Asymptote (United Kingdom)

Cryopreservation is the ability to hold cells at an ultralow temperature such that, at some point in the future, they can be returned to normal culture conditions and resume cell function, growth and division. Cryopreserved cells may survive for short periods at higher temperatures such as -80°C for the purposes of quarantine or shipment, but this cannot be used for long-term storage. According to the commonly accepted theory of ‘colligative’ action, a key function of these cryoprotective agents (CPAs) in cell preservation is their ability to increase solute concentration both within the cell and extra-cellular, which suppresses intracellular ice formation. It is clear that professional biobanking systems must address a number of issues related to implementation of cryopreserved storage. In some protocols, nucleation is initiated by tapping the pre-cooled container of cryopro-tected cells or adding a nucleating agent with the cryoprotectant. Inappropriate ice nucleation is a significant variable in controlled rate freezing methods for cryopreservation.

The freezing step influences lyophilization efficiency and protein stability. The main objective of this work was to investigate the impact on the primary drying stage of an ultrasound controlled ice nucleation technology, compared with usual freezing protocols. Lyophilization cycles involving different freezing protocols (applying a constant shelf cooling rate of 1 degrees C/min or 0.2 degrees C/min, putting vials on a precooled shelf, and controlling nucleation by ultrasounds or by addition of a nucleating agent) were performed in a prototype freeze-dryer. Three protective media including sucrose or maltodextrin and differing by their thermal properties and their ability to preserve a model protein (catalase) were used. The visual aspect of the lyophilized cake, residual water content, and enzymatic activity recovery of catalase were assessed after each lyophilization cycle and after 1 month of storage of the lyophilized product at 4 degrees C and 25 degrees C. The freezing protocols allowing increasing nucleation temperature (precooled shelf and controlled nucleation by using ultrasounds or a nucleating agent) induced a faster sublimation step and higher sublimation rate homogeneity. Whatever the composition of the protective medium, applying the ultrasound technology made it possible to decrease the sublimation time by 14%, compared with the freezing method involving a constant shelf cooling rate of 1 degrees C/min. Concerning the enzyme activity recovery, the impact of the freezing protocol was observed only for the protective medium involving maltodextrin, a less effective protective agent than sucrose. Higher activity recovery results were obtained after storage when the ultrasound technology or the precooled shelf method was applied. Controlling ice nucleation during the freezing step of the lyophilization process improved the homogeneity of the sublimation rates, which will, in turn, reduce the intervial heterogeneity. The freeze-dryer prototype including the system of controlled nucleation by ultrasounds appears to be a promising tool in accelerating sublimation and improving intrabatch homogeneity.