|Grusak, Michael - Mike|
|Erdman, jr., John|
Submitted to: In Vitro Cellular and Developmental Biology - Plants
Publication Type: Peer reviewed journal
Publication Acceptance Date: 8/11/2003
Publication Date: 1/1/2004
Citation: Grusak, M.A., Rogers, R.B., Yousef, G.G., Erdman, J.W., Lila, M.A. 2004. An enclosed-chamber labeling system for the safe 14C-enrichment of phytochemicals in plant cell suspension cultures. In Vitro Cellular and Developmental Biology - Plants. 40(1):80-85. Interpretive Summary: Fruit and vegetables contain several chemical compounds that have been linked to the promotion of good health and the prevention of various diseases in humans. Flavonoids are one such group of these compounds. They are found in foods such as berries, grapes, and teas and are considered beneficial because they have antioxidant properties. Although we know which foods contain flavonoids, we unfortunately know little about how flavonoids are absorbed in the human gut, or where they go once they've entered the blood stream. We have a poor understanding in this area because we lack good tools to follow or track flavonoids from our food supply. One way to get around this problem is to label or tag flavonoid molecules with radioactive carbon atoms and track them in animal feeding trials. To make these radioactive compounds safely and efficiently, we have developed and tested a plastic and glass enclosure that is used to grow liquid cultures of isolated plant cells, while these are fed radioactive sugars. The enclosure and connected absorbent traps allow us to capture radioactive carbon dioxide that is released from the growing cells; these cells also use the radioactive sugars to manufacture labeled flavonoids. These molecules can be extracted from the cell mass after a short period of growth, and once purified can be used in absorption studies with animals. The enclosure and accessory equipment described in this paper will provide an optimal environment for the growth and flavonoid production of many cell types. Its use also will help to ensure a safe workplace for laboratory personnel.
Technical Abstract: Various plant secondary products have been implicated in the promotion of good health or the prevention of disease in humans, but little is known about the way they are absorbed in the gut, or in which tissues they are deposited throughout the body. While these issues could be studied if the phytochemicals were isotopically labeled, generating labeled molecules often is problematic because many compounds of interest can be synthesized only in planta at present. In order to generate 14C-labeled phytochemicals of high radioactive enrichment, we developed an enclosed-chamber labeling system in which cell suspension cultures can be safely and efficiently grown when supplied 14C-enriched precursors. The system is designed to hold culture flasks within a clear, polyacrylic compartment that is affixed to the top of a rotary shaker. The flow-through gas exchange nature of the system allows for oxygen replenishment and complete capture of respired 14CO2 throughout the entire period of cell culture. Air is circulated internally with the aid of a small fan, and chamber air temperature is monitored continuously with an internal temperature probe and data logger. Production runs of 12 to 14 d with Vaccinium pahalae (ohelo berry) and Vitis vinifera (grape) suspension cultures, using 14C-sucrose as the carbon source, demonstrated a 20 to 23% efficiency of 14C incorporation into the flavonoid-rich fractions. Further studies with ohelo cell cultures showed that flavonoids were produced with either sucrose or glucose as the carbohydrate source, although flavonoid productivity (measured as anthocyanins) was higher with sucrose. This comprehensive chamber system should have broad applicability with numerous cell types and can be used to generate a wide array of labeled phytochemicals.