**2. Slow freezing**

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 vitrification.

Overview and Innovation 3

Technologies for Cryopreservation: Overview and Innovation 529

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

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

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

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

In order to overcome to problems connected with vibrations of Stirling Engines, a programmable freezer based on a Pulse Tube cryocooler is being developed in "Sapienza" University of Rome Laboratory of Mechanical Engineering, in collaboration with *MES - Microconsulting Energia & Software S.c.a.r.l.*<sup>3</sup> and *LABOR S.r.l.*4. Alike the Stirling Engine, the Pulse Tube machine is a closed cycle system and it does not require liquid-nitrogen. The Pulse Tube cryocooler is able to rich temperatures below -150◦ making the refrigerator fluid (that is generally helium or nitrogen) move oscillatory. The fluid motion is obtained using a compressor and a rotative valve. The Pulse Tube offers low vibrations, as discussed by (Ikushima et al., 2008; Riabzev et al., 2009; Suzuki et al., 2006; Wang & Hartnett, 2010).

Next to the *cold head* (the cooling part of the Pulse Tube), the refrigerator fluid absorbs heat from the test tube, cooling it. The Pulse Tube cryocooler is characterized by a higher cooling rate than the ideal one for cell freezing (0.1◦/min ÷ 10◦/min) in the temperature range used for cryopreservation (+30◦ ÷ -60◦). The cooling rate is reduced in the proposed solution

A heater is placed by the test tube older (Fig. 2). The power dissipated through the heater for

1. *On-Off* **regulation**. A threshold control system has been implemented: the heater is activated when the real temperature is more than 1◦ below the desired temperature, and it is turned off when the real temperature is more than 1◦ over the desired temperature. Using this control system, oscillations of ±6◦ around the desired temperature were

<sup>2</sup> The Asymptote EF600 can be connected to a conventional 240V electricity supply or to a car battery

or electrical energy is required2 in order to rich temperature below - 100◦.

(as reported by (De Santis et al., 2007; Edgar, 2009; Rosendahl et al., 2011)).

through a control system that can supply heat to the cryorefrigerator.

Joule effect varies according to two different control systems proposed:

obtained, as it is illustrated in Fig. 3 and Fig. 4.

<sup>4</sup> Tecnopolo Tiburtino, Via G. Peroni 386 - 00131 Roma, Italy

<sup>3</sup> Via A. Panzini, 3 - 00137 Roma, Italy

of the proposed system (Faszer et al., 2006; Morris et al., 2006).

be used for slow freezing applications.

**2.3 Pulse tube cryocooler**

## **2.1 Programmable freezers**

Currently, programmable freezers are the most common technology for slow freezing process. Programmable freezers are based on liquid nitrogen technology, but their use is denied in areas without availability of nitrogen or during long transport. Cooling rate is controlled by a heater (Asymptote EF600, Cryologic CL8800) or by the synchronous use of two valves.

Main characteristics of the most common programmable freezers are shown in Tab. 1. <sup>1</sup>


Table 1. Programmable freezers main characteristics
