060 Towards large scale GMP cryopreservation of the conditioned liver cell biomass for a bioartifical liver machine: Delivery of process control for the freezing protocol

Introduction: It is increasingly being recognised that the cold-chain step is of essential importance in the delivery pathway for regenerative medicine products. Equally, clinical translation requires often requires products of sufficiently large scale to support adult human physiology, and at the same time deliver cryopreservation which will comply with Good Manufacturing Process (GMP). Whilst stirling motor cooling technologies permit GMP controlled-rate freezing (CRF) at small scale using the EF600 cryocooler, successful large scale GMP cryopreservation is more challenging because of heat transfer issues and control of ice nucleation – complex biophysical events which impact on cryopreservation success. Methods: We have developed a proto-type scaled-up cryocooler based CRF (VIA Freeze) capable of processing larger volumes and have evaluated it using alginate encapsulated liver cell spheroids (ELS) which will form the active component of the UCL bioartifical liver support system. Sample temperatures and Stirling cryocooler power consumption were recorded throughout cooling runs for both small (500 μl) and large (200 ml) volume samples. ELS recoveries were assessed using viability (FDA/PI staining with image analysis), cell number (nuclei count) and function (protein secretion). In addition, cryo-SEM and freeze substitution techniques were applied to identify ultrastructural effects of the protocols. Slow cooling profiles with a Me 2 SO-based cryoprotectant (CPA) were successfully applied to samples in both the EF600 and the VIA Freeze, and a number of cooling and warming profiles were evaluated. The importance of controlled ice nucleation was also investigated. Results: The nucleation of ice can was important for high post-thaw recoveries and could be detected in the VIA Freeze by recorded changes in power output during release of latent heat of ice formation. An optimised non-linear cooling profile (in the range from ice nucleation to −60 °C, followed by transfer to −150 °C) was implemented in both the EF600 and VIA Freeze. It proved possible to reduce the excess CPA supernatant volume after equilibration and just prior to freezing without compromising protocol success. Ultrastructural analyses identified different patterns of ice formation depending on control of ice nucleation, and significant levels of dehydration of the ELS in samples which were compatible with high post-thaw recoveries. The VIA freeze allowed cryopreservation of 200 ml of ELS with post-thaw viabilities at 93.4 ± 7.4 , viable cell numbers at 14.3 ± 1.7 × 106 nuclei/ml alginate and protein secretion at 10.5 ± 1.7 μg/ml/24 h. These values compared well with control non-frozen ELS (viability – 98.1 ± 0.9%; cell numbers – 18.3 ± 1.0 million nuclei/ml, protein synthesis – 18.7 ± 1.8 μg/ml/24 h) and also those achieved in small scale cryopreservation in the EF600 (viability – 95.9 ± 2.3; viable cell numbers – 21.0 ± 2.2). Conclusion: Large volume GMP cryopreservation of ELS is possible with good functional recoveries using the VIA Freeze which additionally provides novel determination of ice nucleation events with potential advantages for Quality Control purposes and active control of heat transfer at this point. The system may find other application in cryopreservation for regenerative medicine. Source of funding: None declared. Conflict of interest: None declared. b.fuller@ucl.ac.uk