R. Robert Balcarcel

Metabolic Screening of Mammalian Cell Cultures Using Well‐Plates

For use in a broad spectrum of cell culture applications, we have devised a novel method, termed High‐Throughput Metabolic Screening (HTMS), with which to more rapidly screen the overall activity of major metabolic pathways of mammalian cells. This current protocol uses adaptations of theoretical and experimental techniques from metabolic and cell culture engineering. First, HTMS makes use of a simplified metabolic network for metabolic flux analysis. Despite its simplicity, the network is capable of generating flux distributions and ATP production rates that are comparable to a more detailed network. Second, HTMS makes use of microtiter well‐plate technology and adaptations of well‐known enzymatic assays to increase precision and throughput for cell culture experiments. Multireplicate, multiparallel cultures in the sub‐milliliter scale yield very precise metabolic rates using common laboratory equipment and at a fraction of the cost and time of traditional experiments with T‐flasks, spinner flasks, or bioreactor systems. The simplicity of the network and the well‐plate assays synergistically comprise a new, extremely useful, broadly applicable, and relatively inexpensive way to probe cell cultures for metabolic effects, screen drugs and toxins, optimize media, and support the development of bioprocesses. The simplified network and cell culture and analytical assays are also useful for undergraduate, graduate, and professional training.

Determination of carbon dioxide production rates for mammalian cells in 24-well plates

In this report, we describe a method for the quantitative determination of carbon dioxide production rates of mammalian cells. Custom-made, reusable, optically clear plugs are used to seal the wells of a 24-well plate. These plugs prevent the loss of CO2 produced by the mammalian cells cultured in bicarbonate-free medium. Measurements of pH, total liquid phase CO2, and viable cell density are used to estimate the average CO2 production rate during a 6-h incubation period. Using this method, four chemicals well-characterized in regards to toxicity, 2,4-dinitrophenol, antimycin A, rotenone, and cyanide, were found to elicit significant changes in CO2 production for given concentrations within 6 h, without inducing a decline in culture viability. Over longer exposure times, similar concentrations caused growth inhibition but not cell death. An assay based on metabolic change corresponding to growth inhibition that is more sensitive than traditional measures of cell death is a feasible complement to existing methods in drug discovery and toxicity testing.