Decellularization of porcine corneas and repopulation with human corneal cells for tissue‐engineered xenografts

Purpose: To evaluate the potential use of decellularized porcine corneas (DPCs) as a carrier matrix for cultivating human corneal cells in tissue engineering. Methods: Corneal cells were isolated from human corneoscleral rims. Porcine corneas were decellularized using hypotonic tris buffer, ethylene diamine tetra‐acetic acid (EDTA, 0.1%), aprotinin (10 KIU/ml) and 0.3% sodium dodecyl sulphate. Haematoxylin–eosin (HE) and 4,6‐diamidino‐2‐phenylindole (DAPI) staining were performed to confirm removal of the corneal cells. Quantitative analysis was performed to determine levels of desoxyribonucleic acid (DNA) using DNA Purification Kit (Fermentas, St. Leon‐Rot, Germany). Alcian blue staining was carried out to analyse the structure of the extracellular matrix (ECM). Corneal stromal cells were injected into the DPCs; limbal corneal epithelial cells and corneal endothelial cells were seeded onto the anterior and posterior surfaces of the DPCs, respectively. Evaluation was undertaken at days 14 and 30. The phenotypical properties of the cultivated corneal cells were investigated using Immunolocalization of type I collagen, keratocan, lumican, cytokeratin 3 (AE5) and type VIII collagen. Results: Haematoxylin–eosin and DAPI staining showed efficient elimination of porcine corneal cells, whereas alcian blue confirmed gross preservation of the ECM. The quantitative analysis of the DNA content showed a significant reduction (mean before decellularization: 75.45 ± 13.71 ng/mg; mean after decellularization: 9.87 ± 2.04 ng/mg, p < 0.001). All three types of corneal cells were efficiently cultured and expanded on the DPCs. Conclusions: Decellularized porcine corneas might serve as a potential scaffold for tissue engineering of the cornea, possibly providing xenogenic substrate for corneal transplantation.     

A comparison of three methods of decellularization of pig corneas to reduce immunogenicity

AIM:To investigate whether decellularization using different techniques can reduce immunogenicity of the cornea, and to explore the decellularized cornea as a scaffold for cultured corneal endothelial cells (CECs). Transplantation of decellularized porcine corneas increases graft transparency and survival for longer periods compared with fresh grafts.METHODS: Six-month-old wild-type pig corneas were cut into 100-200 μm thickness, and then decellularized by three different methods:1) 0.1% sodium dodecyl sulfate (SDS); 2) hypoxic nitrogen (N2); and 3) hypertonic NaCl. Thickness and transparency were assessed visually. Fresh and decellularized corneas were stained with hematoxylin/eosin (H&E), and for the presence of galactose-α1,3-galactose (Gal) and N-glycolylneuraminic acid (NeuGc, a nonGal antigen). Also, a human IgM/IgG binding assay was performed. Cultured porcine CECs were seeded on the surface of the decellularized cornea and examined after H&E staining.RESULTS:All three methods of decellularization reduced the number of keratocytes in the stromal tissue by >80% while the collagen structure remained preserved. No remaining nuclei stained positive for Gal or NeuGc, and expression of these oligosaccharides on collagen was also greatly decreased compared to expression on fresh corneas. Human IgM/IgG binding to decellularized corneal tissue was considerably reduced compared to fresh corneal tissue. The cultured CECs formed a confluent monolayer on the surface of decellularized tissue.CONCLUSION:Though incomplete, the significant reduction in the cellular component of the decellularized cornea should be associated with a significantly reduced in vivo immune response compared to fresh corneas.     

Acellular ostrich corneal stroma used as scaffold for construction of tissue-engineered cornea

AIM: To assess acellular ostrich corneal matrix used as a scaffold to reconstruct a damaged cornea. METHODS: A hypertonic saline solution combined with a digestion method was used to decellularize the ostrich cornea. The microstructure of the acellular corneal matrix was observed by transmission electron microscopy (TEM) and hematoxylin and eosin (H&E) staining. The mechanical properties were detected by a rheometer and a tension machine. The acellular corneal matrix was also transplanted into a rabbit cornea and cytokeratin 3 was used to check the immune phenotype. RESULTS: The microstructure and mechanical properties of the ostrich cornea were well preserved after the decellularization process. In vitro, the methyl thiazolyl tetrazolium results revealed that extracts of the acellular ostrich corneas (AOCs) had no inhibitory effects on the proliferation of the corneal epithelial or endothelial cells or on the keratocytes. The rabbit lamellar keratoplasty showed that the transplanted AOCs were transparent and completely incorporated into the host cornea while corneal turbidity and graft dissolution occurred in the acellular porcine cornea (APC) transplantation. The phenotype of the reconstructed cornea was similar to a normal rabbit cornea with a high expression of cytokeratin 3 in the superficial epithelial cell layer. CONCLUSION: We first used AOCs as scaffolds to reconstruct damaged corneas. Compared with porcine corneas, the anatomical structures of ostrich corneas are closer to those of human corneas. In accordance with the principle that structure determines function, a xenograft lamellar keratoplasty also confirmed that the AOC transplantation generated a superior outcome compared to that of the APC graft.     

异种角膜基质材料的制备和体外细胞种植的实验研究

目的研究异种角膜基质生物支架材料的制备方法和体外种植兔角膜基质细胞后细胞在材料上的生长、增殖情况。方法将York猪全层角膜用去垢剂联合0．25％胰蛋白酶、DNA-RNA酶祛除猪角膜基质细胞并冻干制备成支架材料；将兔角膜组织块用胶原酶消化后，用含10％血清的DMEM培养液体外培养，并做波形丝蛋白，角蛋白免疫组织化学染色检测；将培养的兔角膜基质细胞的2～3代接种在材料上，培养5d后，做HE染色、扫描电镜观察。结果猪角膜基质片经脱细胞处理后，细胞成分祛除干净并保留了角膜组织的三维网格状结构，网状间隙明显增大，利于细胞生长；体外培养的兔角膜基质细胞波形丝蛋白染色为阳性、角蛋白染色为阴性；HE染色、扫描电镜结果显示兔角膜基质细胞在支架材料上生长、增殖良好。结论异源性角膜经祛脱细胞处理而获得的生物支架材料利于异种细胞的黏附和增殖，可进一步用于组织工程角膜的研究。     

Decellularized porcine cornea-derived hydrogels for the regeneration of epithelium and stroma in focal corneal defects

PurposeHydrogels derived from decellularized tissues provide superior biocompatibility, tenability and tissue-specific extracellular matrix (ECM) components. Based on the preparation of decellularized porcine cornea (DPC), here we developed an injectable and transparent hydrogel for the regeneration of epithelium and stroma in focal corneal defects.MethodsThe DPC-derived hydrogel was prepared with N-cyclohexyl-N′-(2-morpholinethyl) carbodiimide metho-p-toluenesulfonate/N-hydroxysuccinimide (CMC/NHS) as cross-linkers. The characteristics of the hydrogel were analyzed and its cytocompatibility was assessed by Live/Dead and Cell Counting Kit (CCK)-8 assays. Immunofluorescence staining, quantitative PCR and Western blot analyses were performed to assess the relative protein and gene expression in corneal fibroblasts on hydrogel. The safety and efficiency of the hydrogel for repairing focal corneal defects in rabbit were measured by slit-lamp, anterior segment optical coherence tomography (AS-OCT), confocal microscopy and histological analyses.ResultsThe DPC-derived hydrogel cross-linked with CMC/NHS assumed favorable transparency, exhibited distinct mechanical properties and preserved the ECM components of native porcine cornea (NPC). In vitro experiments showed that the hydrogel maintained the phenotype, supported the proliferation and promoted the ECM synthesis of corneal fibroblasts. When injected onto rabbit corneas, the hydrogel rapidly covered, solidified and formed a smooth surface on the focal defect. Corneal epithelium was fully regenerated within 3 days. The thickness of the corneal epithelium and stroma was restored at 12 weeks after surgery without significant inflammation or scar formation. Notably, the hydrogel showed no harmful effects on the resident stroma and endothelium.ConclusionsThe DPC-derived hydrogel may represent a promising biomaterial for corneal epithelial and stromal regeneration.     

Efficacy of pig-to-rhesus lamellar corneal xenotransplantation

PURPOSE. To solve the shortage of donor corneas, a decellularizing method based on hypertonic saline treatment was introduced, and a favorable outcome was observed in pig-to-rabbit lamellar corneal transplantation. This study was an investigation of the efficacy of pig-to-nonhuman primate lamellar corneal transplantation, using both decellularized and fresh porcine corneas to assess feasibility as a substitute for human corneas. METHODS. Nine Chinese rhesus macaques underwent lamellar corneal transplantation using both decellularized (n = 5) and fresh (n = 4) porcine corneas. Clinically acceptable graft size (7.5 mm in diameter) and minimal immunosuppression based on topical and systemic corticosteroids were applied. Rejection signs, histology of porcine grafts, and serial changes in recipients' blood profile, including memory T-cell subset, anti-α-Gal and donor pig-specific antibodies, and complement were evaluated. Changes in aqueous complement concentration were also assessed at 4 weeks after transplantation. RESULTS. Of the decellularized porcine lamellar grafts, 80% remained transparent for more than 6 months, whereas half of the fresh porcine lamellar grafts developed chronic rejection. Rejected grafts showed extensive cellular infiltration, predominantly CD8(+) T lymphocytes and macrophages. Immunologic profiles of the recipients with rejected grafts showed a significant increase in the concentration of aqueous complement, an enhancement of memory T cells, and an abrupt increase in donor pig-specific antibodies. CONCLUSIONS. The findings suggested that decellularized porcine cornea could be a promising substitute for human corneal allograft. Fresh porcine cornea may be a feasible option for a substitute if combined with more potent immunosuppression or if obtained from transgenic pigs with complement-regulatory proteins.     

Acellular porcine corneal matrix as a carrier scaffold for cultivating human corneal epithelial cells and fibroblasts in vitro

AIM: To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts, and an acellular porcine cornea matrix (APCM) in vitro. METHODS: The scaffold was prepared from fresh porcine corneas which were treated with 0.5% sodium dodecyl sulfate (SDS) solution and the complete removal of corneal cells was confirmed by hematoxylin-eosin (HE) staining and 4’, 6-diamidino-2-phenylindole (DAPI) staining. Human corneal fibroblasts and epithelial cells were cultured with leaching liquid extracted from APCM, and then cell proliferative ability was evaluated by MTT assay. To construct a human corneal anterior lamellar replacement, corneal fibroblasts were injected into the APCM and cultured for 3d, followed by culturing corneal epithelial cells on the stroma construction surface for another 10d. The corneal replacement was analyzed by HE staining, and immunofluorescence staining. RESULTS: Histological examination indicated that there were no cells in the APCM by HE staining, and DAPI staining did not detect any residual DNA. The leaching liquid from APCM had little influence on the proliferation ability of human corneal fibroblasts and epithelial cells. At 10d, a continuous 3 to 5 layers of human corneal epithelial cells covering the surface of the APCM was observed, and the injected corneal fibroblasts distributed within the scaffold. The phenotype of the construction was similar to normal human corneas, with high expression of cytokeratin 12 in the epithelial cell layer and high expression of vimentin in the stroma. CONCLUSION: Corneal anterior lamellar replacement can be reconstructed in vitro by cultivating human corneal epithelial cells and fibroblasts with an acellular porcine cornea matrix. This laid the foundation for the further transplantation in vivo.     

Antigenicity of porcine cornea as xenograft

PURPOSE: To investigate the antigenicity of porcine corneal stroma as xenograft to man. METHODS: The localization of alpha-gal epitope in the porcine eye was determined using biotinylated Griffonia simplicifolia 1 isolectin B4. Porcine corneal stromal was inserted into corneal stromal pockets of cynomolgus monkeys. Immunohistochemistry was performed to analyze the immunological reaction in the monkey. RESULTS: Immunohistochemistry showed no alpha-gal epitope in the porcine cornea except for several keratocytes in the anterior-most part. Haze and keratic precipitates developed in two corneas out of three corneas that were followed up until 6 months after the surgery. In these two corneas, infiltrating cells included CD4+, CD8+, or HAM56+ cells, suggesting that haze and keratic precipitates were induced by cellular rejection to porcine corneal stroma. CONCLUSIONS: Porcine corneal stroma induces no hyperacute rejection but mild cellular rejection when transplanted in the cornea of cynomolgus monkeys.     

异种角膜脱细胞基质为供体进行角膜前板层移植的初步研究

目的探讨以异种猪角膜脱细胞基质为供体植片,分析兔角膜进行前板层移植后的生物相容性：设计实验研究。研究对象新西兰白兔。方法应用1％TritonX-100及冷冻干燥处理制备猪角膜脱细胞基质载体,切取1／3厚度前板层作为供体角膜植片,对兔眼角膜前板层切除后进行移植,同时以新鲜猪角膜板层为供体对兔进行前板层移植作对照。通过术后角膜透明度、组织结构观察,评价猪角膜脱细胞基质的生物相容性及植片转归的情况。主要指标角膜透明度和组织学HE染色。结果制备的猪角膜脱细胞基质植片作前板层移植到兔眼后,未见明显的新生血管、炎症反应、角膜坏死等排斥现象,观察期内植片较透明;脱细胞基质表面上皮化良好,植片基质板层与植床逐渐融合,植片内有宿主细胞迁入生长,板层结构与正常角膜相似。结论猪角膜脱细胞基质具有良好的生物相容性、安全性和低抗原性,有望成为角膜板层移植的供体材料。     

脱细胞猪角膜基质的生物相容性与组织工程化兔角膜上皮移植的研究

目的 探讨脱细胞猪角膜基质的生物相容性,评价组织工程化角膜上皮组织作供体的可行性,观察支架材料的细胞化情况和种子细胞的存活情况.方法 实验研究.采用完全随机化设计的方法,用Dispase-Triton-X-100处理猪角膜基质,脱去角膜细胞;以角膜基质囊袋内植入的方法,观察异种角膜基质植入后的生物相容性,A组:脱细胞猪角膜基质,B组:新鲜猪角膜基质,C组:空白对照组.以组织工程化雄性角膜上皮组织为供体,同种雌性为受体,作板层角膜移植,观察角膜的混浊、水肿、新生血管等情况;组织病理学和免疫组化方法检测支架材料的细胞化情况,Y染色体性别决定基因(SRY)-聚合酶链反应(PCR)方法追踪种子细胞的存活情况.结果 猪角膜基质植入兔角膜囊袋后,角膜逐渐恢复透明,排斥反应指数＜6,组织病理学观察角膜结构完整,胶原纤维平行排列,少许细胞长入脱细胞猪角膜基质边缘,各组免疫组化检测未见CIM+、CD8+T淋巴细胞浸润.组织工程化角膜上皮作异体板层角膜移植后,3～4 d上皮光滑,10～20 d变为透明;15 d时角膜上皮、基质、内皮完整,上皮细胞约4或5层结构,少许基质细胞长入支架,1个月时可见角膜上皮细胞约7或8层细胞,基质纤维排列规则,多量细胞长入脱细胞角膜基质.上皮细胞表达CK3,支架内新生细胞表达波形蛋白.SRY-PCR结果显示种子细胞可以在受体内长期存活.结论 脱细胞猪角膜基质生物相容性良好,组织工程化角膜上皮可作为板层角膜移植的供体,脱细胞猪角膜基质细胞化良好,种子细胞可以在受体内长期存活.     

Viscoanesthesia. Part I: toxicity to corneal endothelial cells in a rabbit model

PURPOSE: To evaluate the toxicity of a solution combining sodium hyaluronate 1.5% with lidocaine (0.5%, 1.0%, or 1.65%) to the rabbit corneal endothelium. SETTING: Center for Research on Ocular Therapeutics and Biodevices, Storm Eye Institute, Medical University of South Carolina, Charleston, South Carolina, USA. METHODS: Each rabbit cornea was excised, and the endothelium was exposed to 1 of the following solutions for 20 minutes: viscoanesthetic solution (0.5%, 1.0%, or 1.65% lidocaine in sodium hyaluronate 1.5%; 5 corneas each), sodium hyaluronate 1.5% (n = 5), balanced salt solution (BSS(R)) (n = 5), mitomycin-C 0.02% (n = 2), dextran 15% (n = 2), or distilled water (n = 2). The endothelium was then stained with trypan blue and alizarin red. Two corneas were stained immediately after excision. Cell morphology and damage to the corneal endothelium were analyzed by microscopic examination. RESULTS: The endothelium in the corneas of the viscoanesthetic groups was comparable to that in the sodium hyaluronate 1.5% and the BSS groups and to the corneas not exposed to any solution. In some areas of the 1.0% and the 1.65% viscoanesthesia groups, the corneal endothelial cells presented irregular intercellular borders. Staining with trypan blue, which indicates cellular damage, was observed in some linear areas corresponding to corneal folds in all groups. The folds were probably caused during manipulation for corneal excision and staining. The corneal endothelium was destroyed in the mitomycin group. In the dextran and distilled-water groups, morphological alterations probably resulting from osmotic changes were observed. CONCLUSIONS: The 3 concentrations of viscoanesthetic solutions appeared to be safe to rabbit corneal endothelium.     

两种新型无血清培养基器官培养保存兔角膜内皮细胞活性的实验研究

目的研究两种新型培养基-内皮细胞培养基（ECM）和无动物成分培养基（ACF）在无血清器官培养法保存角膜内皮细胞活性方面的效果。方法将64枚兔角膜平均分为4份,分别密闭保存于ECM＋2％胎牛血清（FBS）、ECM和ACF等4种不同的培养基中4周,再以葡聚糖巧oo脱水2d。伊格尔最低基础培养基（MEM）作对照组。检测保存前后角膜内皮细胞密度、角膜厚度、内皮层中肌动蛋白微丝（F-actin）的表达和透射电镜下内皮细胞超微结构的变化。结果保存结束后,ECM＋2％FBS组的兔角膜内皮细胞密度最高（21234-240）个／mm^2,ECM和ACF组结果与其接近,内皮细胞死亡率也较低,对照组内皮细胞密度最低（1732±144）个／mm^2,3个实验组与对照组间差异有统计学意义（F=23．180,P=0．000）。免疫组织化学染色显示F-actin在兔角膜内皮层成功表达。WB半定量检测表明F-actin蛋白在各组角膜内皮层的表达趋势与内皮细胞密度计数结果一致,3个实验组与对照组间差异有统计学意义（F=159．876,P：0．000）。3个实验组内皮细胞的超微结构与正常角膜区别较小。结论在器官培养法保存过程中,无血清的ECM和ACF培养基可以较传统MEM培养基更好地保持角膜内皮细胞的活性和屏障功能,在无血清器官培养保存角膜方面具有应用前景。     

Evaluation of novel decellularizing corneal stroma for cornea tissue engineering applications

AIM: To develop a new decellularization method depended upon the natural corneal structure and to harvest an ideal scaffold with good biocompatibilities for corneal reconstruction. METHODS: The acellular cornea matrix (ACM) were prepared from de-epithelium fresh porcine corneas (DFPCs) by incubation with 100% fresh human sera and additional electrophoresis at 4℃. Human corneal epithelial cells (HCEs) were used for the cytotoxicity tests of ACM. ACM were implanted into the Enhanced Green Fluorecence Protein (eGFP) transgenic mouse anterior chamber for evaluation of histocompatibility. RESULTS: HE and GSIB4 results showed fresh porcine cornea matrix with 100% human sera and electrophoresis could entirely decellularize stromal cell without reducing its transparency. ACM had no cytotoxic effect ex vivo. Animal test showed there was no rejection for one month after surgery. CONCLUSION: These results provide a decellularizing approach for the study of corneal tissue engineering and had the broader implications for the field of biological tissue engineering in other engineered organ or tissue matrix.     

锥兰联合茜素红染色证实保存后的兔角膜内皮细胞活性

我们应用锥兰联合茜素红染色证实保存后的兔角膜内皮细胞活性。我们随机的将离体的新西兰白兔眼球分为四组，每组５只。将房水抽空，随即前房内注入Ｃ３Ｆ８（全氟乙烷，惰性气体）气体，于湿房４℃条件下分别保存５，７，１０，１４天，然后取下带巩膜环的角膜片，内皮面向上放在角膜容器内，将０．２５％锥兰溶液滴于内皮面，１分半钟后将染料洗净，再将０．２％的茜素红溶液滴于内皮面，染色１分半钟，将染料洗净，将角膜片放于２     

Epi-laser in situ keratomileusis: comparative evaluation of epithelial separation with 3 microkeratomes

PURPOSE: To evaluate the cleavage plane, corneal cytoarchitecture, and cell vitality of separated corneal epithelial sheets created with 3 commonly used microkeratomes. SETTING: Laboratories of the Regensburg University Medical Center, Regensburg, Germany. METHODS: Mechanical separation of the epithelial layer in 10 porcine eyes and 2 human eyes was performed with 3 different microkeratomes (Amadeus II, Zyoptix XP, Epivision). Five of 10 porcine corneas and the 2 human corneas treated with each microkeratome were processed for histology, electron microscopy, and immunohistochemistry. In 5 of 10 porcine corneas, the corneal epithelial sheets were separated from the globe and cell vitality was assessed with the trypan blue dye vitality test. RESULTS: A reproducible epithelial separation with a smooth surface was achieved in all eyes. The cleavage plane was located between the lamina lucida and the lamina densa. Damage to epithelial cells was mainly limited to the cut margins. CONCLUSIONS: Mechanical separation of the epithelial sheet in epithelial laser in situ keratomileusis (epi-LASIK) was safe and reproducible with all evaluated microkeratomes. Immunohistochemistry and electron microscopy showed the cleavage plane in epi-LASIK was between the basal epithelium and the basement membrane at the level of the lamina lucida.     

去端肽猪皮Ⅰ型胶原在兔角膜组织中的生物相容性

目的 评价去端肽猪皮Ⅰ型胶原在兔角膜组织中的生物学反应,以探讨其作为角膜支架材料的可行性.方法 大耳白兔40只,分为5组,每组8只,一眼为实验眼,另一眼作为正常对照眼.将去端肽猪皮Ⅰ型胶原植入兔角膜前板层,术后1个月内每周2次、1个月后每月1次行肉眼、裂隙灯观察,并于每次观察后记录角膜是否透明、有无新生血管的评分情况;术后3 d、14 d、1个月、3个月及6个月,取兔角膜行组织病理学观察以及上皮细胞标志蛋白K3的免疫组化检测.评分结果采用Friedman秩和检验行组间比较,两两之间比较行Wilcoxon符号秩和检验.结果 术后角膜透明度逐渐恢复,新生血管化程度逐渐升高,至术后1个月最高,后逐渐减轻并退化;术后6个月,角膜透明度和新生血管化程度与正常眼比较,差异无统计学意义（P＞0.05）.整个观察期间未见角膜坏死、溃疡及植片脱出.结论 去端肽猪皮Ⅰ型胶原具有较好的生物相容性,进一步改进后有望成为一种新型的角膜移植支架材料.     

In-situ porcine corneal matrix hydrogel as ocular surface bandage

PurposeBioactive substrates can be used therapeutically to enhance wound healing. Here, we evaluated the effect of an in-situ thermoresponsive hydrogel from decellularized porcine cornea ECM, COMatrix (COrnea Matrix), for application as an ocular surface bandage for corneal epithelial defects.MethodsCOMatrix hydrogel was fabricated from decellularized porcine corneas. The effects of COMatrix hydrogel on attachment and proliferation of human corneal epithelial cells (HCECs) were evaluated in vitro. The effect of COMatrix on the expressions of the inflammatory genes, IL-1β, TNF-α, and IL-6 was assessed by RT-PCR. The in-situ application and also repairing effects of COMatrix hydrogel as an ocular bandage was studied in a murine model of corneal epithelial wound. The eyes were examined by optical coherence tomography (OCT) and slit-lamp microscopy in vivo and by histology and immunofluorescence post-mortem.ResultsIn vitro, COMatrix hydrogel significantly enhanced the attachment and proliferation of HCECs relative to control. HCECs exposed to COMatrix had less induced expression of TNF-α (P < 0.05). In vivo, COMatrix formed a uniform hydrogel that adhered to the murine ocular surface after in-situ curing. Corneal epithelial wound closure was significantly accelerated by COMatrix hydrogel compared to control (P < 0.01). There was significant increase in the expression of proliferation marker Ki-67 in wounded corneal epithelium by COMatrix hydrogel compared to control (P < 0.05).ConclusionsCOMatrix hydrogel is a naturally derived bioactive material with potential application as an ocular surface bandage to enhance epithelial wound healing.     

组织工程角膜三维构建的实验研究

目的以人胚胎干细胞（hESC）诱导细胞为种子细胞,以脱细胞猪角膜基质（APCM）为支架三维构建生物工程角膜,以期用于穿透性角膜移植,解决角膜供体极度匮乏的难题。方法实验研究。无菌条件下将新鲜猪角膜组织置于0.5% SDS溶液中4 ℃脱细胞 24 h,获取APCM。将hESCs与人角膜基质细胞通过Transwell共培养5 d,获取眼周间充质干细胞（POMPs）,再于人晶状体上皮细胞源性条件培养基继续培养14 d获取角膜内皮样细胞并进行鉴定和筛选纯化。将纯化后扩增的角膜内皮样细胞接种于APCM构建角膜内皮植片,并移植入角膜内皮功能失代偿动物模型进行泵功能评估;采用人角膜缘干细胞（LSCs）来源的条件培养基培养hESCs 12 d,诱导其分化人角膜上皮样细胞并筛选鉴定,将其与APCM构建的角膜上皮植片移植于LSC失代偿动物模型的角膜缘,观察其眼表修复能力。结果诱导的人角膜内皮样细胞表达内皮细胞相关标记物vimentin、N-cadherin、Na+/K+ATP酶和ZO-1。构建的角膜内皮植片能够促使角膜内皮功能失代偿动物的角膜逐渐恢复透明。构建的角膜上皮细胞植片具有4～5层细胞复层结构,类似于正常角膜上皮,且能够一定程度上修复LSC失代偿动物模型眼表。结论采用hESCs诱导分化来源的细胞与APCM构建的人角膜内皮植片和人角膜上皮植片具有类似于正常角膜的功能,为全层生物角膜的构建提供了良好的实验和理论基础,具有良好的临床应用前景。     

无血清器官培养法保存大鼠角膜内皮细胞的活性

目的:研究两种新型培养基-内皮细胞培养基(endothelial cell medium,ECM)和无动物成分培养基(animal compoundfree medium,ACF)在无血清器官培养法保存角膜内皮细胞活性方面的应用效果。方法:将大鼠角膜密闭保存于ECM和ACF培养基中4wk,再经葡聚糖T500脱水2d。以含低浓度牛血清的MEM基础培养基作为对照。计数保存前后角膜内皮细胞密度,检测保存结束大鼠角膜内皮层中胞质紧密粘连蛋白1(zonula occludens-1,ZO-1)的表达水平,评估内皮细胞层的屏障功能。结果:保存结束后,MEM+20mL/L FBS对照组大鼠的角膜内皮细胞密度值最低,ECM+20mL/L FBS组则高达2250±202个/mm2,ECM和ACF组结果与ECM+20mL/LFBS组接近,细胞死亡率也较低,组间差异有统计学意义(F=28.965,P=0.000)。冰冻切片显示ZO-1在保存后大鼠角膜内皮层成功表达。RT-PCR检测表明ZO-1mRNA在各组的表达趋势与内皮细胞密度结果一致(F=592.751,P=0.000)。结论:无血清ECM和ACF培养基可以很好地保持器官培养法保存角膜内皮细胞的活性和紧密连接的屏障功能。