Cryoprotective enhancing effect of very low concentration of trehalose on the functions of primary rat hepatocytes

• Published in: Regenerative therapy 2020-12, Vol.15, p.173-179
• Main Authors: Yoshida, Kozue, Ono, Fumiyasu, Chouno, Takehiro, Perocho, Bual Ronald, Ikegami, Yasuhiro, Shirakigawa, Nana, Ijima, Hiroyuki
• Format: Article
• Language: English
• Subjects: Hepatocytes cryopreservation; Ice crystal; Liver function; Original; Osmotic stress; Very low concentration of trehalose
• ISSN: 2352-3204; 2352-3204
• DOI: 10.1016/j.reth.2020.08.003

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. 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. 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. We showed that trehalose at very low concentrations (2.6 μM) improved dramatically viability and liver function of hepatocyte after cryopreservation. •Very low concentration of trehalose could suppress ice crystal formation and protect cell structure.•There was a correlation between osmotic pressure of trehalose and hepatocytes viability.•Very low concentration of trehalose improved viability and liver function of hepatocyte after cryopreservation.