Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Insect cell – per se
Reexamination Certificate
2001-07-18
2003-07-29
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Insect cell, per se
C435S325000, C435S368000, C435S383000, C435S384000, C435S391000, C435S392000, C435S404000, C435S405000, C435S406000, C435S407000, C435S408000
Reexamination Certificate
active
06599740
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the culture of tissue, including cells and organs, and more specifically to the culture of mammalian tissue using at least one component of plasma derived from fish. The method has significant advantages over the more commonly used technique of utilizing serum or plasma components derived from humans or cows, or the more recently developed technique of utilizing whole serum or plasma from fish.
BACKGROUND OF THE INVENTION
Tissue culture, the production of living tissue in vitro, permits numerous applications that would be difficult or impossible in a living organism. These applications include in vitro applications such as diagnosing disease and assessing toxicity, and more recently, the production of therapeutics, including vaccines and recombinant proteins, and growing human tissue, including cells and organs, for therapeutic applications (tissue engineering).
The culture of animal tissue usually requires animal biologics: either whole serum, most commonly fetal calf serum (FBS), or plasma components, for “serum-free” media or biological gels. Current methods for deriving mammalian serum or plasma components are well-known. The raw material is human or bovine blood from which the cellular portion is removed by centrifugation. If an anticoagulant is used, the liquid portion is plasma; if the blood is allowed to clot, the liquid portion is serum. The most widely used method of fractionating human or bovine plasma is the Cohn process (Cohn et al., 1946), which utilizes adjustments of temperature, pH, and ethanol to separate plasma proteins.
However, the risk of mammalian infectious organisms in mammalian plasma or serum products used in tissue culture for therapeutics is an increasing concern. Some plasma proteins can be manufactured by recombinant technology, others, especially the glycoproteins, must be obtained from humans or animals. Although various viral-inactivation treatments for plasma or serum components are frequently used, problems remain in achieving 100% inactivation without compromising quality. An even more serious concern is the emergence of transmissible spongiform encephalopathies (TSEs) such as “mad cow disease”, and the possibility of prions or infectious proteins in plasma or serum derivatives. The later problem is especially difficult, since at present, it is not possible to predict which individual blood donors, human or bovine, may years later develop a prion disease.
In order to improve the safety profile of animal products used in mammalian cell culture, Sawyer et al. (U.S. Pat. Nos. 5,426,045 and 5,443,984) developed a method using fish whole serum to replace FBS or other animal serum. This fish serum provided the important advantage of a low probability of mammalian infectious agents, and successfully replaced FBS by promoting growth in a few cell lines. However, it was toxic to many mammalian cells, and ineffective for others.
Sawyer et al., in the '045 patent, identified (among several factors) the high lipid content of fish serum as “potentially inhibiting” to mammalian cell growth. Therefore, we attempted to overcome the toxicity problem by removing some of the lipid.
Using known methods (Condie, 1979: Ando, 1996), we separated lipids and lipoproteins from the plasma of Atlantic salmon (Salmo salar). The delipidated plasma was used to replace FBS on several mammalian cell lines. In each case, the material proved toxic to the mammalian cells.
This toxicity pointed to a similar problem with the removed lipid. Furthermore, cell culture teaches a like-to-like match or species-specificity of biological materials used and cells being cultured (Hewlett, 1991). Since fish lipids are significantly different from mammalian lipids (Babin and Vernier, 1989), it seemed unlikely that the fish lipid would promote mammalian cell growth. Nonetheless, we tried the salmon lipid as a media supplement for a mammalian cell line (Vero). The unexpected result was enhanced growth of the mammalian cells.
Because of the success of the lipid component, we attempted to overcome whole plasma toxicity by separating (purifying) other components from the whole plasma, in particular, plasma proteins, which might be useful in mammalian tissue culture. This approach presented the problem of dissimilar structure between fish and mammalian plasma proteins, and therefore a low probability that a given protein would function in a similar manner to its mammalian homologue. Doolittle (1987) studied fish plasma proteins from the perspective of comparative physiology and evolution, and found only partial identity in amino acid sequence to their mammalian homologues. For example, lamprey fibrinogen is less than 50% homologous to human fibrinogen, and salmon transferrin has only a 40-44% amino acid sequence identity with human transferrin (Denovan-Wright, 1996). This and similar data on percent homology for other plasma proteins such as fish albumin (28% homology) would dissuade those skilled in mammalian cell culture from attempting to use the fish homologue.
We encountered additional difficulties since the usual method of fractionating mammalian plasma protein (Cohn et al., 1946) could not be used with salmon plasma. The Cohn process is the most widely used method of separating, or fractionating, serum or plasma into its components. Although this process has been improved and modified considerably, it achieves basic separation and precipitation of plasma fractions by cold temperature, and adjustments in pH and ethanol concentration. Since salmon blood is often at a temperature of 4° C. or less when it is drawn from the fish during winter, temperature separation of proteins was not a consistent or reliable method.
Sawyer et al., (U.S. Pat. No. 6,007,811), extracted two proteins, fibrinogen and thrombin, from salmon plasma for use as a sealant for hemostasis. However, immunoblots and SDS-PAGE showed a different primary structure for human (lane 1.) vs. salmon (lane 2.) fibrinogen (FIG.
1
). Furthermore, this application is unrelated to cell culture, and provided no indication that these proteins would be less cytotoxic than the salmon whole plasma.
Fibrinogen and thrombin form a fibrin gel, and an optimal environment for certain mammalian cells, especially neurons, is a three-dimensional matrix, usually a gel made from mammalian proteins. We used methods known for mammalian plasma to purify fibrinogen and thrombin from salmon plasma. We chose mouse spinal cord neurons as test cells for the fish fibrin gel, since they are a model for human neuron regeneration, and very sensitive to toxicity.
When the survival and process outgrowth of these neurons was compared in human, bovine, and salmon fibrin gels, the unexpected result was the superior performance of the neurons in the fish material. Since mammalian fibrin gels are already being used to grow neurons for therapeutic purposes, the improved neuron process outgrowth and safety profile of the fish gels would make them an attractive alternative. Additional advantages of the salmon gel were its ease of preparation (lyophilized salmon fibrinogen can be resolublized at room temperature instead of at 37° C.), and resistance to changes in pH and osmolality (FIG.
2
).
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a tissue culture process that overcomes the cytotoxicity problem of whole fish serum or plasma for mammalian tissue culture.
It is a further object of the present invention to provide a method of mammalian tissue culture that does not use mammalian blood components in the culture medium.
It is an additional object of the present invention to provide a method of mammalian tissue culture that uses fish plasma components in the cell culture medium.
The present invention overcomes the cytotoxicity of fish whole serum or plasma, provides material with unique, advantageous properties for cell culture, and retains the important safety profile of fish biologics over the more commonly used serum or plasma components derived from humans
Flanagan Lisa A.
Janmey Paul A.
Sawyer Evelyn A.
Guzo David
IP Strategies P.C.
Sea Run Holdings, Inc.
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