Protein therapeutics have significantly widened the scope of difficult-to-treat diseases which were not treatable with small molecule drugs. Nowadays, the cell and gene therapy is providing solutions to medical conditions, beyond proteinaceous medicines. Cell therapy products (CTPs) are however, highly complex systems, which face substantial challenges in manufacturing, distribution and storage. The storage is mostly based on cryopreservation in liquid nitrogen, which has several drawbacks. Besides being expensive, it also demands from clinicians to be skilled in often complex thawing protocols and in other post-manufacturing procedures. This involves elimination of dimethylsulfoxide (DMSO) from thawed formulations. DMSO is currently a standard cryoprotectant, utilized in numerous CTPs due to its good protection against low temperature, even though it is not safe in humans. Development of an alternative preservation model with cells stored at temperatures above those of liquid nitrogen for long periods, without the use of DMSO, would make these therapies much safer and cost efficient. A potential alternative could be freeze dried CTPs, where the cells are in dried formed and are reconstituted before administration. However, a successful formulation and process, that would provide sufficient cell recovery after freeze drying, is yet to be developed.
The aim of this study was to screen the cell survival of freeze-thawing process in a number of formulations and establish, if it’s possible to lyophilize them. For this purpose, SK-N-AS cells were used as a model adherent cancer cell line. The research firstly focused on the DMSO replacement with polyols, optimization of tonicity, testing the effect of cell medium supplements and introduction of trehalose intracellularly. A good post-thaw cell survival was obtained with several designed formulations, however the visual inspection showed a poor appearance of freeze dried products. Secondly the cells were successfully cultured in 2D on the glass vial surface and in 3D hydrogel scaffolds. Cultures were then freeze-thawed and the function of cell attachment in cell preservation at -80°C storage was tested. Next, the lyophilisability of cell scaffolds was evaluated by visual inspection. Addition of polyol/trehalose cryoprotectant formulations to the scaffold negatively impacted the lyophilization of the product. Preliminary results showed some protective ability of hydrogels and a very good lyophilisability.