Specifying a cryobiology storage system is an extremely important decision for any life sciences laboratory. A poor choice can put irreplaceable research at risk and also add unnecessary costs.
Two primary considerations are the temperature required at the top of the freezer and if the products are required to be immersed in liquid nitrogen (LN2) or stored in the vapour phase. Also, there is remarkedly better cell viability of products stored below the glass transition temperature of -130°C.
Two primary reasons to use vapour phase storage (storing above the LN2) versus immersing in LN2 are: the risk of cross contamination is reduced; and there is no dripping LN2 when the samples are then retrieved from the cryogenic freezer systems.
Customers can select from a wide range of cryogenic refrigerators and freezers. Both are double walled vessels with super insulation in the vacuum space.
Methods of storing the product in the freezer vary with applications however, and common storage methods are:
*Vials on aluminium canes and stored in canisters
*Vials in boxes and stored in racks
*Bags in cassettes and stored in racks
*Straws used in the artificial insemination industry stored in canes or goblets.
Cryogenic refrigerators are manufactured from aluminium and can typically store as many as 6000 2ml vials.
Generally, products are immersed in LN2 and the refrigerators are filled manually from a liquid cylinder using a transfer hose and phase separator. The larger aluminium refrigerators can be fitted with an autofill controller which will automatically fill the unit and provide -190°C vapour phase storage.
There are two styles of stainless steel cryogenic freezers for larger storage requirements:
1. The ‘wide mouth’ style cryogenic freezers have vacuum and super insulation on the circumference and bottom, with capacities ranging from 10,000 to 38,000 2ml vials.
2. The ‘limited access’ freezers with smaller openings are used for -190°C temperatures in vapour phase with capacities ranging from 20,000 to 94,000 2ml vials.
These units have vacuum and insulation surrounding the product. The ‘limited access freezers’ have ergonomic rotating turntables. All the racks are retrieved from the front of the freezer. This significantly reduces the risk of injury for the operator as there is no stretching to retrieve the racks at the rear of the freezer. The temperature graph shows the capability of these ‘limited access freezers’ with very low uniform temperatures at the top of the racks in the freezer.
Even with the lid open for three hours, the temperature at the top rack under the lid opening is -169°C. Operators in the laboratory may need aluminium dewars to decant small amounts of LN2 to work with samples on the bench. In this case, the aluminium dewar can be set-up with a withdrawal device.
When the customer requires LN2, the liquid valve is opened and product is safely withdrawn through the phase separator. Larger quantities of LN2 can be supplied from a liquid cylinder, cryogenic transfer hose and phase separator.
Security is extremely important as many of the products stored are irreplaceable. Automatic filling will provide the system with low pressure LN2 at less than 1.5 bar (22 psi) regardless of the time – day or night.
When the level falls to a pre-determined level, the controller opens the solenoid and fills to a preset level.
Typical alarms are low level, high level, high temperature, low LN2 supply, and sensor errors. These alarms should always activate the remote alarm as the laboratories seldom have staff working 24/7.
Some customers may require all the data to be recorded on a PC and have two-way communication with the freezers. The controller on each unit can be connected to a PC via an ethernet port. Two-way communication between the controller and PC is important in giving customers remote capabilities to identify immediately what freezer is in alarm, what alarm is activated, and initiate an immediate course of action. Customers can even start an LN2 fill remotely themselves.
Many customers select a battery back-up for autofill freezers. In the event of a power outage the battery back-up will keep the system running for three to five days and indicate to the user that there is no mains power at the unit.
The customer’s product is not in immediate danger to power outages and they do not need expensive emergency power generators with the battery back-up controller. A back-up controller generally uses less energy than a light bulb.
If the temperature falls below a preset level, a redundant alarm can be activated.
It is good practice to have a redundant system, when considering that these are usually irreplaceable life sciences products being stored. One option is an independent 4-20ma signal.
LN2 storage & filling
Liquid cylinders, microbulk or bulk tanks are commonly used to store and supply LN2.
Smaller refrigerators are filled from liquid cylinders (160-240 litres) using transfer hoses and phase separators. The liquid cylinder is replaced when empty.
Usually an individual freezer will be connected to one liquid cylinder. If the liquid cylinder runs out over a weekend or holiday, an alarm may be activated. Quite often, liquid cylinders are changed out if it there is not enough LN2 to get through the weekend or holiday. Another option is to have a liquid cylinder switcher so that when one liquid cylinder or bank of liquid cylinders runs out of LN2 it will automatically change to another full cylinder.
For multiple freezers in one location, these are typically connected to a vacuum jacketed (VJ) line with the supply source being either a microbulk or bulk tank. There is no movement of liquid cylinders and gas companies can fill the microbulk or bulk tank 24/7.
For large installations, of up to 20-plus freezers, the VJ line may be a keep-full line and the freezers call for LN2 independently.
Another option is sequence filling. When one unit calls for LN2 a solenoid valve opens at the end of the line and vents the gas to the outside. This valve will close when LN2 is at the end of the line. The sequencer will then open the solenoid on the first freezer and it will fill, then the second unit and so on. The piping is cooled down once, conserving LN2.
Customers may need to ship the products to other locations at temperatures below the glass transition temperature of -130°C.
Cryogenic dry shippers have an adsorbent that adsorbs the LN2 and therefore the samples are kept in vapour. An additional reason to use a dry shipper is the LN2 will not spill out of the unit onto the transport’s floor if it was inadvertently turned on its side.
For the end-user, it is better to schedule regular service calls than wait until a problem occurs.
Scheduling emergency service calls during the evening, holiday or weekend can be frustrating and ultimately expensive. A cryobiology end-user’s equipment and/or gas supplier will be able to recommended regular maintenance and service schedules for their cryogenic equipment.
As with all walks of life and industrial gas practices, safety is of paramount importance.
It is the customer’s responsibility to safely handle the material stored in the refrigerators or freezer systems. These materials may or may not be infectious. Gas companies meanwhile, provide their cryogenic expertise for safely handling and distributing LN2.
One very important factor that may sometimes be overlooked is asphyxiation. If there are small areas where LN2 is being used, then the customer may need an oxygen monitor to observe the atmosphere of this environment.
One litre of liquid nitrogen will become 696 litres of nitrogen gas and may quickly fill a small room. With an oxygen monitor employed however, this will typically activate at a level of around 19.5% oxygen.
There may be both a flashing light and horn in the room and a flashing light outside the room, to warn a customer before they enter this room.
LN2 refrigerators and freezers provide the best economic solution to achieve the preservation of a customer’s research at temperatures below -130°C, whether in liquid or vapour phase.