Low Temperature Preservation Unit, National University Medical Institutes, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Cryobiology. 2012 Dec;65(3):289-300. doi: 10.1016/j.cryobiol.2012.07.080. Epub 2012 Aug 24.
This work explores the design of a vitrification solution (VS) for scaled-up cryopreservation of hepatocytes, by adapting VS(basic) (40% (v/v) ethylene glycol 0.6M sucrose, i.e. 7.17 M ethylene glycol 0.6M sucrose), previously proven effective in vitrifying bioengineered constructs and stem cells. The initial section of the scale-up study involved the selection of non-penetrating additives to supplement VS(basic) and increase the solution's total solute concentration. This involved a systematic approach with a step-by-step elimination of non-penetrating cryoprotectants, based on their effect on cells after long/short term exposures to high/low concentrations of the additives alone or in combinations, on the attachment ability of hepatocytes after exposure. At a second stage, hepatocyte suspension was vitrified and functions were assessed after continuous culture up to 5 days. Results indicated Ficoll as the least toxic additive. Within 60 min, the exposure of hepatocytes to a solution composed of 9% Ficoll+0.6M sucrose (10⁻³ M Ficoll+0.6 M sucrose) sustained attachment efficiency of 95%, similar to control. Furthermore, this additive did not cause any detriment to the attachment of these cells when supplementing the base vitrification solution VS(basic). The addition of 9% Ficoll, raised the total solute concentration to 74.06% (w/v) with a negligible 10⁻³ M increase in molarity of the solution. This suggests main factor in inducing detriment to cells was the molar contribution of the additive. Vitrification protocol for scale-up condition sustained hepatocyte suspension attachment efficiency and albumin production. We conclude that although established approach will permit scaling-up of vitrification of hepatocyte suspension, vitrification of hepatocytes which are attached prior to vitrification is more effective by comparison.
这项工作旨在设计一种用于扩大规模的肝细胞玻璃化溶液 (VS)，通过调整之前在玻璃化生物工程构建体和干细胞中证明有效的 VS(basic) (40% (v/v) 乙二醇 0.6M 蔗糖，即 7.17 M 乙二醇 0.6M 蔗糖)。在扩大规模研究的初始阶段，选择非渗透添加剂来补充 VS(basic) 并增加溶液的总溶质浓度。这涉及一种系统的方法，根据单独或组合暴露于高/低浓度添加剂后对细胞的影响，逐步淘汰非渗透冷冻保护剂，其方法是基于对细胞的影响，根据细胞的附着能力来选择。在第二阶段，将肝细胞悬浮液玻璃化，并在连续培养长达 5 天后评估其功能。结果表明，Ficoll 是毒性最小的添加剂。在 60 分钟内，将肝细胞暴露于由 9% Ficoll+0.6M 蔗糖组成的溶液中（10⁻³ M Ficoll+0.6 M 蔗糖），可以维持 95%的附着效率，与对照组相似。此外，当补充基础玻璃化溶液 VS(basic) 时，这种添加剂不会对这些细胞的附着造成任何损害。添加 9% Ficoll 将总溶质浓度提高到 74.06%（w/v），而溶液的摩尔浓度仅增加了微不足道的 10⁻³ M。这表明，对细胞造成损害的主要因素是添加剂的摩尔贡献。扩大规模条件下的玻璃化方案维持了肝细胞悬浮液的附着效率和白蛋白的产生。我们得出结论，尽管已建立的方法将允许扩大规模的肝细胞悬浮液的玻璃化，但与玻璃化前附着的肝细胞相比，这种方法更有效。
