Cryopreservation is important for enabling long-term cell preservation. However, physical damage due to ice crystal formation and membrane permeation by dimethyl sulfoxide (DMSO) severely affects ...cryopreserved cell viability. To ensure cell survival and functional maintenance after cryopreservation, it is important to protect the cell membrane, the most vulnerable cell component, from freeze-thaw damage. This study aimed to create a glycolipid derivative having a positive interaction with the cell membrane and cytoprotective effects. As a result, we synthesized a novel trehalose derivative, oleyl-trehalose (Oleyl-Treh), composed of trehalose and oleyl groups. Its use led to increased viable cell counts when used with DMSO in a non-cytotoxic concentration range (1.6 nM–16 μM). Oleyl-Treh significantly improved viability and liver-specific functions of hepatocytes after cryopreservation, including albumin secretion, ethoxyresorufin-O-deethylase activity (an indicator of cytochrome P450 family 1 subfamily A member 1 activity), and ammonia metabolism. Oleyl-Treh could localize trehalose to the cell membrane; furthermore, the oleyl group affected cell membrane fluidity and exerted cryoprotective effects. This novel cryoprotective agent, which shows a positive interaction with the cell membrane, provides a unique approach toward cell protection during cryopreservation.
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Liver transplantation plays an important role in the medical field. To improve the quality of a donor liver, there is a need to establish a preservation system to prevent damage and maintain liver ...function. In response to this demand, machine perfusion (MP) has been proposed as a new liver preservation method instead of the conventional static cold storage. There is controversy about the optimal MP temperature of the donor liver. Since the oxygen consumption of the liver differs depending on the temperature, construction of a system that satisfies the oxygen demand of the liver is crucial for optimizing the preservation temperature. In this study, an MP system, which satisfies the oxygen demand of liver at each temperature, was constructed using an index of oxygen supply; the overall volumetric oxygen transfer coefficient, the amount of oxygen retention of perfusate and oxygen saturation. Both subnormothermic MP (SNMP, 20–25 °C) and normothermic MP (NMP, 37 °C) could maintain liver viability at a high level (94%). However, lactate metabolism of the liver during NMP was more active than that during SNMP. Furthermore, the ammonia metabolism of liver after NMP was superior to that after SNMP. Hence, NMP, which maintains the metabolic activity of the liver, is more suitable for preservation of the donor liver than SNMP, which suppresses the metabolic activity. In summary, normothermia is the optimal temperature for liver preservation, and we succeeded in constructing an NMP system that could suppress liver damage and maintain function.
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.
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