The Importance of Controlling the Rate of Freezing

Controlled-rate Freezing

The rate at which cell and gene therapies are frozen is important, as the freezing process affects the rate of formation and size of ice crystals, as well as the solution effects that occur during freezing. Different types of cells may require different cooling rates; however, a uniform cooling rate of 1°C per minute from ambient temperature is effective for a wide variety of cells and organisms. Generally, the larger the cells, the more critical slow cooling becomes. Most bacteria and spore-forming fungi will tolerate less-than-ideal cooling rates and can be frozen by placing the material at -80°C for a period of time. More fastidious bacteria and non-sporulating fungi require more uniform rates of cooling. Protists, mammalian cells, and plant cells often require even greater control of the cooling rate, including special manipulation to minimize the detrimental effects of undercooling and the heat liberated during the phase change from water to ice.

Despite the control applied to the cooling of cells, most of the water present will freeze at approximately -2°C to -5°C. The change in state from liquid to crystalline form results in the release of energy in the form of heat; this is known as the “latent heat of fusion.” Warming of the sample occurs until the equilibrium freezing point is reached, at which temperature ice continues to form. To minimize the detrimental effects of this phenomenon, undercooling must be minimized by artificially inducing the formation of ice. This can be accomplished by seeding the suspension with ice or some other nucleating agent, or by rapidly dropping the temperature of the external environment to encourage ice crystal formation.

To achieve uniform, controlled cooling rates, use a programmable controlled-rate freezer (CRF) such as the Thermo Scientific™ CryoMed™ CRF. Unlike the CryoMed, manual/simpler units allow only the selection of a single cooling rate for the entire temperature range. The CryoMed, however, allows a selection of variable freezing rates for different portions of the cooling curve.

The CryoMed controlled-rate freezer provides a simple-to-use system designed to achieve a rate of cooling of 1°C per minute. Typical cooling rates for homemade freezing systems lead to uncontrolled cooling that averages 1°C per minute but the cells actually experience more rapid rates of cooling during some parts of the cooling curve.¹ Homemade freezing systems are also non-repeatable and their performance cannot be validated.

Storage

The temperature at which frozen preparations are stored affects the length of time after which the material can be recovered. The lower the storage temperature, the longer the viable storage period. Ultimate stability of frozen cells cannot be assured unless the material is maintained below -130˚C.²Some bacteria and spore-forming fungi may tolerate storage temperatures of -60˚C to -80˚C for long periods of time. However, more fastidious cells, such as mammalian tissue cultures, hybridomas and stem cells must be maintained below -130˚C to assure long-term stability. It has been demonstrated that some cells survive for less than one year when stored at -80˚C.²For ultimate security and maximum stability, living cells and embryos should be stored in liquid nitrogen freezers. However, there are risks associated with immersing vials directly into liquid nitrogen, so liquid nitrogen units that provide all-vapor storage are ideal as long as the working temperature at the opening of the unit remains below -130˚C.

To assure that a liquid nitrogen freezer maintains the proper working temperature, the volume of liquid nitrogen in the unit should be adjusted to a level that results in a temperature of -150°C just above the stored material when the lid of the unit is removed³. An adequate working temperature can be attained in most liquid nitrogen freezers; however, the design of some models requires that the amount of liquid nitrogen necessary to attain the proper working temperature will reduce the amount of usable storage space. If vials are to be immersed in the liquid phase of liquid nitrogen, they must be correctly sealed intubing to prevent penetration by liquid nitrogen. Improper use may cause entrapment of liquid nitrogen inside the vial and lead to pressure build up, resulting in possible explosion or biohazard release. Liquid phase LN penetration can also be a source of contamination for submerged samples not properly protected by their tubing. In most cases, vapor phase storage at –130˚C is adequate and avoids the hazards of liquid phase storage.

Improper handling of material maintained at cryogenic temperatures can have a detrimental effect on the viability of frozen cells. Each time a frozen vial is exposed to a warmer environment, even briefly, it experiences a dramatic change in temperature. Storage systems should be designed to avoid exposure of stored material to warmer temperatures, as well as minimizing prolonged exposure of personnel during specimen retrieval. Box stacking systems (i.e. stainless steel racks) necessitate exposure of boxes at the top to warmer temperatures when retrieving boxes at lower temperatures. When box stacking systems are used, maintain a small number of vials of each preparation in the top box of the rack and store the remaining vials of each preparation in lower boxes. By doing this, a vial of one preparation can be retrieved without exposing all vials of any particular culture or lot.⁴ To maximize the available space in liquid nitrogen freezers and minimize exposure of material during retrieval, use small storage boxes or aluminum canes. Press the vials onto the canes, putting no more than one lot of one culture on each cane. Canes provide a flat surface for coding their position and easy identification during retrieval. Place the canes into cardboard or plastic sleeves to eliminate the potential for vials to fall from the canes. When retrieving vials from canes, the cane should be lifted only to a level that exposes the first available vial, without removing the remaining vials from the working temperature of the freezer.⁴

Discoveries in gene and cell therapies are happening every day. The burden of making the transition from the discovery phase to the production phase can be eased by using The Thermo Scientific CryoMed controlled-rate freezer. The CryoMed delivers consistent and repeatable performance that can be validated and is designed to support research and medical applications.

References:

Simione, F.P., P.M. Daggett, M.S. MacGrath and M.T. Alexander. 1977. The use of plastic ampoules for freeze preservation of microorganisms. Cryobiology 14: 500-502.
Simione, F.P. 1992. Key issues relating to the genetic stability and preservation of cells and cell banks. J. Parent. Science and Technology 46: 226-232.
Simione, F.P. and J.Z. Karpinsky. Points to Consider BeforeValidating a Liquid Nitrogen Freezer, In: Validation Practices for Biotechnology Products, ASTM STP 1260, J.K. Shillenn, Ed., American Society for Testing and Materials, 1996, pgs. 24-30.
Simione, F.P. Cryopreservation: Storage and Documentation Systems, In: Biotechnology: Quality Assurance and Validation, Drug Manufacturing Technology Series, Vol. 4, Interpharm Press, Buffalo Grove, Illinois, 1999, pgs. 7-31.