Periodic Reporting for period 1 - CryoDES (Nature-inspired Cryopreservation Systems using Natural Deep Eutectic Systems)
Reporting period: 2022-10-01 to 2024-03-31
Cell therapies are foreseen as the next generation therapeutics for some of the chronic diseases with highest global economic and social burden. The market for human cell therapies has been estimated to be 8 B€ in 2021 with an expected annual growth rate of 26% until 2028. The key aspect for the success of a cell therapy is the preservation of cells. Currently, cells must be cryopreserved using a cryoprotectant agent (CPA) at -196ºC, in a liquid nitrogen tank. The gold standard CPA is a chemical organic compound, dimethyl sulfoxide (DMSO) highly toxic. CryoDES is a cryoprotectant agent, non-toxic and composed of generally regarded as safe components. These systems have proven to be able to replace the gold standard CPA, presenting the same or better performance indicators in different immortalized cell lines. However, CryoDES has never been tested in clinically relevant cells. This was the main aim of this project, which also focused on relevant cells for the veterinary market.
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.
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.
The results of CryoDES demonstrate that the use of DES in the cryopreservation of cells with therapeutic potential does not have a significant improvement over the gold standard conventionally used, which is DMSO. This project has hence shown that the technical feasibility of CryoDES is impaired for this application.
However, this is not the case for cells with therapeutic potential in veterinary. The results on the veterinary stem cells indicate that CryoDES have a great potential to be used for veterinary purposes. However, cells must be frozen first in DMSO, thawed and then frozen with DES as cryoprotectant agent. Once thawed, these cells can be directly injected into the animal without further toxicity as indicated by the in vitro studies reported herein.
In order to ensure further uptake by the market, particularly the veterinary stem cell market, further research in different cells should be pursued. Additionally, an in depth market research should be carried out with this market segment. In parallel, the scaling-up and regulatory aspects related to the commercialization of the cryoprotectant agent should be assessed.
However, this is not the case for cells with therapeutic potential in veterinary. The results on the veterinary stem cells indicate that CryoDES have a great potential to be used for veterinary purposes. However, cells must be frozen first in DMSO, thawed and then frozen with DES as cryoprotectant agent. Once thawed, these cells can be directly injected into the animal without further toxicity as indicated by the in vitro studies reported herein.
In order to ensure further uptake by the market, particularly the veterinary stem cell market, further research in different cells should be pursued. Additionally, an in depth market research should be carried out with this market segment. In parallel, the scaling-up and regulatory aspects related to the commercialization of the cryoprotectant agent should be assessed.