For many years, the impact of hyper- and hypothermia on mammalian cells has been examined. With the exception of short, low temperature storage, which has uses in areas such as preservation for transplantation or regenerative medicine, advantages for the use of low temperature treatment in hepatocytes have not been previously reported. We have observed that alginate-encapsulated HepG2 liver spheroids that are cryopreserved or experience a cold reduction in temperature (≤10°C) for periods between 1 and 90 min display an enhanced cell proliferation during culture 7-16 days post-treatment compared with untreated samples. Following 8-12 days post-treatment, alginate-encapsulated liver spheroids experienced a cell density of 1.71 ± 0.35 times that of control samples (p < 0.001). This effect occurred in samples with a variety of cold treatments. This low temperature treatment offers a simple method to rapidly increase cell proliferation rates for extended culture systems, such as bioartificial liver devices. This would allow the manufacture of required biomass more rapidly, and to a higher cell density, reducing final required biomass volume. This could enable bioartificial liver devices to be prepared more cheaply, making them a more cost effective treatment.