The application of heat conduction microcalorimetry to study the metabolism and pharmaceutical modulation of cultured mammalian cells

Abstract

The heat produced by animal cells in culture can be used as the primary indicator of the kinetics of their metabolism because the scalar flux of it is a function of the metabolic flux. The validity of the relationship between heat and metabolism was demonstrated theoretically through the concept of thermal advancement and in experiments by the use of continuous cultures. This validation permitted the application of heat flux as a probe of the metabolic state of cells in culture. It consisted of an on-line heat conduction microcalorimeter that measures the instantaneous heat flow and dividing the smoothed signal with one obtained simultaneously using a dielectric spectrometer that records the change in capacitance as an estimate of the amount of viable biomass. In this mini-review, it is shown with Chinese hamster ovary cells (CHO320) genetically engineered to produce interferon-γ (IFN-γ) that heat flux is an early signal of deteriorating metabolism in cultures that produce considerable amounts of toxic lactate under fully aerobic conditions. The early detection favours the use of heat flux as the control variable in fed-batch cultures. This is a particularly useful finding in the context of the pharmaceutical industry because it will help to ensure the high fidelity of the cytokines, antibodies and vaccines produced in large-scale cultures. The monotonic relationship between the fluxes for heat and metabolism means that the enthalpy balance method can be employed to test the validity of the growth reaction for cells in culture. This showed that the crucial ratio between the substrates, glucose and glutamine, in the culture medium was incorrect at 5.5:1 instead of about 3:1, depending on the phase of the culture. Together with other changes to the medium composition, an improved formulation was made that ensured faster cell growth and greater specific rate (flux) of IFN-γ constitutive secretion while decreasing glucose utilisation and, most importantly, halving the excretion of lactate, that is toxic to the cells and harmful to the fidelity of their secondary products. Indirect calorimetry (oxygen uptake rate, OUR) is often favoured over the direct technique, but the former only measures aerobic metabolism. The environmental conditions in cultures favours lactate production even under fully aerobic conditions. Developments in measuring OUR mean that the stationary liquid phase balance can be used successfully to make the calorimetric:respirometric (CR) ratio a valuable tool in optimising cell culture to grow cells that synthesise the maximum amounts of the high fidelity secondary products. Besides the value of heat flux in improving the cultures of animal cells producing heterologous products, three different techniques are examined that should be valuable in the testing the many compounds that are produced on a speculative basis as potential drugs. They are: (i) a thin-film thermopile transducer as an immunosensor; (ii) infra-red imaging of cells cultured in multi-well microtitre plates and (iii) integrated circuit (IC) calorimetry for small samples and low detection limit. One or more of these methods could well find favour with industry in the near future.