Organ culture of rabbit cornea: morphological analyses

Tissues maintained in vitro often undergo changes in the pattern of protein synthesis that result in the deposition of macromolecules quantitatively or qualitatively unrelated to those normally synthesized. In these preliminary studies, we modified published techniques to maintain adult and neonate rabbit corneas in vitro for 24 to 48 h. Measurements of corneal wet weight, and histologic and ultrastructural analyses were made to determine the success of maintaining rabbit corneas in culture. The results show that rabbit corneas can maintain normal corneal hydration and tissue structure for at least 48 h when incubated in Coon's modification of Ham's F12, 5% fetal or newborn calf serum, 2 mM glutamine, and 2% chondroitin sulfate or 2% 50 kDa dextran sulfate at 37 degrees C in a 5% CO2/air atmosphere. In addition, we confirmed previous observations that corneal explants must have a 1 to 2 mm rim of limbal sclera, and the organ placed in the culture dish with the epithelial side down to guard against damage and insure endothelial functioning. Normal ultrastructure of neonate rabbit cornea is also maintained when organ-cultured with these procedures. Moreover, neonate corneas continue to synthesize collagen in culture.     

组织工程角膜片移植治疗无菌性角膜溃疡的实验研究

目的探讨组织工程角膜片治疗角膜溃疡的可行性及疗效。方法将16只新西兰大白兔制作成角膜溃疡模型,随机分为2组,每组8只,分别作单纯羊膜移植和以羊膜为载体的组织工程角膜片移植。术后进行大体观察、裂隙灯观察、组织学观察、扫描电镜观察。结果羊膜为载体组织工程角膜片移植治疗角膜溃疡,平均7.5周溃疡完全愈合,角膜恢复透明性和屈光性,较单纯羊膜移植组(平均9周)角膜溃疡修复快,效果好结论羊膜为载体构建的组织工程角膜片可以有效治疗角膜溃疡。     

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.     

Effect of donor cornea preservation in tissue culture and in McCarey-Kaufmann medium on corneal graft rejection and visual acuity

In a retrospective study the rate of rejection and clouding of the donor cornea for other reasons was investigated in 230 penetrating keratoplasties performed between 1984–1986. Donor corneas were stored in McCarey-Kaufmann Medium (MK) at 4 °C or in a modified Minimal Essential Medium (MEM) at 31 °C. No statistical differences in rejection rate, cloudiness due to other causes or visual acuity was found between MEM- and MK-stored donor corneas.     

人脐带血清器官培养液保存猪角膜的实验研究

目的 观察胎牛血清器官培养液和人脐带血清器官培养液对猪角膜内皮细胞形态学、组织学、超微结构、酶活性及代谢等的影响。方法 器官培养方法：4周以内31℃密闭培养,之后脱水24h。选择猪眼113只,其中100只角膜分为两组进行配对器官培养保存。第1组(50只角膜)：应用培养液Ⅰ(含10％胎牛血清);第2组(50只角膜,第1组的对侧角膜)：应用培养液Ⅱ(含10％人脐带血清)。13只角膜作为正常对照。器官培养每周每组各取出12只角膜进行内皮细胞形态学分析、HE染色、酶组织化学染色。保存2周、4周时行扫描电镜检查。检测保存前后培养液的pH值和葡萄糖、乳酸浓度。器官培养过程中行微生物学检测。结果 器官培养期间角膜内皮细胞单层保持完整,多形性增加,两组之间角膜内皮细胞密度、细胞面积的变异系数和六边形细胞比例在保存4周内差异无显著意义。保存4周的猪角膜与正常猪角膜相比,平均内皮细胞丢失率第1组为10．98％,第2组为10．85％。两组角膜器官培养后的组织学、超微结构、酶活性无明显区别。扫描电镜显示内皮细胞层完整,细胞形态改变。酶组织化学染色显示上皮、内皮细胞酶活性旺盛,基质细胞的酶活性随保存时间的延长而降低。角膜代谢状态良好。器官培养液污染率为6％。结论 两种器官培养液均可以保持角膜内皮活性达4周,推测人脐带血清能够替代胎牛血清作为角膜器官培养液的成分。(中华眼科杂志,2004,40：533-538)     

The influence of tissue culture on corneal immunogenicity

In this study we examined whether immunization with heterotopic corneal graft can be suppressed by usage of cultured corneal tissue. Starting from the hypothesis that the corneal antigenicity might change during long-time storage, we compared, in a mouse model, the immunization obtained with fresh and> stored corneas. Heterotopic (chest wall) mice corneal allografts were exchanged between donors and hosts: (1) mismatched at multiple minor H loci and (2) only H-Y mismatched animals. Median survival time (MST) of primary and secondary skin grafts exchanged between mentioned donors and hosts was recorded. Recipient mice were immunized with either: (a) tail-skin graft, (b) fresh cornea graft or (c) corneal graft stored for three weeks in tissue culture. Three weeks later, recipients were challenged with skin graft placed at the opposite side of the chest wall and MST of these skin grafts was recorded. MST of secondary skin grafts in animals that had been immunized by skin served as a control. In case of multiple minor H disparity, MST of a first-set skin graft was 12 days, as compared to 9 days in case of secondary skin graft (P<0.05). MST of secondary skin graft following immunization by both fresh and stored corneas was 10 days. These data suggest that stored corneas don't loose ability to sensitize the multiple minor H disparate host. It also show that both cultured and fresh corneas, when placed in non-privileged site, have same immunizing capacity as skin (MST of 10 and 9 days, respectively; P>0. 1). When only H-Y disparate animals were used, MST of a first-set skin grafts was 26 days and of secondary skin graft 11 days (P<0.01). In case of H-Y disparity, MST obtained after immunization with fresh and stored corneal tissue (19 and 18 days, respectively) was significantly longer as compared to skin (P<0.05). However, no significant difference in MST of secondary skin grafts between recipients of fresh (19 days) and stored corneal grafts (18 days) was recorded. According to our results, the ability of corneal tissue to immunize both multiple minor H mismatched, as well as only H-Y mismatched host, was not influenced by storage in a tissue culture.     

Preserving the cornea: corneal storage media

PURPOSE OF REVIEW: The aim of this article is to review the recent literature regarding corneal storage media since the last review that discussed this topic in substantial detail in this journal. RECENT FINDINGS: During the last few years, despite the development of new corneal storage media and the addition of new additives to established corneal storage media, Optisol GS (Bausch & Lomb, Irvine, California, USA) has continued to remain the popular choice among storage media used in the United States, and traditional organ culture methods are still used in Europe. Recognizing that persistent epithelial defects after corneal transplantation can be a serious complication, however, attention has started to focus on not only preserving the endothelium, but also the epithelium. In addition, there has been more research towards antimicrobial prophylaxis. SUMMARY: With recent evidence suggesting that longer storage times may allow better outcomes in high-risk grafts because of the depletion of donor T cells from the donor cornea into the storage media, storage media will need to be optimized for preserving the endothelium for longer periods of time. In addition, because the epithelium is typically unable to be sustained for longer than 1 week in storage media, research toward preserving the epithelium will also be essential.     

Evaluation of corneal cell growth on tissue engineering materials as artificial cornea scaffolds

The keratoprosthesis (KPro; artificial cornea) is a special refractive device to replace human cornea by using heterogeneous forming materials for the implantation into the damaged eyes in order to obtain a certain vision. The main problems of artificial cornea are the biocompatibility and stability of the tissue particularly in penetrating keratoplasty. The current studies of tissue-engineered scaffold materials through comprising composites of natural and synthetic biopolymers together have developed a new way to artificial cornea. Although a wide agreement that the long-term stability of these devices would be greatly improved by the presence of cornea cells, modification of keratoprosthesis to support cornea cells remains elusive. Most of the studies on corneal substrate materials and surface modification of composites have tried to improve the growth and biocompatibility of cornea cells which can not only reduce the stimulus of heterogeneous materials, but also more importantly continuous and stable cornea cells can prevent the destruction of collagenase. The necrosis of stroma and spontaneous extrusion of the device, allow for maintenance of a precorneal tear layer, and play the role of ensuring a good optical surface and resisting bacterial infection. As a result, improvement in corneal cells has been the main aim of several recent investigations; some effort has focused on biomaterial for its well biological properties such as promoting the growth of cornea cells. The purpose of this review is to summary the growth status of the corneal cells after the implantation of several artificial corneas.     

A tissue culture assay of corneal epithelial wound closure

Experimental assays have been developed using cultured tissue derived from rabbit corneal epithelium to study migration of epithelial sheets during wound closure and cell-substrate adhesion. To study wound closure, epithelial defects, 6 mm in diameter, were produced in vitro in 24 well multiplates by a local freezing technique, and the size of the remaining defect was quantitated over time by staining. To study adhesion, cultured cells were labeled with 3H-leucine, suspended, and added to fresh culture plates. At various times, adherent cells were lysed and the radioactivity of the lysate was determined. Serum enhances the closure of experimental defects, but laminin and fibronectin have no effect. Agents which alter mitotic rate, such as epidermal growth factor and 5-fluorouracil, do not influence the rate of wound closure in this assay. Compounds which elevate intracellular levels of cyclic AMP inhibit wound closure but promote cell-substrate adhesion. Thus, cultured corneal epithelial cells may be used to assay for influences on the migratory events governing closure of superficial epithelial wounds.     

Characterization of rat corneal epithelium maintained in tissue culture

Using Dispase-treated rat corneas, primary cultures of corneal epithelium have been established which are free of contaminating cell types. Cultures were maintained for periods up to 36 days and were monitored with morphological and electrophysiological methods. Phase and scanning electron microscopy revealed a mosaic of polygonal epithelial cells which migrated over the culture plate. Actively migrating cells at the periphery of the culture presented a complex border of ruffles and filopodia. Surface specializations of the epithelial cells, i.e. microvilli, reflected those seen in vivo. Transmission electron microscopy revealed many cytological features common to the intact cornea: bundles of tonofilaments, desmosomes between adjacent cells and glycocalyx-covered microvilli on all free surfaces. These features developed over time in culture. Membrane potentials of the cultured epithelial cells could be recorded intracellularly. Therefore, it appears that pure cultures of rat corneal epithelium which maintain the morphological characteristics of their in vivo counterparts can be grown for considerable periods of time and that their electrophysiological properties can be monitored. This system offers the possibility of studying, under the controlled conditions of tissue culture, a wide variety of factors which might influence the integrity of the corneal epithelium or alter its susceptibility to disease.     

Cyclic nucleotides in rabbit corneal endothelial cells: fresh tissue vs. tissue culture.

Quantities of adenosine 3′,5′-cyclic monophosphate were measured in rabbit corneal endothelial cells taken from fresh tissue and tissue culture. Higher levels of both nucleotides were observed when the cells were initially frozen in liquid nitrogen compared with those that were not. Adenosine 3′,5′-cyclic monophosphate was 14 times lower in the primary cultures compared to the fresh tissues while subcultures were about two-thirds that of the fresh tissue amount. Guanosine 3′,5′-cyclic monophosphate was four times higher in the primary cultures compared to the fresh tissue while subcultures were about two-thirds that of the fresh tissue amount. It is emphasized that the ratios of adenosine 3′,5′-cyclic monophosphate to guanosine 3′,5′-cyclic monophosphate were similarly high in both fresh tissue and subcultured cells approaching confluency, but quite low in primary outgrowths.     

Autosomal recessive cornea plana: in vivo corneal morphology and corneal sensitivity

PURPOSE: Autosomal recessive corneal plana (RCP) is a rare corneal anomaly with unknown pathogenesis and a high incidence in Finland. The aim was to examine corneal sensitivity and the morphology of different corneal layers and subbasal nerves in RCP patients. METHODS: Three patients with a diagnosed autosomal recessive cornea plana were examined. Corneal sensitivity to different modalities of stimulation was tested in four corneas using noncontact esthesiometry. Tissue morphology of three corneas was evaluated, and in two corneas thickness of corneal layers was measured using in vivo confocal microscopy. RESULTS: Corneas of RCP patients appear to have mechanosensory, polymodal, and cold-sensitive nerve terminals. RCP patients had normal sensation thresholds for chemical, heat, and cold stimulation but a high threshold for mechanical stimulation. Their capacity to discriminate increasing intensities of stimulus was reduced, except for cold stimuli. Thickness of the epithelial layer was reduced, whereas total corneal and stromal thicknesses were slightly reduced or close to normal values. In all cases Bowman's layer was absent. Subbasal nerves had abnormal branching patterns. The arrangement of anterior keratocytes was altered, showing clustered and irregularly shaped nuclei. Increased backscattering of light in confocal microscopy through focusing (CMTF) profiles was observed throughout the stroma. Epithelial and endothelial cells appeared to be regular in shape. CONCLUSIONS: The present study revealed qualitative and quantitative alterations in corneal sensitivity, cellular morphology, and the thickness of corneal layers in RCP patients.     

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)     

角膜缘组织定位培养和冷冻后培养的实验研究

目的验证角膜缘干细胞的组织学定位,探讨低温冷冻保存对其增殖活性的影响。方法取新鲜角膜缘上皮组织和相应部位浅层巩膜组织各10块进行体外细胞培养,对比观察细胞生长情况。取冷冻保存的角膜缘上皮组织14例,观察体外培养后细胞生长情况。通过免疫组化方法检测冷冻保存的角膜缘上皮细胞的增殖活性和培养后单层细胞K3角蛋白的表达。结果10例新鲜角膜缘上皮组织培养后,7例有上皮细胞生长,1周形成细胞单层;10例浅层巩膜组织培养后未见细胞生长。14例冷冻角膜缘上皮组织培养后,4例有上皮细胞生长,9d形成细胞单层。5例冷冻角巩膜环组织冰冻切片中,3例可见角膜缘上皮基底细胞PCNA表达阳性。培养细胞对K3角蛋白特异性的AE-5单克隆抗体免疫反应阳性。结论角膜缘干细胞定位于角膜缘上皮基底部,低温冷冻保存的角膜缘干细胞组织可以保持增殖活性,体外培养后生长分化成为角膜上皮。     

The growth of endothelium from human corneal rims in tissue culture

The authors report the successful in vitro growth of human corneal endothelium derived from 12 of 31 corneal-scleral rims obtained following corneal transplantation. The average age of donors whose endothelium grew was 17.6 +/- 3.2 years. The average interval between death and culturing was 51.5 +/- 10.1 hr. The cells migrated from the explants within 3-7 days and were characterized by a flattened, polygonal shape with a centrally located nucleus. Cell growth exhibited a doubling time of 72-96 hr in the second and third tissue culture passages. A reduced growth rate was observed by cell lines maintained in vitro for over 6 months. Fibroblast growth factor, epidermal growth factor, and endothelial cell growth supplement all exerted a positive influence on cell proliferation. Corneal-scleral rims can be a valuable source of endothelial cells for corneal research.     

Comparison of limbal and peripheral human corneal epithelium in tissue culture

Peripheral human corneal epithelium grows better in tissue culture than central epithelium, but it is not known whether ocular limbal epithelium grows even better than does the peripheral corneal epithelium. In this work we compared the growth kinetics of limbal and peripheral human corneal epithelial cells in tissue culture. Four 1-2 mm2 explants, removed from the limbus or from peripheral cornea (1-2 mm inside the limbus) of eye bank eyes, were grown to confluence in primary culture. Cells were then passaged at 2 X 10(5) cells per dish. At intervals thereafter, the cells were counted in a hemocytometer to determine plating efficiency and growth curves. Mitotic activity was determined 4 days after passaging by labeling cultures with 3H-thymidine and counting aliquots using the hemocytometer and scintillation counter. In the primary cultures, limbal epithelium grew as small, uniformly polygonal cells. Peripheral corneal cells grew to a variety sizes. The 24 hr plating efficiency and doubling time of limbal epithelial cells were 47 +/- 8% and 80 +/- 14 hr, respectively, while those of peripheral corneal cells were 41 +/- 10% (P less than 0.1) and 131 +/- 25 hr (P less than 0.001). The mitotic activity of limbal cells was significantly higher than that of peripheral (2.9 +/- 1.2 vs. 0.8 +/- 0.6) (P less than 0.01). These results indicate that human ocular limbal epithelium grows better in culture than does peripheral human corneal epithelium.     

Comparison of central and peripheral human corneal epithelium in tissue culture

Past attempts to grow human corneal epithelium in culture had limited success, with confluence rarely attained. This work is to determine whether different areas of human corneal epithelium grow better in tissue culture. We compared the extent, the mitotic rates, and morphology of outgrowths and histology of explants from central and peripheral human corneas in culture. Explants, 2 mm in diameter, removed from eye bank eyes, were placed epithelial side up on a culture dish with modified SHEM tissue culture medium (Jumblatt et al, 1983). After 7 days, the tissues were fixed, stained and the area of outgrowths from explants measured using an image processor. For eight eyes from donors averaging 66 yr old, the average area of central outgrowths was 7.8 +/- 1.1 mm2, while that of peripheral outgrowths was 52.8 +/- 5.2 mm2 (P less than 0.001). The mitotic rate of outgrowths of central epithelium was significantly less than that of peripheral epithelium (1.1 +/- 0.5% vs 18.8 +/- 0.8%) (P less than 0.001). After 14 days, central outgrowths had not attained confluence and consisted of large cells. Peripheral outgrowths had attained confluence and consisted of small polygonal cells. Histology of explants showed that only one layer of epithelium remained on the stroma in central explants, but several layers were present on the peripheral explants. Thus, peripheral human corneal epithelium grows better in culture than does central human corneal epithelium.