A scale down process for the development of large volume cryopreservation

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 24 h 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 48 h or 72 h 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.

Engaging Cold to Upregulate Cell Proliferation in Alginate-Encapsulated Liver Spheroids

“…This has been done either to preserve ELS once a sufficient cell density has been achieved, or to explore the possibility that alginate encapsulation may offer some cryoprotection. It was in the course of our work that we observed a stimulatory effect on cell proliferation within ELS following freezing and thawing (9,(12)(13)(14). In this study, the potential for low temperatures to induce proliferation of alginate encapsulated cells upon return to culture conditions has been examined in detail.…”

“…The cryopreservation of these ELS has been studied previously (8,9), and groups have examined the cryopreservation of other alginate encapsulated cell lines (7,10,11). This has been done either to preserve ELS once a sufficient cell density has been achieved, or to explore the possibility that alginate encapsulation may offer some cryoprotection.…”

Advances in the slow freezing cryopreservation of microencapsulated cells

“…By contrast, following spontaneous ice nucleation, intercellular ice was formed provoking cell injury ( Figure 3C-2,4 [17]. Therefore, cell-based products should be chilled to avoid CPA induced [63]. Two different heat transfer modules designed for an electrical Stirling cooling machine were used to cryopreserve microencapsulated liver spheroids in cryovials.…”

“…A similar study using cryopreserved microencapsulated porcine hepatocytes in FLF induced mice was also performed [123]. The Liver Group, from University College of London (UCL), is developing a bioartificial liver (7x10 10 cells biomass) for the treatment of FLF [83,88,125], studying for that aim, the slow freezing cryopreservation of alginate microencapsulated liver HepG2 spheroids [12,63,64,74,79,126,127]. To reduce contamination risks in the process, they compared a CRF without the use of liquid coolants with another CRF that use liquid nitrogen for slow cooling cryopreservation [126].…”

“…Interestingly, with the aim of cryopreserving a whole bioartificial liver (2.3L biomass) the process of ice formation and propagation was investigated in the slow freezing cryopreservation [63]. In this scenario, the water-ice phase change can be dominated by the network solidification process, which is typically present in small samples such as cryostraws or cryovials, or progressive solidification, which is more often observed in large volumes or environmental freezing.…”