Cryopreservation of Encapsulated Liver Spheroids for a Bioartificial Liver: Reducing Latent Cryoinjury Using an Ice Nucleating Agent
Cryopreservation of Encapsulated Liver Spheroids for a Bioartificial Liver: Reducing Latent Cryoinjury Using an Ice Nucleating Agent
复制标题
DOI：
10.1089/ten.tec.2010.0394
复制
发表时间：
2011-07-01
影响因子：
3
通讯作者：
Fuller, Barry
中科院分区：
文献类型：
--
作者：
Massie, Isobel;Selden, Clare;Fuller, Barry
由于供体器官短缺，急性肝衰竭具有很高的死亡率。生物人工肝脏可以“弥补”移植或自然恢复之间的差距。海藻酸盐包封HepG2细胞，使细胞形成球形，从而提供足够的功能性生物量。这些球体的低温保存（CryoP）将允许在不可预测的紧急情况下使用现成的能力。CryoP过程中的细胞死亡通常是由于过冷后细胞内结冰造成的。一种冰成核剂（INA），结晶胆固醇，被用来减少过冷和随后的冷冻损伤。材料和方法：球体在12%二甲亚砜/Celsior +/- INA的控制速率冷冻机中冷却，并全程记录样品温度。通过图像分析的荧光染色评估细胞活力，通过细胞核计数评估细胞数量，通过检测肝脏特异性蛋白质合成和分泌，3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四唑（MTT）还原和广谱细胞色素P450活性来评估功能。结果：无INA冷冻保存的球体在解冻后的前6小时表现出潜在的冷冻损伤。INA减少了CryoP期间的过冷和潜在的冷冻损伤。在CryoP期间，当INA存在时，解冻后72h的细胞数量、活力和功能都得到改善。
Introduction: Acute liver failure has high mortality due to donor organ shortages. A bioartificial liver could "bridge the gap" to transplant or spontaneous recovery. Alginate encapsulation of HepG2 cells enables cell spheroid formation, thus providing sufficient functional biomass. Cryopreservation (CryoP) of these spheroids would allow an off-the-shelf capability for unpredictable emergency use. Cell death during CryoP often results from intracellular ice formation, after supercooling. An ice nucleating agent (INA), crystalline cholesterol, was trialled to reduce supercooling and subsequent cryoinjury.Materials and Methods: Spheroids were cooled in a controlled rate freezer in 12% dimethylsulfoxide/Celsior +/- INA, and sample temperatures were recorded throughout. Viability was assessed using fluorescent staining with image analysis, cell number by nuclei count, function using assays to detect liver-specific protein synthesis and secretion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction, and broad-spectrum cytochrome P450 activity.Results: Spheroids cryopreserved without INA displayed latent cryoinjury in the first 6 h after thawing. INA reduced supercooling during CryoP and also latent cryoinjury. Cell numbers, viability, and function as measured over 72 h post-thaw were all improved when INA was present during CryoP.