**3.1.4 Cryoleaf**

Cryoleaf is an open device for embryos and oocytes vitrification and storage (Fig. 8). Developed by Dr. Chian and Prof. Tan at McGill University, Montreal, the system uses PROH, EG and sucrose as cryo-protectants in the cooling phase, while in the warming procedure media contains sucrose. The recommended maximum load of the McGill Cryoleaf is 2-3 oocytes or embryos.

Oocytes or embryos are prepared for vitrification according to laboratory protocols. The outer cover of the McGill Cryoleaf is plunged into the liquid nitrogen bath, allowing the air to come out. Vitrified oocytes or embryos are quickly loaded into the McGill Cryoleaf using a suitable pipette. The excess of media, that must be less then 1 *μl*, has to be removed. The McGill

Overview and Innovation 11

Technologies for Cryopreservation: Overview and Innovation 537

after vitrification of human embryos and after cryopreservation of human oocytes worldwide.

Reference Cell n % Survival % Pregnancy Teramoto 2004 Blastocysts 197 100 57.7 Kuwayama 2005 Oocytes 64 91 41.3 Kuwayama 2005 PN Embryos D14 5881 100 - Kuwayama 2005 Embryos D3 897 98 27 Kuwayama 2005 Blastocysts 6328 90 53 Lucena 2006 Oocytes 159 97 56.5 Antinori 2007 Oocytes 330 91 32.5 Cobo 2008 Oocytes 243 97 65.2 Cobo 2008 Oocytes 797 96 63.2

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,

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

DCV cryopreservation method, explored on mousse ovarian, has demonstrated to be highly efficient at increasing morphologically normal and viable follicles from cryopreserved ovarian

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

Fig. 9. Kitazato Cryotop - Kitazato Industries

Table 2. Results achieved with Cryotop vitrification in human

tissue, compared with slow freezing and conventional vitrification.

Clinical results are shown in Tab. 2

**3.1.6 Direct Cover Vitrification - DCV**

15% DMSO and 0.5 M sucrose for 2 min.

placed into a liquid nitrogen tank.

**3.1.7 Solid Surface Vitrification - SSV**

Fig. 8. McGill Cryoleaf Open System for Vitrification

Cryoleaf is inserted, with oocytes or embryos, directly into liquid nitrogen. Then, the Cryoleaf is blocked, sliding the protective sleeve over the tip.

### **3.1.5 Cryotop**

Cryotop method, developed by Kuwayama in the Advanced Medical Research Institute of Kato Ladies Clinic, is based on the assumption that minimizing the volume<sup>6</sup> of the vitrification solution, increases both cooling and warming rates, also decreasing the chance of ice crystal nucleation/formation. Moreover the high-rate cooling decreases CPA concentration, also reducing chilling injury occurring between +15◦ (in human GV oocytes even +25◦) and -5◦, which can be minimized by passing embryos or oocytes rapidly through this temperature zone. Finally, studies have demonstrated that the use of small devices eliminates embryo fracture damages, especially in open systems.

The Cryotop tool consists of a narrow, thin film strip (0.4 mm wide, 20 mm long 0.1 mm thick) attached to a hard plastic handle for a minimum volume cooling. To protect oocytes and embryo on strip from mechanical damage and virus contamination during storage, a 3 cm long plastic tube cap is attached to cover the film part (Fig. 9).

The tool and the solutions for Vitrification and warming are market by Kitazato Co., Fujinomiya, Japan.

After a two-step equilibration in a vitrification solution containing EG, DMSO and sucrose, oocytes and embryo are loaded with a narrow glass capillary onto the top of the film strip in a volume of <0.1 ml. After loading, almost all the solution is removed so as to leave only a thin layer covering the oocytes or embryos, and the sample is quickly immersed into liquid nitrogen. Subsequently, the plastic cap is pulled over the film part of the Cryotop, and the sample is stored under liquid nitrogen (Kuwayama, 2007).

The minimal volume increases the cooling and warming rates up to 40,000◦/min, contributing positively to the embryos or oocytes survival.

Cryotop vitrification method is applied successfully in various areas of animal technology and now it is indicated as the process which guarantees the highest number of babies born

<sup>6</sup> According to common use, for Minimum Volume is intended less than 1 ml for direct dropping of samples into liquid nitrogen or the open pulled straw (OPS) method.

10 Will-be-set-by-IN-TECH

Cryoleaf is inserted, with oocytes or embryos, directly into liquid nitrogen. Then, the Cryoleaf

Cryotop method, developed by Kuwayama in the Advanced Medical Research Institute of Kato Ladies Clinic, is based on the assumption that minimizing the volume<sup>6</sup> of the vitrification solution, increases both cooling and warming rates, also decreasing the chance of ice crystal nucleation/formation. Moreover the high-rate cooling decreases CPA concentration, also reducing chilling injury occurring between +15◦ (in human GV oocytes even +25◦) and -5◦, which can be minimized by passing embryos or oocytes rapidly through this temperature zone. Finally, studies have demonstrated that the use of small devices eliminates embryo

The Cryotop tool consists of a narrow, thin film strip (0.4 mm wide, 20 mm long 0.1 mm thick) attached to a hard plastic handle for a minimum volume cooling. To protect oocytes and embryo on strip from mechanical damage and virus contamination during storage, a 3

The tool and the solutions for Vitrification and warming are market by Kitazato Co.,

After a two-step equilibration in a vitrification solution containing EG, DMSO and sucrose, oocytes and embryo are loaded with a narrow glass capillary onto the top of the film strip in a volume of <0.1 ml. After loading, almost all the solution is removed so as to leave only a thin layer covering the oocytes or embryos, and the sample is quickly immersed into liquid nitrogen. Subsequently, the plastic cap is pulled over the film part of the Cryotop, and the

The minimal volume increases the cooling and warming rates up to 40,000◦/min, contributing

Cryotop vitrification method is applied successfully in various areas of animal technology and now it is indicated as the process which guarantees the highest number of babies born

<sup>6</sup> According to common use, for Minimum Volume is intended less than 1 ml for direct dropping of

Fig. 8. McGill Cryoleaf Open System for Vitrification

is blocked, sliding the protective sleeve over the tip.

fracture damages, especially in open systems.

cm long plastic tube cap is attached to cover the film part (Fig. 9).

sample is stored under liquid nitrogen (Kuwayama, 2007).

samples into liquid nitrogen or the open pulled straw (OPS) method.

positively to the embryos or oocytes survival.

**3.1.5 Cryotop**

Fujinomiya, Japan.

Fig. 9. Kitazato Cryotop - Kitazato Industries

after vitrification of human embryos and after cryopreservation of human oocytes worldwide. Clinical results are shown in Tab. 2


Table 2. Results achieved with Cryotop vitrification in human
