Comparison of closed vs open vitrification devices

The concept of fast freezing or vitrification was first described more than 80 years ago by Basile J. Luyet, the so-called Father of Cryobiology (Luyet, 1937). He showed that supercooled solutions could be solidified without crystallization, forming a glass-like state. Already then, the potential of the technique and the associated challenges were in the research spotlight. Today, we’ve managed to overcome all methodological-related issues of vitrification. It has evolved into a reliable and efficient method to freeze oocytes and embryos. Vitrification is used for medically assisted reproduction and fertility preservation: the goal is to ensure the maximum survival rate with the highest level of biosafety. In this blog post, we will compare closed and open carrier devices for vitrification.

Why are there two types of vitrification devices?

The main principle of vitrification, to avoid ice crystal formation during cooling, storage and warming, can be equally achieved using a closed or open vitrification device. The key difference is that open devices expose the sample directly to the environment, whereas closed devices protect the sample from potential disease transmission from the environment. The invention of closed devices followed medical requirements for aseptic handling of oocytes and embryos during the entire in vitro fertilization (IVF) procedure.

Liquid nitrogen-mediated transmission of infectious agents was proven under experimental conditions

Sources of contamination during cryopreservation include exposure to non-sterile liquid nitrogen, cross-contamination of pathogens between samples during storage, unsterilized storage tools (canisters, holders) and using dewars that have not been decontaminated. To avoid these risks, biological samples can be successfully vitrified inside sterile straw and then hermetically sealed using the closed Rapid-i vitrification system, which prevents direct contact between oocyte/embryo and liquid nitrogen.

Health authorities around the globe are cautious about possible contamination when using open vitrification devices.

Liquid nitrogen-mediated transmission of infectious agents is not just a theory, but a proven reality under experimental conditions (Bielanski et al., 2000; Bielanski et al., 2003; Bielanski and Vajta, 2009). Furthermore, several cases of hepatitis B virus transmission to patients were reported due to cross-contamination of biological samples stored in liquid nitrogen (Tedder et al., 1995). Considering all the possible contamination sources for vitrified samples, it is surprising that no IVF-related case has been reported (Vajta et al., 2015). But could contamination of vitrified/warmed oocytes/embryos explain why some frozen embryo transfers fail to implant? The use of the closed device eliminates the potential contamination of oocytes/embryos during vitrification and storage. Indeed, global health authorities are cautious about possible cross-contamination during storage or handling when using open vitrification devices (De Munck et al, 2016).

An experimental study using the Cryotop open device demonstrated that 45 % of samples became contaminated after just 10 seconds of contact with contaminated liquid nitrogen (Criado et al., 2011). In the same study, no samples were contaminated when a closed device was used. In the light of the COVID-19 pandemic, it is imperative to follow the highest biosafety protocols and use closed vitrification devices (ESHRE 2020; Adiga et al., 2020).

Using a closed device guarantees 100 % protection from possible contamination.

Some medical centers do not consider liquid nitrogen-mediated contamination to be a risk for frozen biomaterial. Yet, all clinics store gametes and embryos from patients who are hepatitis/HIV positive in a separate dewar to avoid cross-contamination between positive and negative patients. Is “low” an acceptable risk for our patients? Using a closed device guarantees 100 % protection from possible contamination.

Cooling and warming rates

Cooling rates were a key focus for embryologists for many years, as vitrification requires a sufficiently high cooling speed. Carrier devices have evolved from simple electron microscopy grids to a vast array of commercially available carriers including open pulled straws (Vajta et al., 1998), Cryoloop (Lane et al., 1999), Cryotop (Katayama et al., 2003) and Cryolock (Bernal DP et al., 2009). These are open vitrification devices, which provide very high cooling speeds. However, it has long been recognized that even if vitrification is successful, the sample can still be damaged by ice crystal formation during warming (Luyet and Gehenio, 1940). Considering cooling speed only does not guarantee the best success rates.

Rapid-i Kit combines a sufficiently high cooling speed with a very high warming speed

An experimental study on mouse oocytes demonstrated that a relatively moderate cooling rate of 522 °C/min was sufficient to ensure high survival rate if it was followed by a very high warming rate of 2950 °C/min (Seki and Mazur, 2009).  Knowledge of this phenomenon paved the way for the successful clinical use of the closed vitrification device, Rapid-i Kit, which prevents direct contact between the sample and liquid nitrogen and combines a sufficiently high cooling speed with a very high warming speed. Clinical studies comparing vitrification with closed (Rapid-i Kit) and open devices (Cryotop) demonstrated that the survival rates were better for human oocytes (Pujol et al., 2019) and similar for blastocysts (Hashimoto et al., 2013).

Rapid-i Kit as the gold standard of efficient and safe vitrification

The oocyte survival rate was found to be higher using the Rapid-i Kit than the Cryotop device.

Pujol et al., 2019

The cooling rate depends on the shape and volume of the drop of vitrification media containing the sample. Incorrect loading of an open device may reduce the cooling rate if the drop is too big or cause mechanical damage to the sample if the drop’s shape is wrong. 

These problems are not an issue with the Rapid-i Kit. The Rapid-i stick has a  pre-defined aperture, which is easy to fill with the correct volume of vitrification media. Oocytes or embryos are held on the Rapid-i stick by surface tension and do not contact the device itself. Thus, the issue of shape doesn’t exist: the sample is suspended in a thin film of vitrification solution. Using the Rapid-i Kit is the best way to standardize the loading process and reduce variability between operators. Another advantage of the Rapid-i Kit is that it functions as a closed device for cryopreservation, but as an open device for warming. The Rapid-i stick with the sample is directly immersed into warming media ensuring the highest possible warming rate.

Loading with Rapid-i Kit is standardized thanks a pre-defined aperture, which is easy to fill with the correct volume of vitrification media.

The comparison between Rapid-i Kit vs Cryotop

A recent study comparing the Rapid-i Kit with the Cryotop for vitrification/warming of human donor oocytes, demonstrated that the survival rate was significantly higher with the closed system (Pujol et al., 2019). Results from multiple studies using the Rapid-i Kit show excellent survival and clinical pregnancy rates for oocytes, cleavage stage embryos and blastocysts.

Table 3. Oocyte and embryo survival rates using closed devise Rapid-i Kit and open device Cryotop (Pujol et al., 2019).

*p<0.01 vs Cryoptop

Clinical outcomes after vitrification using the Rapid-i Kit

Results from multiple studies using the Rapid-i Kit show excellent survival and clinical pregnancy rates for both oocytes and blastocysts (figures below).

Weighing up the pros and cons of closed and open systems

Vitrification is an important part of IVF treatment and modern technological advances ensure equally high survival rates using both closed and open devices. As only closed devices provide absolute biosafety for vitrified samples, we advocate that they are the best choice for vitrification of your patient’s oocytes and embryos.

• The closed Rapid-i Kit ensures a very high survival rate with 100% protection from possible contamination.
• Regulatory requirements in many countries are shifting towards the use of closed devices.
• The potential effects of SARS-CoV-2 virus on assisted reproduction outcomes are not evident yet, but the use of the Rapid-i Kit avoids possible risks.
  
  

References

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