Review of non-permeating cryoprotectants as supplements for vitrification of mammalian tissues
Introduction
Cryopreservation is a method of preserving living cells and tissues over a long term using subzero temperatures [54,113]. There are two cryopreservation approaches, which are slow freezing and vitrification [112]. Slow freezing involves low cooling rates (e.g., 1 °C/min) to freeze a liquid containing living cells or tissues using a programmable freezer [45]. Vitrification involves an ice-free approach to convert a liquid containing living cells or tissues into a glass-like amorphous solid either using high concentrations of cryoprotectants (CPAs) or rapidly cooling with or without the use of a cryodevice (e.g., cryovial, plastic straw, and cryotop) [83]. Over the past decade, vitrification is becoming increasingly more utilized for the cryopreservation of mammalian tissues. Compared to slow freezing, vitrification aims to avoid ice formation that is harmful to mammalian tissues [50,83]. It has been demonstrated that the formation of extracellular or intracellular ice in mammalian smooth muscle, articular cartilage, and kidney tissues is severely damaging [47,76,77]. Several studies reported that vitrification is better than slow freezing in preserving viability and functional properties of different types of mammalian tissues such as embryos, and ovarian, testicular, and articular cartilage tissues [36,39,89,99,109,114]. Moreover, vitrification is cost-effective as it does not require an expensive programmable freezer [5,58]. To date, vitrification of mammalian tissues is important in the fields of human assisted reproduction, animal reproduction, and regenerative medicine.
In general, vitrification requires a viscous and highly concentrated (6–8 M) cryoprotectant to avoid ice formation during cooling [54,116]. CPAs can be divided into permeating CPAs and non-permeating CPAs. Permeating CPAs, such as ethylene glycol (EG), dimethyl sulfoxide (Me2SO), propylene glycol (PG), and glycerol, are small molecules that can penetrate the plasma membrane and form hydrogen bonds with water molecules to lower the freezing point thereby preventing intracellular and extracellular ice formation [53,94]. The literature on the properties and roles of permeating CPAs in cryopreservation is readily available [29,37,44,[53], [54], [55]]. It is necessary to have at least one permeating CPA for vitrification of mammalian tissues. Because a high concentration of one permeating CPA is extremely cytotoxic, vitrification usually uses a combination of two or more permeating CPAs to lower the concentration of each CPA to reduce cytotoxicity [4,32,108]. Combination of different permeating CPAs has successfully vitrified embryos, and ovarian, testicular, and articular cartilage tissues [10,23,43,99,107]. Despite these positive outcomes, researchers are still concerned with optimizing post-thaw cell viability and functionalities to prevent poor clinical outcomes and faulty reproduction. Therefore, it may be possible that a combination of non-permeating and permeating CPAs in vitrification of mammalian tissues will further diminish cytotoxic effects of permeating CPAs and improve the clinical outcomes of vitrification [70].
Non-permeating CPAs are large molecules that do not penetrate the plasma membrane and remain in the extracellular compartment during cooling to promote glass formation [48]. Non-permeating CPAs include sugars (e.g., sucrose and trehalose) and high molecular weight (MW) polymers (e.g., polyvinyl pyrrolidone and Ficoll) [5]. Compared to permeating CPAs, they are relatively less cytotoxic. Their addition contributes to viscosity and tonicity, allowing lower concentrations of permeating CPAs to be used without compromising vitrification properties [5]. Some studies reported that combining non-permeating and permeating CPAs further improved post-thaw viability and functionalities of vitrified embryos and ovarian tissues [3,24,67,69,115]. For example, in one study, vitrification medium containing sucrose, EG, and Me2SO was found to enhance post-thaw follicular viability in ovarian tissues compared to vitrification medium without sucrose [69]. Despite promising outcomes, many challenges associated with incorporation of non-permeating CPAs in vitrification of mammalian tissues remain to be addressed.
Several review articles are focused on vitrification of embryos and ovarian tissues [5,70,75]. However, there is no review focused on the supplemental effects of non-permeating CPAs toward vitrification outcomes of mammalian tissues, including embryos, and reproductive, cartilage, and kidney tissues. In view of the escalating demand for the use of non-permeating CPAs in vitrification of mammalian tissues, there is a strong need for a timely review on the supplemental effects of non-permeating CPAs toward vitrification outcomes of mammalian tissues. In this review, the roles of non-permeating CPAs including sugars and high MW polymers in vitrification are first discussed. The supplemental effects of non-permeating CPAs on viability and functionalities of mammalian embryos, and ovarian, testicular, articular cartilage, tracheal, and kidney tissues following vitrification are subsequently reviewed. Lastly, the challenges associated with the use of non-permeating CPAs in vitrification of mammalian tissues are briefly discussed.
Section snippets
Sugars
Some sugars, such as sucrose, trehalose, and raffinose, have been used in vitrification of mammalian tissues [15,71,73]. Sucrose is a disaccharide consisting of glucose and fructose. Sucrose is found in nectars, fruits, and roots of many plants [20]. Trehalose is a disaccharide composed of two glucose moieties. It is present in some animals, insects, plants, fungi, and bacteria and used by these organisms to overcome freezing and dehydration [22]. Raffinose is a trisaccharide consisting of
Embryos
Cryopreservation of mammalian embryos is important in three major areas. First, it assists reproduction for couples with infertility problems [56]. Second, it conserves genetic resources of livestock mammals for reproduction to maintain food supplies, especially meat and milk, and wild mammals threatened with extinction [2,98]. Third, it also conserves genetic resources of laboratory mammals to maintain production of laboratory mammals for preclinical studies, such as normal and mutant mouse
Challenges associated with the use of non-permeating CPAs in vitrification of mammalian tissues
There are several challenges associated with the use of non-permeating CPAs in vitrification of mammalian tissues. First of all, non-permeating CPAs, particularly sugars, can cause excessive dehydration to mammalian tissues and compromise vitrification properties of permeating CPAs when used in high concentrations. Therefore, concentration of sugars in both vitrification and warming media should be optimized to minimize freezing injury to cells.
To date, non-permeating CPAs have been clinically
Conclusions
Non-permeating CPAs play many roles in supporting permeating CPAs in vitrification of mammalian tissues. While contributing to viscosity needed for vitrification, addition of non-permeating CPAs in vitrification media replaces a portion of permeating CPAs to lower concentration of each permeating CPA, reducing the cytotoxic effects of permeating CPAs. Moreover, addition of sugars in both vitrification and warming media protects cells from physical damage by extracellular ice and restricts
Declaration of competing interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
N.M. Jomha and J.A.W. Elliott are inventors on US Patent No. 8,758,988: N.M. Jomha, L.E. McGann, J.A.W. Elliott, G. Law, F. Forbes, A. Abazari, B. Maghdoori, and A. Weiss, Cryopreservation of articular cartilage (2014).
Acknowledgements
This work was supported by the Edmonton Orthopaedic Research Committee. J.A.W. Elliott holds a Canada Research Chair in Thermodynamics.
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