**3. Open device**

Following recent studies of cell and tissue contamination in freezing and the recent debates regarding the sterility of LN2 in vitrification processes the devices play an important role in the asepsis of the frozen sample. There are many different types of device and of various materials but from the point of view of sterility, devices can be divided into Open, Closed and Semi-closed devices.

There is a lot of controversy and confusion about the concept of Open device. For most cryobiologists Open devices are devices that allow direct contact of the biological sample to be frozen with the cooling solution but when there is contact with the interior of the device but not with the sample to be frozen it would not be considered an open device. Once inside the common cooling containers the cooling solution enters and leaves the device keeping all frozen samples in contact.

Fig. 2. Risk of Contamination with open devices (47)

Generally, using open devices the achieved cooling rates are approximately 20.000-30.000 ° C / min which favor good vitrification of the sample. The problem is that being in direct contact with the cooling solution there is a risk of pathogen transmission to the biological sample at the time of freezing and a high risk of cross contamination in the common cooling containers. They have been prohibited in many countries for this high risk of contamination and the global trend is to ban them for use with human samples. Recent microbiological studies indicate a 45% pathogen contamination (Pseudomonas and E-coli) with a simple 10-second contact with the open device (Cryotop) with contaminated cooling solution (28).

The most known and used open devices are the following:

#### **Open pulled straw**

114 Current Frontiers in Cryopreservation

appropriately. So stand out from other actions that the sender must ensure the proper identification, packaging, labeling and documentation according to established biosafety guidelines in the "Recommendations of the Committee of Experts of the United Nations Transport of Dangerous Goods" transporting must be kept in appropriate conditions (temperature, light ...) the material from which the sender receives it until it is delivered to your destination and have the appropriate licenses to perform this type of transport, and finally, the recipient must confirm with national authorities that the material can be legally

According to AEBT (21), the possibility of returning a material that hasn't been used should be avoided, as a rule, the return of the semen that has been provided by the Bank, as it will

Following recent studies of cell and tissue contamination in freezing and the recent debates regarding the sterility of LN2 in vitrification processes the devices play an important role in the asepsis of the frozen sample. There are many different types of device and of various materials but from the point of view of sterility, devices can be divided into Open, Closed

There is a lot of controversy and confusion about the concept of Open device. For most cryobiologists Open devices are devices that allow direct contact of the biological sample to be frozen with the cooling solution but when there is contact with the interior of the device but not with the sample to be frozen it would not be considered an open device. Once inside the common cooling containers the cooling solution enters and leaves the device keeping all

only accept the return of the displayed when you meet the following 3 conditions:

2. You can demonstrate the integrity of the packaging (the seals are intact). 3. The temperature of the sample was maintained throughout the transport.

imported.

**3. Open device** 

and Semi-closed devices.

frozen samples in contact.

Fig. 2. Risk of Contamination with open devices (47)

1. The sample wasn't thawed.

In the OPS method, 0.25 mm standard insemination plastic mini-straws were heat-softened over a hot plate and pulled manually, as originally described by Vajta et al. (48). The inner diameter and the wall thickness of the pulled part of the straw are approximately 0.8 and 0.07 mm respectively. Cells are load into the pulled straws by placing the narrow end of the pulled straw in the third droplet of medium and aspirating oocytes within a 2–3 mm long liquid column (1–1.5 μl) using capillarity. The straws are then cooled by being plunged directly into LN2 and stored briefly. For warming, the open end of the straw is immersed vertically into 4.5 ml of the warming solution at 37°C. The solidified vitrification solution became liquid within 1–2 s. A cooling rate of 16.700ºC/min is obtained with this device (49).

Fig. 3. A) The 0.25 ml conventional straw is loaded with 1 cm of vitrification medium, 0.5 cm of air, 2 cm of vitrification medium containing oocytes, 0.5 cm of air, and 3.5 cm of vitrification medium using a syringe. (B) The open pulled straw is loaded with vitrification solution (1–2 μl) containing oocytes by means of the capillary effect by a simple touch.

#### **Cryoloop**

The Cryoloop (Hampton Research, Aliso Viejo, CA, USA ), used as a vessel in vitrification, is a thin nylon loop used to suspend a film of cryoprotectant containing the oocytes and directly immerse them in LN2. Vitrification of oocytes using the Cryoloop has advantages over conventional vitrification procedures in that the open system lacks a thermo insulating

The Problem of Contamination: Open vs. Closed vs. Semi-Closed Vitrification Systems 117

handle specially constructed according to specifications by Kitazato. The droplet volume was estimated from the length of the fluid column within the pipette tip. As soon as the oocyte was placed onto the thin polypropylene strip of the Cryotop, it was immediately submerged vertically into filtered LN2. Then, the thin strip was covered with a hard plastic cover (3 cm long) on top of the Cryotop sheet to protect it during storage in LN2 containers. For warming, the protective cover was removed from the Cryotop while it was still submerged in LN2, and the polypropylene strip of the Cryotop was immersed directly into the solution at 37°C for 1 min. A cooling rate of 23.000ºC/min is obtained with this device

The McGill Cryoleaf™ is very similar to Cryotop but with a number of features designed to improve the loading and storage of cells. Safety during storage has been improved, as the cells are double protected from stress and contamination through a closed cover system but not hermetically sealed leaving cells in direct contact with LN2. The McGill Cryoleaf™ and the vitrification media have been developed by Dr. Chian and Prof. Tan at McGill

(Biodiseño, Colombia) With this device, cells are deposited near the black mark using the minimal amount possible of vitrification solution (2 μl aprox.) The black mark eases the

(49).

Fig. 6. Cryotop

University, Montreal.

Fig. 7. Cryoleaf

**Cryolock** 

**Cryoleaf** 

layer, together with the small volume of <1μl, results in both rapid and uniform heat exchange during cooling. A cooling rate of 20000ºC/min is obtained with this device.

#### Fig. 4. Cryoloop

#### **Hemi-straw**

The Hemi-straw (Astro-Med-tec, Salzburg, Austria) is an embryo carrier that consists of a large gutter on which a small quantity of CPAs (<1µl) containing the cell is deposited. The Hemi-straw is subsequently inserted into a larger pre-cooled 0.5 ml straw (CBS, Cryo Bio System, Grenoble, France) under LN2. Prior to the commencement of the warming process the Hemi-straw is pulled out of the larger straw under LN2 and the tip of the Hemi-straw is immediately immersed into a petri dish containing a sucrose solution. A rapid cooling rate of >20.000ºC/min is achieve by allowing direct contact of the biological material with LN2. (50)

Fig. 5. Scheme of the Hemi-Straw: (A) loading the cell on the tip of the Hemi-Straw; (B) insertion of the Hemi-Straw into a larger 'CBS' straw. (51)

#### **Cryotop**

(Kitazato Supply Co, Fujinomiya, Japan) Individual oocytes were picked up in an extremely small volume (<0.1 μl) of vitrification solution and placed on top of a very fine polypropylene strip (0.4 mm wide × 20 mm long × 0.1 mm thick) attached to a hard plastic handle specially constructed according to specifications by Kitazato. The droplet volume was estimated from the length of the fluid column within the pipette tip. As soon as the oocyte was placed onto the thin polypropylene strip of the Cryotop, it was immediately submerged vertically into filtered LN2. Then, the thin strip was covered with a hard plastic cover (3 cm long) on top of the Cryotop sheet to protect it during storage in LN2 containers. For warming, the protective cover was removed from the Cryotop while it was still submerged in LN2, and the polypropylene strip of the Cryotop was immersed directly into the solution at 37°C for 1 min. A cooling rate of 23.000ºC/min is obtained with this device (49).

Fig. 6. Cryotop
