One of the main problems in the cryopreservation of biological samples is the formation of ice and the consequent mechanical damage to cells and tissues, due to the crystalline structure of ice and its associated mechanical damage. It is necessary to detect this deleterious formation of ice, especially in tissues and organs, because of their large volume and the complexity of their vascular system in the case of bulky organs. In this work, we propose the use of X-ray Computed Tomography (CT) to detect this ice formation inside tissues and organs. To achieve this aim, rabbit kidneys were loaded with cryoprotectant solutions containing Me2SO at low temperatures (below -140°C). Drops of water with a volume between 2 and 8 μL were then introduced inside the organs. Finally, the rabbit kidneys were cooled to -196°C. Volumes of ice of up to 1 μL were detected in our CT device, with a resolution of up to 50 μm, validating the proposed technology. On the contrary, we analyzed bovine ovarian tissues cryopreserved with a controlled-rate slow-cooling protocol. CT images showed the different structure on the extracellular ice formation according to the procedure, and even the intracellular ice that can be formed in the tissues. These positive results have a straightforward application in the control of the formation of ice, of significant importance for the creation of biobanks.
Keywords: X-ray computed tomography; ice detection; organ cryopreservation.