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To date, two novel freeze-responsive proteins, Fr10 and Li16, have been discovered in the wood frog, Rana sylvatica, and likely support freezing survival. Although previous studies have established tissue distribution of each protein, there have been no studies that explore their functional consequences in intolerant cells. To assess the ability of Fr10 and Li16 to confer freeze tolerance, we transfected each protein into a freeze-intolerant silkworm cell line (BmN). Selected controls were the transfection of an unrelated protein (CAT) and a no-transfection sample. Li16 and Fr10 showed 1.8 ± 0.1- and 1.7 ± 0.2-fold, respectively, greater survival after freezing at -6°C for 1 h than did transfection controls. To investigate how these novel proteins protect cells from freezing damage, protein structures were predicted from primary amino acid sequences. Analysis of the structures indicated that Fr10 is a secreted protein and may be a new type IV antifreeze protein, whereas Li16 may have intracellular membrane associated functions. This study shows that freezing protection can be provided to intolerant cells by the overexpression of transfected Li16 and Fr10 frog proteins. Results from this study will provide new insights into adapting intolerant cells for medical organ cryoprotection using a natural vertebrate model of tolerance.

The Japan Aerospace Exploration Agency (JA X A) has be en developing seve ral life s c i e n c e h a r d w a r e c o m p o n e n t s f o r t h e Japanese experiment module 'Kibo' in the International Space Station (ISS). The Cell Biology Experiment Facility (CBEF) is one of these. It contains an environmental control system to regulate variables such as the temperature and humidity. It is also equipped with a centrifuge and has the ability to culture bio-specimens in a temperature range of 15 to 40˚C. In biological experiments that include in vitro cell culture, temperature control is a key factor in the success of the experiment. Thus, we first examined the temperature data in the CBEF on the ground while changing the room temperature and cooling water temperature to investigate the ability of the CBEF. Based on the ground experiment data, we carried out life science experiments onboard Kibo. Judging from the temperature data in the CBEF under microgravity, 12 life science experiments were successfully conducted up until 2011. Considering our present data, we strongly believe that the CBEF is a functional incubator in the ISS and will contribute to future experiments.

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