Cryopreservation Protocol for Chlamydomonas reinhardtii
Cryopreservation is commonly used for storing viable cells, tissues, organs or organisms at ultralow temperatures, and usually involves immersion in liquid nitrogen at -196 °C. Here we provide a detailed cryopreservation protocol for C. reinhardtii based on Crutchfield's work (Crutchfield et al., 1999) , with minor changes (Yang and Li, 2016) . In this study, we compared the cryoprotection effect of two common cryopreservation agents (CPAs), methanol and DMSO. Furthermore, the two-step
... rvation process was divided into five stages to study the factors affecting the survival rate at each stage. We found that the use of methanol as the CPA, combined with the cooling process outlined here (cooling from 25 °C to -55 °C at a rate of 1 °C/min), were indispensable for cell survival after cryopreservation. The thawing process described here (thawing at 35 °C for 5 min) was also important for increasing the survival rate. [Background] Nowadays, cryopreservation is used frequently for the storage of transgenic lines or mutation lines of C. reinhardtii, and for experimental needs involving this organism. Morris et al. (1979) discussed the effects of different CPAs in cooling, the relationships between temperature and survival rate with or without the CPAs, and the cooling rate. They found that with the addition of methanol the half-lethal temperature was the lowest of all the CPAs tested (-14.4 °C), while that of DMSO was -4.9 °C (Morris et al., 1979) . Through storage in 7% (v/v) DMSO overnight at room temperature, followed by storage at -70 °C, Johnson and Dutcher (1993) gained the highest viabilities, nearly 10% in C. reinhardtii cultures. However, the survival rate may be restricted in C. reinhardtii cell lines, especially in the cell line CC-125 we used, with viabilities of only 0.34%, although the authors claim was caused by liquid culturing. Nevertheless, this method was time consuming and resulted in low cell viabilities. Crutchfield et al. (1999) reported a two-step cooling procedure for the cryopreservation of C. reinhardtii using 5% methanol as the CPA, which retained relatively high viability (> 40%). Three different methods for improving survival rate were compared in this study. We followed the protocols mentioned above, and made detailed analyses at each stage of the cryopreservation process. The different effects of methanol and DMSO are also discussed, the results agreeing with the work of previous authors.