Artuyants A. Theoretical and experimental justification of effective cryopreservation conditions of Candida albicans yeast-like fungi

The research object - the processes of cryopreservation and preservation of the viability and biological properties of yeast-like fungi C.albicans. The research aim - development and experimental validation of effective cryopreservation conditions for yeast fungi Candida albicans. Research methods: microbiological, cryobiological, cytological, biochemical, PCR, flow cytometry and scanning confocal microscopy, the methods of mathematical modeling and statistical analysis of the results. Equipment: Software freezer "Cryoson" (Germany); cycler "Tertsik" (DNA Technology, Russia); flow cytometry FACS Calibur (BD, USA); laser scanning confocal microscope LSM 510 meta (Carl Zeiss, Germany). A theoretical evaluation of the effective values depending on the cooling rate of the processes of crystal formation during freezing of intracellular yeast-like fungi C.albicans in solutions of penetrating cryoprotectant DMSO and obtained experimental confirmation of the theoretical results. An effective regimen of cryopreservation protocol of C.albicans fungi has been designed. According to this regimen the cell suspensions were cooled in 5% solutions of DMSO or glycerol at a rate of 3.5 to 7°C/min down to -40°C with following plunging into liquid nitrogen. This regimen ensures the preservation of viability higher than 90% of the cells. It has been shown, that long-term storage of C.albicans fungi in low-temperature bank at a constant temperature does not lead to an additional cell death, and ensures the safety of their genetically determined biological properties. It has been established that the freezing of the developed protocol ensures the safety of C.albicans fungi genome. It is noted that the cells after cryopreservation on developed protocol for C.albicans (as opposed to cells that had been frozen for other modes), had no significant violations of the asymmetric distribution of phospholipids in the membrane, DNA fragmentation of cells, and notes minimal accumulation of ROS in them. Experimentally proved that the cells C.albicans, retained after cryopreservation their proliferative properties had non-lethal and conditionally lethal damage, which need time for the repair. This is evidenced by: an increase in lag-phase and the initial stages of log-phase of periodic cultures, additional cell death due to a modification of the MTC structure and function by nystatin after freezing, a longer fermentation and assimilation of carbohydrates in cryopreserved cells. The magnitude of these changes correlated with the cooling mode. Obtained results are recommended for further use in the practice of banks and collections of microorganisms.