Wenzel Daniel A, Kunzmann Berenike C, Steinhorst Nils A, Spitzer Martin S, Schultheiss Maximilian
Department of Ophthalmology, University Medical Center Hamburg-Eppendorf (UKE);
University Eye Hospital, Center For Ophthalmology, University Hospital Tübingen.
J Vis Exp. 2019 Oct 6(152). doi: 10.3791/60171.
Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet's membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.
角膜内皮细胞的实验研究存在若干困难。人类供体角膜稀缺，通常因移植需要而很少用于实验研究。内皮细胞培养物往往难以很好地转化为体内情况。由于非人类角膜的生物结构特征，培养过程中的基质肿胀会导致大量角膜内皮细胞损失，这使得长时间进行培养变得困难。诸如右旋糖酐等消肿剂被用于抵消这种反应。然而，它们也会导致显著的内皮细胞损失。因此，建立了一种无需消肿剂的离体器官培养模型。使用当地屠宰场的猪眼制备分离的角膜纽扣。在进行部分角膜环钻后，去除角膜的外层（上皮、Bowman层、部分基质）。这显著减少了在较长培养期内由大量基质肿胀和Descemet膜折叠引起的角膜内皮细胞损失，并改善了内皮细胞层的总体保存情况。随后进行完整的角膜环钻，接着从剩余的眼球中取出分离的角膜纽扣并进行培养。使用光学显微镜在制备后长达15天的随访时间（即第1天、第8天、第15天）评估内皮细胞密度。所采用的制备技术通过较少的基质组织肿胀能够更好地保存内皮细胞层，这导致分离的角膜纽扣中的内皮细胞密度下降速率缓慢且呈线性，与人类供体角膜相当。由于这种标准化的器官典型培养研究模型首次能够稳定培养至少两周，它是人类供体角膜的一种有价值的替代物，可用于未来研究各种外部因素对角膜内皮的影响。
