Hempel B, Bednarz J, Engelmann K
Klinik und Poliklinik für Augenheilkunde des Universitätsklinikums Hamburg-Eppendorf, Hamburg, Germany.
Graefes Arch Clin Exp Ophthalmol. 2001 Oct;239(10):801-5. doi: 10.1007/s004170100364.
The success of long-term corneal organ culture is limited by the progressive loss of endothelial cells during culture and the use of culture medium supplemented with fetal calf serum as a possible source of contamination with infectious agents. In this study, we investigated the suitability of a serum-free medium (Endothelial-SFM) to improve preservation conditions for human donor corneas.
Six pairs of corneas were stored in Minimum Essential Medium (MEM) supplemented with 2% fetal calf serum (FCS) for 8-14 days. One cornea of each pair was then further cultivated in Endothelial-SFM supplemented with 2% FCS or in MEM with 2% FCS, respectively. In a second series of experiments, the endothelial cell density of seven pairs of freshly isolated donor corneas was determined during cultivation in Endothelial-SFM with 2% FCS or serum-free Endothelial-SFM.
After precultivation in conventional medium, the endothelial cell density of corneas allocated to cultivation in Endothelial-SFM was 1000-1950 cells/mm2 and that of those subsequently cultured in MEM 1200-2000 cells/mm2. At 9 weeks, cell densities of 900-1500 cells/mm2 were found after cultivation in Endothelial-SFM compared with a total cell loss in MEM. Freshly isolated corneas cultured in Endothelial-SFM with or without FCS supplementation showed a decrease of endothelial cell density of about 20% within the first 2 weeks of storage. During further cultivation cell density remained constant without statistically significant differences between the groups. Glucose consumption of the corneas was higher in Endothelial-SFM than in MEM. Corneas stored in Endothelial-SFM with 2% FCS showed a higher glucose consumption than those preserved in serum-free Endothelial-SFM.
Organ culture of human donor corneas using the serum-free basal medium Endothelial-SFM is superior to conventional culture conditions because the decrease in endothelial cell density can be ameliorated, the culture period can be prolonged and the risk of transmitting infectious agents via serum can be minimised.
长期角膜器官培养的成功受到培养过程中内皮细胞逐渐丧失以及使用添加胎牛血清的培养基作为感染性病原体潜在污染源的限制。在本研究中，我们调查了无血清培养基（内皮细胞生长培养基，Endothelial-SFM）对改善人类供体角膜保存条件的适用性。
将六对角膜保存在添加2%胎牛血清（FCS）的最低必需培养基（MEM）中8 - 14天。然后每对中的一个角膜分别在添加2% FCS的Endothelial-SFM或含2% FCS的MEM中进一步培养。在另一系列实验中，测定了七对新鲜分离的供体角膜在添加2% FCS的Endothelial-SFM或无血清的Endothelial-SFM中培养期间的内皮细胞密度。
在传统培养基中预培养后，分配到Endothelial-SFM中培养的角膜内皮细胞密度为1000 - 1950个细胞/mm²，随后在MEM中培养的角膜内皮细胞密度为1200 - 2000个细胞/mm²。在9周时，在Endothelial-SFM中培养后细胞密度为900 - 1500个细胞/mm²，而在MEM中细胞全部丢失。在添加或不添加FCS的Endothelial-SFM中培养的新鲜分离角膜在储存的前2周内内皮细胞密度下降约20%。在进一步培养期间，细胞密度保持恒定，两组之间无统计学显著差异。角膜在Endothelial-SFM中的葡萄糖消耗量高于在MEM中的。保存在添加2% FCS的Endothelial-SFM中的角膜比保存在无血清Endothelial-SFM中的角膜显示出更高的葡萄糖消耗量。
使用无血清基础培养基Endothelial-SFM进行人类供体角膜的器官培养优于传统培养条件，因为可以改善内皮细胞密度的降低，延长培养期，并将通过血清传播感染性病原体的风险降至最低。
