Suspension cultures of Eschscholtzia californica accumulate the dihydro forms of the benzo- phenanthridine alkaloids sanguinarine, chelirubine, macarpine and chelerythrine, all of which are known to be constituents of the Eschscholtzia plant. Under most experimental conditions dihydrochelirubine was found to be the main constituent of the cultured cells. The specific yields of alkaloids varied from zero to 1.7% on a dry weight basis depending on the media conditions. The highest specific yield was 1.5 mg/g dry weight or 13 mg/1 with the growth medium B5. After transfer of the cells into the induction medium IM2 the alkaloid accumulation increased to 17 mg/g dry weight and 146 mg/1. The induction medium contained increased levels of sucrose, decreased levels of phosphate and was devoid of phytohormones. The effect of the various media conditions on the biosynthesis of phenolics was quite different to those found for the alkaloids.
Dense cell cultivation of the recombinant cell line BHK 21 pSVIL2 was performed in a fluidized bed bioreactor system containing porous borosilicate glass carriers. Experiments were carried out with different medium formulations for a period of 48 days. Due to an effective immobilization of the cells in the reactor, continuous operation was easy to perform. Maximal cell densities and product yields could be maintained, even when protein-free medium was perfused exceeding 2 reactor volumes per day. Final cell densities of magnitude 7.1 x 10(7) mL-1 intrasphere volume were reached, while the interleukin-2 production rate was 0.70 mg day-1. The cell specific productivity reached a value of 1.3 x 10(-10) mg day-1. The first results were presented with a cell line that grows under glutamine-free medium conditions. The use of a glutamine-free medium for the cultivation of the cells resulted in a drastic decrease in cell metabolism. Furthermore, the amino acids lysine and histidine were produced and secreted into the culture supernatant, although these metabolites normally are considered to be essential for animal cells grown in vitro. However, no lethal effect on the cells has been detected, and the total number of cells in the reactor remained constant. The metabolism of threonine has been detected to be directly dependent on the presence of glutamine. Cells grown in glutamine-free culture medium produced glycine yields 6 times higher than those grown in glutamine-containing medium. A bead-to-bead transfer of the cells has also been detected when the cells immobilized within the intrasphere volume of the borosilicate carriers reached the stationary phase.
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