Borderie V M, Lopez M, Lombet A, Carvajal-Gonzalez S, Cywiner C, Laroche L
Banque de Cornées Saint-Antoine, ETS de l'APHP, Hôpital Saint Antoine, Paris, France.
Invest Ophthalmol Vis Sci. 1998 Jul;39(8):1511-9.
To assess the effects of two different concentrations of albumin in a cryoprotective solution and two freezing methods on human corneal keratocyte ctyopreservation.
Isolated keratocytes were used for cryopreservation. Solutions of 10% dimethylsulfoxide with either 2% or 10% human albumin were used as cryoprotective agents. Cells either were transferred directly into a -80 degrees C freezer (freezing rate, 2 degrees C/min) or were cooled in a programmed freezer (1 degrees C/min until -40 degrees C and then 10 degrees C/min), which resulted in four different cryopreservation protocols. Cells were stored at -80 degrees C, then were thawed at 37 degrees C, and subsequently were cultured. Keratocytes were studied by means of trypan blue staining, growth assay, apoptosis assays, transmission electron microscopy, and immunochemistry.
The percentage of cells that were alive after thawing ranged from 80% to 99% by trypan blue staining and from 45% to 60% by flow cytometry. The ratio of the number of living cells at the end of primary culture after cryopreservation to that before cryopreservation was significantly (P=0.04) higher after direct transfer into the -80 degrees C freezer than after controlled-rate freezing, whereas the albumin concentration had no significant influence on this ratio (P=0.45). The percentage of apoptotic cells was significantly higher after cryopreservation than in the control group of noncryopreserved cells; more than 5% 24 hours after thawing. Cryopreservation did not modify the keratocyte ultrastructure. Fibroblast growth factor dramatically decreased the serum-induced cell expression of alpha smooth muscle actin, whereas cryopreservation had no influence on this cell expression.
A freeze-thaw trauma, which was related to cryopreservation-induced cell apoptosis, was revealed during primary culture after thawing. Direct transfer into the -80 degrees C freezer resulted in better postcryopreservation growth in the culture than controlled-rate freezing. A change in albumin concentration from 2% to 10% did not affect the results.
评估冷冻保护溶液中两种不同浓度的白蛋白以及两种冷冻方法对人角膜基质细胞低温保存的影响。
使用分离的角膜基质细胞进行低温保存。将含有2%或10%人白蛋白的10%二甲基亚砜溶液用作冷冻保护剂。细胞要么直接转移至-80℃冰箱（冷冻速率为2℃/分钟），要么在程序降温冰箱中冷却（1℃/分钟直至-40℃，然后10℃/分钟），这产生了四种不同的低温保存方案。细胞储存在-80℃，然后在37℃解冻，随后进行培养。通过台盼蓝染色、生长测定、凋亡测定、透射电子显微镜和免疫化学对角膜基质细胞进行研究。
经台盼蓝染色，解冻后活细胞百分比在80%至99%之间；经流式细胞术检测，该百分比在45%至60%之间。与程序降温冷冻相比，直接转移至-80℃冰箱后，低温保存后原代培养末期活细胞数量与低温保存前活细胞数量的比值显著更高（P=0.04），而白蛋白浓度对该比值无显著影响（P=0.45）。低温保存后凋亡细胞百分比显著高于未进行低温保存的对照组细胞；解冻后24小时超过5%。低温保存未改变角膜基质细胞超微结构。成纤维细胞生长因子显著降低血清诱导的α平滑肌肌动蛋白细胞表达，而低温保存对该细胞表达无影响。
解冻后的原代培养过程中发现了与低温保存诱导的细胞凋亡相关的冻融损伤。与程序降温冷冻相比，直接转移至-80℃冰箱在培养中低温保存后的生长情况更好。白蛋白浓度从2%变为10%不影响结果。
