Date Published: May 23, 2019
Publisher: Public Library of Science
Author(s): Julie Meneghel, Peter Kilbride, John G. Morris, Fernanda Fonseca, Dariush Hinderberger.
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.
Cryopreservation is a key enabling technology contributing to the delivery of cell therapies to the clinic. However, 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 . During equilibrium cryopreservation of a cell suspension, where slow cooling in the presence of a cryoprotectant such as dimethyl sulfoxide (DMSO) is used, ice forms first in the extracellular compartment. This effectively removes water and produces a two-phase system of ice and a residual, freeze-concentrated solution of suspending medium including cryoprotectant and cells [2, 3]. The osmolality of this freeze-concentrated solution increases as the temperature is reduced and more ice forms. As slow cooling progresses the suspended cells will shrink as they lose water to try to remain in osmotic equilibrium with the extracellular solution. Thus, the cells are able to avoid intracellular ice formation. If the cooling rate is increased, a temperature will be reached where cellular water loss is not rapid enough to effectively reduce the increasing osmotic gradient between cells and suspending solution (non-equilibrium freezing). At this point the remaining water within the cell can form lethal intracellular ice . Understanding more about the physical state of the intracellular compartment of cells that avoid intracellular ice formation during equilibrium cryopreservation is clearly of value for optimising the technology and the freezing protocols.
The membrane lipid phase transition observed approximately 5°C above the extracellular compartment’s ice nucleation and melting temperature (Fig 1) is known not to affect cell viability substantially in other mammalian cell types . It is reversible, although with a slight hysteresis, highlighting an increased lipid conformational order during warming than during cooling [22, 23]. During the further, imposed slow cooling in the presence of extracellular ice, the Jurkat cells were subjected to dehydration as extracellular freeze concentration withdrew water from the cell. The endothermic event subsequently occurring (event B, Fig 2) was ascribed to an intracellular glass transition (Tg’i, event B, Fig 2). It should be noted that this vitrification signal detected in the cell suspensions by DSC, was absent from cell-free supernatants and so represents a cellular event. Finally, the extracellular medium vitrified at -123°C. Fig 5 schematically illustrates these sequential events taking place in a suspension of Jurkat cells during equilibrium cryopreservation in the presence of DMSO.