ReviewThe need for novel cryoprotectants and cryopreservation protocols: Insights into the importance of biophysical investigation and cell permeability
Introduction
Cryopreservation offers huge opportunities for both research and medical treatments. Through cryopreservation, blood banks can ensure sufficient supplies, stem cell therapies can be used to treat a range of diseases, and the field of assisted reproductive technology has undergone huge advances [1]. Furthermore, cryopreservation can be used for long-term storage of seeds [2], endangered plants [3] and animals [4]. In the area of plant cryopreservation in particular a lot of effort is being directed not only to optimising the use of CPAs to decrease cell membrane damage and toxicity, but also toward developing new cryoprotection methods and ameliorating damage arising from oxidative stress [5].
Successful cryopreservation relies on more than simply freezing cells, which exposes them to numerous stresses including dehydration and mechanical pressures. Usually, a cryoprotective agent (CPA) is added to minimise freezing damage through inhibiting ice formation, preserving cellular membranes and promoting vitrification [6,7].
The two most commonly used CPAs are glycerol and dimethyl sulfoxide (DMSO). Glycerol was first identified as a CPA in 1949 [8] while DMSO was identified in 1959 [9]. Unfortunately, both have levels of toxicity making them unsuitable for many applications, and often require extensive washing during thawing to prevent cell death or subsequent adverse reactions if used in medical treatments [[10], [11], [12], [13]]. In the decades since their discovery, many other CPAs have been explored, but very few have shown the same efficacy as glycerol or DMSO [14]. Therefore, the search for new, effective, non-toxic CPAs is ongoing.
In addition to the toxicity problems of existing cryoprotectants, current methods of cryopreservation do not work for certain cell types, for example granulocytes [13] and pluripotent human stem cells [15]. Research is on-going to develop efficient cryopreservation protocols for stem cell therapies - Chen et al. [16] examined the effects of various cryoprotectants on human umbilical cord blood stem cells, and a recent review by Hunt [17] highlights the need for a holistic approach to designing stem cell cryopreservation protocols by considering not only the choice of CPA, but also the freezing container, the cooling rate and so on. Current methods are also not suitable for whole organs, or even whole tissue cryopreservation, due to the variety of cell types (and therefore different cryopreservation requirements) [18]. There is also the issue of achieving sufficient CPA penetration into deeper cell layers within tissue, without which deeper tissue layers suffer extensive damage [1].
Thus, there is an obvious need for new CPAs that can overcome the limitations of existing protocols. CPAs may be classified as either non-penetrating—meaning they do not enter the cells and instead act in the extracellular space—or penetrating—meaning that they do enter the intracellular space. Both classes will be discussed later, but the focus of this review is on penetrating CPAs.
In 1969, Karow published a review entitled “Cryoprotectants – A New Class of Drugs” [14]. Now, more than 50 years later, we are following up on that work to discuss the progress made in developing new methods of cryopreservation and in identifying new CPAs. Despite a half a century of research, there is still a lot that remains unknown about CPAs and the search for alternatives to DMSO and glycerol continues. In 2004, Fuller's group detailed the mechanisms of action of known CPAs and re-iterated that many potential CPAs are toxic at the concentrations required for cryopreservation [19]. The same group conducted an extensive review in 2017 of the different types of CPAs, including alcohols, sugars, and polymers [7]. That review again highlighted problems with CPA toxicity and the modes of action of the different CPA groups. The review presented here builds on this by specifically focusing on the rational design of novel CPAs based on our current understanding of how CPAs function. The last few years have seen targeted investigations that have specifically modified potential CPAs to improve their activity e.g. by making them better able to penetrate cells. Other work has focused on using naturally produced molecules such as amino acids as CPAs. The outcomes of these targeted and novel approaches will be summarised here, with an outlook toward rational design of future CPAs.
Section snippets
Mechanisms of damage during freezing
For many cells, low temperatures are not in themselves damaging, but freezing is often lethal. There are a number of different mechanisms that may cause damage during freezing, including mechanical damage due to ice crystals, and solute damage due to changes in concentration of electrolytes [6]. There is some disagreement in the literature about which mechanisms are the most important, however it is likely that the most pertinent mechanism of damage is different for different cell types, and is
Mechanism of action of CPAs based on biophysical properties
Cryoprotective agents (CPAs) are used to protect biological samples from freezing damage during cryopreservation. This is achieved by different mechanisms depending on the CPA, but even for well-known CPAs there is still some doubt as to the exact mechanism of protection [19,28,[40], [41], [42]]. The potential mechanisms are discussed briefly below, but have been reviewed in more depth elsewhere [7,19,40,43].
CPAs are separated into two broad categories: penetrating and non-penetrating [7,14,19,
Rational design of Novel CPAs
As discussed by Elliott et al., despite more than half a century of studies into cryopreservation, the same few CPAs continue to be used [19]. Therefore, there is a need for targeted, rational design of new cryoprotectants.
While non-penetrating CPAs are important to cryopreservation and can greatly improve outcomes when used as an additive, penetrating CPAs offer the greatest potential for freezing new cell types, or even tissues and organs, because they inhibit intracellular ice formation [19,
Conclusion
The most commonly used CPAs (DMSO and glycerol) are not only toxic, which makes them unsuitable or inefficient for many clinical applications, but they are also ineffective for hundreds of cell types. Therefore, new, non-toxic CPAs must be developed.
In order to efficiently design CPAs and cryopreservation protocols, a number of factors must be considered including the biophysical properties of the cells to be preserved, the penetration ability of the CPA, the rate of cooling, and the ice
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors acknowledge the support of the ARC Research Council grants LP160101496 and DP190101010.
R.R. acknowledges the support of an Australian Government Research Training Program Scholarship.
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