Cryo-Light Microscopy To Study The Freezing Behavior of Microalgae Cells

ABSTRACT

Microalgae Dunaliella salina Teod. and Chlorococcum disectum are an important object of biotechnology. The preservation of microalgae by regular reseeding on liquid nutrient media has high risks of contamination of the culture or even becomes impossible in the face of large-scale pandemics, like a COVID-19. Therefore, the most reliable way of long-term storage of plant objects with invariable genetic characteristics is cryopreservation. To develop a cryopreservation protocol, it is important to understand what processes occur in cells at the freezing-warming stage. We have made an attempt to establish a correlation between the processes of dehydration-rehydration of cells, intracellular crystallization and their preservation after freezing. To visualize these processes, we assembled and used a cryo-light microscope. The cryo-light microscope allows determining the most important parameters that are responsible for the safety of cells in the process of cryopreservation temperature parameters of phase transitions, the presence of extra- and intracellular ice, its structure, crystal size and their growth rate. We fixed the temperatures of extracellular crystallization for the control samples (without cryoprotectants) and those after adding cryoprotective mixtures, to evaluate the features of the crystal formation process. Crystal formation at the cooling-warming stages depended on the type and concentration of cryoprotectants or their mixtures (PVS), the cooling rate and the composition of the cell culture medium. The cryo-light microscope can also detect the osmotic reactions occurring in cells within the freezing-warming stage. Thus, at the moment of extracellular crystallization, the Chlorococcum cells in the control samples noticeably dehydrate, maintaining this state until the last ice crystal melts. Cryo-light microscopy also makes it possible to assess the morphological preservation of microalgae samples, as well as their viability for motile forms (D. salina). The data obtained are a promising step towards the creation of effective protocols for cryopreservation of microalgae and other biological objects. Funding: Not applicable Conflict of Interest None to disclose