Issues in Contamination and Temperature Variation in the Cryopreservation of Animal Cells and Tissues

Abstract
The cryopreservation of animal cells and tissues has traditionally been accomplished using liquid nitrogen based storage vessels and freezers. With changing technologies in the life sciences, two major problems have surfaced from this approach, the first being contamination of submersed samples, and second, the observation that vapor phase storage systems may have temperature gradients which rise above the glass transition temperature of water. To remedy these problems, auto-cascade mechanical freezers can be used for the storage of animal cells at temperatures below -130°C, i.e., the glass transition temperature of water. Data shows that the chamber temperature of the Revco cryofreezer is below -130°C as compared to liquid nitrogen vapor which has been documented to be as high as -72°C. Since storage tubes are not submersed in mechanical freezers, contamination concerns are lessened as well. The consistent storage of samples below -130°C without cross-contamination is allowing for the novel application of the cryofreezer to the preservation of cells in microwell plates.

Introduction
The non-destructive preservation of biological materials is dependent upon suspending spontaneous and enzymatic degradation while maintaining form and function. Not only is the integrity of the biological material required, but also the maintenance of spatial arrangement (e.g., protein folding) and biological activity. Basically, both the macroscopic and molecular aspects of the preserved material must be maintained. Several means are available for preservation, and these include low temperatures, the reduction of water activity, and the addition of chemical protectants. These methods can be used alone or in combination. The nature of the biological material, whether it is a protein or tissue sample, will determine the methods used for its preservation.

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.

Contamination Issues
Liquid nitrogen not only serves as a refrigerant, but like water, can also act as a vehicle for the transmission of viruses, bacteria, fungi, and animal cells. The report that infective viruses were found in liquid nitrogen and thus should be treated as a biohazard (Schafer et al., 1976) is indicative of the potential contamination problem of liquid nitrogen. The submersion of screw capped plastic tubes allows for contact between contaminated liquid nitrogen and the sample. Condensation of the atmosphere within the tube creates a vacuum which can draw in the liquid nitrogen. Any contaminants in the liquid nitrogen may contaminate the sample. For example, bone marrow and stems cells harvested from patients undergoing cytotoxic treatment became contaminated with Hepatitis B virus (HBV) as a result of storage in liquid nitrogen and caused a HBV outbreak (Tedder et al., 1995). Of the six patients afflicted, human DNA, Hepatitis B surface antigen A, and HBV DNA matching those patients were found in the liquid nitrogen. The interesting observation is that DNA from the patients, and thus presumably their cells, was found in the liquid nitrogen indicating that contaminants move both in and out of the storage containers. A follow-up study to this HBV outbreak confirmed the human and HBV sources by DNA sequence analysis (Hawkins et al., 1996).

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.

Temperature Gradients
A second problem observed in liquid nitrogen freezers is the lack of temperature homogeneity within the chamber. Liquid nitrogen systems function by filling the lower part of the storage vessel with liquid nitrogen and allowing the vapor to cool the upper chamber. With smaller laboratory dewar type vessels, the entire tank is filled and then allowed to evaporate over a period of time. During this period, the cells stored in the upper racks of the chamber start at -196°C, and as the liquid nitrogen vapor level drops, a temperature gradient develops from the liquid upward. In larger liquid nitrogen freezers, vapor phase gradients have been documented to span the glass transition temperature of water, at times reaching -72°C (White and Wharton, 1984), -70°C (Wolfinbarger, 1998), and -95°C (Rowley and Byrne, 1992). The wide temperature ranges observed with liquid nitrogen storage systems is inherent to their operation.

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.

The difficulties 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.

Liquid nitrogen is not as reliable for cryopreservation of tissue culture cells than mechanical freezers
Fig. 1. Comparison of the temperature distribution in a liquid nitrogen freezer and mechanical cryogenic freezer. The temperature of the liquid nitrogen is -196°C while the freezer is set at -135°C. Numbers represent temperatures (°C) at relative locations within the freezers. Shaded area in the liquid nitrogen freezer represents the liquid phase. Columnar boxes are to designate relative positions of temperature measurements within the chambers.

Mechanical Freezing Alternatives
Traditional laboratory freezers cannot attain temperatures sufficiently low for the long-term storage of animal cells. In the early 1980s, the engineers at Queue Systems developed an alternative technology to liquid nitrogen freezer systems. This technology, known as an auto-cascade freezer, was adopted by Revco and used to reach and sustain temperatures below -130°C for the long-term storage of animal cells. The auto-cascade system is a unique refrigeration system which employs an orbital compressor and multiple mixed refrigerants. Unlike liquid nitrogen freezers, evaporating coils (i.e., cooling elements) of cryogenic freezers surround the chamber walls and remove heat throughout the chamber. In contrast, liquid nitrogen freezers depend on the evaporation of the refrigerant to cool the chamber. In practice, a gradient must form from the liquid nitrogen to the top of the chamber. The practical difference between the systems is that mechanical refrigeration provides a generally uniform chamber environment while liquid nitrogen freezers create a temperature gradient. A comparison of a cryogenic freezer equipped with a scroll compressor and a standard liquid nitrogen freezer demonstrates the differences between the two systems for cryopreservation (White and Wharton, 1984)(Fig. 1). Either periodic or prolonged exposure of cryopreserved cells to temperatures above -130°C is detrimental to cell viability. With liquid nitrogen freezers, high temperatures in the gradient have been observed and cited as a problem on several occasions (White and Wharton, 1984; Wolfinbarger et al., 1996; Rowley and Byrne, 1992). The effects of cell warming may include cell lysis, continued enzyme activity, and cell dehydration. Consequently, for valuable and irreplaceable specimens, such temperature gradients or fluctuations should be strongly avoided. Mechanical freezers provide temperature consistency necessary for the cryopreservation of sensitive cells.

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.

Future Applications
The growth of laboratory automation has created a demand for high density storage of animal cells in microwell plate formats. Studies are underway in our laboratory to develop methods for the storage of animal cells in microwell plates at temperatures below -130°C. This format will allow for high density storage and provide a link between cell storage and laboratory automation. The ultimate goal of this effort is to reduce the variability of cryopreserved cells and their subsequent use. This application has promise in reducing the physiological variability of cells used in cell-based assays.

References
Areman E., R. Sacher, and H. Deeg. 1990. Processing and storage of human bone marrow: A survey of current practices in North America. Bone Marrow Transplant 6: 203-209.

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.

Dr. David Burden served as a consultant for Revco Scientific in the area of cryopreservation
David W. Burden,Ph.D.

President

While serving as the Chairman for Revco's Scientific Advisory Group, Dr. Burden produced several application notes on cell culture preservation and growth. The following is a link to Revco's website and this note:

Revco Note

The Cryogenic Workstation is used to handle cryogenic tissues and vials at room temperature without fear of thawing.