Vitrification Successfully Preserves Hepatocyte Spheroids
Vitrification Successfully Preserves Hepatocyte Spheroids
复制标题
玻璃化冷冻成功保存肝细胞球体
作者：
R. Magalhães;X. Wang;S. Gouk;K. H. Lee;C. M. Ten;H. Yu;L. Kuleshova
这是第一份关于玻璃化低温保存自组装细胞聚集体的报告，玻璃化是一种既省时又省钱的技术。我们开发了一种有效的肝细胞球状体玻璃化（无冰冷冻保存）方案，该方案采用在室温下快速逐步暴露于冷冻保护剂（10.5 min）并直接浸入液氮（-196 ° C）中。为此，制定了三种玻璃化溶液（VS），并检查了它们对玻璃化温热球体的影响。使用乙二醇（EG）-蔗糖VS的冷冻保存显示出优异的保存能力，从而通过共聚焦和扫描电子显微镜成像观察到高度保存的细胞活力和玻璃化球体的完整性，与未处理的对照相比。通过尿素产生和白蛋白分泌来评估EG-蔗糖VS-冷冻保存的球状体的代谢功能，在同一天的观察内与对照组的代谢功能没有显著差异。在玻璃化冷冻组和对照组中，白蛋白分泌始终较高，范围分别为47.57 ± 14.39至70.38 ± 11.29 μg/106个细胞和56.84 ± 14.48至71.79 ± 16.65 μg/106个细胞，尿素产量在培养期间逐渐增加。玻璃化冷冻法保存肝细胞球体功能的有效性并不因引入第二种渗透性冷冻保护剂1，2-丙二醇（PD）而提高。球体冻存与EG-PD-蔗糖VS显示保持细胞活力，然而，在连续培养中，这两个代谢功能的水平低于冷冻保存与EG-蔗糖VS。EG-PD VS，其中非渗透性冷冻保护剂（蔗糖）被排除在外，在冷冻保存过程中提供保护球体较差。这项研究表明，蔗糖在自组装细胞聚集体的有效玻璃化中起着重要作用。从广义上讲，所获得的优异结果表明，所开发的玻璃化冷冻策略（冷冻的替代方案）可有效地用作细胞移植领域的平台技术。
This is the first report on low-temperature preservation of self-assembled cell aggregates by vitrification, which is both a time- and cost-effective technology. We developed an effective protocol for vitrification (ice-free cryopreservation) of hepatocyte spheroids that employs rapid stepwise exposure to cryoprotectants (10.5 min) at room temperature and direct immersion into liquid nitrogen (-196°C). For this, three vitrification solutions (VS) were formulated and their effects on vitrified-warmed spheroids were examined. Cryopreservation using ethylene glycol (EG)-sucrose VS showed excellent preservation capability whereby highly preserved cell viability and integrity of vitrified spheroids were observed, through confocal and scanning electron microscopy imaging, when compared to untreated control. The metabolic functions of EG-sucrose VS-cryopreserved spheroids, as assessed by urea production and albumin secretion, were not significantly different from those of control within the same day of observation. In both the vitrification and control groups, albumin secretion was consistently high, ranging from 47.57 ± 14.39 to 70.38 ± 11.29 μg/106 cells and from 56.84 ± 14.48 to 71.79 ± 16.65 μg/106 cells, respectively, and urea production gradually increased through the culture period. The efficacy of vitrification procedure in preserving the functional ability of hepatocyte spheroids was not improved by introduction of a second penetrating cryoprotectant, 1,2-propanediol (PD). Spheroids cryopreserved with EG-PD-sucrose VS showed maintained cell viability; however, in continuous culture, levels of both metabolic functions were lower than those cryopreserved with EG-sucrose VS. EG-PD VS, in which nonpenetrating cryoprotectant (sucrose) was excluded, provided poor protection to spheroids during cryopreservation. This study demonstrated that sucrose plays an important role in the effective vitrification of self-assembled cell aggregates. In a broad view, the excellent results obtained suggest that the developed vitrification strategy, which is an alternative to freezing, may be effectively used as a platform technology in the field of cell transplantation.
完整和分解的肝细胞球体的零下渗透特性。
影响因子：
2.7
作者：
Korniski,B;Darr,TB;Hubel,A
通讯作者：
Hubel,A