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

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.) using a *Cryotop* support.

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

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virus (BIV), that can be considered a model for retrovirus like the human immunodeficiency virus (HIV). Risk of Hepatitis B contamination is analyzed in (Tedder et al., 1995).

Moreover, vitrification requires a greater amount of CPA (CryoProtectant Agent) than Slow Freezing does, increasing the toxicity of the environment.

In order to reduce the risk of contamination, *closed supports* for vitrification were developed (*Cryotip* and *Isachenko Method*). However, a lower survival rate is obtained than using an *open support*.

A lower survival rate after thawing using Slow Freezing instead of Vitrification was claimed by many authors. (Fadini et al., 2009) reports a survival rate of human oocytes of 78.9% using Vitrification, while it is reduced to 57.9 % using Slow Freezing (*p-value* lower than 0.0001); similar results are shown in (Vutyavanich et al., 2010) (where survival rate of human spermatozoa is measured equal to 64.8% using Vitrification and equal to 50.4% using Slow Freezing, *p-value* equal to 0.0036). However, many authors believe that a better understanding of slow freezing principles will improve its performances (Bianchi et al., 2007; De Santis et al., 2007; Edgar, 2009; Fadini et al., 2009; Mcgrath, 2009).

Both Vitrification and Programmable Freezers (the most common machines use for Slow Freezing) require a supply of liquid-nitrogen, that is a limiting factor in many situations of inefficient or absent nitrogen distribution network, such as small industries, isolated places and during transport of cells. In order to overcome to this limitation, two alternative systems for Slow Freezing (*Asymptote EF600* and *Pulse Tube Cryocooler*) have been developed.

Stirling Engine7 and Pulse Tube Cryocooler are closed-cycle machines, reducing risk of contamination and toxicity. A cells freezing system based upon closed-cycle machines is a viable commercial solution, especially for those markets where liquid nitrogen supply is difficult or excessively expensive, or during transport.

However, Stirling Engine exhibits high vibration, thus the nucleation process can not be inducted manually. Moreover, vibrations might damage cells. Those problems are avoided using a Pulse Tube cryocooler.

The application of a Pulse Tube Cryocooler for cells cryopreservation is under developing at "Sapienza" - University of Rome Laboratory of Mechanical Engineering. A validation of the proposed system with the assessment of cells survival rate after thawing is envisaged as next step. Future work will also focus on the development of a cost effective control system which allows the operator to set a desired cooling rate.
