**3.7 Liquidus tracking equipment**

Because liquidus tracking is a relatively novel technique, there is little choice of equipment to assist with research into its use. Planer plc do manufacture a Liquidus Tracking controller that can be used for research into this approach. The equipment comprises a conventional slow-rate chamber coupled with a liquidus tracking controller and two peristaltic pumps.

The controller cools the sample in a similar manner to the conventional slow-rate freezing process. The cooling profile is typically a simple linear ramp. During the cooling of the sample, the controller monitors the current chamber temperature and adjusts the speeds of the two pumps to dynamically alter the concentration of cryoprotectant surrounding the samples. In the ideal process the concentration of cryoprotectant is maintained just above the liquidus curve. As the temperature decreases, the concentration of cryoprotectant is therefore increased. However, as the temperature of the sample decreases, the toxicity of the cryoprotectant decreases and this allows the sample to tolerate the ever increasing concentrations; see figure 1.

Precision in Cryopreservation – Equipment and Control 521

method is suitable for use with small sample containers and has been used for discs of ovine

The single solution mode is more suitable for use with larger samples. It is illustrated in figure 3. Here a highly concentrated solution is cooled and delivered to the sample container. This increases the concentration of the CPA solution surrounding the sample. To maintain a constant volume within the container, the second pump extracts the excess solution from the container. This technique is suitable for larger samples as it reduces the total volume of cryoprotectant required. For a sample container volume of Vs, a cryoprotectant concentration of Ksol and a target concentration of Kt, the volume of

For a sample container volume of 50 ml, depending on the actual values of Ksol and Kt this approach could require less than 100 ml of concentrated DMSO solution. Because the incoming solution has to be thoroughly mixed within the container, additional stirring equipment running at cryogenic temperatures is required. This results in a mechanically

V = Vs.ln(Ksol / (Ksol – kt)) (1)

articular cartilage ( see Wang et al., 2007).

Fig. 2. Dual solution mode.

cryoprotectant V can be calculated from this equation.

more complex arrangement than the dual solution approach.

Fig. 1. Concentration of sample tracking liquidus

Two specific requirements of this process are the rather large volumes of cryoprotectant required and the need to ensure good mixing around the sample. The Planer Liquidus Tracker supports two modes of operation each with its own advantages and disadvantages; these are the single solution and dual solution modes.

In the dual solution mode, shown in figure 2, a solution containing a high concentration of CPA (typically 72 % w/w DMSO plus isotonic salts) and a solution containing only isotonic salts (nominal 0 % solution) are used. Each solution is pumped through a mixing junction and a heat exchanger into the sample container and thence into a waste collection container. The relative speeds of the pumps are continuously adjusted via a computer program to deliver the correct concentration to the sample. The pump speeds are adjusted to maintain a constant flow rate through the sample container. The dual solution system requires a small volume surrounding the sample so that the incoming, premixed solution is able to displace the existing solution completely as it flows through the container. The total volume of cryoprotectant can be quite large; for example, a run from 0 °C to -70 °C at 0.3 °C/minute requires a total volume of solution equal to 233 times the sample container volume. This

Two specific requirements of this process are the rather large volumes of cryoprotectant required and the need to ensure good mixing around the sample. The Planer Liquidus Tracker supports two modes of operation each with its own advantages and disadvantages;

In the dual solution mode, shown in figure 2, a solution containing a high concentration of CPA (typically 72 % w/w DMSO plus isotonic salts) and a solution containing only isotonic salts (nominal 0 % solution) are used. Each solution is pumped through a mixing junction and a heat exchanger into the sample container and thence into a waste collection container. The relative speeds of the pumps are continuously adjusted via a computer program to deliver the correct concentration to the sample. The pump speeds are adjusted to maintain a constant flow rate through the sample container. The dual solution system requires a small volume surrounding the sample so that the incoming, premixed solution is able to displace the existing solution completely as it flows through the container. The total volume of cryoprotectant can be quite large; for example, a run from 0 °C to -70 °C at 0.3 °C/minute requires a total volume of solution equal to 233 times the sample container volume. This

Fig. 1. Concentration of sample tracking liquidus

these are the single solution and dual solution modes.

method is suitable for use with small sample containers and has been used for discs of ovine articular cartilage ( see Wang et al., 2007).

Fig. 2. Dual solution mode.

The single solution mode is more suitable for use with larger samples. It is illustrated in figure 3. Here a highly concentrated solution is cooled and delivered to the sample container. This increases the concentration of the CPA solution surrounding the sample. To maintain a constant volume within the container, the second pump extracts the excess solution from the container. This technique is suitable for larger samples as it reduces the total volume of cryoprotectant required. For a sample container volume of Vs, a cryoprotectant concentration of Ksol and a target concentration of Kt, the volume of cryoprotectant V can be calculated from this equation.

$$\text{V} = \text{Vs.ln}(\text{Ksol} \mid \text{(Ksol - kt)}) \tag{1}$$

For a sample container volume of 50 ml, depending on the actual values of Ksol and Kt this approach could require less than 100 ml of concentrated DMSO solution. Because the incoming solution has to be thoroughly mixed within the container, additional stirring equipment running at cryogenic temperatures is required. This results in a mechanically more complex arrangement than the dual solution approach.

Precision in Cryopreservation – Equipment and Control 523

The Asymptote EF600, a unique liquid nitrogen free, controlled-rate freezer, is electrically powered by a Stirling Cycle Cryocooler rather than liquid nitrogen. This allows the freezer to be used where liquid nitrogen is in short supply, where extra high air quality is needed,

The Planer Kryo 360 cell freezer controls down to a -180 °C end temperature to ensure sample integrity during transfer to storage. Fully programmable, it allows the use of protocols associated with the most advanced cryopreservation techniques and is widely

The Crysalys controlled rate freezer: programmes, time and temperatures may be entered via a touch screen with up to 100,000 cycles held on the onboard SD card; the data can be retrieved by any PC or Mac computer. A battery back up operates the system for 3 hours; its

portability and 3.2 kg weight make it especially suited to veterinary purposes.

or where there is a risk of LN2 contamination to samples.

Asymptote liquid nitrogen free controlled-rate freezer

used in laboratories around the world.

Planer Kryo 360 programmable cell freezer

Fig. 3. Single solution mode.
