To promote the recovery of cells that undergo intracellular ice formation (IIF), it is imperative that recrystallization of intracellular ice is minimized. Hepatocytes are more prone to IIF than most other mammalian cells; therefore, we sought to evaluate the ability of novel small molecule carbohydrate derivatives to permeate and control rates of intracellular ice recrystallization in hepatocytes. HepG2 monolayers were treated with N-(4-chlorophenyl)-d-gluconamide (IRI 1), N-(2-fluorophenyl)-d-gluconamide (IRI 2), or para-methoxyphenyl-β-D-glycoside (IRI 3). Fluorescent cryomicroscopy was used for real time visualization of intracellular ice recrystallization under isothermal conditions. Both IRI 2 and IRI 3 reduced rates of intracellular recrystallization, whereas IRI 1 did not. IRI 2 and IRI 3, however, demonstrated a marked reduction in efficiency in the presence of the most frequently used permeating cryoprotectants (CPAs): glycerol, propylene glycol (PG), dimethyl sulfoxide (DMSO), and ethylene glycol (EG). Nevertheless, IRI 3 reduced rates of intracellular recrystallization relative to CPA-only controls in the presence of glycerol, PG, and DMSO. Interestingly, IRI preparation in trehalose, a commonly used non-permeating CPA, did not impact the activity of IRI 3; however, trehalose proved to increase the activity of IRI 1 while decreasing that of IRI 2. Together, this data suggests that each of these three compounds could prove relevant in hepatocyte cryopreservation protocols where IIF would be prominent. However, CPA-mediated modulation of intracellular IRI activity is apparent and warrants further investigation.

中文翻译：

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使用小分子碳水化合物衍生物对肝细胞内冰再结晶的冷冻保护剂依赖性控制

为了促进经历细胞内冰形成 (IIF) 的细胞的恢复，必须尽量减少细胞内冰的重结晶。肝细胞比大多数其他哺乳动物细胞更容易发生 IIF；因此，我们试图评估新型小分子碳水化合物衍生物在肝细胞中渗透和控制细胞内冰重结晶速率的能力。用 N-(4-氯苯基)-d-葡糖酰胺 (IRI 1)、N-(2-氟苯基)-d-葡糖酰胺 (IRI 2) 或对甲氧基苯基-β-D-糖苷 (IRI 3) 处理 HepG2 单层）。荧光低温显微镜用于等温条件下细胞内冰再结晶的实时可视化。IRI 2 和 IRI 3 都降低了细胞内重结晶的速率，而 IRI 1 则没有。然而，IRI 2 和 IRI 3，证明在最常用的渗透冷冻保护剂 (CPA) 存在下效率显着降低：甘油、丙二醇 (PG)、二甲基亚砜 (DMSO) 和乙二醇 (EG)。然而，在甘油、PG 和 DMSO 存在下，IRI 3 相对于仅 CPA 的对照降低了细胞内重结晶率。有趣的是，海藻糖（一种常用的非渗透性 CPA）中的 IRI 制剂不会影响 IRI 3 的活性；然而，海藻糖被证明可以增加 IRI 1 的活性，同时降低 IRI 2 的活性。这些数据表明，这三种化合物中的每一种都可以证明在 IIF 会很重要的肝细胞冷冻保存方案中具有相关性。然而，CPA 介导的细胞内 IRI 活性调节是显而易见的，值得进一步研究。