Abstract
The process of ice formation and propagation during cryopreservation impacts on the post-thaw outcome for a sample. Two processes, either network solidification or progressive solidification, can dominate the water–ice phase transition with network solidification typically present in small sample cryo-straws or cryo-vials. Progressive solidification is more often observed in larger volumes or environmental freezing. These different ice phase progressions could have a significant impact on cryopreservation in scale-up and larger volume cryo-banking protocols necessitating their study when considering cell therapy applications.This study determines the impact of these different processes on alginate encapsulated liver spheroids (ELS) as a model system during cryopreservation, and develops a method to replicate these differences in an economical manner.It was found in the current studies that progressive solidification resulted in fewer, but proportionally more viable cells 24h post-thaw compared with network solidification. The differences between the groups diminished at later time points post-thaw as cells recovered the ability to undertake cell division, with no statistically significant differences seen by either 48h or 72h in recovery cultures.Thus progressive solidification itself should not prove a significant hurdle in the search for successful cryopreservation in large volumes. However, some small but significant differences were noted in total viable cell recoveries and functional assessments between samples cooled with either progressive or network solidification, and these require further investigation.
Highlights
A bioartificial liver (BAL) machine can temporarily replace the functions of the liver, allowing a damaged liver to regenerate while protecting the patient’s other organs from the life-threatening damage that ensues during liver failure
While the sample layer adjacent to the cylinder wall reduced in temperature approximately linearly, the central sample layers experienced delayed cooling, non-linearity of temperature change, and eventual solidification, with a temperature plateau existing in the core of the sample for some considerable time at the equilibrium melting temperature before solidification occurred
A planer ice structure is present under conditions of progressive solidification (PS) in samples processed in the acetal module (Fig. 5A), with vertical ice crystals forming in the sample, entrapping encapsulated cell spheroids (ELS) between ice crystals
Summary
A bioartificial liver (BAL) machine can temporarily replace the functions of the liver, allowing a damaged liver to regenerate while protecting the patient’s other organs from the life-threatening damage that ensues during liver failure. While there are reports of the cryopreservation in bags of large volumes (>100 ml) of adult stem cells [25], mammalian tissue culture cells [8,9,12] and ELS [15], the geometry of these samples have been those of a thin slab (2d sample) less than 20 mm in thickness. These experience lesser thermal gradients than in our system
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