**5. Semi-closed device**

124 Current Frontiers in Cryopreservation

comparison with potential contamination of an open device, a bacteriological study would be necessary that could demonstrate that with a high % of contamination, a simple contact of the end of the device with the warming solution is sufficient to contaminate the sample.

Ultravit is a novel device composed of a 0.3 mm internal diameter quartz glass microcapillary tube and a flexible, transparent inert sheath that has been designed to protect and prevent it floating in the LN2. Loading the internal microcapillary tube and removing the cells from the device is very simple and easy using a syringe. Before warming, the protective sheath is cut and the internal microcapillary tube is placed in a sterile medium at 37ºC after the thawing protocol. The open end of the sheath can be sealed ultrasonically in milliseconds without affecting the temperature inside the microcapillary tube, closing the system and ensuring a hermetic seal, thus preventing cross-contamination. The last microbiological control of Ultravit showed that the 5-10 seconds contact of Ultravit's internal part with contaminated LN2 (E.Coli and Psudomonas) is not sufficient to produce direct contact of cells with the cooling solution and does not result in contamination (0% of Ultravit Vs 45% of Cryotop) (28). In this study we didn't find contamination in the microdrops into which we emptied the contents of the microcapillary, also submerging the end of this (0.2mm diameter, 0.01mm of wall thickness and 1 mm in contact with the warming drop). There is a great difference between thawing with an open device (in which the entire strip is submerged in the warming solution with a surface of 42-50 mm2) and thawing with Ultravit with 95.5% less of surface in contact with the warming solution at 37ºC (1.7-1.9 mm2). With Ultravit protocol, only the end of the microcapillary touches the base of the dish used to thaw, but at no time does the external part touch the warming drop (56). The following diagram shows the use of Ultravit, presuming that the cooling solution is

Fig. 19. Risk of Contamination with some closed devices (47)

**Ultravit** 

contaminated:

As a consequence of the necessity of a device which avoids contact of the biological sample with the cooling solution but that would achieve cooling rates high enough to ensure a high rate of vitrification, semi-closed devices were designed. Gabor Vajta was one of the first to hypothesize the enclosure of open carriers (after direct contact of cells/LN2) in pre cooled hermetical containers (48). There are systems in which there is direct contact of the biological sample with the cooling solution only at the time of vitrification. Once the sample is vitrified , the device is placed in a protective sheath which is hermetically sealed before being passed to the communal container. This ensures no cross-contamination in the tanks. As stated earlier in this chapter, microbiological studies performed (23) showed that many of the Identified bacteria isolated in the commercial tanks are ubiquitous environmental micro-organisms and are rare opportunistic pathogens of low significance in producing disease in humans or animals so these devices are an important tool for high survival of biological samples to avoid cross-contamination. Theoretically all open systems can become semi-closed systems to protect the biological sample from cross-contamination with a high cooling rate but the most important is:

#### **OPS safe method**

Vajta in 1997 devised a vitrifying system with OPS, but once submerged in LN2, the OPS straw is transferred to a 0.5ml CBS straw. Using this method a cooling rate of approximately 16.700 ºC/min is achieved, but once passed into the 0.5ml CBS straw it is protected from cross-contamination in the common tanks.

The Problem of Contamination: Open vs. Closed vs. Semi-Closed Vitrification Systems 127

needed reducing the detrimental effect of the inherent toxicity of CPAs and increasing the

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

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

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

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

Kinetic vitrification and concentrations of CPA's typical of Slow freezing.

there is a possibility of detaching from the device.

overall success of the procedure (50).


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

Table 2. Different cooling rates
