Cryopreservation has become an indispensable step in the daily routine of scientific research as well as in a number of medical applications, ranging from assisted reproduction and transplantations to cell-based therapies and biomarker identification. It is hardly possible to picture todays scientific and medical advancements without this technique.The successful development and implementation of all the therapeutic and scientific discoveries involving cryopreservation relies on the correct and safe translation of the method from the laboratory to the clinical and manufacturing scale.

With the need to correctly use this technique, more research is focusing on optimizing cryopreservation methods and investigating what the long-term effects and consequences are on the physiology of the cryopreserved material.

An important part of cell therapy research is focused on adult stem cells (ASCs). ASCs can be derived from different sources such as peripheral blood, bone marrow or adipose tissue and display strong promises because of their capacity to differentiate into any cell type of the human body.In recent work3, the team of Michael Pepper at the Institute for Cellular and Molecular Medicine in Pretoria, South Africa, explored the effects of cryopreservation on the differentiation ability of adipose tissue-derived stem cells (ADSCs). After analyzing gene expression of key adipogenic genes and the degree of differentiating cells, characterized with high levels of CD36 and intracellular lipid droplets, the scientists reported that slow freeze cryopreservation of cells shortly after their isolation causes no alterations on their ability to differentiate. Pepper is convinced of the necessity to perform such analysis when cryopreserving important cell pools: It is critical to do a post-thaw analysis of cell function to determine how the cryopreservation may have affected the cells.His team is analyzing the effects of cryopreservation on other cell types largely used in cell-based therapies such as hematological stem cells and peripheral blood mononuclear cells (PBMCs). Although they didnt observe major alterations in terms of immunophenotyping or the post-thaw proliferation of the cells, Pepper expresses his concern that more subtle characteristics might be affected.

Correct cryopreservation of cells intended for therapeutic use is crucial. This is very important particularly as cells may persist for a long time in the recipient. This area of cell therapy research definitely requires more attention, Pepper says. Moreover, his words reflect on the need to evaluate not only the direct post-thaw recovery, but to look deeper into the late-onset effects cryopreservation might have and ensure that transplanted cells have preserved their therapeutic properties.

In contrast to slow freezing, vitrification relies on the fast freezing of the material by putting it in high concentration of cryoprotectant and in contact with liquid nitrogen. This method allows the direct transition of water from liquid to solid state without crystal formation. The highly concentrated cryoprotectant prevents ice formation and therefore there is no need for slow cooling.

Although vitrification has a great potential, there are a couple of parameters that are a point of concern. The quick and drastic freeze is possible thanks to the high concentration of cryoprotectant, but the latter is also associated with higher toxicity. In some cases, an additional limitation is the direct contact of the sample with liquid nitrogen which is a predisposition for viral or bacterial contamination.The team of Christiani Amorim at the Institute for Experimental and Clinical Research in Louvain, Belgium, is approaching the challenges of vitrification in the context of ovarian auto-transplantation. Ovarian auto-transplantation consists of preserving a piece of ovarian tissue with active follicles from the pre-therapeutic ovary of a cancer patient, as chemotherapy often has damaging effects on the reproductive organs. This tissue sample will be conserved and auto-transplanted onto the patients ovary when she has recovered and wishes to become pregnant.In their recent research4, the authors used stepped vitrification, in which the concentration of the cryoprotectant is gradually increased while simultaneously temperature decreases. This avoids ice crystal formation and also prevents cryoprotectant toxicity.Although stepped vitrification has previously given good results in bovine ovarian tissue5, this was not the case for human ovarian tissue. The scientists didnt detect normal follicles following thawing and linked this to high cryoprotectant toxicity. Indeed, they observed all signs of dimethyl sulfoxide (DMSO)-related cell membrane damage: significant organelle damage, cell membrane disintegration and apoptosis. These observations imply on the variability of outcomes that the method could give when applied to the same type of tissue but from a different organism.Amorim is positive about the future of their method and recognizes the need for further research on the topic: I can see a great potential in the stepped vitrification approach, but I also believe that there is a lot we still need to learn before thinking about using it as method of choice for human ovarian tissue cryopreservation. The high cryoprotectant concentration that should be applied in this approach is my first concern. () Our study clearly showed that 50% DMSO is too high, so we need to try lower concentrations or combine it with other cryoprotectants.

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Freezing Life: The Current Trends in Cryopreservation - Technology Networks