Cryopreservation using cryoprotective agents (CPAs) is the standard for storing biospecimens for a variety of purposes, including cell-based therapeutics. The most common CPA, dimethyl sulfoxide (Me2SO), demonstrates dose-dependent cytotoxicity, impacting cryopreserved cells as well as potentially producing side effects in patients. For this reason, the development of non-toxic protective strategies for cryopreservation is beneficial.
One emerging possibility is to harness intrinsic cellular mechanisms that play protective roles, like heat shock proteins (HSPs). These highly conserved proteins operate as molecular chaperones, binding to cellular proteins under stress in order to reduce the impact of stress-induced misfolding. Another promising possibility is the inclusion of compatible solutes evidencing cryoprotective effects, including ectoine and L-proline, in freezing media to enhance cell survival.
In this study, Hofmann et al. (2015) used human pulmonary microvascular endothelial cells pretreated via hyper- or hypo-thermal activation before cryopreservation with Me2SO and in combination with compatible solutes.1 The team heat-shocked (43ºC CO2 incubator or 45ºC water bath) or cold-shocked (4ºC water bath) the cells before incubation (37ºC) at three recultivation periods (4 hours, 10 hours, 12 hours) then froze them with various CPA concentrations (Me2SO 0%, 1%, 2.5%, 7.5%). They report that for heat-treated cells, the 10-hour recultivation period produced the best results in terms of membrane integrity but not for efficiency of recultivation. This measure appeared observable after 12 hours for all Me2SO concentrations after both hyper- and hypo-thermal activation.
For heat-shocked cells, RT-PCR analysis showed an upregulation of HSP70 gene expression at both the 4-hour and 12-hour time points, with the 43ºC thermal pretreatment evidencing greater expression than the 45ºC thermal pretreatment. The cold-shocked cells revealed a slight increase in HSP70 gene expression after 12 hours. The team used Western blotting (with BCA Protein Assay Kit for protein concentration and SuperSignal West Pico Chemiluminescent Substrate for detection, Thermo Scientific) to confirm. They found the highest levels of heat shock protein expression after thermal activation at the 12-hour time point.
Overall, the team could increase the efficiency of recultivation by >45% via hyperthermal activation (heat shock) when compared with untreated controls. Further, they were able to reduce Me2SO concentration to 2.5% while achieving a 20% improvement for efficiency of recultivation in comparison with controls. They required a 12-hour recultivation period for the best cell viability outcomes. The authors report that hypothermal pretreatment (cold shock) was less effective. In contrast with their previous study wherein supplementing freezing media with compatible solutes allowed for reduced Me2SO concentrations, here the team reports no additional benefit from the inclusion of compatible solutes. Hofmann et al. call for further studies using HSPs as protective agents as well as identifying other underlying mechanisms that may play a role in the effectiveness of thermal pretreatment.
1. Hofmann, N., et al. (2015) “Thermal pretreatment improves viability of cryopreserved human endothelial cells,” Biopreservation and Biobanking, 13 (5). doi: 10.1089/bio.2015.0024.