**6. Cooling rate Vs closed systems**

Today the differences between Slow freezing and Vitrification are known worldwide. We all know that slow freezing is characterized by a prolonged cooling curve and the use of low concentrations of cryo-protectors generally ''non-toxic'' for the cells (1–2 M) with cell injury due to ice formation (58) and that Vitrification is characterized by the rapid procedure and the use of a high concentration of cryo-protectors (4-6M) to prevent cell damage that is toxic to most mammalian cells (59-67).Thus, vitrification with a semi-close devices have a better cooling rate without cross-contamination or novel cyopreservation techniques are needed that allow rapid cooling to achieve vitrification in the absence of high concentration of CPA or if is possible without CPA.

The requirements and relationships for conditions to achieve satisfactory vitrification in the area of mammalian ART are well displayed in the equation of Yavin and Arav (68)

> Probability of vitrification Cooling and warming rates × Viscosity (CPA concentration) = Volume

Fig. 21. Probability of vitrification by Yavin and Arav (68)

The main points to be gathered from this relationship are that the smaller volume of the vitrification solution in which the cellular material is placed for the vitrification process, the faster cooling and warming rate that can be achieved and the lower concentration of CPAs

DEVICE VOLUME COOLING RATE CRYOLOOP >1 µl 20.000 ºC/min HEMI-SATRAW >1 µl >20.000 ºC/min CRYOLEAF >1 µl 23.000 ºC/min VITRI-INGA 1 µl 20.000 ºC/min CVM-RING >1 µl 10.000 ºC/min VITRISAFE >1 µl 1.300 ºC/min HSS 0.5 µl 2.000 ºC/min

0.25 ML STRAW 25 µl 2.500 ºC/min

Today the differences between Slow freezing and Vitrification are known worldwide. We all know that slow freezing is characterized by a prolonged cooling curve and the use of low concentrations of cryo-protectors generally ''non-toxic'' for the cells (1–2 M) with cell injury due to ice formation (58) and that Vitrification is characterized by the rapid procedure and the use of a high concentration of cryo-protectors (4-6M) to prevent cell damage that is toxic to most mammalian cells (59-67).Thus, vitrification with a semi-close devices have a better cooling rate without cross-contamination or novel cyopreservation techniques are needed that allow rapid cooling to achieve vitrification in the absence of high concentration of CPA

The requirements and relationships for conditions to achieve satisfactory vitrification in the

The main points to be gathered from this relationship are that the smaller volume of the vitrification solution in which the cellular material is placed for the vitrification process, the faster cooling and warming rate that can be achieved and the lower concentration of CPAs

Cooling and warming rates ×

Viscosity (CPA concentration) = Volume

area of mammalian ART are well displayed in the equation of Yavin and Arav (68)

Probability of vitrification

Fig. 21. Probability of vitrification by Yavin and Arav (68)

OPS 1 µl 16.700 ºC/min CRYOTOP 0.1 µl 23.000 ºC/min CRYOTIP 1 µl 12.000 ºC/min RAPID-I 0.5 µl 1.200 ºC/min CRYOPETTE 1.2µl 23.700 ºC/min ULTRAVIT 0.2 µl 250.000 ºC/min

A summary of the cooling rates obtained with each device is as follows:

Table 2. Different cooling rates

or if is possible without CPA.

**6. Cooling rate Vs closed systems** 

needed reducing the detrimental effect of the inherent toxicity of CPAs and increasing the overall success of the procedure (50).

Fig. 22. necessary cooling rate to have a good probability of vitrification

What would happen if We could vitrify without CPA's or with a low concentration of CPA's? What would happen if We could combine the advantages of Slow freezing and Vitrification and vitrify with low concentrations of CPA's with a secure and free contamination device? That is Kinetic vitrification (Ultra-vitrification). Perfecting the techniques of Vitrification has been achieved a morphological survival rate comparable to normal Vitrification protocol (30) or a 59.1% of blastulation rate in mouse embryo (69) with Kinetic vitrification and concentrations of CPA's typical of Slow freezing.

Previous studies have tried to achieve high cooling rates for cell vitrification. However, none of them utilized low CPA concentrations (1.5-2 M). In 1985, Rall and Fahy successfully vitrified mouse embryos in 6.5 M cryoprotectant cocktail solution (70). In that case the method consisted in a 0.25 ml straw container plunged into LN2; the cooling rate was 2.500 ºC/min. When this container was plunged into Slush nitrogen, the cooling rate increased up to 4000 ºC/min (71). The use of OPS (instead of the 0.25 ml straw) in LN2 increases this cooling rate up to 5.300 ºC/min (71) and to 10.000–20.000 ºC/min if plunged in Slush nitrogen (71,72). Similar cooling rates were achieved in the case of a Cryoloop quenched in Slush nitrogen (73). The use of electron microscope copper grids has also been investigated, but the cooling rates were in the same order of magnitude that the afore mentioned works: 11.000–14.000 ºC/min in the case of plunging the grid in LN2 (74) and 24.000–30.000 ºC/min if plunged in Slush nitrogen (74,75). From Boutron's theory, none of these approaches reaches the critical cooling rate to achieve vitrification with low concentration of CPA (1.5- 2M). It´s impossible to use open devices with Slush nitrogen as the cell is on the outside and there is a possibility of detaching from the device.

Adjusting to Yavin and Arav formula the Ultra-vitrification technique arose achieving a cooling rate above 250.000 ºC/min and of 90.000 ºC/min in thawing. This rate is one order of magnitude higher than the highest cooling rate achieved in different strategies (electron microscope copper grids in Slush nitrogen (74,75), whilst keeping all the advantages of a

The Problem of Contamination: Open vs. Closed vs. Semi-Closed Vitrification Systems 129

In a thermal performance of quartz capillaries for vitrification done by Risco and his group (57) a commercially available version of the OPS (MTG Medical Technological Vertriebs, GmbH) was used. The thermal conductivity of these PVC straws was 0.19 W m\_1 K\_1. The inner diameter is 0.800 mm and the thickness of its wall is 0.075 mm (Fig. 1a). The QC used (The Charles Supper Company, Inc.) have an inner diameter of 0.180 mm and a wall thickness of 0.010 mm. These geometrical improvements (4.44 times smaller in diameter and 7.50 times thinner) translate not only into a faster heat transfer, but also into a 20 times reduction in volume of the contained solution (for a given height). This is beneficial because the thermal conductivity of the quartz glass is 1.3 W m\_1 K\_1, that is almost one order of

Fig. 24. (a) Thermal history for the OPS when filled with a 1.5M propane-1,2-diol and 0.3M sucrose cryoprotectant solution quenched in LN2 and then thawed in a water bath at 37ºC.

Fig. 23. Xiaoming He et al (77)

magnitude higher than that of PVC.

straw-like form for the container and being in the range of the necessary cooling rate to achieve vitrification. To have this increase in the cooling rate a few changes were made to the normal vitrification process:

#### **Slush Nitrogen**

As a cooling agent this technique uses Slush nitrogen, much colder than LN2 (-196ºc Vs - 210ºC) and with the property of avoiding the Leiderfrost Effect. When something is submerged in LN2, bubbles rise to the surface through the device, varying the thermal conductivity from the outside into the inside of the device. This does not happen with Slush nitrogen. Slush nitrogen is achieved with a vacuum pump in 5 to 10 minutes and it remains slush for a further 5 – 10 minutes before returning to liquid.

It was shown for oocytes and embryos that increasing the cooling rate would improve survival rates by up to 37% (76)


Table 3. Effect of cooling rate on survival; comparison between LN2 and Slush nitrogen (75)

#### **Quartz Micro-capillary**

Another determining factor to achieve a high cooling rate is the device used. To increase the thermal conductivity and minimize the volume, this technique has used a quartz microcapillary. This has a 0.2-0.3 diameter allowing to ultra-vitrify 0.1-0.2 µl with a 0.01mm wall, a lot thinner than any other device (0.075 mm in OPS). Another important characteristic is the material it is made from: Quartz. The thermal conductivity of quartz glass is a lot higher than that of plastic of which other devices are made of. This converts it in one of the materials that best conducts the temperature (77)

Fig. 23. Xiaoming He et al (77)

straw-like form for the container and being in the range of the necessary cooling rate to achieve vitrification. To have this increase in the cooling rate a few changes were made to

As a cooling agent this technique uses Slush nitrogen, much colder than LN2 (-196ºc Vs - 210ºC) and with the property of avoiding the Leiderfrost Effect. When something is submerged in LN2, bubbles rise to the surface through the device, varying the thermal conductivity from the outside into the inside of the device. This does not happen with Slush nitrogen. Slush nitrogen is achieved with a vacuum pump in 5 to 10 minutes and it remains

It was shown for oocytes and embryos that increasing the cooling rate would improve

Table 3. Effect of cooling rate on survival; comparison between LN2 and Slush nitrogen (75)

Another determining factor to achieve a high cooling rate is the device used. To increase the thermal conductivity and minimize the volume, this technique has used a quartz microcapillary. This has a 0.2-0.3 diameter allowing to ultra-vitrify 0.1-0.2 µl with a 0.01mm wall, a lot thinner than any other device (0.075 mm in OPS). Another important characteristic is the material it is made from: Quartz. The thermal conductivity of quartz glass is a lot higher than that of plastic of which other devices are made of. This converts it in one of the

the normal vitrification process:

survival rates by up to 37% (76)

**Quartz Micro-capillary** 

materials that best conducts the temperature (77)

slush for a further 5 – 10 minutes before returning to liquid.

**Slush Nitrogen** 

In a thermal performance of quartz capillaries for vitrification done by Risco and his group (57) a commercially available version of the OPS (MTG Medical Technological Vertriebs, GmbH) was used. The thermal conductivity of these PVC straws was 0.19 W m\_1 K\_1. The inner diameter is 0.800 mm and the thickness of its wall is 0.075 mm (Fig. 1a). The QC used (The Charles Supper Company, Inc.) have an inner diameter of 0.180 mm and a wall thickness of 0.010 mm. These geometrical improvements (4.44 times smaller in diameter and 7.50 times thinner) translate not only into a faster heat transfer, but also into a 20 times reduction in volume of the contained solution (for a given height). This is beneficial because the thermal conductivity of the quartz glass is 1.3 W m\_1 K\_1, that is almost one order of magnitude higher than that of PVC.

Fig. 24. (a) Thermal history for the OPS when filled with a 1.5M propane-1,2-diol and 0.3M sucrose cryoprotectant solution quenched in LN2 and then thawed in a water bath at 37ºC.

The Problem of Contamination: Open vs. Closed vs. Semi-Closed Vitrification Systems 131

on cryopreservation protocols and devices to ensure a major benefit and patients' safety during procedures. We need to find a method that combines a high cooling and warming rate, high survival and function of cells and tissues and is made in a way that ensures

We must be clear that survival after a vitrification process is a "morphological survival" and that this cell has to fertilize perfectly and develop normally to rule out any damage in the

With so much variety of devices and many different protocols, laboratories have to find the protocol and the device that best suits their skills, provided they ensure the sterility of vitrified samples and prevent cross-contamination in general containers. It has created an exaggerated paranoia about the vitrification cell and tissue contamination of the cooling solution that everything can contaminate our samples. In my opinion we must think scientifically, leaving aside the commercial interests of many of us and worrying about more logical things and research data: the probability of contamination must be demonstrated with % of contamination and leave a little aside science fiction and theoretical assumptions. For many cryobiologists avoiding cross-contamination of samples in the general containers is the most important matter as it has been proven that commercial LN2 does not have a high enough risk to have to sterilize it. The use of a closed or semi-closed device that would allow us a high cooling rate and a sealing prior to being deposited in the general container

should be enough to ensure sterility and good survival and development results.

We must centre all of our interest in more practical things like finding a vitrification protocol that allows us to a vitrify without cryoprotectant, discovering new cooling solutions, discovering new materials, new devices, new procedures in which we can safely freeze samples without cryoprotectants toxicity problems, thus ensuring a good development cell after thawing, all in secure systems which ensure sterility of the sample and avoid crosscontamination. Finding an "Universal Protocol" risen several times by Dr. Katkov (79), allowing us to use the same protocol worldwide, as movement of frozen specimens around the world has increased dramatically and the lack of component preparation in the laboratory that is to thaw the frozen sample with a protocol and a device different to those they know, would not give us 100% guarantee of sample survival. Today freezing has become a luxury and not all patients can afford the excessive cost of the freezing products, if we could find a protocol without the need of cryoprotectants or a universal protocol that all could use, the price of products would decrease and the patients would benefit

The author thanks Ms Ana Yus and Ms Cristina Gonzalez for the critical and technical

[1] Karrow AM, Critser JK. Reproductive tissue banking: scientific principles. San Diego:

[2] Vajta G, Nagy ZP. Are programmable freezers still needed in the embryo laboratory?

Review on vitrification. Reprod Biomed Online 2006;12:779–96.

process of cryopreservation and to consider a real survival.

patient safety.

economically.

support.

**9. References** 

**8. Acknowledgment** 

Academic Press, 1997.

A clear heat release peak is present during cooling as well as melting during rewarming. (b) Thermal history for QC when filled with a 1.5M propane-1,2-diol and 0.3M sucrose cryoprotectant solution quenched in LN2 and then thawed in a water bath at 37ºC. Crystallization of water is not obvious during cooling, but melting is shown during rewarming. (c) Thermal history for OPS when filled with a 1.5M propane-1,2-diol and 0.3 M sucrose cryoprotectant solution quenched in Slush nitrogen and then thawed in a water bath at 37ºC. In this case, crystallization during cooling and melting during rewarming was not recorded. However, visual inspection reveals the presence of ice. (d) Thermal history for QC when filled with a 1.5Mpropane-1,2-diol and 0.3 Msucrose cryoprotectant solution quenched in Slush nitrogen and then thawed in a water bath at 37ºC. The sample keeps its transparency over all the cooling–rewarming cycle, an indication of the capability of this approach to vitrify the studied solution.

All these changes have allowed us to maintain a concentration of cryoprotectors typical of slow freezing, 2 M PrOH+0.5 M sucrose, obtaining a morphological survival rate of 92 % in human oocytes (31). Dr. Ho-Joon Lee et al (69) tested this new technique on mouse ooctyes and they saw that using Ultra-vitrification with low concentrations of cryoprotectors improved the fertilization rate and above the blastulation rate. Only the use of Ultravit device in this technique ensures the non contamination of the sample or cross-contamination in communal containers.


Table 4. Comparison between slow Freezing, Vitrification and Ultravitrification (78, 69, 31)

This comparison demonstrates the use of low concentration of cryoprotectant in the Ultravitrification protocol favours the morphological survival (92%) and increases the blastulation rate (59.1%). Thus confirming the hypothesis that cryoprotectants are toxic to the biological sample and if we could find a vitrification protocol that would allow us to vitrify without cryoprotectant, we would achieve a better embryo development and a greater chance of pregnancy in the case of freezing eggs or embryos. A lot more studying is needed regarding this new technique but a priori the results indicate that we can hopefully lower the concentration of the cryoprotectants decreasing the toxicity in cells.
