**2.3 Pulse tube cryocooler**

2 Will-be-set-by-IN-TECH

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>

Producer Planer plc Planer plc CryoLogic Fisher Scientific Control range [◦] +40 to -180 +30 to -180 +40 to -120 +50 to -180 Cooling rates [◦/min] -0.01 to -50 -0.01 to -50 -0.04 to - 10 (at -40 ˛aC) -0.1 to -50 Heating rates [◦/min] 0.01 to 10 0.01 to 10 — 0.1 to 10 Capacity [*l*] 1.7 or 3.3 16 11.5 17 or 48

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

Kryo 360 Kryo 560M Cryo-Logic 8800 + Thermo Scientific

Fast CryoChamber Forma 94741

**2.1 Programmable freezers**

**2.2 Stirling engine cryocooler**

or in other borderline applications).

Table 1. Programmable freezers main characteristics

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

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

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

A heater is placed by the test tube older (Fig. 2). The power dissipated through the heater for Joule effect varies according to two different control systems proposed:

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

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

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

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

Overview and Innovation 5

Technologies for Cryopreservation: Overview and Innovation 531

Fig. 3. *On-Off* regulation - Temperature inside the test tube vs time. A desired cooling rate of

Fig. 4. *On-Off* regulation - Temperature inside the test tube vs time. A desired cooling rate of



Fig. 2. Representation of the experimental apparatus. *Ttt* represents the temperature inside the test tube, measured by a thermocouple, *Q* is the heat absorbed by the refrigerating fluid and *W* is the dissipated power.

However, the oscillation might be reduced optimizing the threshold parameters. The *On-Off* regulation can be easily implemented, and it does not require the regulation of the power dissipated through Joule effect.

2. *Predictive model* **regulation**: the cooling slow-down is achived by providing an amount of heat, variable with the time, that will be able to raise the temperature of the PT cold head to the desired value (Cipri et al., 2010). The amount of heat is calculated using a predictive and adaptive model. Using this regulation modality, oscillation can be removed. However, it requires the regulation of the power dissipated through Joule effects, increasing the cost of the hardware. Moreover, more computational power is required in order to calculate the amount of heat which has to be dissipated.<sup>5</sup>

Results are shown in Fig. 5 and Fig. 6.

In the determination of the *Predictive model* a lot of simplifying assumptions were made (Cipri et al., 2010), and we believed that the system should have better results if the model was set in more accurate way. Further researches are fostering investigation at Sapienza Laboratory.

At this very moment, the system is not yet commercially viable.

<sup>5</sup> An *On-Off* regulation is still used before the transition phase, marked by the abrupt rise of temperature typical of the subcooling

4 Will-be-set-by-IN-TECH

Fig. 2. Representation of the experimental apparatus. *Ttt* represents the temperature inside the test tube, measured by a thermocouple, *Q* is the heat absorbed by the refrigerating fluid

However, the oscillation might be reduced optimizing the threshold parameters. The *On-Off* regulation can be easily implemented, and it does not require the regulation of

2. *Predictive model* **regulation**: the cooling slow-down is achived by providing an amount of heat, variable with the time, that will be able to raise the temperature of the PT cold head to the desired value (Cipri et al., 2010). The amount of heat is calculated using a predictive and adaptive model. Using this regulation modality, oscillation can be removed. However, it requires the regulation of the power dissipated through Joule effects, increasing the cost of the hardware. Moreover, more computational power is required in order to calculate

In the determination of the *Predictive model* a lot of simplifying assumptions were made (Cipri et al., 2010), and we believed that the system should have better results if the model was set in more accurate way. Further researches are fostering investigation at Sapienza Laboratory.

<sup>5</sup> An *On-Off* regulation is still used before the transition phase, marked by the abrupt rise of temperature

and *W* is the dissipated power.

typical of the subcooling

the power dissipated through Joule effect.

the amount of heat which has to be dissipated.<sup>5</sup>

At this very moment, the system is not yet commercially viable.

Results are shown in Fig. 5 and Fig. 6.

Fig. 3. *On-Off* regulation - Temperature inside the test tube vs time. A desired cooling rate of -0.5◦/min was selected.

Fig. 4. *On-Off* regulation - Temperature inside the test tube vs time. A desired cooling rate of -1◦/min was selected.

Overview and Innovation 7

Technologies for Cryopreservation: Overview and Innovation 533

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

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

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:

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

this method has been demonstrated to increase the survival rate after thawing.

and especially oocytes, through application of slow-rate freezing process.

or establishing modern solutions that include non-penetrating additives.

**3. Vitrification**

cooling rate is increased.

Fig. 5. *Predictive model* regulation - Temperature inside the test tube vs time. A desired cooling rate of -0.7◦/min was selected.

Fig. 6. *Predictive model* regulation - Temperature inside the test tube vs time. A desired cooling rate of -1◦/min was selected.
