Prevention of hypothermic haloing extends the preservation time of hepatocytes at non freezing temperatures

Factors limiting the hypothermic preservation of hepatocytes on gelatin gels at 10 °C were investigated. Following 4 days of preservation, uniform morphological changes started to appear. The cells exhibited halos that increased in size. The particulate matter of the cell was confined to the central region. Cell viability was reduced from day 7 onwards. Neither fresh media changes nor the use of conditions to minimise free radical formation improved cell survival. However, haloing was decreased by short term temperature elevation to 37 °C (3 h), to reactivate the cells, and could be prevented completely by a stepwise increase in the sucrose concentration of the medium. The addition of sucrose in increments of 50 mM, at four day intervals, was found to inhibit morphological change. Prevention of haloing enabled the cells to be preserved for at least two weeks. The preserved cells attached to supports and spread as if freshly isolated. The procedure allows extended preservation of cells at non-freezing temperatures.