006 The importance of spatial location of cells during large volume cryopreservation and contrast between larger and smaller cryopreservation volumes

Abstract

The differences between large and small volume freezing on alginate encapsulated liver spheroids (ELS) have been studied. When small volumes are cryopreserved, they tend to follow a pre-set thermal profile. The whole sample will be approximately thermally homogeneous – no great temperature gradients will exist within. As the sample falls below its equilibrium melting point, the sample will become uniformly supercooled (US), followed by ice transition and associated release of latent heat. Ice will form rapidly throughout the sample homogeneously, before the temperature profile of the run continues. With a large volume (in our case 1500–2000 ml in a cylindrical chamber) different conditions are experienced by different areas of biomass, dependant on location inside the cryopreservation chamber. The biomass close to the edge of the chamber (i.e. closest to the surface and cooling plate), will follow a profile similar to small volumes. The core will be different however. Ice will grow relatively slowly through the sample (controllable ice formation (CIF), dependant on surface conditions) at its equilibrium melting point, without a temperature peak. This ice front preferentially exclude solutes, resulting in the biomass furthest from the chamber walls experiencing increasingly hypertonic solutions for protracted periods of time. ELS were cryopreserved using either one of the above conditions, in a 38% Viaspan, 12% Me 2 SO, and 50% ELS solution (by volume). A modified flat-plate EF600 CRF was used with special inserts – both holding 6 ml scintillation vials. One insert was made of aluminium and one of acetal (good/bad conductors of heat respectively). The aluminium insert mimicked the cryopreservation profile of small volumes – cooling equally from its bottom and sides inducing US. The acetal insert mimics a large volume – only the vial’s base was cooled, resulting in a large temperature gradient. Nucleating beads were added at the vial’s base. The EF600 plate was cooled from 4 to −80 °C at −0.5 °C/min. Samples were held at −80 °C for 1 h, before being stored in a −80 °C freezer for 1 week and then thawed rapidly and re-cultured. Functional tests were carried out a set time points up to 72 h post-thaw. A cell membrane viability and metabolic activity study have been carried out. There was no statistically significant difference at any time point between the two sets. The samples within the acetal insert showed a non-significant lower post-thaw cell density (at 6;h post-thaw – 6.2 ± 2.3 million cells/ml for the controlled ice growth (CIF) sample, 8.1 ± 1.6 million cells/ml for the US; by 48 h post-thaw 10.1 ± 2.6 million cells/ml for the CIF, 10.8 ± 2.7 million cells/ml for the US, albeit with similar MTT function per cryopreserved volume. Another set of ELS were cryopreserved in a large 1500 ml volume CRF developed by Asymptote. Results show the location within the large volume is important to cell survival– those at the extremes (centre of chamber or on the edge of the chamber), have lower post-thaw survival, whereas those in the centre show better survival. These larger volume effects become crucially important when considering cryopreservation of larger biological constructs and whole organ cryopreservation. Source of funding: The Medical Research Council and The Liver Group Charity. Conflict of interest: None declared. peter.kilbride.11@ucl.ac.uk