Method of using fish plasma components for tissue culture

Abstract

A tissue culture substrate for culturing mammalian stem cell tissue includes one or more specific fish plasma fractions, isolated from any plasma remainder, in a nutrient medium. The plasma fractions are extracted from plasma obtained from the blood of one or more fish that are progeny of domesticated broodstock reared under consistent and reproducible conditions.

Description

CROSS-REFERENCE TO RELATED APPLICATION [0001] This is a continuation-in-part of co-pending U.S. patent application Ser. No. 09 / 907,443, filed on Jul. 18, 2001.FIELD OF THE INVENTION [0002] 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 [0003] 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 recombinan...

AI Technical Summary

Benefits of technology

"The present invention provides a way to use fish plasma components in tissue culture without causing cytotoxicity. By using a fish that is a progeny of domesticated broodstock and has been reared under consistent and reproducible conditions, the invention provides a safer alternative to commonly used serum or plasma components derived from humans or cows. The fish plasma components that are used include fibrinogen, thrombin, lipids, transferrin, albumin, plasma proteins, and enzymes. The process involves obtaining blood from the fish, separating the plasma, and extracting specific components of the plasma. The extracted components can then be used to culture mammalian stem cell tissue, such as human stem cell tissue, without causing cytotoxicity. The process includes rendering the fish to a level of loss of reflex activity, drawing blood from a caudal blood vessel, centrifuging the blood to separate the plasma, and adding an antioxidant to the plasma. The invention provides a unique and advantageous material for cell culture that retains the safety profile of fish biologics."

Problems solved by technology

However, the risk of the presence of mammalian infectious organisms in mammalian plasma or serum products used in tissue culture for therapeutics is an increasing concern.

Although various viral-inactivation treatments for plasma or serum components are frequently used, problems remain in achieving 100% inactivation without compromising quality.

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.

However, it was toxic to many mammalian cells, and ineffective for others.

In each case, the material proved toxic to the mammalian cells.

This toxicity pointed to a similar problem with the removed lipid.

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.

We encountered additional difficulties since the usual method of fractionating mammalian plasma protein (Cohn et al., 1946) could not be used with salmon plasma.

Since the temperature of salmon blood is often 4° C. or less when it is drawn from the fish during winter, temperature separation of proteins was not a consistent or reliable method.

1). Furthermore, this application is unrelated to cell culture, and provided no indication that these proteins would be less cytotoxic than the salmon whole pla

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