**2.4 Warming and thawing**

It is usual to thaw cryopreserved or vitrified samples rapidly – typically by plunging them in a 37 °C water bath. The warming rate does have an effect on the recovery of living cells but this is not as great an influence as cooling rate is during cooling. In fact, optimum cooling rates have usually been determined using rapid warming so it is hardly surprising that rapid warming then gives the highest recovery! However, there are circumstances when the warming rate is of importance in its own right. The first is when the sample has been vitrified but is nucleated without a significant amount of ice being present. This is an unstable situation and in such circumstances the warming must be rapid to avoid intracellular freezing during warming. This consideration argues for rapid warming. The other situation occurs when the frozen material contains a significant amount of vitrified material, as is always the case in conventional cryopreservation. Glasses are brittle and the hazard here is that rapid warming will generate thermal stresses and cause the vitreous material to fracture. This will not matter greatly with cell suspensions where a fracture running through the sample is unlikely to traverse many cells but it is very important when the extracellular matrix must be intact – as it must, for example, in grafted blood vessels and heart valves. The solution here is to warm through the vitreous zone, that is from -196 °C to -123 °C, relatively slowly: once above the Tg there is no hazard from fractures and the sample can be warmed as rapidly as you like. A convenient way to do this is to allow the sample to warm slowly in a -80 °C refrigerator or packed in solid CO2 until its temperature is at above -100 °C. Alternatively the sample can be surrounded by a layer of insulation during the initial stage of warming in room air. A warming rate of around 50 °C/minute up to -100 °C was 'slow' enough to prevent fractures in cryopreserved rabbit carotid arteries (Pegg et al., 1997).

#### **2.5 Liquidus tracking – A new method**

The controlled-rate freezing process achieves its results by preventing the formation of intracellular ice. In some samples, however, even extracellular ice can be severely damaging.

Precision in Cryopreservation – Equipment and Control 517

released during this liquefaction is dissipated to the environment via heat exchanger coils. The liquid is then passed through cooling coils within the freezer chamber and absorbs energy from the chamber as it vaporizes. It is the vaporization process that creates the cooling effect. As lower temperatures are required, lower liquid point gases must be employed. In order to liquefy these gases, higher pressures are required and often the liquefaction cannot be completed in a single process; this results in larger, multiple

The most commonly used freezers for cryogenic purposes are upright, front-opening freezers with a cold point at a nominal -80 °C. It should be noted that there is no biological significance for this temperature, merely a physical significance since it approximates to the sublimation temperature of dry ice (solid carbon dioxide, -79 °C). This type of freezer can be employed to store biomaterial in which living cells are not a prime concern, or when it is to be stored for only a short period of time. This type of equipment is intended to be for transactional storage – holding material required daily and which will either be consumed or transferred to more appropriate conditions within a short time - 6 to 12 weeks typically. The front opening design, while adding considerable convenience, creates a significant issue with temperature stability and variability. Because cold air is significantly heavier than warm air, opening the door causes massive air exchanges and temperature rises in the sample area in a short period of time. In addition, because the compressor systems run on a very high cycle time, there is little spare capacity to effect a cooling after the temperature has risen and it can take some time to return to equilibrium after a warming event. This property is similarly exhibited when the freezer is in normal operation and as has been previously noted, there can be significant temperature variations. The use of deep drawers within the refrigerator for the storage of samples is helpful in reducing the loss of cold air

Because of the high cycle times, compressor failures are quite common and expensive to repair. It should also be noted that as the energy removed from the sample area is 100 % dissipated into the room in which the freezer is located, the term cost of operating a unit such as this should take into account not only the electricity consumption required for the compressor system, but also the significant air conditioning costs associated with the expelled heat from the freezers. If this energy is not removed by air-conditioning, the freezers become less efficient as room temperature rises, compressors are required to cycle even longer, power usage rises and compressors fail more quickly. Environmental

These commonly used laboratory units are essentially refrigerated circulators. A reservoir of cooling medium (normally an alcohol) is passed through a cooling system and re-enters a reservoir, reducing the temperature. The degree of refrigeration applied and the flow rate through the cooling coils determine the derived temperature of the reservoir. The relatively large volume of cooling liquid creates two noticeable effects: temperatures are very stable due to the large heat capacity of the available fluid and cooling rates can be controlled very accurately for a similar reason. The corollary to this however is that the rates achievable are very low and so rapid (> 1 °C/minute) rates are very hard to achieve. In addition, alcohol

management at a macro as well as micro level is therefore important.

bath freezers are normally limited to temperatures above -80 °C.

compressors being employed.

when the door is opened.

**3.2 Alcohol bath freezers** 

An example of this is articular cartilage. Isolated chondrocytes can be cryopreserved using conventional techniques (Pegg et. al., 2006a) but results when attempting to cryopreserve chondrocytes in situ have proven to be very disappointing. It was found that traditional cryopreservation results in the formation of ice crystals within the chondrons and not just in the acellular matrix (Pegg et. al., 2006b) which might have been expected from experience with conventional cryopreservation. In articular cartilage it is important to prevent both intracellular and extracellular ice. With this requirement in mind, the most appropriate cryopreservation approach would appear to be vitrification; that is the prevention of any ice formation at all. However, it will be clear that conventional vitrification is out of the question because of the heat transfer problems with bulky samples. Liquidus tracking (LT) provides a new approach to this problem.

During conventional cryopreservation, with a moderate concentration of CPA (say 10 %w/w) and relatively slow cooling (say 1 °C/minute), the cells are exposed to gradually increasing concentrations of cryoprotectant as progressively more extracellular ice is formed. The instantaneous CPA concentration is determined by the temperature according to the phase diagram of that specific system. The idea of LT is to control the instantaneous concentration of CPA throughout the cooling process so that the CPA concentration follows the liquidus line by external control rather than by progressive freezing of the medium. In this way the medium remains just above its freezing point at all times and no ice is formed. It is important to note that the cells are exposed only to the concentrations of CPA that they would experience during conventional cryopreservation. And we know that isolated chondrocytes in suspension can be cryopreserved by standard methods. In effect, the LT process takes advantage of the decrease in cytotoxicity of cryoprotectants as the temperature is decreased: hence, rather than starting with a very high concentration of cryoprotectant, the LT approach controls the concentration dynamically throughout the cooling process. In this way, vitrification can be achieved without using the extremely high concentrations of cryprotectant at the start of the process and without the need for rapid cooling. Of course, allowance has to be made for the time that diffusion of CPA into the tissue takes and this can be very considerable. On the other hand, if an organ can be perfused with the cryoprotectant solution, via the vascular system during cooling, then the diffusion distances will be very short and mass transport delays much less significant. In practice, when designing an LT process for a particular tissue, it is crucial to determine the concentration that is actually achieved in the tissue as the process continues and to adjust the concentration/ temperature/ time program to achieve the desired tissue concentration at all stages of the process. This necessitates slow cooling, commonly of the order of 0.1 to 0.3 °C per minute. The cooling of the samples can be achieved in a conventional controlled rate cooler and the solution composition can be controlled by standard peristaltic pumps, the whole system being under computer control – see section 3.7 below for a discussion of the methods that are now available for research use.
