Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Culture medium – per se
Reexamination Certificate
2000-10-27
2003-05-13
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Culture medium, per se
C435S001100, C435S002000, C435S374000, C435S391000, C424S537000, C424S559000
Reexamination Certificate
active
06562621
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the culture and preservation of cells, and more specifically to the culture and preservation of mammalian cells using the ovarian fluid produced by spawning fish. The method has significant advantages over conventional methods, which use whole serum or plasma components derived from mammals or fish.
BACKGROUND OF THE INVENTION
The culture of animal cells requires a defined medium containing a specific quantity of certain chemicals, and for the culture of most mammalian cells, an undefined nutrient supplement (medium) is also required. From the earliest attempts at cell culture to the present, this nutrient medium has been derived exclusively from animal or human blood. Fetal bovine serum (FBS) remains the most common nutrient medium used for the culture of animal cells. However, in order to eliminate certain risks, and to better quantify media components, a recent trend is to use so-called “serum-free” media. These formulations are not truly serum-free, but rather contain specific quantities of certain serum or plasma components, such as albumin or lipids, in place of whole serum.
This reliance on the use of blood components as nutrients in mammalian cell culture has led to an increased concern over the presence of possible infectious agents in the cells. For example, problems remain in achieving 100% viral inactivation in blood products without compromising quality. An equally serious concern is the emergence of transmissable spongiform encephalopathies (TSEs) in mammalian blood and plasma components. The latter problem is especially difficult to overcome, since at present it is not possible to predict which donors, animal or human, will later develop a prion disease. Although some plasma proteins can be produced by recombinant technology, others, such as many glycoproteins, cannot. In addition, recombinant proteins are generally very expensive.
In order to reduce the risks from mammalian infectious agents, a whole serum product has been developed from fish, particularly salmon and trout, for mammalian and insect cell culture. Although this material is a satisfactory substitute for FBS in a few cell lines, its usefulness is limited by high levels of non-protein nitrogen (NPN), by large amounts of lipids that are easily oxidized, and especially, by proteases that are active at 4° C. The latter problem contraindicates the use of salmonid serum as a medium for preservation and storage of cells at low temperature.
In addition to fish whole serum for mammalian cell culture, fish plasma components, specifically antifreeze glycoproteins (AFGPs) from polar fishes, have been used successfully in low temperature preservation of cells such as human platelets. Although these AFGPs appear promising, their availability is severely limited by the relatively small numbers and size of polar fishes, their natural source, and the difficulty of producing the recombinant glycoprotein.
There is therefore a great need for a nutrient medium that overcomes or minimizes the foregoing problems, and which is also practical for widespread use.
SUMMARY OF THE PRESENT INVENTION
While the field of cell culture has recently come to include blood products from fish, the ovarian fluid of fish has been of interest only to those in fishery science. The ovary of salmonid fishes lacks an ovarian membrane, and ovulated eggs are free in the body cavity surrounded by the slightly viscous fluid. At spawning, the eggs and ovarian fluid are expelled through the genital pore. Ovarian fluid has been studied for its role in salmonid reproduction and for its chemical composition, its novel proteins, and its utility in testing for the presence of fish disease.
According to the present invention, this ovarian fluid produced by spawning female salmon or trout is used to replace whole serum or plasma components as a nutrient medium in mammalian cell culture. The use of this substance as a nutrient medium in the place of whole serum or plasma components promotes the growth and proliferation of the mammalian cells (for example, NIH 3T3 and M2 cells). Use of fish ovarian fluid eliminates the risks and uncertainties inherent in the use of mammalian blood products, as well as the limiting factors of NPN, lipids, and proteases associated with fish serum or plasma when used as nutrient media.
Further, according to the present invention, salmonid ovarian fluid is used in low-temperature preservation of mammalian cells, especially human platelets. With present technology, these cells must be stored at or above 22° C., as lower temperatures produce “activation”, that is, a process marked by aggregation and change in shape, and resulting in irreversible cellular damage. Storage at 4° C. could dramatically increase the useful life of the mammalian cells, now limited to less than a week at 22° C.
Several factors have hindered, or have led those of skill in the art away from, the previous use of fish ovarian fluid for mammalian cell culture and preservation. For example, the field of cell culture and storage, including platelet preservation, is distinctly different from the field of fishery science; those skilled in working with mammalian cells would be unlikely to know of the existence of ovarian fluid and therefore could not speculate on its utility. Certainly, there has been no suggestion to motivate those working with mammalian cells to expend the time, effort, and expense necessary to experiment with fish ovarian fluid to culture or preserve mammalian cells.
Further, utilization of blood products as the nutrient supplements in mammalian cell culture is firmly established. Use of a nutrient fluid from a source other than blood would appear unlikely to be advantageous to one skilled in the art of animal cell culture, particularly absent of any suggestion by those in the field of an advantage.
In addition, the presence of ovarian fluid in wild fish is relatively brief and seasonal, unlike that of blood. Also, spawning time in wild fish under natural conditions varies among stocks and individual fish. These and other factors make it logistically difficult to obtain practically usable quantities of ovarian fluid from wild fish.
Salmonid ovarian fluid contains very small amounts of the components generally considered essential for cell growth, and therefore would appear to be a poor choice for a nutrient medium. For example, the standard nutrient medium FSB contains about 4 g/dL protein and 40 mg/dL cholesterol. Similarly, salmonid serum contains about 4 g/dL protein and more than 200 mg/dL cholesterol. A representative sample of pooled ovarian fluid has been analyzed and was found to contain only 0.2 g/dL total protein and 6 mg/dL cholesterol. It also has been found that ovarian fluid from chum salmon contained only about 10% of the protein and cholesterol found in the plasma. Known and hypothesized functions of ovarian fluid include protection of the eggs from physical or osmotic damage, and maintenance of sperm motility. These functions do not suggest a role for ovarian fluid in mammalian cell culture or preservation.
The use of blood products from the coldwater fishes for mammalian cell culture or storage offers safety from mammalian infectious agents. However, and despite the indications otherwise, it has been found in the extensive research leading to the development of the present invention that the use of ovarian fluid from these fish provides significant advantages over the use of fish blood products.
For example, analyses of salmonid ovarian fluid show very low levels of NPN, lipids and protease activity, factors that have proven inhibitory in the use of fish serum for culture or preservation of mammalian cells.
Further, Salmonid ovarian fluid contains protease inhibitors. As a result of the research, it has been determined that trout ovarian fluid contains a thrombin inhibitor, and anti-proteolytic activity has been observed in fish ovarian fluid.
In addition, ovarian fluid can be obtained from farmed salmonids in at least twice the quantity of serum. Only about 50
Janmey Paul A.
Sawyer Evelyn S.
Sawyer Philip J.
IP Strategies PC
Lankford , Jr. Leon B.
Sea Run Holdings, Inc.
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