Issues in Contamination and Temperature Variation in the Cryopreservation of Animal Cells and Tissues
291 Route 22 East
Lebanon, NJ 08833
TEL: (908) 253-3444
FAX: (908) 575-1660
David W. Burden, Ph.D.
In the case of many microorganisms and biochemicals, preservation is often accomplished by removing water to halt biological activity and degradative reactions. This can be done by lyophilization, i.e., the process of freeze drying. Though widely applied, lyophilization does not work with more complex systems such as cultured mammalian cells. In this case, reducing water activity is accomplished by freezing, which also has the added advantage of reducing the rate of reactions, both spontaneous and enzymatic. Cells and tissues from higher eukaryotes, such as sperm, cultured cells, embryos, cord blood, and tissue products, are typically preserved in liquid nitrogen or mechanical freezers designed specifically for cryogenic storage.
Although scientists generally categorize refrigeration as 4°C, -20°C, and -80°C, for prolonged storage cryogenic temperatures are required, i.e., temperatures below the glass transition temperature of water. This is the temperature at which all biological activity ceases, and is generally accepted as -130°C (Committee on Germ Plasm Resources, 1978). It is well known that "freezing" biological samples in itself is not adequate for preservation since profound changes can occur in frozen samples (e.g., freezer burn). Biological and chemical activity can persist as long as water activity exists, however below -130°C all activity ceases. These cryogenic temperatures can be achieved and maintained by both liquid nitrogen and mechanical refrigeration. A 1990 survey of bone marrow storage programs found that half used liquid nitrogen while the other half used liquid nitrogen vapor or mechanical refrigeration (Areman et al., 1990).
For decades liquid nitrogen storage vessels and freezers have been used for the preservation of tissue culture. Storage originally involved sealing actively growing cells in glass ampoules, controlling the rate of cell freezing down to approximately -80°C, and then immersing the ampoules in the liquid nitrogen. The only significant change to this method was the introduction of plastic cryogenic tubes which permitted freezing with less effort. This aside, methods have remained basically unchanged and accepted. However, within the last decade, the application of animal cell culture to the production of pharmaceuticals and its use as therapeutic agents has demanded a closer examination of the storage methods.
The methods employed for the successful long-term storage of cells and tissues of higher eukaryotes not only require temperatures below -130°C, but must also prevent any adulteration of the sample. Like any process, the lack of adequate controls in cryopreservation has been demonstrated as being detrimental. In particular, several investigators have reported difficulties associated with liquid nitrogen freezer systems, specifically the potential for sample contamination and the formation of temperature gradients resulting in storage temperatures above -130°C.
Foutain et al. (1997) conducted a survey of fungal and bacterial contamination of liquid nitrogen freezers used to store hematopoietic stem cells. Of the 583 cultures tested, 1.2% were found to be contaminated by microorganisms. However, four of five freezers examined contained low level microbial contamination, while the fifth freezer was heavily contaminated with Aspergillus. The microbial contamination found in the freezers was similar to the microbes found in the contaminated cultures. Though not citing the liquid nitrogen as the microbial source, other reports demonstrate the common occurrence of microbial contamination of cryopreserved stem cells (Prince et al., 1995; Lazarus et al., 1991; Stroncek et al., 1991; Webb et al., 1996). In our laboratory, a small liquid nitrogen storage tank was found to be predominantly contaminated by Bacillus.
Though the potential contamination of stored tissue culture cells is a threat to their integrity, prolonged storage at temperatures above the glass transition temperature of water will ensure the loss of viability. Below -130°C, even the most temperature sensitive cells are estimated to survive for hundreds of years. However, above this temperature the longevity of cells is reduced to months.
of storing samples in liquid nitrogen is further created by
the paradox that 1) storing cells in vapor poses risking loss
in viability, and 2) storing tubes submersed in liquid
nitrogen increases the risk of sample contamination. An
alternative is mechanical refrigeration which can be used to
store samples below the glass transition temperature without
the same fear of contamination and temperature instability.
Until the development of the mixed refrigerant auto-cascade
freezer in the 1980s, laboratory freezers were incapable of
reaching and maintaining temperatures below the glass
transition temperature of water. These units are now available
and applied to cryogenic storage.
The contamination problem observed with the submersion of storage tubes in liquid nitrogen can also be remedied with cryogenic freezers. The seepage of liquid nitrogen into submersed tubes occurs due to the formation of a vacuum caused by the condensation of the gaseous nitrogen. The pressure in such a tube will drop from 1 atmosphere to below 0.01 atmospheres. Consequently, any poorly sealed tubes will draw the liquid (and contaminants) inward. With a cryogenic freezer, the drop in pressure is much less significant, from 1 atmosphere to 0.48 atmospheres, resulting in a weaker vacuum. Though data has not been collected on the contamination of the freezer atmosphere, generally the density of environmental airborne contaminants is lower than those in the liquid phase. Hence, contamination of vials stored in a freezer should be significantly lower than comparable tubes submersed in liquid nitrogen.
Committee on Germ Plasm Resources. 1978. Conservation of germplasma resources: An Imperative 7: 79-84.
Fountain D., M. Ralston, N. Higgins, J. Gorlin, L. Uhl, C. Wheeler, J. Antin, W. Churchill, and R. Benjamin. 1997. Liquid nitrogen freezers: A potential source of microbial contamination of hematopoietic stem cell components. Transfusion 37: 585-591.
Hawkins A., M. Zuckerman, M. Briggs, R. Gilson, A. Goldstone, N. Brink, and R. Tedder. 1996. Hepatitis B nucleotide sequence analysis: Linking an outbreak of acute Hepatitis B to contamination of a cryopreservation tank. J. Virol Methods 60: 81-88.
Lazarus H., M. Magalhaes-Silverman, R. Fox, R. Creger, and M. Jacobs. 1991. Contamination during in vitro processing of bone marrow for transplantation: Clinical Significance. Bone Marrow Transplant 7: 241-246.
Prince H., S. Page, A. Keating, R. Saragosa, N. Yukovic, K. Imrie, M. Crump, and A. Stewart. 1995. Microbial contamination of harvested bone marrow and peripheral blood. Bone Marrow Transplant 15: 87-91.
Rowley S. and D. Byrne. 1992. Low-temperature storage of bone marrow in nitrogen vapor-phase refrigerators: Decreased temperature gradients with an aluminum racking system. Transfusion 32: 750-754.
Schafer T., J. Everett, G. Silver, and P. Came. 1976. Biohazard: Virus-contaminated liquid nitrogen (letter). Science 191: 24-26.
Stroncek D., S. Fautsch, L. Lasky, D. Hurd, N. Ramsay, and J. McCullough. 1991. Adverse reactions in patients transfused with cryopreserved marrow. Transfusion 31: 521-526.
Tedder R., M. Zuckerman, A. Goldstone, A. Hawkins, A. Fielding, E. Briggs, D. Irwin, S. Blair, A. Gorman, K. Patterson, D. Linch, J. Heptonstall, and N. Brink. 1995. Hepatitis B transmission from contaminated cryopreservation tank. Lancet 346: 137-140.
Webb I., F. Coral, J. Anderson, A. Elias, R. Finberg, L. Nadler, J. Ritz, and K. Anderson. 1996. Sources and sequelae of bacterial contamination of hematopoietic stem cell components: Implications for the safety of hematotherapy and graft engineering. Transfusion 36: 782-788.
White W and K. Wharton. 1984. Development of a cryogenic preservation system. American Laboratory Oct. 65-76.
Wolfinbarger, L., V. Sutherland, L. Braendle, and G. Sutherland. 1996. Engineering aspects of cryobiology, in Advances in Cryogenic Engineering, 41: 1-12.
Wolfinbarger L. 1998. The basics of laboratory-scale mammalian cell cryopreservation. BioPharm October 1998: 35-39.
While serving as the Chairman for Revco's Scientific Advisory Group, Dr. Burden produced several application notes on cell culture preservation and growth.
The CryoCooler is used to handle cryogenic tissues and vials at room temperature without fear of thawing.