**3.1.6 Direct Cover Vitrification - DCV**

The Direct Cover Vitrification - DCV is a new cooling method base on the minimum use of concentrated cryoprotectans and direct application of liquid nitrogen to the ovarian tissue. This way, the toxicity derived by cryoprotectants is reduced and the ice crystal injury is prevented. The ovary is immersed in a vitrification solution (0.8 ml) consisting of 15% EG, 15% DMSO and 0.5 M sucrose for 2 min.

The ovary is put in a 1.8-ml plastic standard cryovial, placed on a piece of gauze to remove the surrounding vitrification medium. Liquid nitrogen is directly applied onto the ovary for vitrification. The cap of the cryovial is closed. The lid does not have a hole. The vial is then placed into a liquid nitrogen tank.

DCV cryopreservation method, explored on mousse ovarian, has demonstrated to be highly efficient at increasing morphologically normal and viable follicles from cryopreserved ovarian tissue, compared with slow freezing and conventional vitrification.

#### **3.1.7 Solid Surface Vitrification - SSV**

The Solid Surface Vitrification - SSV has been developed at the Department of Animal Science, University of Connecticut. The method aims at defining an effective protocol to cryopreserve

Overview and Innovation 13

Technologies for Cryopreservation: Overview and Innovation 539

In the *Isachenko Method* (Isachenko et al., 2005), embryos are located inside a open-pulled straws (OPS). The OPS is placed inside a sterile insemination straw (indicative size 90-mm), manufactured from standard 0.5-mL insemination straws. One end of sterile insemination straw is previously sealed using a hand-held sealer. The open end is hermetically closed by a metal ball and this container (OPS and sterile insemination straw) is plunged into liquid nitrogen ("straw in straw" vitrification). The *Isachenko Method*, applied to biopsied mouse pronuclear embryos is resulted efficient as conventional vitrification, guaranteeing a complete isolation of embryos from liquid nitrogen and avoiding potential contamination by

A new solution to increase the cooling rate reducing the use of cryoprotectants consists in the physical reduction of liquid nitrogen temperature, as happens in the **Vit-Master**, a new device developed at IMT, Israel. In order to avoid the vaporization of N2, the temperature of liquid nitrogen is reduced until - 210◦ (boiling point of nitrogen), applying a negative pressure (Arav et al., 2002). The evaporative cooling causes the nitrogen to partially solidify, thus creating a nitrogen slush. Samples immersed in nitrogen slush cool more rapidly because they come into contact with liquid nitrogen sooner than those immersed in normal liquid nitrogen (Cai et al., 2005). The *Vit Master* vitrification machine can provide a very high cooling rate (up to 135,000◦/min). The cooling rate is especially enhanced in the first stage of cooling (from 20 to -10◦), when it is six, four or two times higher with 0.25-ml straws, open pulled straws (OPS) or electron-microscope (EM) grids, respectively. Between -10 and -150◦, the cooling rate is only about doubled by use of the *Vit Master*, but that was found to be enough to reduce the chances

Research about using a **Pulse Tube** for Vitrification is ongoing at "Sapienza" - University of

Ultra-rapid freezing can be considered a midway technique between slow freezing and Vitrification. It is quicker than the slow-freezing technique, does not involve the use of programmable machines and requires lower concentrations of cryoprotectant agents (CPA)

Experimental results demonstrate that this technique has lower performances than slow

Vitrification is an attractive freezing technique: supports required are cost effective and experimental data show an high survival rate after thawing. For example, a survival rate of 99% was quoted in (*KITAZATO BioPharma Co., Ltd. - http://www.kitazato-biopharma.com/*, n.d.)

However, vitrification exposes cells to a high risk of contamination, since cells are generally plunged directly into liquid-nitrogen. Risk of contamination is reported in (Bielanski et al., 2000), where cells frozen using vitrification were exposed to the bovine immunodeficiency

**3.2.2 Isachenko Method**

pathogenic microorganisms.

Rome.

**4. Ultra-rapid freezing**

using a *Cryotop* support.

than those used in vitrification.

**3.3 Innovative vitrification devices**

of devitrification and recrystallization during warming.

freezing's and vitrification's ones (AbdelHafez et al., 2010).

**5. Comparison between vitrification and slow freezing**

bovine oocytes for research and practice of parthenogenetic activation, in vitro fertilization and nuclear transfer.

Bovine oocytes matured in vitro are transferred to a vitrification solution (35% EG, 5% polyvinyl-pyrrolidone, 0.4 M trehalose inTCM 199 and 20% FBS). A metal cube covered with aluminum foil is partially submersed into liquid nitrogen (Fig. 10): the surface reaches the temperature of -150◦. Microdrops of vitrification solution, containing the oocytes, are dropped onto the cold upper surface of the metal cube and are instantaneously vitrified. The vitrified microdrops are then stored in liquid nitrogen (Dinnyés et al., 2000).

Fig. 10. The solid surface vitrification (SSV) device
