**4. Acknowledgement**

We would like to thank Dr. Damian Marshall, Principal Scientist, LGC Ltd, Teddington, UK and Mr Ian Pope, Coreus Consulting, Faribault, MN, USA, for their valuable assistance with this chapter.

Additional material can be found in 'Cryopreservation and Freeze-Drying Protocols'; Day, J.; Stacey, G. (Eds.); 2nd ed., 2007 Humana Press Cryopreservation of Animal and Human Cell Lines; See

Chapter 3; Pegg,D.E. Principles of Cryopreservation. Chapter 16; Morris, C; Cryopreservation of Hematopoietic Stem/Progenitor Cells; Chapter 17; Watt S., Austin E., Armitage S.,

**1. Introduction**

1. slow freezing 2. vitrification

processes.

vitrification.

**2. Slow freezing**

3. ultra-rapid freezing

sperms can be reassumed in three categories:

with bacterium, mushroom and virus.

The proposed chapter investigates methods, devices and technologies for cryopreservation, explaining the most used cooling processes, as well as conventional and innovative technologies adopted. Main processes used for cryopreservation of oocytes, embryos and

**Technologies for Cryopreservation:** 

Edoardo Lopez, Katiuscia Cipri and Vincenzo Naso

**Overview and Innovation** 

*"Sapienza" University of Rome, Rome* 

**19**

*Italy* 

Research is not intended to be exhaustive, but is aimed at covering most of relevant topics. Slow freezing involves step-wise programmed decrease in temperature. The procedure is lengthy and requires the use of expensive instrumentation. The process does not exclude ice

In the vitrification process, the use of CryoProtectant Agents and the increasing of cooling rate (from 2,500 ◦/min to 130,000 ◦/min) avoid the ice crystal formation, increasing the embryos and oocytes survival. Unfortunately, common cryoprotectants are toxic and the immersion of solution directly in liquid nitrogen can be cause of contamination of embryos and oocytes

Ultra-rapid freezing can be considered a midway technique between slow freezing and vitrification, but its application has demonstrated lower performances than the other two

Necessary condition for slow freezing is freezing cells with a cooling rate equal or lower than 1◦/min, before storaging them at -130◦ or lower (De Santis & Coticchio, 2011). If cell is cooled down very slowly, it will be exposed to growing concentrations of cellular solutes due to ice formation inside the solution, with a PH variation and cellular dehydration. If it is cooled down too fast, crystal nucleuses will form in the solution and inside the cell, with the destruction of cell membrane. Usually at temperature below -60◦, the samples can be immersed directly in liquid nitrogen or transferred to freezer of maintenance without further loss of viability. Slow freezing generally lasts one or two hours. However, a greater amount of cells can be frozen at a time (Ha et al., 2005), and lower quantity of CPA are used than in

crystal formation, which can have extremely deleterious effects (Pegg, 2005).

## **5. References**

