Culture of rabbit corneal stromal cells on thermo-sensitive materials by the radiation polymerization / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To explore the synthesis of thermo-sensitive poly N-isopropylacry-lamide(PNIPAAm)and the petri dish grafted with PNIPAAm hydrogels by the electron accelerator, as well as the growth conditions and the biological characteristics of rabbit corneal stromal cells on thermo-sensitive PNIPAAm hydrogels, and the cell sheets obtained from the PNIPAAm hydrogels.METHODS: NIPAAm monomer was dissolved in 2-propanol at concentrations of 55% with 0.5% N,N'-Methylenebisacry-lamide(MBA). Solution(70 μL)was added and spread uniformly over 35 mm petri dish. These dishes were immediately subjected to irradiation. After follow-up treatment, rabbit corneal stromal cells were cultured on thermo-sensitive petri dish in vitro.RESULTS: According to the monomer formula and radiation synthesis scheme in this experiment, PNIPAAm can be synthesized on the surface of the petri dish. Rabbit corneal stromal cells grew well in the thermo- sensitive surface and can be separated into sheets.CONCLUSION: The single and multilayer carrier-free cell sheets can be obtained from the use of thermo-sensitive petri dish.

Application of CRISPR/Cas9 genome editing technology in hereditary retinal diseases / 中华实验眼科杂志

Several mutant genes for inherited retinal diseases have been identified, but effective treatments are still lacking.The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system can edit human genomic DNA by nonhomologous end joining or homology-directed repair, offering more possibilities for the treatment of hereditary retinal diseases.CRISPR/Cas9 not only can genetically correct patient-derived induced pluripotent stem cells (iPSCs) to observe their differentiation into retinal cells thereby, thereby exploring the pathogenesis of the disease and implementing cell therapy, but can also be delivered to the body via vectors and directly act on target cells to achieve in vivo gene editing.CRISPR/Cas9 gene editing technology in hereditary retinal diseases has been mainly used in retinitis pigmentosa, hereditary X-linked juvenile retinoschisis, and Leber congenital amaurosis 10, of which the in vitro application of CRISPR/Cas9 for Leber congenital amaurosis 10 has entered the clinical trial stage.In this paper, we reviewed the mechanism and key advances of CRISPR/Cas9 and provided an overview of gene editing in IRDs.

Preparation and drug release effect evaluation of drug-loaded cross-linked decellularized corneal stromal lenticules in vitro / 中华实验眼科杂志

Objective:To prepare a drug release system of drug-loaded cross-linked decellularized corneal stromal lenticules and evaluate its drug release characteristics in vitro. Methods:Lenticules were obtained during femtosecond laser-assisted small incision lenticule extraction (SMILE) surgery in Chongqing Aier Ophthalmology Hospital.Decellularized corneal stromal lenticules were prepared using high concentration sodium chloride (NaCl) combining nuclease.The decellularized corneal stromal lenticules were randomly divided into normal group, 0.5% levofloxacin group, 3% levofloxacin group and 5% levofloxacin group, with 4 lenticules in each group.The lenticules did not receive any treatment in the normal group, and drug-loading those were soaked in different doses of levofloxacin solution for three hours according to grouping.In the crosslinking test, 12 decellularized corneal stromal lenticules were randomly divided into non-crosslinking group, 0.01 mmol 1-(3-dimethylamino) propylimine (EDC) group, 0.05 mmol EDC group and 0.25 mmol EDC group.The lenticules for cross-linking were soaked in different contents of mixed solution of EDC with N-hydroxysuccinyl (NHS) for four hours respectively according to grouping, and then in 3% levofloxacin solution for three hours.Only 3% levofloxacin solution soaking was carried in the non-crosslinking group.High performance liquid chromatography (HPLC) was employed to detect the drug release concentration of the lenticules, and spectral scanning method was performed to measure light transittance of the lenticules.The surface ultrastructure of the decellularized lenticules among different cross-linking groups was examined and compared with scanning electron microscope.The use of the human corneal lenticules was approved by an Ethics Committee of Chongqing Aier Ophthalmology Hospital (No.2019012). Written informed consent was obtained from each patient before surgery.Results:The release concentrations of decellularized corneal stroma lenticules were significantly different at 1 day, 7, 14, and 21 days among 0.5%, 3%, and 5% levofloxacin group ( P<0.05) or also among the 0.01 mmol EDC, 0.05 mmol EDC, and 0.25 mmol EDC cross-linked groups ( P<0.01). The drug release concentrations in 0.05 mmol EDC group were the highest at various time points, and the release time of the three cross-linked groups lasted until 21 days after release concentrations of decellularized corneal stroma lenticules.The drug release concentrations in cross-linked groups and non-crosslinking group were gradually declined with the prolong of drug-loading time, showing a significant difference at different time points ( P<0.05). The transmittance of the lenticules was (88.68±1.19)% and (91.55±1.16)% in the non-crosslinking group and normal group, respectively, with no significant difference ( P>0.05). The average transmittance of the lenticules was significantly reduced in the drug-loaded groups compared with the normal group ( P<0.05). The smaller collagen fiber voids and closely arranged collagen fibers were displayed in the cross-linking groups under the scanning electron microscope with the best effect in the 0.25 mmol EDC group. Conclusions:EDC/NHS cross-linking can improve the drug-loading effect of decellularized corneal stromal lenticules probably by lessening collagen fiber voids.The drug-loaded cross-linked decellularized corneal stromal lenticules have a good drug release effect in vitro.

Research progress on ophthalmologic optogenetics / 中华实验眼科杂志

Optogenetics is a genetic technique that applies illumination with certain wavelength to modulate the biological activity of cells or subcellular components accurately.This technique is friendly to researchers of ophthalmology due to characteristics of the eyes including transparency, accessibility and comparative independence.Optogenetics can be used in retinal neurons, such as retinal ganglion cells (RGCs), even extended to vascular endothelial cells, immune cells and other ocular cells or cell substructures, which can further our understanding of ocular physiology and provide potential, therapeutic approaches for neurodegenerative diseases, vascular diseases, inflammation and other eye-related diseases.Improvement in effectiveness, safety and comfort is pivotal for this technique to expand application in ophthalmology and for its function to reach the physiology state of nomal eyes.In this review, a comprehensive analysis of optogenetics progress in ophthalmology was performed.Challenges in imaging including light sensitivity, spatial resolution and temporal resolution, and problems in expression involving local and systemic safety, specificity and persistence were reviewed.

Role of ROCK inhibitor in corneal reconstruction：promoting the proliferation, migration and adhesion of keratocytes / 中国组织工程研究

BACKGROUND:With the deepen understanding on the biological function of Rho/ROCK pathway, new ROCK inhibitors continue to be discovered, and ROCK inhibitors show good promoting effects on the survival, proliferation and migration of keratocytes. Research on ROCK inhibitors wil provide more donor materials or seed cels for regenerative medicine and clinical cel transplantation. OBJECTIVE:To summarize and explore the progress in the treatment and application of corneal disease using the ROCK inhibitors Y-27632 and Y-39983. METHODS:The PubMed database and CNKI database were retrieved by computer to search the relevant literature published between 2008 to 2015 using the key words of “corneal endothelial cel, corneal epithelial cel, ROCK inhibitor, Y-39983, Y-27632” in English and Chinese, respectively. Relevant articles in line with the theme were screened and analyzed. RESULTS AND CONCLUSION:Totaly 264 papers were initialy searched. At last, 45 papers were selected. Currently there are two main ROCK inhibitors: Y-27632 and Y-39983, but both of which are stil in basic research stage and clinical testing stage. Y-27632 promotes the proliferation and activity of corneal epithelial stem cel after resuscitation; Y-39983 as a novel ROCK inhibitor can be better to inhibit Rho kinases activity than Y-27632, thereby more effectively promoting the healing of the corneal endothelium. There are many studies on the application of ROCK inhibitors in corneal treatment, but not a stable method established to obtain seed cels. Each method has its own advantages and disadvantages, and how to overcome these disadvantages and to find fast and stable access to seed cels is the future direction of development.

Influence of maxadilan on human adipose-derived stem cells / 中国病理生理杂志

[ ABSTRACT] AIM:To investigate the effect of maxadilan, which specifically activates pituitary adenylate cycla-se-activating polypeptide type I receptor (PAC1 receptor), on the proliferation, apoptosis and differentiation potential of human adipose-derived stem cells ( ASCs) .METHODS:ASCs from human adipose tissue were isolated by enzymatic di-gestion and cultured.ASCs were confirmed by the analysis of the markers for cell phenotypes by flow cytometry ( FCM) and adipogenic/osteogenic induction.The effect of maxadilan on ASCs viability was analyzed by CCK-8 assay and FCM.ASCs were irradiated by ultraviolet C ( UVC) at 254 nm and the absorbance of apoptotic ASCs induced by various doses of UVC was measured by CCK-8 assay.ASCs were exposed to 702 J/m2 UVC for 24 h to induce apoptosis.The effect of maxadilan on ASC apoptosis was analyzed by FCM and the determination of caspase 3 and caspase 9 levels.RESULTS:Adipose-de-rived stem cells were confirmed by the detection of the positive expression of cell phenotypes including CD29, CD44, CD59 and CD105 by FCM.The data of CCK-8 assay revealed that ASCs treated with maxadilan (80 nmol/L) had the strongest ability of proliferation.The data of FCM also demonstrated that the addition of 80 nmol/L maxadilan to ASCs in experimen-tal group markedly improved the proliferation capacity of the cells compared with control group (P<0.05).The apoptosis of ASCs exposed to 702 J/m2UVC was dramatically inhibited by the treatment with maxadilan (80 nmol/L).Such process involved the caspase signaling pathway including caspase 3 and caspase 9.There was statistical significance (P<0.05) between experiment group ( ASCs irradiated by UVC and supplemented with maxadilan) and control group ( ASCs only irra-diated by UVC) .Meanwhile, adipogenic and osteogenic differentiation potentials were both positive in experiment group and control group.CONCLUSION:Maxadilan promotes proliferation and inhibits apoptosis of the ASCs.The differentia-tion potential of ASCs toward adipogenic and osteogenic lineages wouldn’ t be altered by maxadilan.Maxadilan would bene-fit to growth and expansion of ASCs in vitro.

The viability and property of rabbit corneal endothelial cells by spheroid culture / 中华实验眼科杂志

Background The construction of tissue-engineered corneal endothelium and corneal endothelial cells (CECs) therapy need abundant seed cells,so how to culture a large amount of CECs with high viability and original cell properties is an urgent issue to be solved.Objective This study was to establish three-dimensional spheroid culture of CECs and explore the cellular biological characteristics.Methods Primary rabbit CECs were isolated with trypsin and subcultured.Low attachment and shaking culture was applied to form CECs spheres.Cultured cells were identified under the inverted microscope.The surface features of the cells were examined under the scanning electron microscope.The viability of the cells were assayed by acridine orange (AO) staining and CCK-8 kit.Then CECs spheres were incubated to 6-well plate for 1 week,and immunofluorescence staining was used to identify the expression of zonula occludens-1 (ZO-1) and Na+/K+-ATPase in the cells.The cell proliferation value of spheroid culture method was compared with that of regular culture method.Results CECs grew into aggregation after cultured with the hexagonal or polygonal shape and tight connection among the cells.The cells converged into single layer and slabstone-like arrangement 1 week later.Cells migrated out of the CECs sphere and formed an uneven spherical surface.The living cells showed green fluorescence for AO with the survival rate 90％.The absorbance (A450) of the cells was 1.524±0.013 and 1.265 ±0.021 in the spherical culture group and conventional culture group,respectively,showing a significant difference between them (t =-3.436,P=0.010).The positive cells of ZO-1 and Na+/K+-ATPase showed the green fluorescence for FITC on cell membrane and blue fluorescence for DAPI on cell nucleus.Conclusions Spherical culture method maintains a high viability and proliferation ability of the cells and remains phenotype of CECs,which is superior to conventional culture method.This culture method provides better seeding CECs for the establishment of tissue engineering cornea endothelial layer and CECs therapy.

Three-dimensional spherical culture of adipose-derived stem cells / 中国组织工程研究

BACKGROUND:Many types of mammalian cells aggregate and display three-dimensional multicellular spheroids when they are in normal physiological conditions. In order to observe and explore cellular natural states, many researchers try to use spherical cellculture in vitro, a common three-dimensional culture pattern. OBJECTIVE:To use three different methods for spherical culture in vitro of adipose-derived stem cells and to observe their biological features. METHODS:Adipose-derived stem cells were confirmed by the analysis of the markers for cellphenotypes as wel as adipogenic and osteogenic differentiation potential assays. Three different methods of sphere cultures were used as fol ows:(1) ultra low attachment culture;(2) hanging-drop culture and (3) Eppendorf tube culture. The sphere formation was compared among above three methods. We used Imagej to calculate mean areas of these spheres. And we used Viability/Cytotoxicity Assay Kit for Animal Live&Dead cells to detect their vitality. RESULTS AND CONCLUSION:(1) Adipose-derived stem cells were confirmed by the analysis of the markers for cellphenotypes, CD29, CD44, CD59 were positive, as wel as adipogenic and osteogenic differentiation potential assays were positive. The conventional monolayer cultures of adipose-derived stem cells showed spindle and cloning growth within three passages. (2) Ultra low attachment culture, hanging-drop culture, Eppendorf tube culture al could elicit adipose-derived stem cells spherical growth. However, spherical size, shape and uniformity differed depending on cellnumbers, culture time and spherical culture methods. The ultra low attachment culture was comparatively difficult to control spherical shape and uniformity of adipose-derived stem cells. But hanging-drop culture and Eppendorf tube culture were able to form even cellspheres. (3) Spherical formation of adipose-derived stem cells using our three methods displayed good cellvitality.

Transwell contact co-culture promotes growth and differentiation of sin-gle-dissociated iPSCs / 中国病理生理杂志

[ABSTRACT]AIM:ToinvestigatethepromotingroleofTranswellcontactco-culturesysteminthegrowthand differentiation of single-dissociated induced pluripotent stem cells (iPSCs).METHODS:Bovine corneal endothelial cells (CECs) at passage 1~2 (P1~2) were seeded on the underside of Transwell inserts placed into culture plates and were cultured in 37 ℃and 5%CO2 for 8 h.Accutase digestion and 40μm filter process disaggregated colony-aggregated iPSCs into single-dissociated iPSCs , and the cells were seeded on the inside of Transwell inserts with CECs in medium of mTeSR 1 for 3 d and then in low-glucose DMEM supplemented with 10% FBS for 2 weeks.The characteristics and differentiation markers were evaluated by real-time fluorescence quantitative polymerase chain reaction ( qPCR ) , immunofluorescence staining, live&dead cell staining and alkaline phosphatase (ALP) staining.The group of iPSCs cultured in conventional medium was used as control group 1.The group of single-dissociated iPSCs co-cultured with CECs was set as experimental group, while single-dissociated iPSCs without co-culture were as control group 2.RESULTS: The bovine CECs showed typical hexagonal cobblestone shape .iPSCs showed colony-like growth , while became single-dissociated cells after Tran-swell contact co-culture with bovine CECs for 3 d.The single-dissociated iPSCs positively expressed the undifferentiated markers, Nanog and Oct4.The mRNA expression levels of Nanog , Oct4 and Sox2 between experimental group and control group 1 were both positive and had no statistical significance difference (P>0.05).The dead cells in experimental group decreased significantly, and there was statistically significant difference compared to control group 2 (P<0.01).After 14 d of induced differentiation co-culture , the single-dissociated iPSCs showed rather uniform polygonal morphology , increased dimension and no obvious colony existence .Negative ALP staining, positive immunofluorescence staining for ZO-1, AQP1 and CD31, and negative for CD34 and CD133 were also observed.The results of qPCR showed that the mRNA expression of Oct4, Nanog and Sox2 significantly decreased , and had statistically significant difference compared with control group 1 (P<0.01).CONCLUSION: When co-cultured with bovine CECs, iPSCs morphologically changed to endothelial-like cells and expressed some markers of CECs .Transwell contact co-culture system not only enhances the growth of single-dis-sociated iPSCs , but also promotes their differentiation .

Research progress in reprogramming induced pluripotent stem cells / 中国病理生理杂志

Induced pluripotent stem cells ( iPSCs) have been first induced from mouse fibroblasts since 2006, and the research on iPSCs has made great progress in the following years .iPS cell lines were established from different so-matic cells through DNA , RNA, protein, and small molecule compounds and various methods of transduction , making the induction of iPSCs more secure and effective , and more attractive prospect of clinical application .In this review , different somatic cell reprogramming , different levels of reprogramming , different transduction pathways , and prospect of application are discussed .

Preparation and identification of recombinant PTD-maxadilan / 生物工程学报

In order to construct a novel fusion protein PTD-maxadilan (PTD-MAX) that can enter the blood-brain barrier (BBB) efficiently, a new gene encoding PTD-MAX was synthesized and cloned into the expression vector pKYB. The recombinant vector pKYB-PTD-MAX was transformed into Escherichia coli ER2566. The expression of fusion protein consisting of PTD-MAX, intein and chitin binding domain was induced by IPTG and the target PTD-MAX protein was purified using Intein Mediated Purification with an Affinity Chitin-binding Tag system. The molecular weight of PTD-MAX determined by the laser flight mass spectrometry was coherent with its theoretical value. The results of the experiment in vivo indicated that the recombinant PTD-MAX can permeate into BBS and inhibitory effects on the food intake were more significantly than maxadilan (P<0.05). The preparation of PTD-MAX lay the foundation for its further application.

Advances in materials of keratoprosthesis and tissue engineering cornea / 中国病理生理杂志

Although allograft corneal transplantation has made great progress,keratoprothesis implantation is the only one hope, for both the patients who has fleshy pannus corneal vitiligo and the patients with the failure corneal transplantation surgery whose far sight is only light perceptions to recover their eye sight. To gain success and reduce complications, a lot of work has been done in keratoprosthesis materials and type, but heterogeneity and bio-compatibility are not solved totally. Because of development in the cell culture and tissue engineering, cornea ex vivo culture has gain a critical success. From cell option to carrier using, reconstructed three dimensional culture and epithelium equivalent mimicked human corneas in key morphology and function and were used in basic and clinical investigation. This article reviews the development of materials of keratoprosthesis and tissue engineering cornea.

Effects of rhTGF-?_1 and TGF-?_1 gene transfection on the proliferation of cultured rabbit corneal endothelial cells in vitro / 中国病理生理杂志

AIM:To investigate the effects of rhTGF-?1 and TGF-?1 gene transfection on the proliferation of cultured rabbit corneal endothelial cells in vitro.METHODS:Cell growth induced by various concentrations of rhTGF-?1 was determined by MTT proliferation assay.Under the induction of liposomes,recombinant pSecTag2-TGF-?1MP vectors were transferred into the corneal endothelial cells.Morphological changes of transfected cells were observed by HE staining.The expression levels of TGF-?1 were assessed by ELISA.Cell cycle analysis was assessed by flow cytometry.DNA fragment analysis was used to confirm the presence of apoptosis.RESULTS:rhTGF-?1 in concentrations of 5-20 ?g/L showed a significant suppressive effect on the proliferation of corneal endothelial cells,0.5-1 ?g/L had no effect,0.05-0.1 ?g/L facilitated cell growth,as compared with negative controls.The morphous of transfected corneal cells had no significant abnormality compared with normal cells.According to the result of ELISA,the concentration of TGF-?1 in the supernatant was calculated to be(98?3)ng/L.Flow cytometry assay showed that S and G2/M phase of transfected cells decreased significantly compared with that of control group,but the cell cycle recovered normally after adding 10 ?g/L EGF into the culture medium.Agarose electrophoresis didn't show marked ladders in transfected group.CONCLUSION:Effects of rhTGF-?1 on the proliferation of corneal endothelial cells are different with various concentrations.TGF-?1 gene transfection shows suppressive effect on the proliferation of cultured corneal endothelial cells,but does not induce cell apoptosis.EGF is the antagonist of this suppressive effect.

Effect of diterpenoid compound 5F of Peris semipinnata L on apoptosis of human pterygium body fibroblasts in vitro / 中国病理生理杂志

AIM: To observe the effect of diterpenoid compound 5F of Pteris semipinnate L on apoptosis in cultured human pterygium body fibroblasts(HPFs).METHODS: Fibroblasts collected from human pterygium body were cultured in vitro with 5F at different time point.Transmission electron microscope(TEM) was used to observe the ultrastructure changes of the HPFs.The changes of the cell cycle and apoptosis of HPFs were evaluated with flow cytometry(FCM).RESULTS: 5F at the concentration of 32 mg/L induced HPF apoptosis at various time points.CONCLUSION: 5F induces HPFs apoptosis.Apoptotic cells are evolved to necrosis with the prolongation of the time.These findings indicate that 5F has a potential clinical value.

Immunosupressive mechanism of cornus officinalis glycosides on the corneal allograft rejection in rats / 中国病理生理杂志

AIM: To investigate the influence of the cornus officinalis glycosides (COG) on immunological function of corneal transplantation model of rats, and to clarify the immunosuppressive mechanism of COG through observing the activation of lymphocytes in blood. METHODS: Wister rats were used as recipients and SD rats were used as corneal graft donors, then the corneal allografts transplantation model on the closed colony rats were set up. Splenocytes proliferation and mixed lymphocyte reaction of Wister rats activated by ConA were observed. The phenotype change of CD4, CD8, CD25 in blood in different time postoperatively were observed by the di-sign flow cytometry, and the rate of CD4/CD8 was calculated. RESULTS: 1. The COG suppressed the proliferation of T lymphocytes and one-way mixed lymphocyte reaction on the corneal allografting. 2. The phenotype change of lymphocytes in boold was as follows: there was no significant difference between the different time of the CD4, CD8 expression and the CD4/CD8 rate in blood of the control group. The CD4 positive cells expressed CD25 postoperatively increased obviously. The CD4/CD8 rate of medicine group had the tendency to decrease. The CD4 positive cells expressed CD25 postoperation in the medicine group were less than that in the control group obviously. CONCLUSION: The suppression of the T lymphocyte proliferation, mixed lymphocyte reaction, CD molecule expressed by the activated T lymphocytes and the IL-2 receptor expression may be the main immunosuppressive mechanisms of Cornus officinalis glycosides on the cell-mediated immunity.

Effect of heterogenic corneal stroma transplantation on peripheral T cells of rats / 中国病理生理杂志

0.05), the expression of CD4+ was increased in rabbit corneal stroma (P

Effect of cornus officinalis glycosides ophthalmic solution on the corneal allograft rejection / 中国病理生理杂志

AIM: To observe the effect of cornus of ficinalis glycosides(COG) ophthalmic solution on the corneal allograft rejection by topical instillation. METHODS: The corneal transplantation model on the closed colony rats was established. The rejection time of all animals was recorded and compared by slit-lamp microscope. The pathologic changes were measured by immunohistochemistry and scanning electron microscope.RESULTS: The histopathological and immunohistochemistry findings showed that the lymphocytes, neovascularity and the expression of ICAM-1 in COG-treated group were significantly fewer than that in control group at 15 d after operation.CONCLUSION: COG ophthalmic solution prevents and suppresses the corneal allograft rejection.

Mechanisms of oral tolerance and the treatment of autoimmune diseases / 中国病理生理杂志

Oral tolerance describes the phenomenon that orally administered proteins induce systemic hyporesponsiveness to the protein fed. The primary mechanisms by which oral tolerance is mediated include clonal deletion, clonal anergy and active cellular suppression through gut-associated lymphoid tissue(GALT). Low doses favor active suppression mediated by Th2 and Th3 cells, whereas high doses favor deletion and anergy mediated by Th1 and Th2 cells. Oral tolerance is an effective and specific approach without toxicity. In recent years, it has been used successfully to treat autoimmune diseases in model animals and patients. The article discussed the mechanisms and advances of oral tolerance for the purpose of providing new ways of treat autoimmune diseases.

Studies of cell sheet engineering on fibrin glue cultured with three kinds of rabbit corneal cells / 中国病理生理杂志

AIM:To study whether three kinds of rabbit corneal cells can grow well on fibrin glue (FG) constructed in vitro, and investigate the feasibility of FG for the scaffold of cell sheet engineering. METHODS: Three kinds of corneal cells were seeded on FG which was produced in vivo. Cell growth on FG was examined as follows: (1) by inverted microscope; (2) histologically by hematoxylin and eosin; (3) by scanning electron microscopy. RESULTS: Fibrin glue prepared was smooth and transparent. With cell growth, FG degradated partly, then obtained cell sheet engineering only with a small amount of FG. Corneal cells generated well on the fibrin glue in vitro and maintained the physiological state of cells. Corneal epithelial cells formed unilaminar and stratified layers and cellular joins. Corneal endothelial cells formed round or polygon, the same size cells and lined up tightly. Corneal stroma cells formed triangle and arborization, cell-cell junction was obvious, and formed network link. CONCLUSION: Fibrin glue is well compatible with three kinds of corneal cells, which can construct tissue engineered cell sheet with fibrin glue, so as to reconstruct ocular surface.