**4. Closed device**

Closed systems were born as a need to solve this direct contact with the open devices. The pioneer and first major proponent of close systems for freezing embryos, oocytes or sperm was F. Ostashko in 1960 as an aseptic alternative to the Cassou method. In such systems the biological sample is not in contact with the cooling solution at the time of freezing or at the time of storage in the common containers. This prevents contamination by contact and cross contamination from shared containers. The main feature is that the cooling rate is much lower with these closed devices. By lowering the cooling rate most vitrification protocols with closed systems have a high concentration of cryoprotectants to prevent crystal formation making them "dangerous" protocols for the cell due to the cytotoxicity of the cryoprotective substances. Many comparative studies of open and closed devices listing very similar results.

In the market there are many closed systems to vitrify and more appear daily due to the emphasis that cryobiologists put into to finding the perfect vitrification system that will prevent contamination of the sample and cross-contamination allowing a survival and cell viability with a protocol free or low of cryoprotective substances. The closed devices can be closed or sealed in many ways but most importantly a hermetic seal must be made, preventing entry to the inside and leakage of pathogens to the outside. Thermo seal, radiofrequency seal and ultrasound seal are some of the most used systems that ensure that the stalled sample remains suspended in time.

The Problem of Contamination: Open vs. Closed vs. Semi-Closed Vitrification Systems 121

surface makes contact with LN2 thus "contaminating" the surface, which could then

Rapid-i (Vitrolife, Sweden) is based on the same principle as the open vitrification system of the Cryoloop meaning that the embryos are place in a minute volume of vitrification solution in a hole and held there by surface tension. The Rapid-i™ holding the embryos is in turn placed in a pre-cooled RapidStraw™ sitting in the container filled with LN2. This unique feature of the Rapid-i™ vitrification System means that vitrification actually takes place in super-cooled air reducing contamination risks. The straw is sealed after vitrification making the critical time frames of the dehydration steps easier to keep and creating an aseptic vitrification system without any contact between vitrified material and LN2. The

main problem is that a cooling rate of 1.200ºC/min is achieved with this device (54).

Is a modification of the previous Hemi-straw vitrification plug that allows a complete insertion in high security 0.5 ml straw. The Vitrisafe consists of a large gutter that is totally inserted into a larger pre-cooled 0.5 ml straw (CBS, Cryo Bio System, Grenoble, France). Only after welding both ends of the 0.5 ml straw to ensure the complete isolation of the biological sample is the complete straw plunged into LN2. For warming, the gutter is removed from the outer straw without contact with LN2 and the tip containing the biological material is directly plunged into the dilution solution in order to archive a rapid

warming. A cooling rate of 1.300ºC/min is archived in the vitrification process (50).

provoke a contamination of the sample.

Fig. 13. CVM Ring Fibre Plug

**Rapid-i™** 

Fig. 14. Rapid-i

**Vitrisafe** 

Fig. 11. Risk of Contamination with closed devices (47)

Amongst the most commonly used closed systems are the following:

#### **25 to 0.5 ml Straw**

This was one of the first devices used to freeze semen, oocytes, embryos or tissues. If the device is not hermetically sealed they are open devices, but if they are hermetically sealed they are closed systems, as the sample does not come into contact with the cooling solution. The main problem is the cooling rate, as the device is constructed from PVC or ionomeric resin and having a substantional wall thickness there is little temperature transmission (CBS). The cooling rate achieved by these straws is approximately 2.500 ° C / min (49)

Fig. 12. straw

### **CVM Ring Fibre Plug**

CVM™ (Cryologic, Australia) involves the rapid cooling of specimens without their immersion in, or direct contact with the cooling solution. This reduces the risk of any potential contamination by pathogenic microorganism that may be present in the cooling solution. The specimens are put into a droplet which is transferred to the hook at the end of a custom designed fibre called a Fibreplug™. The Fibreplug™ is then transferred to the specially treated surface of a CVM™ Block that has been chilled to LN2 temperature. The droplet vitrifies into a glassy bead and the Fibreplug™ is placed securely into a pre-cooled CVM™ sleeve. A cooling rate of 10.000ºC/min is obtained with this device. Besides the cooling rate, another main problem with this method is that to cool the CVM block the surface makes contact with LN2 thus "contaminating" the surface, which could then provoke a contamination of the sample.

#### Fig. 13. CVM Ring Fibre Plug

#### **Rapid-i™**

120 Current Frontiers in Cryopreservation

Fig. 11. Risk of Contamination with closed devices (47)

**25 to 0.5 ml Straw** 

Fig. 12. straw

**CVM Ring Fibre Plug** 

Amongst the most commonly used closed systems are the following:

This was one of the first devices used to freeze semen, oocytes, embryos or tissues. If the device is not hermetically sealed they are open devices, but if they are hermetically sealed they are closed systems, as the sample does not come into contact with the cooling solution. The main problem is the cooling rate, as the device is constructed from PVC or ionomeric resin and having a substantional wall thickness there is little temperature transmission (CBS). The cooling rate achieved by these straws is approximately 2.500 ° C / min (49)

CVM™ (Cryologic, Australia) involves the rapid cooling of specimens without their immersion in, or direct contact with the cooling solution. This reduces the risk of any potential contamination by pathogenic microorganism that may be present in the cooling solution. The specimens are put into a droplet which is transferred to the hook at the end of a custom designed fibre called a Fibreplug™. The Fibreplug™ is then transferred to the specially treated surface of a CVM™ Block that has been chilled to LN2 temperature. The droplet vitrifies into a glassy bead and the Fibreplug™ is placed securely into a pre-cooled CVM™ sleeve. A cooling rate of 10.000ºC/min is obtained with this device. Besides the cooling rate, another main problem with this method is that to cool the CVM block the Rapid-i (Vitrolife, Sweden) is based on the same principle as the open vitrification system of the Cryoloop meaning that the embryos are place in a minute volume of vitrification solution in a hole and held there by surface tension. The Rapid-i™ holding the embryos is in turn placed in a pre-cooled RapidStraw™ sitting in the container filled with LN2. This unique feature of the Rapid-i™ vitrification System means that vitrification actually takes place in super-cooled air reducing contamination risks. The straw is sealed after vitrification making the critical time frames of the dehydration steps easier to keep and creating an aseptic vitrification system without any contact between vitrified material and LN2. The main problem is that a cooling rate of 1.200ºC/min is achieved with this device (54).

Fig. 14. Rapid-i

#### **Vitrisafe**

Is a modification of the previous Hemi-straw vitrification plug that allows a complete insertion in high security 0.5 ml straw. The Vitrisafe consists of a large gutter that is totally inserted into a larger pre-cooled 0.5 ml straw (CBS, Cryo Bio System, Grenoble, France). Only after welding both ends of the 0.5 ml straw to ensure the complete isolation of the biological sample is the complete straw plunged into LN2. For warming, the gutter is removed from the outer straw without contact with LN2 and the tip containing the biological material is directly plunged into the dilution solution in order to archive a rapid warming. A cooling rate of 1.300ºC/min is archived in the vitrification process (50).

The Problem of Contamination: Open vs. Closed vs. Semi-Closed Vitrification Systems 123

an ultra fine tip and a protective metal cover sleeve. This device has been optimized as a closed system for cryopreservation procedures. For freezing aseptically remove one CryoTip when ready to use. Aseptically attach the wide end of the CryoTip to an aspiration tool, such as a luer tip syringe, using the Connector. When specimens are ready to load into the CryoTip, aseptically slide the metal cover sleeve carefully along the straw to expose the fine tip end. Gently load the specimens into the CryoTip by aspiration using the plunger on the syringe to control the uptake of medium and specimens. Heat seal the fine tip below the 1st mark, then slide the metal cover sleeve down over the fine tip to protect it and plunge the sealed CryoTip into the LN2 reservoir. A cooling rate of 12.000ºC/min is obtained with this

(Origio) It is derived from the original STRIPPER® family of denudation tools. It includes a sterile STRIPPER® tip with an integrated bulb to facilitate loading. This eliminates awkward external handles, rods, and pick-up tools required with other devices and guarantees simplicity, speed and ease of use. The bulb is designed to deliver the sample to the desired location every time. The maximum load volume is 1.2 µl, producing a cooling rate of

These last two devices have been criticized by Parmegiani and his group's latest articles and in the last CRYO congress 2011, for the "potential danger" they have in his opinion: In the thawing process the external part of the device is in direct contact with the warming solution and any pathogen that could be on the exterior could pass onto the sample. In my opinion it is a very remote hypothesis and the probability that this could occur is minimal in

device (49)

Fig. 17. Cryotip

Fig. 18. Cryopette

approximately 23.700ºC/min.

**Cryopette®** 

Fig. 15. Vitrisafe

#### **High security vitrification straw**

The CBS™ High Security vitrification straws are made from an ionomeric resin that is chemically inert, biocompatible and has physical characteristics resistant to ultra low temperatures and pressures created by expanding liquids and LN2. Sealed straws are resistance tested to 150 kg/cm2 (2133 lb/sq.inch), both the seals and the material should resist in order to have the batch approved. The HSV (High Security Vitrification) kit is composed of a High Security ionomeric resin straw, a capillary tube with a pre-formed gutter attached to a colored handling rod and a blue plastic insertion device. For freezing the sample is deposited into the gutter a few millimeters from the end using a micropipette. The drop holding the sample must be under 0.5 µL. immediately place the capillary rod and handler into the straw and push until the rectangular portion of the handler comes in contact with the flared end of the straw. While still holding the straw in place, seal the open end, hold the straw using tweezers in the area of the handling rod and quickly plunge the entire straw into LN2 vertically. For thawing lift the straw enough to expose the colored handling rod. Make sure the end with the sample remains immersed in the LN2. Holding the straw, use the opening device for HSV kit to section the straw and immediately (within 2 seconds), plunge the gutter into the first dilution media. A cooling rate of 2.000ºC/min is archived in the vitrification process (55).

Fig. 16. High security vitrification straw

#### **CryoTip**

(Irvine Scientific) A plastic straw container which can be sealed as a closed device to hold gametes or embryos in a specialized medium during cryopreservation procedures and subsequent long term storage in a LN2 tanks. CryoTip consist of a drawn plastic straw with an ultra fine tip and a protective metal cover sleeve. This device has been optimized as a closed system for cryopreservation procedures. For freezing aseptically remove one CryoTip when ready to use. Aseptically attach the wide end of the CryoTip to an aspiration tool, such as a luer tip syringe, using the Connector. When specimens are ready to load into the CryoTip, aseptically slide the metal cover sleeve carefully along the straw to expose the fine tip end. Gently load the specimens into the CryoTip by aspiration using the plunger on the syringe to control the uptake of medium and specimens. Heat seal the fine tip below the 1st mark, then slide the metal cover sleeve down over the fine tip to protect it and plunge the sealed CryoTip into the LN2 reservoir. A cooling rate of 12.000ºC/min is obtained with this device (49)

#### Fig. 17. Cryotip

#### **Cryopette®**

122 Current Frontiers in Cryopreservation

The CBS™ High Security vitrification straws are made from an ionomeric resin that is chemically inert, biocompatible and has physical characteristics resistant to ultra low temperatures and pressures created by expanding liquids and LN2. Sealed straws are resistance tested to 150 kg/cm2 (2133 lb/sq.inch), both the seals and the material should resist in order to have the batch approved. The HSV (High Security Vitrification) kit is composed of a High Security ionomeric resin straw, a capillary tube with a pre-formed gutter attached to a colored handling rod and a blue plastic insertion device. For freezing the sample is deposited into the gutter a few millimeters from the end using a micropipette. The drop holding the sample must be under 0.5 µL. immediately place the capillary rod and handler into the straw and push until the rectangular portion of the handler comes in contact with the flared end of the straw. While still holding the straw in place, seal the open end, hold the straw using tweezers in the area of the handling rod and quickly plunge the entire straw into LN2 vertically. For thawing lift the straw enough to expose the colored handling rod. Make sure the end with the sample remains immersed in the LN2. Holding the straw, use the opening device for HSV kit to section the straw and immediately (within 2 seconds), plunge the gutter into the first dilution media. A cooling rate of 2.000ºC/min is

(Irvine Scientific) A plastic straw container which can be sealed as a closed device to hold gametes or embryos in a specialized medium during cryopreservation procedures and subsequent long term storage in a LN2 tanks. CryoTip consist of a drawn plastic straw with

Fig. 15. Vitrisafe

**High security vitrification straw** 

archived in the vitrification process (55).

Fig. 16. High security vitrification straw

**CryoTip** 

(Origio) It is derived from the original STRIPPER® family of denudation tools. It includes a sterile STRIPPER® tip with an integrated bulb to facilitate loading. This eliminates awkward external handles, rods, and pick-up tools required with other devices and guarantees simplicity, speed and ease of use. The bulb is designed to deliver the sample to the desired location every time. The maximum load volume is 1.2 µl, producing a cooling rate of approximately 23.700ºC/min.

#### Fig. 18. Cryopette

These last two devices have been criticized by Parmegiani and his group's latest articles and in the last CRYO congress 2011, for the "potential danger" they have in his opinion: In the thawing process the external part of the device is in direct contact with the warming solution and any pathogen that could be on the exterior could pass onto the sample. In my opinion it is a very remote hypothesis and the probability that this could occur is minimal in

The Problem of Contamination: Open vs. Closed vs. Semi-Closed Vitrification Systems 125

Our work has demonstrated that the microorganisms that may be in the cooling solution, on the outside of the microcapillary, cannot come into contact with ultra-vitrified cells inside due to the loading procedure, the contact time with the cooling solution and the diameter and surface of Ultravit making it a secure device and is enough to exclude the theoretical danger of contamination. The cooling rate obtained with Ultravit was 250.000 Cº/min (57) with Slush nitrogen allowing ultra-vitrification with low concentration of CPA (1.5-2 M) and a morphological survival rate of 92% of human mature oocytes and 59.1% of blastulation

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

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

Fig. 20. Risk of Contamination with Ultravit

rate in mouse embryos.

**5. Semi-closed device** 

cooling rate but the most important is:

cross-contamination in the common tanks.

**OPS safe method** 

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.

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

#### **Ultravit**

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 contaminated:

Fig. 20. Risk of Contamination with Ultravit

Our work has demonstrated that the microorganisms that may be in the cooling solution, on the outside of the microcapillary, cannot come into contact with ultra-vitrified cells inside due to the loading procedure, the contact time with the cooling solution and the diameter and surface of Ultravit making it a secure device and is enough to exclude the theoretical danger of contamination. The cooling rate obtained with Ultravit was 250.000 Cº/min (57) with Slush nitrogen allowing ultra-vitrification with low concentration of CPA (1.5-2 M) and a morphological survival rate of 92% of human mature oocytes and 59.1% of blastulation rate in mouse embryos.
