From a Backup Technology to a Strategy-Outlining Approach

The Success Story of Cryopreservation

Gábor Vajta; Anikó Reichart; Filippo Ubaldi; Laura Rienzi

Disclosures

Expert Rev of Obstet Gynecol. 2013;8(2):181-190. 

In This Article

The Two Main Approaches

Currently, techniques used for cryopreservation of ova or embryos in domestic and experimental mammals as well as in humans belong to two major groups: the traditional slow-rate freezing or vitrification. For the establishment, theoretical background and technical features of these techniques, the authors refer to the many reviews published recently;[1–7] in this article, the authors only provide a short practical summary.

In traditional freezing, after equilibration with a relatively low concentration of permeable cryoprotectants, embryos or oocyes are loaded into 0.25 ml standard plastic straws and are, typically, placed into a freezing machine. The instrument is designed to provide very accurately regulated, consistent and repeatable cooling parameters for the samples. The steps of the cooling process include a relatively rapid cooling to around -7°C. Because of the presence of cryoprotectants and lack of mechanical trauma, ice formation does not start spontaneously; it has to be induced, typically with a metal tool (forceps) immersed briefly into liquid nitrogen. By touching the surface of the straws far away from the sample with this tool, a small white spot occurs inside the straw, indicating the start of ice formation. The process is called seeding and is usually performed manually, although more sophisticated machines can already induce the proper seeding automatically. In the subsequent period, as the result of a consistent slow cooling with 0.3–1.0°C/min, the ice crystal slowly grows and eventually reaches the sample. However, at that moment, the growing ice removes most of the water from the solution and induces a rapid increase concentration of the cryoprotectants around as well as inside the sample. Eventually, this high concentration decreases the probability of ice crystal formation, and a kind of amorphous solidification of the solution occurs on both sides of the cellular membrane.

The amorphous solidification is also called vitrification (i.e., vitreous, glass-like transformation) because the solution remains transparent, although hardly detectable in the minuscule volume in and around the sample, surrounded by ice crystals. On the other hand, the term itself has already been reserved for another approach in cryobiology, where the phenomenon is not only presumed, but definitely should occur, and in most cases should be easy to detect, even with the naked eye, in the whole solution that holds the sample, without any sign of ice crystal formation. Although the process also occurs in nature (and not only in water, but in most diverse solutions – that of common sugar, silicon dioxide, and so on), its induction requires special conditions. In mammalian embryology, the most feasible strategy is to increase the concentration of both permeable and nonpermeable cryoprotectants to a much higher level than used in traditional slow-rate freezing, and the application of rapid cooling and warming rates. For the latter, the simplest way is the direct immersion of the sample into liquid nitrogen (with minimal or no thermo-insulating layer), and the use of a small volume of the solution that surrounds the sample.

At first glance, traditional freezing seems to be a much more professional approach, with sophisticated and expensive computer-regulated machines with multiple displays, various, versatile adjustment possibilities and fully automated cooling processes. Compared to that, vitrification is a primitive manipulation with extremely simple carrier tools, foam boxes that have been used for delivery of some sensitive chemicals years ago, sometimes still with the old labels and mailing addresses on the external surface and with the shockingly simple cooling step. It is only the outcome that justifies the existence of this younger approach, and qualifies vitrification as the method of choice by an increasing number, probably the majority, of human embryology laboratories.

The authors have to admit that the characteristics are not always sharply different, and the borders are not always entirely clear between the two procedures. Basically, the purpose of both traditional freezing and vitrification is to decrease or rather totally eliminate ice crystal formation inside and around the sample, and two different approaches are used to achieve this goal. However, there are many ways for technical realization of both, and there is a third, although rarely applied, version of cryopreservation between the two major groups called rapid freezing, reportedly successful for a great variety of samples in mammalian embryology. In rapid freezing, the applied cryoprotectant concentration is not enough to achieve vitrification, and the quick cooling process does not allow the slow growth of ice crystals that absorb water and practically dehydrate the sample. Paradoxically, high survival and subsequent developmental rates were reported by using this third strategy as well; however, the consistency and reliability is low.[8]

Another problem is the definition of vitrification and slow-rate freezing. The presence or absence of freezing machines and foam boxes simply cannot be the basis of scientific categories. The presence or absence of ice crystals in the solution is an important criterion, but difficult to detect when minuscule amounts of solutions – for example a film layer – are used. We need our imagination and trust of scientific calculations, but the certainty at the given technique may not be directly obtainable. Additionally, transitional ice crystal formation, the so-called 'devitrification', may also occur at warming of vitrified samples, transforming the sharp qualitative difference to a hardly measurable quantitative one. Even terms suggested previously to separate the two methods, for example, freezing and thawing for the traditional slow-rate process and cooling and warming for vitrification,[9] are randomly used now by both scientists and companies, and purist attempts to clarify what we are discussing here are regarded by some scientists as awkward and obsolete.

Accordingly, the best way to differentiate vitrification from traditional freezing is to focus on the different approach. In traditional freezing, our intervention is rather mild and passive. We use the growing ice to perform water removal and to make crystal formation eventually impossible in and around the sample. At vitrification, on the other hand, we perform a drastic and active intervention by using high cryoprotectant concentration and/or high rates of temperature change, and eliminate in this way all possibility of ice formation – at least during the cooling and storage phase.

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