脱水保存角膜基质为载体培养角膜内皮细胞的实验研究

目的探讨以脱水保存角膜基质/后弹力层为载体培养角膜内皮细胞,构建组织工程化角膜内皮细胞移植膜的可行性及其机理。设计实验性研究。研究对象体外培养的兔角膜内皮细胞和脱水保存角膜基质/后弹力层。方法兔角膜经中性蛋白酶37°C孵育5min,去除内皮细胞保留后弹力层和角膜基质,无水氯化钙脱水后低温保存,使用前磷酸盐缓冲液复水。纯化的角膜内皮细胞接种于基质载体的后弹力层上进行体外培养,直至生长融合为细胞单层,倒置显微镜下观察细胞形态学变化。在不同时间点(1、2、4、6d)收集植片进行HE染色和电镜检测,分析组织结构的变化。主要指标角膜内皮细胞在脱水保存角膜基质/后弹力层载体上形成单层时间和生长特性,组织工程化角膜内皮细胞移植膜的三维结构和超微结构。结果角膜内皮细胞在载体上快速贴壁生长并增殖,体外培养6～7d即融合成单层,复合角膜内皮组织由基质/后弹力层和单层扁平内皮细胞组成,与生理状态下的角膜内皮组织相近。电镜下组织培养的兔角膜内皮细胞间连接紧密,细胞为多边形,胞核清晰,具有正常兔角膜内皮细胞的超微结构。结论角膜内皮细胞能够在干燥脱水保存基质/后弹力层载体上良好生长,并形成形态结构与正常角膜内皮组织相似的细胞单层,为角膜内皮细胞移植提供了新的载体选择。(眼科,2006,15:164-168)     

异种角膜脱细胞基质和几丁糖载体构建角膜基质层的比较

目的探索异种角膜脱细胞基质和几丁糖为载体构建生物角膜组织的可行性，评价二者的结构、功能和生物相容性。方法l％TritonX-100及冷冻干燥处理获得猪角膜脱细胞基质，网状几丁糖材料制备成似角膜片状形态，将体外培养的兔角膜基质细胞种植于两种载体，体外培养2周，形成细胞载体复合物。对构建的角膜组织进行苏木精-伊红染色、扫描电镜观察；同时将两种生物材料移植到兔角膜基质囊袋内，观察其生物相容性。结果角膜基质细胞在两种载体上皆可黏附生长，并分泌细胞外基质。脱细胞基质载体内细胞形态不规则，位于胶原板层间，形成类似正常组织的角膜基质结构；细胞可在几丁糖网状纤维上黏附并包绕生长。两种载体移植到兔角膜囊袋后，脱细胞基质降解较慢，与宿主角膜组织相融性良好，未发生明显排斥及毒性反应；几丁糖载体降解较迅速，但降解产物诱导较严重的排斥反应。结论异种角膜脱细胞基质保留正常组织的胶原板层结构，并具角膜的韧性和良好的生物相容性，更适于作为体外构建生物角膜的载体，而几丁糖材料则需要在构成结构和性能等方面进一步改进．     

以羊膜为载体培养角膜内皮细胞的实验研究

目的 以羊膜为载体培养角膜内皮细胞并形成单层组织，为以后内皮细胞层移植提供方法和材料来源。方法 胰酶消化去除羊膜上皮细胞，保留其底膜作为载体，种植兔角膜内皮细胞，体外培养形成单层内皮层，并进行形态，组织学，超微结构观察。结果 角膜内皮细胞在羊膜上黏附生长并增殖，体外培养6-7d即融合成单层，其形态及生长密度优于单纯培养皿上培养的内皮细胞。复合角膜内皮组织由羊膜基底膜和单层扁平内皮细胞组成，与生理状态下的角膜内皮组织相近。结论 以羊膜为载体培养角膜内皮细胞可形成单层细胞层，有望成为角膜内皮移植良好的材料来源。     

以异种角膜脱细胞基质为载体构建角膜上皮-载体-内皮复合物的实验研究

目的探讨以异种角膜脱细胞基质为载体,体外构建生物角膜的可能性和方法。方法应用去垢剂1％TritonX-100冷冻干燥处理制备猪角膜脱细胞基质载体,在其上皮面和内皮面分别接种兔角膜上皮细胞和内皮细胞,体外培养2周。将复合物制成组织切片,在光镜下观察组织形态（HE染色）,采用免疫组织化学方法检测角膜上皮特异性细胞角蛋白3（CK3）,使用锥虫蓝联合茜素红染色观察内皮细胞,在扫描电镜下观察上皮面和内皮面的超微结构。结果体外培养生物角膜获得上皮、无细胞基质和内皮三层复合结构。4或5层复层上皮中以扁平细胞为主,胞质内特异性CK3表达阳性;内皮层为一连续的单层扁平细胞,细胞活性良好,锥虫蓝联合茜素红染色显示组织呈典型的蜂巢样镶嵌结构。扫描电镜下观察,在载体的上皮面细胞呈复层样生长,形态近似扁平与梭形之间;内皮面为多边形单层细胞,表面具有微绒毛结构。结论构建的生物角膜为上皮一脱细胞基质载体一内皮复合物,初具角膜的雏形结构。异种角膜脱细胞基质提供了良好的细胞生长界面,有望成为体外构建角膜的载体材料。     

家兔角膜内皮细胞的快速培养

目的:短期内体外扩增获得角膜内皮细胞。方法:采用揭膜法与消化法相结合获取原代角膜内皮细胞。并采用本实验室自行研制的角膜内皮细胞培养体系,在24孔培养板中分别以1×106,5×105,1×105,5×104,1×104,5×103细胞/孔浓度传代培养细胞,观察细胞的生长情况。结果:以1×105～5×105个细胞/孔浓度的细胞悬液并添加消化后的Descemet膜原代培养细胞,4d后细胞形成与在体细胞相似的密集单层,可以快速获取纯化的角膜内皮细胞。传代培养时,虽然不同接种浓度在4d的细胞增殖倍数差异不显著,但以1×105～5×105个细胞/孔的浓度传代,3d就可生长成密集单层并可继续传代,现已传至第10代,但传至第4代,细胞形态开始发生改变;而以5×105个细胞/孔以上的浓度传代细胞时,1d的细胞贴壁率下降;以5×104个细胞/孔以下浓度传代,需7d方可长满皿底,并且细胞开始老化,体积变大,不能继续传代培养。或者细胞不能铺满皿底就衰老死亡。结论:本实验的培养体系可在短期内获得实验用角膜内皮细胞。角膜内皮细胞体外培养的增殖能力与其培养浓度和增殖的空间有关,1×105～5×105个细胞/孔的浓度是角膜内皮细胞快速增殖的适宜培养浓度。     

人胚肺成纤维细胞饲养兔角膜上皮和内皮细胞的体外培养

目的 研究人胚肺成纤维饲细胞体外培养兔角膜上皮和内皮细胞。方法 人胚肺成纤维细胞系细胞。经丝裂霉素C处理成饲细胞,兔角膜上皮和内皮细胞组织块原代培养,消化接种于人胚肺饲细胞瓶传代培养,体外培养的角膜上皮和内皮细胞行常规病理形态学检查。角膜上皮细胞行角蛋白,内皮细胞行神经烯醇化酶免疫组化染色检测。结果 人胚肺成纤维细胞形态均一。易于分辨,经丝裂霉素处理后推动增殖能力。角膜上皮和内皮细胞原代培养5-7天。细胞密集,经人胚肺饲细胞共培养5代。细胞无衰老现象,检测角膜上皮细胞角蛋白,内皮细胞神经烯醇化酶表达阳性,人胚肺饲细胞表达阴性。结论 人胚肺饲细胞能够明显增强角膜上皮和内皮细胞的体外生存能力。提高细胞的增殖能力。     

角膜内皮细胞改良培养技术的初步探讨

目的 评价角膜后弹力层撕除联合酶消化法分离角膜内皮细胞的效率,分析细胞体外生长的生物学特性.方法 将兔角膜带有内皮细胞的后弹力层完整撕下,用胰蛋白酶-EDTA联合酶消化,纯化的角膜内皮细胞进行体外培养.观察细胞形态,用神经元烯醇化酶抗体进行细胞表型鉴定.苏木精-伊红染色以及茜素红-台盼蓝联合染色分析细胞活性,流式细胞仪检测细胞体外生长过程中Anne xiv-PE的表达情况,透射电镜和扫描电镜进行细胞超微结构形态的观察.结果 带角膜内皮细胞的后弹力层撕除联合酶消化法可快速获得大量纯化的角膜内皮细胞,快速贴壁生长并增生,体外培养3～4d即融合成单层细胞,且表达神经元烯醇化酶抗体阳性,苏木精-伊红染色及活性染色提示细胞功能良好,Annexiv-PE抗体表达水平微弱.结论 角膜后弹力层撕除联合酶消化法可提高角膜内皮细胞的获取和培养效率,为工程化角膜内皮移植膜的构建提供稳定的种子细胞来源.     

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。     

保存人羊膜对人脐静脉内皮细胞生长状态的影响

目的:观察保存人羊膜对体外培养及移植状态下人血管内皮细胞生长状况的影响,研究羊膜移植治疗角膜新生血管性疾病的机制。方法:取健康产妇胎盘羊膜,以酶消化加机械分离法去上皮,-80℃甘油保存。使用时复水,平铺于培养板底,干燥备用。1g/L胰蛋白酶灌流消化法收获人脐静脉内皮细胞,以1～1.7×108/L分别接种于培养板及铺有羊膜的培养板孔内培养。倒置显微镜及电镜进行形态学观察。待羊膜上细胞生长成完整单层后,将载有血管内皮细胞的羊膜移植到去内皮细胞的猫眼角膜内皮侧,3mo后取角膜植片行组织病理学观察。结果:羊膜及培养板上培养的细胞均在24h完全贴壁,2～3d形成单层,细胞为铺路石样,Ⅷ因子染色阳性。植入猫眼内的人脐静脉内皮细胞在长达3mo的观察期内仍存活,有些甚至增生成多层排列。结论:体外培养及移植状态下,保存人羊膜都不能抑制血管内皮细胞增殖,其抑制角膜新生血管形成的机制可能不是通过内皮细胞途径。     

以异种后弹力膜基质为载体培养角膜内皮细胞的研究

目的　探讨以异种后弹力膜 (DM) /基质为载体培养角膜内皮细胞的可行性 ,为培养内皮细胞的临床移植提供新的方法和供体材料。方法　取厚 10 0 μm的猪角膜后层 ,冻存解冻后置中性蛋白酶 3 7℃孵育 5min ,去除内皮细胞 ,保留DM及薄层基质 ,置纯甘油中脱水保存 6个月 ;使用前复水 ,紫外线二面灭菌 ,制备成载体 ;接种兔角膜内皮细胞于载体的DM面并体外培养 ,行细胞形态、密度、组织学、超微结构的观察。结果　兔内皮细胞在猪DM/基质载体上贴附生长 ,形成紧密连接的单层 ,形态近似六角形分布 ;细胞密度达 ( 3 62 0± 110 2 3 )个 /mm2 ,具有正常兔角膜内皮细胞的超微结构。结论　角膜内皮细胞能够在异种DM/基质载体上良好生长 ,该载体有可能成为培养内皮细胞应用于临床移植的供体材料     

Descemet's membrane substrate from human donor lens anterior capsule

Background: To study the potential use of human donor anterior lens capsule as a Descemet's membrane substrate. Methods: Anterior lens capsules were recovered from the lenses of 30 cornea donors. Human corneal endothelial cells were recovered from the remaining corneal sclera rims of 15 donor corneas used for penetrating keratoplasty. Samples were sorted into three groups. Group 1 consisted of 10 samples in which the endothelial cells were allowed to grow on anterior lens capsules. In Group 2 human corneal endothelial cells grew on a collagen membrane and in Group 3 on polystyrene culture plates. Cell density, morphology and adherence of the cell–capsule complex were evaluated at 1, 4, 7 and 14 days with a phase‐contrast microscope, a scanning electron microscope and by histology. Cell viability was quantified by a microscopic live–dead assay. Expression of zonula occludens‐1, Na⁺/K⁺‐adenosine triphosphatase, tissue transglutaminase and vimentin were investigated by immunohistochemistry. Results: A mean diameter of 10.05 ± 0.13 mm of anterior capsule was obtained as a substrate for cell culture. Endothelial cell density of Group 1 was measured at 2455.4 ± 283.8 cells/mm², which was also comparable with the cell density of the control group. Cell viability was 95% or superior in all groups and multiple cellular interconnections developed between growing cells. Immunohistochemical analysis demonstrated strongly positive staining for all investigated proteins. Electron microscopy confirmed the adherence and monolayer growth of the endothelial cells. Conclusions: Human donor anterior lens capsule might therefore be a potential scaffold for the ex vivo expansion of human corneal endothelial cells.     

改良揭膜消化法分离培养恒河猴角膜内皮细胞

目的　分析改良揭膜消化法分离培养恒河猴角膜内皮细胞的可行性。方法　揭取恒河猴角膜后弹力层和内皮层,反复剪碎,用１ｍｇ·ｍＬ－１胶原酶Ａ消化１５ｍｉｎ,离心后重悬接种。待原代细胞长满８０％ ～９０％时,以４０×１０６个·Ｌ－１的密度传代。观察记录原代及传代细胞生长情况、传代时间,进行细胞鉴定,并与组织贴壁法相比较。结果　改良揭膜消化法获取的原代细胞生长较慢,２１ｄ融合成单层铺路石样结构,无基质细胞污染;传代细胞生长较快,５～６ｄ可传一代;神经元特异烯醇化酶表达阳性。组织贴壁法分离组织２４ｈ后组织片体积变小,透亮度明显下降,疑似溶解,高倍镜下观察显示,后弹力层纤维排列紊乱,细胞观察不清;继续培养至第５天,细胞完全溶解,组织片碎裂,轮廓不清,未见角膜内皮细胞爬出。结论　改良揭膜消化法能简易高效地获得大量恒河猴角膜内皮细胞。     

角膜内皮细胞移植

角膜内皮细胞在体外培养的条件下可以在同种异体或异种角膜后弹力层上成功生长,培养的上皮细胞的生长情况及特性与供体年龄、内皮细胞来源部位、载体材料、培养条件、以及是否应用生长因子有密切关系。人角膜内皮细胞培养的成功,以及在非人类的灵长目动物眼上的成功移植,为内皮移植技术的治疗作用提供了实验基础。培养传代的角膜内皮细胞移植到活体上后具有正常内皮细胞的功能,可以作为穿透性角膜移植的替代方法之一加以选择。     

In vitro system of corneal endothelium

A method for preparation of highly ordered monolayer cultures of the corneal endothelium was described. The technique of agar and palladium double-coating of culture plates was utilized for demarcation of the haptotactic area. The culture plate was prepared by precoating with agar and subsequent coating with palladium to define areas where growth of corneal endothelial cells was allowed. A micro-mass culture of corneal endothelial cells on the area defined by the palladium coating demonstrated their active growth and their reconstitution of the typical cellular pattern of the corneal endothelium.     

Human corneal endothelial cells: isolation, characterization and long-term cultivation

Long-term cultures of human corneal endothelial cells have been established. In culture, these cells form a dense monolayer (about 500,000 cells cm-2), similar to that found in vivo, and synthesize an extracellular matrix containing laminin, entactin, and fibronectin. Factor VIII and angiotensin-converting enzyme were not found in either the cultured or native corneal endothelium. Cells were obtained by scraping corneal buttons that had been preincubated in the culture medium supplemented with endothelial cell mitogen. The human corneal endothelium was grown under conditions virtually the same as those used for cultivation of human vascular endothelial cells, namely, on fibronectin- or gelatin-coated tissue culture plastic in Medium 199 supplemented with 20% human serum and 400 micrograms ml-1 endothelial cell growth supplement. Human corneal endothelial cells from the culture obtained can be used for transplantation onto human corneas, for studying repair of damaged corneal endothelium in situ, as well as for in vitro studies of cell growth regulation.     

Serum-free Media for Culturing and Serial-Passaging of Adult Human Retinal Pigment Epithelium

The ability of a chemically-defined serum-free culture medium to support the attachment, growth and serial passaging of primary adult human retinal pigment epithelial (RPE) cells was studied. Primary cultures of adult human RPE were established in a chemically-defined serum-free culture medium on both bare or bovine corneal endothelial extracellular matrix-coated tissue-culture plastic. Confluent cells were serially passaged in chemically-defined serum-free culture medium three times by trypsinization, and trypsin activity was quenched with aprotinin. First passage RPE cells were plated onto tissue-culture plastic precoated with bovine corneal endothelial extracellular matrix or uncoated tissue-culture plastic in 24 well plates at a density of 50 viable cells mm⁻². Cells were maintained either in chemically-defined serum-free culture medium, DMEM without serum, or DMEM with 15% fetal bovine serum. For each medium plating, efficiencies were determined 24 hours after plating, and growth rates were determined on the first, third and seventh days after plating. Morphometric image analysis was performed on cells cultured for up to 6 weeks and three serial passages. Seeding efficiency on bovine corneal endothelial extracellular matrix-coated tissue-culture plastic and treated tissue-culture plastic were higher for chemically-defined serum-free culture medium (88.9±2.7% and 47.1±4.1%, respectively) and DMEM with serum (87.2±5.6% and 52.9±10.5%, respectively) than DMEM without serum (59.2±5.6% and 33.1±6.9%, respectively;P<0.01). The RPE proliferation rate in chemically-defined serum-free culture medium was comparable to DMEM with serum on both substrates within the first 3 days, although cells in DMEM with serum had a higher proliferation rate on day 7. Cells cultured in DMEM without serum, eventually decreased in number. RPE maintained in chemically-defined serum-free culture medium maintained a consistent proliferation rate, reached confluence, and retained an epitheloid morphology on either extracellular matrix or tissue-culture plastic for up to 6 weeks and three serial passages. Primary RPE reached confluence at 12±3 days on bovine corneal endothelial extracellular matrix-coated tissue-culture plastic and 21±5 days on treated tissue-culture plastic. Confluent cultures were composed of small hexagonal cells with epitheloid morphology on both substrates. We concluded that primary adult human RPE can be cultured in this chemically-defined serum-free culture medium. RPE will proliferate, reach confluence, retain their epitheloid morphology and can be serially passaged in the absence of serum.     

Effects of intraocular irrigating solutions on the spreading of rabbit corneal endothelial cells on extracellular matrices

The effects of four commercially available irrigating solutions on the spreading of rabbit corneal endothelial cells on various extracellular matrices were studied. Cultured rabbit corneal endothelial cells, suspended in one of the following intraocular irrigating solutions, Opeguard MA, BSS, BSS Plus, lactated Ringer solution (Lactec) or physiological saline, were placed on uncoated tissue culture plates or on plates coated with extracellular matrices (fibronectin, laminin, collagen type I, or collagen type IV). The cell area was measured after 45 minutes' incubation. The cells spread on all of the extracellular matrices examined but not on the uncoated tissue culture plates. On the fibronectin or laminin matrix, the cell area was significantly greater with Opeguard MA or BSS Plus. On laminin and collagen type IV, the cell area was the greatest with Opeguard MA. On collagen type I, the cell area was significantly greater with Opeguard MA, BSS, or BSS Plus. These results demonstrated that the rabbit corneal endothelial cells responded to the extracellular matrices, and that Opeguard MA or BSS Plus provided more favorable conditions for the spreading of these cells. These results indicated that both Opeguard MA and BSS Plus might aid the spreading of corneal endothelial cells during wound-healing immediately after intraocular surgery.     

Analysis of angiogenesis induced by cultured corneal and oral mucosal epithelial cell sheets in vitro

The aim of this study was to compare angiogenesis-induction capabilities of cultured corneal epithelial cells (CCE) and cultured oral mucosal epithelial cells (COE) in vitro, and identify candidate factors that induce corneal neovascularization after transplantation of COE sheets. Rabbit corneal and oral mucosal epithelial cells were co-cultured with mitomycin C-treated NIH/3T3 cells on culture plates and inserts. After CCE and COE were multilayered, culture medium was replaced by basal medium and incubated. Angiogenic potential was examined by invasion, migration and tube formation assays with human umbilical vein endothelial cells (HUVECs). Protein secretion of fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF), angiopoietin-1 and transforming growth factor beta1 was assessed in conditioned medium by ELISA. Gene expression of FGF2 and VEGF was also quantified by real-time RT-PCR and neutralizing antibodies against FGF2 and VEGF were employed for blocking assays. COE induced significantly greater invasion, migration and tube formation of HUVECs, when compared to CCE. CCE secreted a significantly lower amount of FGF2 than COE, while amounts of VEGF were approximately equal in both culture media. Similarly, significantly higher expression of FGF2 mRNA was observed with COE, while no significant difference in VEGF mRNA expression was observed between COE and CCE. Only anti-FGF2 neutralizing antibody significantly suppressed HUVEC invasion and migration induced by COE, without suppression in CCE. In conclusion, angiogenic potential of COE is greater than that of CCE and FGF2 is a candidate involved in the induction of corneal neovascularization after COE sheet transplantation.     

Construction of a complete rabbit cornea substitute using a fibrin-agarose scaffold

PURPOSE: To construct a full-thickness biological substitute of the rabbit cornea by tissue engineering. METHODS: Ten rabbit corneas were surgically excised, and the three main cell types of the cornea (epithelial, stromal, and endothelial cells) were cultured. Genetic profiling of the cultured cells was performed by RT-PCR for the genes COL8 and KRT12. To develop an organotypic rabbit cornea equivalent, we used a sequential culture technique on porous culture inserts. First, endothelial cells were seeded on the base of the inserts. Then, a stroma substitute made of cultured keratocytes entrapped in a gel of human fibrin and 0.1% agarose was developed. Finally, cultured corneal epithelial cells were grown on the surface of the scaffold. Stratification of the epithelial cell layer was promoted by using an air-liquid culture technique. Corneal substitutes were analyzed by light and electron microscopy. RESULTS: All three types of corneal cells were efficiently cultured in the laboratory, expanded, and used to construct a full-thickness cornea substitute. Gene expression analyses confirmed that cultured endothelial cells expressed the COL8 gene, whereas epithelial cells expressed KRT12. Microscopic evaluation of the cornea substitutes demonstrated that epithelial cells tended to form a normal stratified layer and that stromal keratocytes proliferated rapidly in the stromal substitute. The endothelial monolayer exhibited a pattern similar to a normal corneal endothelium. CONCLUSIONS: These findings suggest that development of a full-thickness rabbit cornea model is possible in the laboratory and may open new avenues for research.