Physical events occurring during the cryopreservation of immortalized human T cells

Abstract

Cryopreservation is key for delivery of cellular therapies, however the key physical and biological events during cryopreservation are poorly understood. This study explored the entire cooling range, from membrane phase transitions above 0°C to the extracellular glass transition at -123°C, including an endothermic event occurring at -47°C that we attributed to the glass transition of the intracellular compartment. An immortalised, human suspension cell line (Jurkat) was studied, using the cryoprotectant dimethyl sulfoxide. Fourier transform infrared spectroscopy was used to determine membrane phase transitions and differential scanning calorimetry to analyse glass transition events. Jurkat cells were exposed to controlled cooling followed by rapid, uncontrolled cooling to examine biological implications of the events, with post-thaw viable cell number and functionality assessed up to 72 h post-thaw. The intracellular glass transition observed at -47°C corresponded to a sharp discontinuity in biological recovery following rapid cooling. No other physical events were seen which could be related to post-thaw viability or performance significantly. Controlled cooling to at least -47°C during the cryopreservation of Jurkat cells, in the presence of dimethyl sulfoxide, will ensure an optimal post-thaw viability. Below -47°C, rapid cooling can be used. This provides an enhanced physical and biological understanding of the key events during cryopreservation and should accelerate the development of optimised cryobiological cooling protocols.

Automated dry thawing of cryopreserved haematopoietic cells is not adversely influenced by cryostorage time, patient age or gender

“…The data presented in Fig 3 indicates that extending frozen storage from 3 months to 17 years has no significant effect on the post-thaw outcome of the samples. To ensure the safe, long-term storage of apheresis samples it is critical that the samples are held, continuously, below the glass transition temperature of the cryoprotectant solution, some -120°C for DMSO-based solutions [ 6 , 32 ]. Studies with other biological systems have shown this to be effective, and necessary, for decades [ 4 , 33 – 37 ].…”

Compared DNA and RNA quality of breast cancer biobanking samples after long-term storage protocols in − 80 °C and liquid nitrogen

“…This effect is used in vitrification processes for cryo-conservation without damaging ice crystals 40 . For our studies Tg can only be estimated, but Meneghel et al demonstrated a Tg around − 47 °C for human T-cells in suspension 41 . Although our cells were not in suspension and no T-cells, cytoplasm might be of similar composition.…”

Cryopreservation of Human Midbrain Dopaminergic Neural Progenitor Cells Poised for Neuronal Differentiation

“…Cooling rates were varied from 0.5 to 2 • C/min using a VIA Freeze TM Duo controlled-rate freezer starting from 4 • C and stopping at −80 • C before transferring vials to vapor phase liquid nitrogen storage (Figure 2). The vapor phase of liquid nitrogen (below −170 • C) was chosen since this is below the glass transition temperature of cells in cryoprotectant (∼−120 • C), and biological material is known to be highly stable at this temperature with no changes in cell viability for over 1 year (Silani et al, 1988;Massie et al, 2013;Meneghel et al, 2019). The recovery of mDA neural progenitor cells was performed by either thawing vials slowly in air at 4 • C or rapidly in a water-bath set to 37 • C. Cell viability was assessed by Trypan blue exclusion immediately upon thawing prior to plating in mDA neuronal differentiation conditions, and again at 24 h post-thawing.…”