Effect of three different media on serum free culture of donor corneas and isolated human corneal endothelial cells
Effect of three different media on serum free culture of donor corneas and isolated human corneal endothelial cells
复制标题
DOI：
10.1136/bjo.85.12.1416
复制
发表时间：
2001-12-01
影响因子：
4.1
通讯作者：
Engelmann, K
中科院分区：
文献类型：
--
作者：
Bednarz, J;Doubilei, V;Engelmann, K
背景：由于血清成分的可变性和潜在的感染风险，从器官培养基中去除牛血清是必要的。两种特定的内皮细胞培养基（F99和内皮- sfm）与常规使用的培养基MEM进行比较，用于无血清培养人角膜内皮细胞。细胞（HCEC）和供体角膜。方法：将hcec培养于血清含量增加或不增加的三种培养基中，进行生长测定。7对供体角膜分别在3种培养基中培养3周，其中1对角膜添加血清，1对不添加血清。内皮细胞密度每周测定1次。3周后对内皮细胞进行台盼蓝染色，对角质细胞进行活体染色。结果：三种培养基在添加血清时均能促进培养的HCEC的增殖。供体角膜内皮细胞密度在不同培养基中培养3周后具有可比性。只有在没有血清的培养基中培养的角膜内皮细胞损失更高。培养后内皮细胞台盼蓝染色显示内皮- sfm培养基中角膜损伤细胞数量最少。内皮- sfm培养的角膜形成细胞密度最高，MEM培养的角膜形成细胞密度最低。结论：即使在无血清条件下，在内皮- sfm中培养后，角膜的内皮细胞存活率、细胞膜完整性和角质细胞密度都有更高的质量。这种培养基可以取代MEM，后者在欧洲眼库中常规使用，但需要补充血清。
Background-Removal of bovine serum from organ culture medium is necessary because of the variability in serum composition and the potential risk of infection. Two specific endothelial cell media (F99 and Endothelial-SFM) were compared with the routinely used medium MEM for their use in serum free cultivation of human corneal endothelial. cells (HCEC) and donor corneas.Methods-HCEC were incubated in three test media with or without increasing serum content and a growth assay was performed. Seven pairs of donor corneas were cultured in each of three media for 3 weeks, one cornea with serum supplementation and one without. Endothelial cell density was determined once each week. Trypan blue staining of the endothelium and vital staining of keratocytes was performed after 3 weeks.Results-All three media promoted proliferation of cultured HCEC when supplemented with serum. Endothelial cell density of donor corneas was comparable after 3 weeks of cultivation in the different media. Only corneas cultured in medium MEM without serum exhibited a higher endothelial cell loss. Trypan blue staining of the endothelium after cultivation revealed the lowest number of damaged cells on corneas cultured in the medium Endothelial-SFM. The highest densities of keratocytes were found in corneas cultured in Endothelial-SFM and the lowest densities occurred after culture in MEM.Conclusion-After incubation in Endothelial-SFM even under serum free conditions corneas were found to be of higher quality with respect to endothelial cell survival, cell membrane integrity, and keratocyte density. This medium may replace MEM, which is routinely used in European eye banks but requires supplementation with serum.