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.

Fig 1. Membrane lipid phase transition of Jurkat cells identified by following the position of the symmetric CH₂ stretching vibration (νCH₂) of membrane fatty acids chains by Fourier transform infrared spectroscopy.

Measurements were carried out during cooling from 37°C to -50°C (thick black) and warming to 60°C (thick grey) at rates of 2°C min^-1. Derivatives of these curves were calculated (thin lines) and their maxima were taken as the membrane lipid phase transitions: solidification on cooling (thin black, Ts), observed at -1.0°C ± 0.8°C and melting on warming (thin grey, Tm), observed at 5.8°C ± 0.2°C (n = 3).

Fig 2

A typical DSC trace of Jurkat cells (solid lines) and cell-free supernatant (dotted lines) during warming, and their first-order (grey) and second-order (black) derivatives. Both samples were separated by centrifugation from a suspension of Jurkat cells in culture medium with 10% (v/v) dimethyl sulfoxide. They were initially cooled at 2°C min^-1 to -150°C. Three distinct thermal events are apparent: the extracellular glass transition (A), the intracellular glass transition (B), and bulk melting of the extracellular solution (C). Note that transition B, characterized by its onset, maximum and endset, is absent in the DSC trace of the cell-free supernatant.

Fig 3. First-order derivatives of the DSC traces of mixtures of CryoStor10 and serum albumin during warming at 10°C min^-1 following cooling at -10°C min^-1.

The following percentages, by weight, of albumin dissolved in CryoStor10 are identified by trace colour and are: black, 0%; grey, 10%; grey-blue, 20%; light blue, 30%; blue, 40%; dark blue, 50%. The raw DSC trace of CryoStor10 is also included (dotted black line).

Fig 4

Viable cell count (A, C), and metabolic activity (B, D) of Jurkat cells cooled down at 1°C min^-1 to separate endpoints before plunging into liquid nitrogen. Viable cell count was measured through fluorescein diacetate staining and metabolic activity was evaluated through the reduction of resazurin to the fluorescent resorufin, at 24 h (grey), 48 h (white), and 72 h (black), post thaw (n = 5 ± SD). For metabolic activity, fluorescent intensities were normalised to 1 at the -50°C, 24 h time point.

Fig 5. Schematic of the physical transitions occurring during cryopreservation of a Jurkat cell in the presence of dimethyl sulfoxide.

As temperature decreases (from left to right), the cell membrane first undergoes a liquid-crystal to gel phase transition, with its midpoint at approximately 0°C. At approximately -7°C ice starts to nucleate in the extracellular compartment and with ice crystal growth the extracellular solute concentration increases. Intracellular water flows out of the cells in response to the osmotic gradient. As cell dehydration takes place, the intracellular compartment becomes more and more crowded (the blue and red dots symbolising various macromolecules), until it undergoes a glass transition, starting at -47°C. Intracellular macromolecules now form a network (linked dots) analogous to a colloidal glass, that behaves as a molecular sieve in which nanopores still remain liquid. Upon further cooling, the extracellular compartment and the intracellular nanopores undergo a glass transition at -123°C (darkening of the blue shade of the intra- and extracellular compartments). Below this temperature, molecular mobility ceases. Jurkat cell images adapted from Walter et al. [24].