The ERC PoC focused on the exploitation of the technical viability of CryoDES in order to leverage the TRL level of the technology towards TRL 5, validation in relevant environment. To achieve this, several contacts with industrial partners were done and four main collaboration protocols were established. The work carried out targeted not only the development of CryoDES as cryoprotectant for stem cells with human therapeutic potential but also cell for veterinary applications.
One major company in the market has tested three different types of cells using CryoDES. Induced pluripotent stem cells (iPSC), natural killer cells (NK) and human breast tumoroids. Cells were frozen with CryoDES and DMSO (control) and the preliminary results obtained after thawing were not very encouraging. There was a significant loss of viability after thawing, which despite being recovered over time compromises our solution as the CryoDES did not show a similar performance to the gold standard DMSO, hence the studies were not continued.
The work carried out with another company aimed to study human adipose-derived stromal/stem-like cells (hASCs). The results obtained demonstrate that the biocompatibility of CryoDES was similar to DMSO; the cryopreservation efficiency was also similar for short storage (1 and 7 days). However, for longer cryopreservation periods only CryoDES at 10% (v/v) represents a viable alternative to DMSO. In a second step we have evaluated the effect of cryopreservation of hASCs, in terms of cell identity and stemness potential.
The viability of hASCs cryopreserved in CryoDES is significantly lower than that of cells frozen in DMSO at the same temperature (-80 °C), negatively impacting the number of cells recovered after freezing. Nevertheless, flow cytometry analysis of this cell population revealed similar expression patterns of mesenchymal stem cells identity markers across the tested conditions. That is to say that no major qualitative differences were observed regarding the impact of each cryoprotectant on hASCs' clonogenic ability. Similarly, the ability of hASCs to differentiate towards adipogenic and osteogenic lineages remained unaffected by CryoDES.
CryoDES does not alter hASCs during the cryopreservation process, but the lack of benefits compared to DMSO-based solutions and the lower number of viable cells recovered after thawing pose challenges for widespread commercial applications.
The application of CryoDES in cells for veterinary applications was carried out in dog and cat stem cells. Cells were cryopreserved in the presence of CryoDES and DMSO, for different time periods at -80 ºC. Once cells arrived in the lab, they were cryopreserved. After thawing, cells were allowed to reach confluency, especially those cryopreserved in DMSO, and then frozen again in the presence of the same CryoDES and DMSO. Post-thawing cell viability was determined and overall, the results showed that CryoDES were more effective in dog stem cells than in cat cells, however this was probably due to a delay in the delivery of cat stem cells caused by the transporter.
Within the framework of another collaboration established with a partner expert in veterinary stem cells, CryoDES was tested in bovine primary cells (FMMB1). After two months of freezing at -80 ºC the results show that the viability after thawing is much lower with CryoDES than with DMSO, however there is a gradual recovery and after three passages and the viability and metabolic activity is equiparable. In terms of populations doubling, which quantifies the growth of a cell population over time in cell culture, the results are identical in both CryoDES and DMSO.
