Introduction

Cells have various applications in biomedical research. Cryopreservation is a cell-preservation technique that provides cells for such applications. After cryopreservation, sensitive cells, such as primary hepatocytes, suffer from low viability due to the physical damage caused by ice crystals, highlighting the need for better methods of cryopreservation to improve cell viability. Given the importance of effectively suppressing ice crystal formation to protect cellular structure, trehalose has attracted attention as cryoprotectant based on its ability to inhibit ice crystal formation; however, trehalose induces osmotic stress. Therefore, to establish a cell-cryopreservation technique, it is necessary to provide an optimal balance between the protective and damaging effects of trehalose.

Methods

In this study, we evaluated the effects of osmotic stress and ice crystal formation on the viability and function of primary rat hepatocytes at wide range of trehalose concentration.

Results

There was no osmotic stress at very low concentrations (2.6 μM) of trehalose, and 2.6 μM trehalose drives the formation of finer ice crystals, which are less damaging to the cell membrane. Furthermore, we found that the number of viable hepatocytes after cryopreservation were 70% higher under the 2.6 μM trehalose-supplemented conditions than under the dimethyl sulfoxide-supplemented conditions. Moreover, non-cryopreserved cells and cells cryopreserved with trehalose showed comparable intracellular dehydrogenase activity.

Conclusions

We showed that trehalose at very low concentrations (2.6 μM) improved dramatically viability and liver function of hepatocyte after cryopreservation.

中文翻译：

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极低浓度海藻糖对大鼠原代肝细胞功能的冷冻保护作用

介绍

细胞在生物医学研究中有多种应用。冷冻保存是一种细胞保存技术，可为此类应用提供细胞。冷冻保存后，原代肝细胞等敏感细胞由于冰晶造成的物理损伤而活力低下，这凸显了需要更好的冷冻保存方法来提高细胞活力。鉴于有效抑制冰晶形成以保护细胞结构的重要性，海藻糖因其抑制冰晶形成的能力而作为冷冻保护剂受到关注。然而，海藻糖会诱导渗透压。因此，为了建立细胞冷冻保存技术，有必要在海藻糖的保护作用和破坏作用之间提供最佳平衡。

方法

在这项研究中，我们评估了在广泛的海藻糖浓度范围内渗透应力和冰晶形成对原代大鼠肝细胞活力和功能的影响。

结果

在极低浓度 (2.6 μM) 的海藻糖下没有渗透应力，而 2.6 μM 海藻糖可促使形成更细的冰晶，对细胞膜的损害较小。此外，我们发现在添加 2.6 μM 海藻糖的条件下，冷冻保存后的存活肝细胞数量比添加二甲亚砜的条件下高 70%。此外，非冷冻保存的细胞和用海藻糖冷冻保存的细胞显示出相当的细胞内脱氢酶活性。

结论

我们发现，极低浓度 (2.6 μM) 的海藻糖可显着改善冷冻保存后肝细胞的活力和肝功能。