Robust Alternative To Cryobags For Large Volume Cryopreservation

Large-volume cryopreservation

Cryopreservation of small sample volumes is common in many scientific applications. However, there is increasing demand for large-volume cryopreservation, especially in cell and gene therapies, where treatments require large cell numbers.

Although cryobags are generally a good choice for the cryopreservation of large-volumes, cryovials provide a more robust alternative. They are also considerably easier to fill and empty, and are available in volumes up to 50 mL to meet high volume needs. However, the thermal performance of large-volume cryovials differs markedly from small vials and cryobags, which have wall thicknesses of typically 100 to 300 µm and so enable rapid heat transfer in and out of a biological sample.

By comparison, large-volume vials require thicker walls, typically up to 1 mm, to maintain structural security during cooling, thawing, storage, and transportation, and are made from polymers with poor thermal conductivity. This combination results in delayed heat transfer in and out of a sample, which can lead to thermal gradients within a large-volume sample.

The resulting cooling inconsistencies mean cells in the center of a vial can freeze more slowly than those nearer the edge, affecting their viability and function (1).

Such inconsistent cooling is also a consideration when freezing cells with liquid nitrogen (LN2), often the default choice for cryopreservation. Samples nearest to where the liquid nitrogen is pumped into the cooling chamber cool at a different rate to samples further away. As well as potential sample variability, liquid nitrogen gives rise to concerns about contamination and sterility, safety, and high running costs.

This study demonstrates an optimized cooling protocol for cryopreserving large-volume cryovials in a VIA Freeze Quad controlled-rate freezer, avoiding the need for liquid nitrogen. Using a new sample plate, developed to accommodate large cryovials and increase the capacity of the VIA Freeze Quad freezer to 16 vials and a total of 480 mL, this protocol produces consistent cooling profiles. Samples are cooled evenly below the -60°C necessary for cryopreservation and show rapid recovery and post-thaw viability.