**2.1 Freezing in mechanical freezers**

Passive cooling uses insulation to moderate the cooling rate of samples that have been equilibrated with low concentrations of cryoprotectant and then placed inside traditional electromechanical refrigerators at -20 °C, -40 °C, -80 °C or even at lower temperatures. The cells are dessicated slowly during the cooling process. This method can be used for most robust cells but even under the best circumstances the post-thaw recovery rates may not be ideal. In addition, in most cases no instrumentation monitoring or recording of the process is provided. The variation of temperature within mechanical refrigerators is well known with one study reporting values of -43.5 °C to -90 °C in upright freezers (Su et. al., 1996). Since there is no active control during the process, it follows that the poor repeatability of the process can affect the cooling rate and hence the efficiency of the whole procedure. Variability might be improved if the local environment were more stable and protected from instantaneous variation due to external factors such as door openings etc. It is generally preferable to use a liquid nitrogen gas phase freezer for this approach since the internal temperature variation is small and the environment disturbed less frequently.

This approach to cooling and freezing is increasingly being used for material provision in pharmaceutical drug screening programmes as they move from supply by continuous culture towards a "cells-as-reagents" concept. In this approach, the cells are insulated in polystyrene containers as they are cooled initially to -80 °C and then transferred for cryogenic storage into liquid or vapour phase nitrogen. The need for rapid use of the cells for drug assays following cryopreservation, places an increased importance on the postthaw quality of the cells. In recent work carried out at LGC (Teddington,UK) this has been shown to be compromised by this type of freezing where the cooling rates are not actively controlled but rely on the passive characteristics of the system. In particular, temperature fluctuations within the polystyrene container and the storage time at -80 °C can significantly impact the post-thaw recovery of the cells and their biological function.

Precision in Cryopreservation – Equipment and Control 515

samples with very small volumes, ideally those with very high surface area-to-volume ratios; for example cryogenic straws can fit this description. It is important to be aware of the Leidenfrost effect where a sheath of vapour will surround a warm sample when plunged into liquid cryogen, essentially insulating the sample for a short period of time. For many vitrification protocols however, even this short additional time period before the sample is vitrified has proven fatal to the cells due to increased toxic exposure to the CPA and decreased cooling rates. In conventional vitrification, very high cooling rates are achieved by exposing small samples directly to the liquid nitrogen. The sample is surrounded with as little physical material as possible to achieve the maximum cooling

Although vitrification is normally associated with cooling rates in the tens of thousands of degrees Celsius per minute, slower techniques have been reported such as the S3 vitrification technique for blastocysts (Stachecki & Chen, 2008); this uses rates <200 °C/minute. But in fact vitrification does not necessarily require rapid cooling at all. It all depends on the dependence of the critical cooling rate required to prevent freezing on the concentration of the cryoprotectant. (Sutton, 1991). As the following section describes.

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

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.

rates. With large samples, however, such high cooling rates are impracticable.

vitrification can be produced at really low cooling rates.

cryopreserved rabbit carotid arteries (Pegg et al., 1997).

**2.5 Liquidus tracking – A new method** 

**2.4 Warming and thawing** 
