Evaluation of potential organ culture media for eye banking using human donor corneas
Evaluation of potential organ culture media for eye banking using human donor corneas
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DOI：
10.1136/bjo.85.9.1075
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发表时间：
2001-09-01
影响因子：
4.1
通讯作者：
Engelmann, K
中科院分区：
文献类型：
--
作者：
Moller-Pedersen, T;Hartmann, U;Engelmann, K
目的-评价不同市售细胞培养液在生理温度下3周“封闭系统”器官培养期间保存人类供体角膜的能力。进行这种筛选，试图建立一个合理的基础上发展的无血清器官培养基eyebanking.Methods-72正常人供体角膜器官培养21天，在31 degreesC在8个不同的测试媒体（9角膜在每组）。基础培养液包括：最低必需培养基（MEM）、含有稳定L-谷氨酰胺的MEM、M199、DIF-1000、SFM、F99和含有抗坏血酸、胰岛素、bFGF、转铁蛋白、硒和脂质（称为F99-Sr）的F99。除MEM外，所有培养基均补充有2%胎牛血清（FCS），MEM也在8% FCS下进行了研究。评价参数包括：（1）使用台盼蓝染色评价的内皮细胞损失;（2）使用放射自显影和数字图像分析评价的角膜细胞和内皮细胞将氚化尿苷掺入RNA的能力;（3）使用ELISA评价的免疫原性硫酸角质素的渗漏;结果SFM诱导的内皮细胞损失率最低，为14%（SD 2%），而添加2%FCS的所有试验溶液的RNA合成率最高。储存在SFM中的角膜也显示出硫酸角质素的最少泄漏和最高的葡萄糖消耗和乳酸盐产生。在5种培养基（含2% FCS的MEM、含稳定L-谷氨酰胺的MEM、M199、F99和F99-Sr）中，观察到器官培养的相当和中等潜力，内皮细胞损失为16- 19%。相比之下，29%（4%）的内皮细胞在DIF 1000中储存后丢失。有趣的是，使用8%FCS（在MEM中）对内皮有明显的保护作用，其显示出最高的RNA合成活性，细胞损失仅为11%（4%），而2%FCS为19%（6%）（p
Aim-To evaluate the ability of different commercially available cell culture solutions to preserve human donor corneas during 3 weeks of "closed system" organ culture at physiological temperature. This screening was performed in an attempt to establish a rational basis for the development of a serum-free organ culture medium for eye banking.Methods-72 normal human donor corneas were organ cultured for 21 days at 31 degreesC in eight different test media (nine corneas in each group). The basic culture solutions included: minimal essential medium (MEM), MEM with stabilised L-glutamine, M199, DIF-1000, SFM, F99, and F99 with ascorbic acid, insulin, bFGF, transferrin, selenium, and lipids (termed F99-Sr). All media were supplemented with 2% fetal calf serum (FCS), except for MEM, which was also studied at 8% FCS. The evaluation parameters included: (1) the endothelial cell loss as evaluated using trypan blue staining; (2) the ability of keratocytes and endothelial cells to incorporate tritiated uridine into RNA as evaluated using autoradiography and digital image analysis; (3) the leakage of immunogenic keratan sulphate as assessed using ELISA; and (4) changes in storage medium pH, glucose, and lactate content.Results-SFM induced the lowest endothelial cell loss of 14% (SD 2%) and the highest RNA synthesis rates of all test solutions supplemented with 2% FCS. Corneas stored in SFM also showed the least leakage of keratan sulphate and the highest glucose consumption and lactate production. In five media (MEM with 2% FCS, MEM with stabilised L-glutanuine, M199, F99, and F99-Sr), comparable and intermediate potentials for organ culture were observed with endothelial cell loss of 16-19%. By contrast, 29% (4%) of the endothelium was lost after storage in DIF1000. Interestingly, the use of 8% FCS (in MEM) had a marked protective effect on the endothelium, which showed the highest RNA synthetic activity combined with a cell loss of only 11% (4%), compared with 19% (6%) at 2% FCS (p