**3. Vitrification**

6 Will-be-set-by-IN-TECH

Fig. 5. *Predictive model* regulation - Temperature inside the test tube vs time. A desired

Fig. 6. *Predictive model* regulation - Temperature inside the test tube vs time. A desired

cooling rate of -0.7◦/min was selected.

cooling rate of -1◦/min was selected.

A criticality of common cryopreservation methods consists in the formation of ice crystals that drastically reduces the survival of treated embryos and oocytes. Vitrification process produces a glasslike solidification of living cells which completely avoids ice crystal formation. The process is based on the principle that water, characterized by high cellular viscosity increased by the adding of CryoProtectant Agent (CPA), and frozen using a high cooling rate, is not capable of forming ice. The main limits of Vitrification process are represented by: use of potentially toxic cryoprotectant; risk of contamination of embryos and oocytes with bacterium, mushroom and virus when directly immersed in liquid nitrogen or during the storage phase. Studies have demonstrated that reduced quantity of CPA can be used if the cooling rate is increased.

A freezing rate of 2,500◦/min and CPA concentration of 5-7 M is reached with the immersion of embryos and oocytes in micro-capillary straws, while in the pulled straws the cooling rate is about 20,000 ◦/min (Kuleshova & Lopata, 2002). Theoretically, the reaching of a cooling rate of 107◦/sec should allow to vitrify also in pure water, but this rating is not practicable at the moment. Several studies are also oriented to formulate nontoxic and more efficient Vitrification solutions, also combining different cryoprotectants such as sugars and polymers or establishing modern solutions that include non-penetrating additives.

Moreover, the implementation of *Minimum Volume methods* has allowed to reduce the concentration of cryoprotectant. EG (ethylene glycol), characterized by low toxicity, is an important component of vitrification solution, commonly combined with DMSO or PROH (propanediol). In particular, non-permeable cryoprotectans (such sucrose or PVP) can be added in the solution on order to reduce the concentration of permeable cryoprotectans and facilitate dehydration and vitrification. Researches oriented to improve the characteristics of cryoprotectans have been carrying on in order to reduce toxicity. An EG and sucrose (non-permeable cryoprotectans) solution has been tested for cryopreservation of all preimplantation stages of *in vivo* generated mouse and day-6 sheep embryos. Experiments have not shown a loss of viability in vitro or in vivo. The same solution has been proved for vitrification of human oocytes, attaining high surviveal rates using conventional straws.

Another solution used to reduce toxicity is to equilibrate the cryoprotectant using a two-step method: the pretreatment solution, named *equilibration solution*, contains 20-50% concentrations of permeating cryoprotectans. The lower concentration of permeating cryoprotectans in the equilibration solution is much less toxic than the vitrification solution. The permeating cryoprotectant enters into the cells and facilitytes the intracellular vitrification. The cells pretreatment with equilibration solution is used in oocytes vitrification: this method has been demonstrated to increase the survival rate after thawing.

Main devices, commonly use in vitrification, are *Open Supports*: *Pulled Straws, CryoLoop, CryoEM, Cryoleaf* and *CryoTop*. The risk of contamination, due to the use of *Open Supports* for vitrification, limits the use of this process for human cells and tissues, according to the European regulations. In order to reduce contamination risks, *Close Supports* have been introduced: unfortunately their use decreases the cooling rate with consequently need to improve the quantity of CPA for guaranteeing the same survival rate. Vitrification process has demonstrated high performance in term of survival after thawing, comparable to slow cooling and it has become a promising alternative in cryopreservation of mammalian embryos and especially oocytes, through application of slow-rate freezing process.

Overview and Innovation 9

Technologies for Cryopreservation: Overview and Innovation 535

Fig. 7. CryoLoop Properly Installed in a CrystalCap Copper Magnetic - *Hampton Research*

Biological material is spread on an electron microscopy grid and is preserved in a frozen-hydrated state by rapid freezing (about 3,000◦/min), usually in ethane slush close to liquid nitrogen temperature. Specimens, maintained at liquid nitrogen temperature or colder, are contained into the high-vacuum of the electron microscope column. The frozen sample grid is then kept at liquid nitrogen temperature in the electron microscope and digital micrographs are collected with a camera. Images obtained from the cryo-electron microscopy are usually very noisy and have very low contrast. It is necessary to smooth the noise as well

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 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

*Corporation*

as enhance the contrast.

oocytes or embryos.

**3.1.4 Cryoleaf**
