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Three issues are critical for successful cryopreservation of multicellular material: gases dissolved in liquid, thermal conductivity of the tissue, and localization of microstructures. Here we show that heat distribution is controlled by the gas amount dissolved in liquids and that when changing the liquid into solid, the dissolved gases either form bubbles due to the absence of space in the lattice of solids and/or are migrated toward the concentrated salt and sugar solution at the cost of amount of heat required to be removed to complete a solid-state transition. These factors affect the heat distribution in the organs to be cryopreserved. We show that the gas concentration issue controls fracturing of ice when freezing. There are volumetric changes not only when changing the liquid into solid (volume increases) but also reduction of the volume when reaching lower temperatures (volume decreases). We discuss these issues parallel with observations of the cryosurvivability of multicellular organisms, tardigrades, and discuss their analogy for cryopreservation of large organs.

Autonomous temperature data loggers were used to measure the temperature profile within a growing ice cover and in the water below. The ice formed under natural conditions over the pond. We observed the presence of distinct layers of gas bubbles throughout the ice thickness. Temperature measurements allowed us to determine growth rates (μm s−1) and cooling rates (°C s−1) of the ice and demonstrated that these bubble layers formed during the peak ice growth rates from 0.58 to 0.92 µm s−1. The growth rates, leading to the formation of layers of bubbles, were more than an order of magnitude lower than for bubbles produced in controlled laboratory conditions (from 3 to 80 µm s−1). This observation introduces the possibility that solid impurities play a role in natural waters and that they must lower the limit of growth rates required for bubble occurrence. Data revealed a decrease in ice growth rates while cooling rates increased. We interpret this observation as an effect of the heat flux from the water to the ice (8.34–34.11 W m−2), and of gas concentration changes in the water below. Calculations of the ice thickness using traditional methods showed the necessity to include the heat flux from the water to the ice and the effect of gas bubbles within the ice and near the ice–water interface.

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