Massie I, Selden C, Morris J, Hodgson H, Fuller B
Royal Free Campus, UCL, Royal Free Hospital, London, UK.
Cryo Letters. 2011 Mar-Apr;32(2):158-65.
Acute liver failure has high mortality with unpredictable onset. A bioartificial liver, comprising alginate-encapsulated HepG2 spheroids, could temporarily replace liver function but must be cryopreservable. For clinical use, contamination risks from liquid coolants for cryopreservation and storage should be minimized. A cryogen-free cooler was compared to nitrogen vapour-controlled cryopreservation of alginate-encapsulated liver cell spheroids (AELS). AELS were cooled using a multi-step, slow-cooling profile in 12 percent v/v Me2SO Celsior and stored in liquid nitrogen; temperatures were recorded throughout, and the AELS were assayed at 24, 48 and 72 hours post-warming and results compared to unfrozen control values. Viability was assessed by fluorescent staining and quantified using image analysis; cell numbers were quantified using nuclear counts, and cell function using albumin synthesis. The cryogen-free cooler performed the cooling profile as desired, apart from one step requiring a rapid cool ramp. Viability, cell numbers and function were similarly decreased in both cryopreserved groups to about 90 percent, 70 percent and 65 percent of the controls respectively. This technology offers a clinic alternative to liquid nitrogen-coolant cryopreservation.
急性肝衰竭死亡率高，发病难以预测。一种由藻酸盐包裹的HepG2球体组成的生物人工肝可以暂时替代肝功能，但必须可冷冻保存。对于临床应用，应尽量降低冷冻保存和储存所用液体冷却剂带来的污染风险。将无制冷剂冷却器与藻酸盐包裹的肝细胞球体（AELS）的氮气蒸汽控制冷冻保存进行了比较。AELS在含12%（体积/体积）二甲基亚砜的Celsior中采用多步缓慢冷却曲线进行冷却，并储存在液氮中；全程记录温度，并在复温后24、48和72小时对AELS进行检测，结果与未冷冻的对照值进行比较。通过荧光染色评估活力，并使用图像分析进行定量；使用细胞核计数对细胞数量进行定量，使用白蛋白合成对细胞功能进行定量。除了有一步需要快速冷却斜率外，无制冷剂冷却器按要求完成了冷却曲线。两个冷冻保存组的活力、细胞数量和功能均同样下降，分别降至对照组的约90%、70%和65%。该技术为液氮冷却剂冷冻保存提供了一种临床替代方法。
