Thermally Induced Introduction of Trehalose into Primary Rat Hepatocytes

Abstract

Trehalose was introduced into suspended primary rat hepatocytes through pathways resulting from thermally induced alterations of the cellular membrane. The hepatocytes were suspended in a diluted hepatocyte culture medium (medium:dH₂O = 1:2) with 0.4 M trehalose during thermal treatments. A significant amount of cytoplasmic trehalose (0.07 M) was detected using high-performance liquid chromatography (HPLC) after heating hepatocytes to 39°C for 10 min in trehalose-supplemented medium. High cell viability (approximately 90%) was retained. The cytoplasmic trehalose concentration reached a plateau (approximately 0.16 M) after heating for 1–2 h. However, the cell viability decreased significantly after 30 min of heating (< approximately 72%). It was further found that by repetitive heating between 0°C and 39°C every 10 min for 1 h (0–39°C, 1 h), high cell viability (approximately 83%) could be maintained and a high cytoplasmic trehalose concentration (approximately 0.13 M) could be obtained. The trehalose-laden hepatocytes (0–39°C, 1 h) were cultured in a double-collagen gel sandwich system for 15 days. They retained normal morphology and produced a normal distribution of F-actin filaments. Furthermore, the hepatospecific functions of urea production and albumin synthesis were similar to those of control hepatocytes kept in fresh medium on ice for one hour. In short, trehalose can be introduced effectively into primary rat hepatocytes by challenging the cells with super-zero to mild hyperthermic (39°C) temperatures. Future studies will focus on the development of effective protocols for both cryopreservation and lyopreservation of trehalose-laden hepatocytes.