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.
Highlights
Cryopreservation is a key enabling technology contributing to the delivery of cell therapies to the clinic
In previous studies of equilibrium cooling of microorganisms in their natural environment, and under conditions relevant for cryopreservation, we have shown that the intracellular compartment becomes increasingly viscous and eventually undergoes a colloidal-like glass transition with continued cooling [6, 7]
The first cellular, physical transition observed during cooling of a suspension of Jurkat cells in dimethyl sulfoxide (DMSO) occurred within the cell membrane (Fig 1)
Summary
Cryopreservation is a key enabling technology contributing to the delivery of cell therapies to the clinic. Many details of critical, cellular responses to cryopreservation stresses are not well understood, which limits the pace of development of improved and efficient cell preservation protocols. A significant area concerns the formation of intracellular ice which is, typically, a lethal event for the cell [1]. Advanced Therapies for Patients in Manchester, Innovate UK Project Number: 6239 (https://applyfor-innovation-funding.service.gov.uk/competition/ search). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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