The goal of this work was to find alternatives to DMSO for the cryopreservation, through the vitrification method, of mammalian cells and potentially for other cell types and tissues. In literature it is possible to find only a reports for the vitrification of mammalian cells, mostly using advanced technologies, such as electroporation.27 Therefore, the results presented in this manuscript show natural cryoprotectants can be used as vitrification media, using a simple methodology, for numerous applications in cryobiology.
It is known that for the prevention of the injury caused by the formation of intracellular ice, a CPA must be added to modulate the rate and extent of cellular dehydration during freezing-induced membrane phase transition.28 Hence, ideally a permeating agent must be present in the vitrification solution. However, in this work we were able to show that this is not actually mandatory and that large molecules (non-penetrating) are, in fact, able to protect the cells during the vitrification process.
Our work focused on the use of NADES as CPA for the vitrification of mammalian cells. These NADES are mainly composed by primary metabolites commonly found in animals and organisms that live in extremely cold environments, as well as, common components of culture media, such as sugars, polyols, and amino acids (Table 1)18.
Most of the systems prepared contain the mono- and disaccharides, glucose, trehalose and sucrose which exert considerable osmotic effects as smaller non-permeating molecules, such as betaine and many amino acids. In fact, the incorporation of a small saccharide promoted the cell dehydration and its vitrification, reducing the concentration of intracellular cryoprotectant and consequently its toxicity, as observed during a study where sucrose alone was able to significantly decrease the toxicity of a vitrification solution for the cryopreservation of mouse embryos.29
Then, 50% (w/v) of NADES were used as CPA for the vitrification of mammalian cells. The reason of using 50% (w/v) instead of higher percentages of NADES is highly related to their high toxicity at those concentrations, as well as, their high viscosity which would difficult the diffusion process during freezing.
Vitrification is known to be achieved when the samples are frozen at freezing rates of 15,000–30,000 ºC/min, generally, achieved by submersing them directly in liquid nitrogen. However, to the extent of our knowledge there are no reports using this method to vitrify mammalian cells in cryovials, as is usually performed using the slow-freezing method. Herein, we have shown that it is, in fact, possible to successfully cryopreserve mammalian cells in cryovials using the vitrification method, using NADES as CPA. For that, cryovials were immediately immerse in a container with liquid nitrogen before transferring them to the − 80 ºC freezer. This method allowed the immediate freezing of the samples. Although requiring extra care during the handling of liquid nitrogen it is a simple methodology, not requiring any state-of-the-art equipment.
An initial screening was performed, in two cell lines, L929 and HacaT cells, to select the most promising systems (Fig. 2). From the results we have seen that for L929, NADES exerted a much more pronounced cryoprotective effect than DMSO. On the other hand, for HacaT cells only one eutectic system (BetTreW) showed promising results. However, HacaT cells frozen with 50% (v/v) DMSO were not able to survive in this method. In general, this method resulted in considerable better results for L929 cells, which is a more robust and resilient cell line. The results can be explained by the thermal behaviour of the aqueous mixtures of NADES and DMSO. While 50% (w/v) aqueous mixtures of NADES are able to freeze, although in an amorphous state, in the conditions reported in this study, the 50% (v/v) aqueous mixtures of DMSO cannot completely freeze in the same conditions. This fact contributes to cells to die due to the high toxicity of DMSO.
In this work, we have also shown that after thawing, the vitrification media does not need to be removed and a simple dilution is sufficient to reduce the toxicity of the CPAs. Then, cells can be directly cultured in T-flasks and/or plated which can be a major advantage because the centrifugation step or washing step to remove the CPA can cause a certain degree of cell death. This can be particularly relevant if this method is used during cell therapy or transplantation procedures.
The systems TreGluSorW, BetTreW and BetSucProW showed the most promising results towards L929 cells, while for HacaT, the system BetTreW was the only system showing favourable results. Hence, further studies were carried out with these systems, including thermal behaviour, morphology and proliferation studies. The thermal studies, using a DSC/POM equipment have shown that the 50% (v/v) aqueous solution of DMSO does not freeze in the experiment conditions, but the aqueous solutions of the selected NADES showed a significant decrease on the Tc and Tm when compared with pure distilled water. Additionally, the shape of the crystals is completely different between the NADES aqueous solutions and water. The major advantage of the method used herein is the fact that we used polarized light which helps to distinguish crystalline and amorphous states. In the case of NADES aqueous solutions we observed black images confirming the amorphous state of the samples, while with water bright and coloured crystals were observed.
The NADES studied in this work did not affect the morphology of the cells after freezing and thawing as showed by the morphology studies after 72h of the thawing (Fig. 2-C1 and Fig. 3-C1). In terms of proliferation, we observed that NADES did not interfere with the normal proliferation of L929 cells but, on the other hand, DMSO significantly compromised the cell growth (Fig. 2-A, C2). Furthermore, DMSO contributed to a very low DNA content while with NADES the complete opposite was observed, after 72h of thawing.
Regarding the HacaT cells it was possible to see that the system BetTreW allowed a successful post-thawing viability, and after 72h very large colonies were observed (Figs. 2-A, C2) and after 5 days cells were completely confluent (not showed). In terms of DNA content, the same trend was observed.
Both L929 and HacaT that showed significant post-thawing survival were cultured up to 3 passages, and the same studies were conducted for different passages (P1 and P2). The results showed that after the first passage, cells presented high values of viability and an improvement of proliferation, suggesting that NADES do not affect cellular activity (Figure S2 and S3).
In summary, we have shown that NADES are an emerging class of CPA that can be used for the cryopreservation of mammalian cells using the vitrification method. Our study showed that the L929 cells were able to be cryopreserved in the presence of the eutectic systems TreGluSorW, BetTreW, and BetSucProW. Additionally, the system BetTreW was able to confer cryoprotective properties to HacaT resulting in a successful cryopreservation. Nevertheless, the possible combinations of primary metabolites able to be used as CPA are endless and therefore in further studies other systems might be studied. Furthermore, it is important to highlight that the major advantage of these new materials is the fact that they contribute for cell survival without their removal from the growth media.
The results presented herein show that NADES are potential CPA for the vitrification of other biological materials such as stem cells, sperm, embryos, tissues, etc.