Engelmann K, Drexler D, Böhnke M
University of Hamburg, Department of Ophthalmology, Germany.
Cornea. 1999 Mar;18(2):199-206. doi: 10.1097/00003226-199903000-00010.
To develop a method for grafting endothelial cells isolated from organ-cultured adult human corneas onto the denuded Descemet's membrane of human recipients.
Adult human or porcine corneal endothelial cells were isolated and maintained in monolayer cultures before seeding. Recipient corneas were stripped of their own endothelium by one of three different methods (mechanical, chemical, or physical) and the completeness of removal assessed after vital staining. The utility of each method was evaluated by monitoring the quality of attachment of the seeded-cell population. The seeding density of transplanted cells required for optimal results also was determined and the final numeric cell density achieved on recipient corneas after culturing for 7-20 days ascertained. The influence of incubating source cells with fibroblast growth factor (FGF), both on this latter parameter and on cell morphology, also was evaluated. The functional integrity of regrafted endothelium was assessed in 24-h perfusion experiments.
The seeding of between 150,000 and 700,000 cells onto recipient corneas, followed by gentle centrifugation to improve attachment, yielded maximal final numeric cell densities of 3,450/mm2 and 1,850/mm2 in porcine and human lines, respectively. Recipient corneas were most effectively denuded of their own endothelium by freezing-and-thawing. The newly established endothelial monolayer remained stable for up to 20 days in organ culture (longest period monitored). FGF treatment did not enhance the final numeric density of cells attained on recipient corneas, but it did have a beneficial effect on their morphology. Only those recipient corneas that exhibited a well-differentiated monolayer of seeded endothelial cells underwent stromal deswelling near to physiologic levels.
A practical working model has been developed, whereby recipient corneas stripped of their own endothelium can be furnished with a "new," near-normal endothelium by appropriate manipulations of the seeded-cell population. This now paves the way for a realistic tackling of the problem of endothelial cell paucity in donor corneas destined for transplantation.
开发一种将从器官培养的成人人类角膜分离的内皮细胞移植到人类受体剥脱的Descemet膜上的方法。
成人人类或猪角膜内皮细胞在接种前分离并维持在单层培养中。通过三种不同方法（机械、化学或物理）之一去除受体角膜自身的内皮，并在活体染色后评估去除的完整性。通过监测接种细胞群体的附着质量来评估每种方法的效用。还确定了获得最佳结果所需的移植细胞接种密度，并确定培养7 - 20天后受体角膜上达到的最终细胞数量密度。评估了用成纤维细胞生长因子（FGF）孵育源细胞对后一参数和细胞形态的影响。在24小时灌注实验中评估重新移植内皮的功能完整性。
在受体角膜上接种150,000至700,000个细胞，然后轻轻离心以改善附着，猪和人类细胞系的最终细胞数量密度分别达到最大值3,450/mm²和1,850/mm²。通过冻融最有效地去除受体角膜自身的内皮。新建立的内皮单层在器官培养中长达20天保持稳定（监测的最长时间）。FGF处理未提高受体角膜上获得的细胞最终数量密度，但对其形态有有益影响。只有那些表现出良好分化的接种内皮细胞单层的受体角膜才会发生接近生理水平的基质消肿。
已开发出一种实用的工作模型，通过对接种细胞群体进行适当操作，可使剥脱自身内皮的受体角膜配备“新的”、接近正常的内皮。这现在为切实解决用于移植的供体角膜内皮细胞缺乏问题铺平了道路。
