**2.2 Stirling engine cryocooler**

The **Asymptote EF600** is the first commercially available programmable freezer which does not require liquid-nitrogen. The absence of liquid-nitrogen reduces drastically risk of contamination, and allows to freeze cells where nitrogen is not available (i.e. during transport or in other borderline applications).

Fig. 1. Asymptote EF600 (http://www.asymptote.co.uk/)

<sup>1</sup> Research is not intended to be exhaustive

The Asympotote EF600's cooling source is a Stirling Engine, a closed cycle machine in which the refrigerant working fluid is contained inside the machine, and only a source of mechanical or electrical energy is required2 in order to rich temperature below - 100◦.

Studies on human spermatozoa (linear cooling at -2◦/min until nucleation followed by linear cooling at -10◦/min to -100◦), embryonic stem cells (linear cooling at -2◦/min until nucleation followed by linear cooling at -1◦/min to -45◦) mouse embryos (linear cooling at -2◦/min until nucleation followed by linear cooling at -0.3◦/min to -35◦ and at -10◦/min to -100◦) and horse semen (linear cooling at -2◦/min until nucleation followed by linear cooling at -4◦/min to -80◦) were carried on, considering survival rate as a parameter for assessing the performances of the proposed system (Faszer et al., 2006; Morris et al., 2006).

Results show that Stirling Engine cryocooler can established the desired time-temperature profile inside the test tubes and the viability after thawing data confirm that the system can be used for slow freezing applications.

However, Stirling Engines are affected by vibrations, as stated by (Hughes et al., 2000) and (Suárez et al., 2003). Vibrations might damage cells; furthermore, manual nucleation cannot be performed at a desired temperature, since vibrations generally start the nucleation process (as reported by (De Santis et al., 2007; Edgar, 2009; Rosendahl et al., 2011)).
