | 2017-06-18 | 2017-09-15 | 广东博溪生物科技有限公司 | The 3D models and its construction method of a kind of utilization serum-containing medium external structure |
| CN108126240A (en) * | 2017-12-29 | 2018-06-08 | 山东省立医院 | A kind of vitro construction method of bioengineering holostrome people's cornea |
| CN108467853A (en) * | 2018-04-08 | 2018-08-31 | 中科政兴（上海）医疗科技有限公司 | A kind of method of stem cell directional culture |
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| CN107075469A (en) * | 2014-06-27 | 2017-08-18 | 加利福尼亚大学董事会 | Mammal limbal stem cell, its production method and its purposes of culture |
| CN106011065B (en) * | 2016-07-15 | 2019-08-02 | 江苏大学 | Human eye bulbar conjunctiva triangular mass of mucous membrane growing from the inner corner of the eye stem cells strain |
| CN106011065A (en) * | 2016-07-15 | 2016-10-12 | 江苏大学 | Human eye bulbar conjunctiva pterygium stem cell strain |
| CN106591235A (en) * | 2017-01-24 | 2017-04-26 | 吴欣怡 | Method for promoting function and characteristic of corneal endothelial cells |
| CN107164309A (en) * | 2017-06-18 | 2017-09-15 | 广东博溪生物科技有限公司 | The 3D models and its construction method of a kind of utilization serum-containing medium external structure |
| CN108126240A (en) * | 2017-12-29 | 2018-06-08 | 山东省立医院 | A kind of vitro construction method of bioengineering holostrome people's cornea |
| CN108467853A (en) * | 2018-04-08 | 2018-08-31 | 中科政兴（上海）医疗科技有限公司 | A kind of method of stem cell directional culture |
| CN109363801A (en) * | 2018-10-09 | 2019-02-22 | 黄国富 | A kind of method that femtosecond laser technology auxiliary prepares porcine cornea endothelium plant piece |
| CN109363801B (en) * | 2018-10-09 | 2021-01-26 | 黄国富 | Method for preparing porcine corneal endothelial implant under assistance of femtosecond laser technology |
| CN113736735A (en) * | 2020-05-27 | 2021-12-03 | 深圳华大生命科学研究院 | Method and kit for inducing limbal stem cells in vitro |
| CN113736735B (en) * | 2020-05-27 | 2024-02-20 | 深圳华大生命科学研究院 | Method and kit for in vitro induction of limbal-like stem cells |
| CN111973808A (en) * | 2020-09-04 | 2020-11-24 | 安徽惠恩生物科技股份有限公司 | Biological membrane material adopting cell transplantation as carrier |
| 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 |
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