Transplantation of cultured adult human or porcine corneal endothelial cells onto human recipients in vitro. Part II: Evaluation in the scanning electron microscope.
Transplantation of cultured adult human or porcine corneal endothelial cells onto human recipients in vitro. Part II: Evaluation in the scanning electron microscope.
复制标题
将培养的成人或猪角膜内皮细胞体外移植到人类受体上。
作者：
M. Böhnke;P. Eggli;K. Engelmann
目的
用扫描电子显微镜（SEM）观察内皮细胞单层移植到剥脱后的后弹力膜上的形态学变化。
方法
在本研究中评价了来自本研究第I部分所述每个实验组的材料。通过机械、化学或物理清创术剥脱其天然内皮的角膜被检查，以评估每种技术在杀死和去除细胞方面的有效性。将猪或人供体角膜内皮细胞维持在单层培养物中达10代，然后在存在或不存在成纤维细胞生长因子（FGF）的情况下将其接种到受体的裸露后弹力膜上。在SEM中检查由此建立的单层，并将单个细胞的形态状态与在器官培养中维持4周的正常人供体角膜（参考对照）中表现出的形态状态进行比较。将分离培养的人角膜细胞移植到受体角膜的剥脱后弹力层上作为非内皮对照标本。根据标准技术处理组织以在SEM中检查。
结果
用于剥离受体角膜的天然内皮的三种方法中的每一种都是有效的，并且没有引起对后弹力膜的总体结构损伤。然而，在后一层中观察到一些小的局灶性缺陷，这些缺陷在机械清创后比化学或物理剥离后遇到的频率更高。将猪或人内皮细胞接种到受体角膜的裸露后弹力层上，形成稳定的单层。再移植细胞的形态学状态与接种前单层培养中表现出的形态学状态相对应，猪的细胞总是比人的细胞更分化。低分化的人内皮细胞具有细长的成纤维细胞样外观，而高分化的人内皮细胞表现为宽的、平坦的、多边形轮廓。单层覆盖整个角膜表面，并在不同程度上影响到小梁网，在交界处的细胞总是假设一个较低的分化形态。FGF持续影响分化状态的增加，随着分化状态的增加，单层细胞侵犯角膜-小梁网边界的能力相应地受到抑制。接种的角膜细胞形成致密的多层鞘，类似于角膜后膜，横跨整个角膜表面、小梁网和虹膜根部。组成细胞的表面特征与内皮细胞所表现的特征截然不同，即使是分化最差的内皮细胞。
结论
将人角膜内皮细胞重新移植到受者裸露的后弹力层上，形成稳定的单层。由于接种细胞的形态学状态与移植前单层培养中表现出的形态学状态非常相似，因此可以预期，改进和优化培养条件的努力将在再移植后改善该参数。如果这些期望能够实现，那么在受体角膜上成功建立用于临床移植的“新的”功能性内皮的可能性很可能在不久的将来实现。
PURPOSE
To evaluate the morphology of endothelial monolayers, which have been regrafted onto the denuded Descemet's membrane, with scanning electron microscopy (SEM).
METHODS
Material derived from each of the experimental groups described in part I of this investigation was evaluated in the current study. Recipient corneas, denuded of their native endothelium by mechanical, chemical, or physical debridement, were examined to assess the effectiveness of each technique in killing and removing cells. Porcine or human donor corneal endothelial cells maintained in monolayer culture for up to 10 passages then were seeded onto the denuded Descemet's membranes of recipients in the absence or presence of fibroblast growth factor (FGF). The monolayers thereby established were examined in the SEM, and the morphologic status of individual cells compared with that manifested in normal human donor corneas maintained for 4 weeks in organ culture (reference control). Isolated and cultured human keratocytes regrafted onto the denuded Descemet's membranes of recipient corneas served as nonendothelial control specimens. Tissue was processed for examination in the SEM according to standard techniques.
RESULTS
Each of the three methods used to strip recipient corneas of their native endothelium was effective and elicited no gross structural damage to Descemet's membrane. Some small focal defects within this latter layer were, however, observed, these being encountered at higher frequency after mechanical debridement than after chemical or physical stripping. Porcine or human endothelial cells seeded onto the denuded Descemet's membranes of recipient corneas formed stable monolayers. The morphologic status of regrafted cells corresponded to that manifested in monolayer cultures before seeding, porcine ones always being more differentiated than their human counterparts. Poorly differentiated human endothelial cells had a slender, elongated, fibroblast-like appearance, whereas more highly differentiated ones manifested broad, flat, polygonal profiles. Monolayers covered the entire corneal surface and impinged to a variable degree onto the trabecular meshwork, at which juncture cells always assumed a less well-differentiated morphology. FGF consistently effected an increase in differentiation status, and as this became augmented, the capacity of monolayers to violate the corneal-trabecular meshwork border was correspondingly repressed. Seeded keratocytes formed dense, multilayered sheaths, resembling retrocorneal membranes, across the entire corneal surface, trabecular meshwork, and iris root. The surface characteristics of the constituent cells were quite distinct from those manifested by endothelial cells, even the least well-differentiated ones.
CONCLUSION
Regrafting of human corneal endothelial cells onto the denuded Descemet's membranes of recipients resulted in the formation of stable monolayers. Because the morphologic status of seeded cells closely mimicked that manifested in monolayer cultures before transplantation, it may be anticipated that efforts to refine and optimize culturing conditions would yield improvements in this parameter after regrafting. If these expectations can be realized, then the possibility of successfully establishing a "new" and functional endothelium on recipient corneas destined for clinical grafting may well be brought to fruition in the not-too-distant future.