**Cryoleaf**

116 Current Frontiers in Cryopreservation

layer, together with the small volume of <1μl, results in both rapid and uniform heat

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)

(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

insertion of the Hemi-Straw into a larger 'CBS' straw. (51)

exchange during cooling. A cooling rate of 20000ºC/min is obtained with this device.

Fig. 4. Cryoloop

**Hemi-straw** 

**Cryotop** 

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 University, Montreal.

#### **Cryolock**

(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

The Problem of Contamination: Open vs. Closed vs. Semi-Closed Vitrification Systems 119

A serum Tube (Sumitomo Bakelite, Tokyo) was employed as a vessel for cryopreservation. A clear polyethylene terephthelate film (50 mm in thick) was cut into a T-shaped piece. As shown in Fig. 10, the horizontal arm of the "T" shape was rolled and fit securely to the inner wall of the cap. After equilibration with cryo-medium, the embryo for vitrification was placed at the center of a plastic blade, the vertical limb projected from the cap, and five embryos were the maximum allowed on one blade. The width of the plastic blade was significantly wider than that of the Cryotop which was commercially available tool for the storage of the vitrified human embryo. The blade was submerged directly into LN2 and inserted into the tube that was pre-cooled with LN2, then the cap was fastened on the serum tube, which accomplished preservation in the LN2 container. For warming, the serum tube that contained the plastic blade was submerged under LN2, and the serum tube was opened and the plastic blade containing vitrified cells was removed from the LN2 and placed

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

directly into the well of the base medium at 37°C. (53)

the stalled sample remains suspended in time.

**Plastic-blade** 

Fig. 10. Plastic-Blade

**4. Closed device** 

cover up. The Cryolock® is immediately plunged into LN2, whilst holding the Cryolock®. After this the cap is grasped with forceps and plunged into LN2 until bubbling stops, be aware to not take the Cryolock® out of the LN2 whit covered up, twist and lock gently. Finally, place the Cryolock® in the goblet with the cap downward facing and store for the desired time. For warming, remove the patients canister form the dewar and place in a styrofoam box completely cover the Cryolock® with LN2. Grasp the Cryolock® body at the indentation with forceps and remove from the goblet. Grasp the cap at the indentation with forceps, twist and pull down without taking the Cryolock® body out of the LN2, it must always remain in LN2. Finally remove the Cryolock® from the LN2 quickly and pass into thawing solution at 37ºc and follow the protocol.

Fig. 8. Cryolock®

#### **Vitri-Inga**

The Vitri-Inga vitrification strip is an apparatus that consists of a fine, very thin polypropylene strip (0.7 mm thick) with a specially designed round tip, in which there is a minute hole to receive the cell; the strip is connected to a hard and thicker plastic handle. Vitri-Inga´ plastic sheaths are 0.5 ml semen straws with a cut in the middle. The total time from when the oocyte was placed into the vitrification solution till its immersion into LN2 is between 50 and 60 seconds. The plastic sheaths, which had been previously cooled for at least 2 min in LN2 vapor on the metal rack inside in the Vitri-Equip, are vertically immersed into LN2. The Vitri-Inga strip with the vitrified oocyte is then inserted into the plastic sheaths for storage, and transferred to a LN2 tank. (52)

Fig. 9. Vitri-Inga

#### **Plastic-blade**

118 Current Frontiers in Cryopreservation

cover up. The Cryolock® is immediately plunged into LN2, whilst holding the Cryolock®. After this the cap is grasped with forceps and plunged into LN2 until bubbling stops, be aware to not take the Cryolock® out of the LN2 whit covered up, twist and lock gently. Finally, place the Cryolock® in the goblet with the cap downward facing and store for the desired time. For warming, remove the patients canister form the dewar and place in a styrofoam box completely cover the Cryolock® with LN2. Grasp the Cryolock® body at the indentation with forceps and remove from the goblet. Grasp the cap at the indentation with forceps, twist and pull down without taking the Cryolock® body out of the LN2, it must always remain in LN2. Finally remove the Cryolock® from the LN2 quickly and pass into

The Vitri-Inga vitrification strip is an apparatus that consists of a fine, very thin polypropylene strip (0.7 mm thick) with a specially designed round tip, in which there is a minute hole to receive the cell; the strip is connected to a hard and thicker plastic handle. Vitri-Inga´ plastic sheaths are 0.5 ml semen straws with a cut in the middle. The total time from when the oocyte was placed into the vitrification solution till its immersion into LN2 is between 50 and 60 seconds. The plastic sheaths, which had been previously cooled for at least 2 min in LN2 vapor on the metal rack inside in the Vitri-Equip, are vertically immersed into LN2. The Vitri-Inga strip with the vitrified oocyte is then inserted into the plastic

thawing solution at 37ºc and follow the protocol.

sheaths for storage, and transferred to a LN2 tank. (52)

Fig. 8. Cryolock®

Fig. 9. Vitri-Inga

**Vitri-Inga** 

A serum Tube (Sumitomo Bakelite, Tokyo) was employed as a vessel for cryopreservation. A clear polyethylene terephthelate film (50 mm in thick) was cut into a T-shaped piece. As shown in Fig. 10, the horizontal arm of the "T" shape was rolled and fit securely to the inner wall of the cap. After equilibration with cryo-medium, the embryo for vitrification was placed at the center of a plastic blade, the vertical limb projected from the cap, and five embryos were the maximum allowed on one blade. The width of the plastic blade was significantly wider than that of the Cryotop which was commercially available tool for the storage of the vitrified human embryo. The blade was submerged directly into LN2 and inserted into the tube that was pre-cooled with LN2, then the cap was fastened on the serum tube, which accomplished preservation in the LN2 container. For warming, the serum tube that contained the plastic blade was submerged under LN2, and the serum tube was opened and the plastic blade containing vitrified cells was removed from the LN2 and placed directly into the well of the base medium at 37°C. (53)

Fig. 10. Plastic-Blade
