Magnus Jansson Ph.D. CSO SymCel Sverige
Isothermal microcalorimetry assays can help speed up identification and quantification.
The cell-based assay arena is entering center stage due to the major and growing medical problems facing the world—both financial (i.e., drug development) and curative (i.e., antibiotic resistance).
In its modern 3D holistic format (a total metabolic response) the mature technique of calorimetry can answer some of these problems and serve as a time- and cost-efficient complement to existing assays.
By monitoring the heat flow in the cell over time (J/s or W), significant biological information can be obtained. Calorimetry-based assays are true measurements of the cellular phenotypic response. Changes in external stimuli, nutrient status, or the environment are reflected in the metabolic status of the cell, directly monitored by the calorimetric assay.
This article focuses particularly on isothermal microcalorimetry (IMC) where the samples are kept at a constant temperature. IMC measures the metabolic status and response of a cellular system, thereby giving the potential for rapid identification and quantification. This makes IMC a phenotypic assay where little information is required about the system to be studied, neither of pathways nor of receptors involved in a process. Instead, novel systems can be studied directly, leading to faster results in cell research and drug discovery.
IMC measures continuously and the power-over-time curve is a specific footprint for the cellular events involved. The power curves are different for necrosis and apoptosis, and the effects of different compounds can thus be classified based on the kinetic profile of a specific compound, thereby opening the door for rapid identification and quantification.
Possibility of 3D
The prime area of IMC cell-based assays is genotypic research complemented with phenotypic results. This current research direction in drug discovery is moving away from target-based drug development, which has shown limited benefit and output in the discovery of new chemical entities. IMC links primary high-throughput screening (HTS) to in vivo results and allows the study of a range of disease models, creating cost-efficiency and better predictability in drug development.
The strong predictive power of cell-based assays links target-binding potential to the true phenotypic response of the human organism. Calorimetry is unique in that it is completely independent of cell morphology and media composition.
Calorimetry assays can be run using 2D cell growth and of 3D cell models on matrices or synthetic tissue models, as well as tissue samples directly from donors. IMC is a continuous and nondestructive technology that increases the value of the assays, since it is not necessary to know the timeframe for the cellular event. IMC also makes it possible to apply whole-body calorimetry—essential especially in metabolic research, where energy expenditure is key.
The demand for open-platform solutions in the assay world is rapidly increasing. Exchanging current, highly specialized, locked-in equipment for a new technology–more than one assay type–is a better and more cost-effective use of scarce research resources. IMC, which is an open platform with the sole limitation being the creativity of the scientist, can easily be adapted for compound testing of novel antibiotics. The same IMC can then be used to evaluate cellular toxicity for lead compounds in mammalian cell cultures. IMC also can be used to quantify the effects of novel antibiotics on bacterial viability and growth. It is clear that IMC has direct advantages for metabolic drug development since the kinetic profiling of the cellular metabolism makes it possible to distinguish between different cellular events. IMC is also well suited for finding and distinguishing between apoptotic and necrotic mechanisms and for grouping antibody behavior-based killing kinetics and efficacy.
Closed-ampule IMC captures basic pharmacologic information, e.g., minimum inhibitory concentration of an antibiotic needed to stop growth of a given organism. In addition, it can simultaneously provide dynamic growth parameters—lag time and maximum growth rate.
With IMC it is possible to use whole intact organisms, thereby providing a novel tool for antiparasite drug development. Parasites of the Helminth type fit in modern, high-throughput IMC, and the drug efficacy can be tested on the whole organism.
Bacteriological Disease Control
IMC is a diagnostic tool, especially in the field of bacteriological disease control. Slow-growing organisms can be rapidly detected with considerable time and cost savings. In personalized medicine IMC can potentially be used in prescreening for the treatment of diseases like leukemia. The direct drug effect can be studied in individual patient material prior to treatment.
Drug development aimed at parasitic worm diseases is time consuming, labor intensive, and uncertain—and therefore a match for IMC as the method improves assay performance. The use of IMC makes it possible to monitor the total metabolism of the parasite, continuously allowing better drug kinetic predictions. IMC also enables isolation of the heat produced as a function of parasite movement, providing novel insights into drug action.
A current laboratory problem is detection of slow-growing pathogenic microorganisms, such as the tuberculosis-causing mycobacteria, which has been highlighted as a major disease issue by the World Health Organization. There is an urgent need for faster, lower-cost detection processes. To detect mycobacterial infection, the presence of an infection and of possible antibiotic resistance in the strain must be determined. IMC can determine mycobacterial growth and antibiotic resistance, which makes it suitable for novel clinical and antibiotic development pathways.
The workflow for modern cell-based IMC is based on low volumes and presterilized, single-use plasticware performed in a closed ampule with no gas or moisture exchange during the experiment. The latest technology available on the market is supplied with 48-well standard-size cell culture plates with presterilized individual plastic inserts. The inserts can be used for mammalian cell growth or for the inoculum of prokaryotic samples.
IMC cell assays provide important time-resolved data. It is nondestructive, has few limits in morphology other than size, is sensitive and fast, has a low cost to run, and is nonspecific.
Calorimetry should no longer be seen as an obscure and esoteric measurement method for the few; it should be thought of as label-free cell monitoring instead of calorimetry. The technique can serve as a good complement to the many current trends in drug discovery and prioritized research areas.