Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of storing cells in a viable state
Reexamination Certificate
2001-03-19
2003-03-04
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of storing cells in a viable state
C435S404000, C435S366000
Reexamination Certificate
active
06528309
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides methods and compositions for the protection and storage of cells. In particular, the present invention provides methods and compositions for the vacuum-mediated desiccation protection of cells.
BACKGROUND OF THE INVENTION
Initially devised as a method of studying the behavior of animal cells in a system that is free of systemic variations, tissue culture has been in use since the early 1900s (See e.g., Freshney, I.,
Culture of Animal Cells: A Manual of Basic Technique
, Alan R. Liss, Inc., New York [1983], at page 1). Although the first work focused on tissues maintained in vitro, cultures of cells have become more commonly used over time. The development of tissue culture has contributed significantly to the fields of virology and oncology, although it has also played an essential role in elucidating various intracellular activities (e.g., DNA transcription, protein synthesis, etc.), intracellular flux (e.g., RNA movement, translocation of hormone receptor complexes, fluctuations in metabolite pools, etc.), cellular and organismal ecology (e.g., infection, drug interactions, and population kinetics, membrane flux, etc.), and cell-cell interactions (embryonic induction, cell population kinetics, cell-cell adhesion, etc.).
Cell cultures provide means to work with cells in controlled environments. For example, the pH, temperature, osmotic pressure, O
2
tension, CO
2
tension and other variables are controllable within the cell culture environment. With advances in media formulations, the culture media used to grow cell cultures may also be defined and controlled. Among their many advantages over the use of experimental animals, cell cultures facilitate the direct observation of effects of compounds/reagents on cells, typically at a lower and defined concentration. Thus, cell cultures are more economical than animal studies, in which losses of the test compound under investigation often occur due to excretion and/or distribution to other tissues.
However, there are disadvantages associated with cell cultures. The procedures must be carried out under aseptic conditions to prevent contamination by bacteria and fungi. In addition, unlike bacteria, cells from multicellular animals do not normally exist in isolation and require a complex environment in order to sustain their existence in vitro. Furthermore, cell cultures maintained in in vitro tend to be unstable. Short-term cultures tend to be heterogenous with regard to growth rate and other characteristics, even if the cells are genetically stable. This can lead to variability between passages of the culture. Indeed, selection and phenotypic drift occur in cultures, although by about the third passage, the cell culture tends to be become more stable. However, if transformed cells are present in the culture, they will overgrow their normal counterparts.
Indeed, during the development of a cell line from a primary culture and during subsequent maintenance of the culture, phenotypic and genotypic instability is typically observed. This instability is the result of culture condition variations, selective overgrowth of some cells in the population, and genetic variation. As it is important to standardize the culture so that the cell population remains as stable as possible over time, seed stocks of the cell culture are often preserved. Cell preservation minimizes the genetic drift in cultures, as well as serving to avoid senescence and guarding against contamination, as well as providing a stock culture, should the “working” culture become contaminated, change, or otherwise unusable.
Freezing is a commonly used method to store cell cultures. In freezing, water is made unavailable to the cells, and the dehydrated cells are maintained at low temperatures. Damage may be caused to the cells during the cooling stage and/or the subsequent thawing. This damage may be caused either by the concentration of electrolytes through removal of water as ice, or by the formation of ice crystals that shear the cells. Damage may be somewhat limited by adjusting the cooling and warming rates, as well as by adding cryoprotectants (e.g., dimethyl sulfoxide [DMSO], etc.) to the cell suspension. Although various temperatures have been used to store frozen cultures (e.g., −20° C., −30° C., −40° C., −70° C., −140° C., and −196° C.), poor results are usually observed at temperatures above −30° C.
Freezing in liquid nitrogen has been widely used for many organisms and cell cultures and is currently recommended for storage of valuable seed stock cultures. There are numerous advantages to this method, as in many cases, no loss of viability occurs during storage (although some cells may die during cooling and warming). In general, there is no genetic change or loss of characters; and the longevity and stability tends to be maintained. Typically, cells are frozen in small aliquots and maintained in liquid nitrogen or at −70° C. The frozen cells are then thawed and revived for use as needed. However, there are potential problems associated with freezing of cells, as cell viability is affected by the freezing medium used, as well as the temperature of storage (e.g., significant deterioration may occur at storage temperatures as low as −70° C.), and the method of thawing and revival. In addition, improperly sealed glass ampules present an explosion risk during thawing. Additional disadvantages of freezing cultures in liquid nitrogen include the need to continually replenish the liquid nitrogen, the high cost of equipment, and the inconvenience of storing and distributing of large numbers of cultures (e.g., storage space may be problematic).
Desiccation has been widely used as a method to preserve microorganisms. A variety of methods are used, although all depend upon the removal of water from the culture and prevention of rehydration. Although drying methods have been more commonly used with molds than bacteria, some bacteria and yeasts have been successfully preserved using these methods. In the most commonly used methods, the cultures are dried in soil, sand, kieselguhr, and/or silica gel, dried onto paper or gelatin strips or discs, or formed into pre-dried plugs.
Freeze-drying involves the removal of water by sublimination from a frozen culture. Organisms are grown on a suitable growth medium, aliquots are suspended in an appropriate freeze-drying liquid in ampules or vials, and placed in the freeze-drying apparatus, where they are frozen, and exposed to a vacuum. The water vapor from the culture is typically trapped in a refrigerated condenser unit or in phosphorous pentoxide. After freeze-drying, the cultures are sealed in their vials, often under vacuum or in an inert gas, and are stored at room temperature, refrigerated, or frozen. Two methods of freeze-drying are commonly used in industry, namely centrifugal and shelf freeze-drying (See, R. H. Rudge, “Maintenance of Bacteria by Freeze-Drying,” in Maintenance of Microorganisms, 2d ed., B. E. Kirsop and A. Doyle (eds.), Academic Press, London, [1991], pp. 31-44).
Although freeze drying has been widely used to preserve various organisms, there are problems associated with this method. For example, glass ampules are generally sealed closed with a flame (e.g., a torch), requiring some care in order to avoid injury to the operator, and some ampules are very difficult to open, requiring filing in order to sufficiently weaken the glass so that the ampule can be broken. This presents risks of contamination of the culture through the introduction of contaminants through the filed area of the ampule, as well as risk of injury to the operator, should the ampule unexpectedly break. Thus, there are major safety considerations associated with the use of currently used freeze drying methods. In addition, these methods have not found use with cell cultures.
Indeed, none of the drying methods has found universal acceptance, as their efficacy appears to be culture-specific (i.e., some cells m
Ketter James
Medlen & Carroll LLP
Sullivan D
The Regents of the University of California
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