The cell-based assay arena is slowly 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 a modern 3-D holistic format, the mature technique of calorimetry is the answer to some of these problems.
This article describes how and why measurement of the total metabolic response of a biological system is a valuable proposition and a true time- and cost-efficient complement to existing assays.
Power and metabolic activity, and a bit of history
The French scientist Antoine-Laurent Lavoisier conducted the first calorimetric experiment in 1782. He concluded that respiration in an animal was equal to a slow combustion. Thus the beginning of calorimetric measurement was in the field of physiology.
All living cells produce heat by the chemical and physical processes of life. By monitoring the heat flow over time (J/s or W), a significant amount of information can be obtained regarding the biological system. 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, which is directly monitored by the calorimetric assay. There are different types of calorimetric types available; this article focuses on isothermal microcalorimetry (IMC). IMC is specific in that its samples are kept at a constant temperature during the course of the experiment.
Calorimetry: A specific footprint of cellular events
IMC measures the total metabolic status and response of a cellular system, thereby giving the potential for rapid identification and quantification. This makes IMC a true phenotype assay with great benefit in that very little information is required about the system to be studied. Because there is no need to know the pathways or receptors involved in a process, novel systems can be studied directly, leading to faster results in drug discovery and cell research.
IMC is a continuous measurement and the power over time curve is comparable to a specific footprint for the cellular events involved. As an example, 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 the specific compound. For prokaryotes, there are different growth patterns for different species and different growth patterns for different nutritional status. This can potentially be used for rapid identification and quantification.
Possibility of 3-D: A holistic approach and a new dawn for drug discovery
The prime area of calorimetry cell-based assays is genotype-based research complemented with phenotype results. This research direction in drug discovery is replacing the previous focus on target-based drug development. Target-based research, with its large investments and reliance on different “’omics,” has shown limited output and the number of new chemical entities (NCEs) has, if anything, decreased.
Calorimetry, and especially IMC, has some unique properties linking high-throughput screening (HTS) primary screens to in vivo results. IMC, being a continuous and nondestructive technology, allows the study of disease models from 2-D to 3-D to tissue, paving the way for cost-efficiency and better predictability in drug development.
Novel cell-based assays with better predictive power links target binding potential to the true phenotype response of the organism, the human. Calorimetry is unique in that it is completely independent of cell morphology and media composition.
Most cell-based assays are limited to 2-D cell cultures or specific growth media. Calorimetry assays can be run using conventional 2-D cell growth and various types of 3-D 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. Also, postexperimental analysis of protein and RNA levels can be performed.
These properties make IMC extremely relevant for studying disease models from 2-D to 3-D to tissue. It is also possible to apply whole body calorimetry to correlate between in vitro models and in vivo results, especially in metabolic research, where energy expenditure is key.
IMC: An open platform that changes scientific development
There is an increasing demand for open platform solutions in the assay world, especially at the higher end of the market. Expensive lab equipment that can be used for more than one assay type is a better and more cost-effective use of scarce resources than the current, highly specialized, locked-in equipment. It can easily be argued that IMC is about as open as a platform can be. The sole limitation is the creativity of the scientist, not the technology. A typical example is the development of novel antibiotic substances in which IMC can easily be adapted for novel compound testing. The same IMC can then be used to evaluate cellular toxicity for lead compounds on mammalian cell cultures.
Example applications
More than metabolic drug discovery
The most obvious use for an energy expenditure assay is metabolic disease drug discovery, but it is in no way limited to this field. 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 mechanism and grouping antibody behavior-based killing kinetics and efficacy.
Antibiotic resistance
Recently there have been several alarming reports on the increasing spread of antibiotic-resistant bacteria. The medical/ pharmaceutical world is facing an enormous challenge to overcome this by rapidly developing novel antibiotic classes. IMC can easily be used to quantify the effects of novel antibiotics on bacterial viability and growth. Closed-ampule IMC captures basic pharmacologic information, e.g., minimum inhibitory concentration (MIC) 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. IMC provides a rapid, cost-efficient development scheme for novel antibiotics.
Antiparasite drugs
With IMC it is possible to use whole intact organisms, thereby providing a novel tool for antiparasite drug development. Parasites of the Helmint type easily fit in modern, high-throughput IMC, and the drug efficacy can be tested on the whole organism. Manual input is minimal compared to the current microscopy assays used.
Bacteriological disease control
It is reasonable to envision the use of IMC as a diagnostic tool, especially in the field of bacteriological disease control. IMC has very high sensitivity, and slow-growing organisms such as in tuberculosis can be rapidly detected with considerable time and cost savings. In the future, when personalized medicine has matured, 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 with minimal lag, increasing the likelihood of a successful treatment scheme.
Case studies
Parasitic worm movement
The development of drugs targeted at parasitic worm diseases is time and labor intensive, and therefore a perfect match for IMC because it can improve assay performance. The current assay technique is based on time-consuming manual microscopy inspection of the worm movement, and generates a great deal of operator-based uncertainty. The use of IMC makes it possible to monitor the total metabolism of the parasite, continuously allowing better drug kinetic predictions while also completely removing the labor and uncertainty of current assays.1 IMC also enables isolation of the heat produced as a function of parasite movement,2 providing novel insights into drug action.
Tuberculosis diagnostics and resistance assessment
A current laboratory problem is detection of slow-growing pathogenic microorganisms, such as the tuberculosis causing mycobacteria, which has been highlighted by the World Health Organization (WHO).3 Since the current methods used are both time consuming and expensive, 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. It has been shown4 that IMC provides a very fast and cost-effective way to determine mycobacterial growth and antibiotic resistance, which makes it suitable for novel clinical and antibiotic development pathways.
Ease of working with IMC
Up until now, calorimetry has been virtually hidden in the scientific community and has had a rather esoteric ring to it. Most students do not come across the technology during basic training. Calorimetry has been considered difficult to understand and required an interest in thermodynamic equations to be really useful. This, and the lack of equipment adapted to the needs of cell biologists, has kept IMC confined to a small community. The IMC community was previously focused on fundamental basic biology research rather than drug and assay development. In addition, the equipment available was hampered by the necessity for large sample volumes and low throughput.
Novel technological developments now make IMC more adaptable to cell biology research. The combined benefits of increased throughput; presterilized, cell growth-compatible disposables; higher sensitivity; and decreased use of cells and chemicals, combined with easier data interpretation, are too important to disregard. Together with outstanding cost-efficiency, this positions IMC as a state-of-the-art technology and increases the IMC-based assay usage in cell biology research.
Isothermal microcalorimetry workflow
The workflow for modern cell-based IMC is based on low volumes and presterilized, single-use plasticware. Typically, IMC has been performed on the milliliter scale and is not really suitable for cell-based assays.5,6 An IMC experiment is performed in a closed ampule with no gas or moisture exchange during the experiment. It is therefore essential to be able to prepare cell samples prior to measurement in a more cell-adapted environment. The calScreener system (SymCel Sverige AB, Kista, Sweden) is supplied with 48-well standard-size cell culture plates with presterilized
Figure 1 – Heat flow: integrated heat accumulation over time and total heat for IMC experiments.individual plastic inserts. The inserts can be used for mammalian cell growth or for the inoculum of prokaryotic samples. The media volume used is normally 200 μL/sample.
When performing an experiment, the cell growth inserts are transferred to a medical-grade titanium ampule that is hermetically sealed to allow correct measurement. The insertion and thermal equilibrium phase is 1 hr, during which no readout is possible. After thermal equilibrium is reached, the experiment can be monitored as long as necessary, usually between a few hours for fast-growing prokaryotes up to weeks for slow-growing mammalian cells.
The collection software samples the energy flow (J/s), continuously providing the raw readout. Data analysis can be performed either by direct comparison of the heat flow curves or by plotting the accumulated integrated heat (J) over time. The integrated heat has a shape similar to a standard growth curve and can be used to determine lag time and exponential growth rates. The total integrated heat (J) is a measure of the complete energy turnover of the cells in the system and can be used as a measure of the cellular metabolism of treated and untreated systems (Figure 1).
Conclusion
Isothermal microcalorimetry-based cell assays offer significant advantages; after all, all living cells produce metabolic heat. IMC is a label-free technology, continuously provides important time-resolved data, is nondestructive, has few limits in morphology other than size, is sensitive and fast, is low cost to run and is nonspecific.
Potential drawbacks of IMC may be its sensitivity and the fact that it is unspecific. By carefully designing and posing the right questions in each study, IMC is no different than any other type of assay.
In conclusion, there is a great deal of valuable information available in heat, information that previously was not given much weight due to preconceived perceptions and lack of tools.
Isothermal calorimetry complements a wide number of cell-based assays, from manual microscopic inspection to oxygen consumption and capacitance measurements. The only limit to possible application areas is creativity. Recent advances in equipment make calorimetry more accessible for the cell environmentalist. The holistic approach, in which 3-D cultures and phenotype response are poised to provide predictable models, requires novel tools.
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. Calorimetry is a good complement to the many current trends in drug discovery and prioritized research areas.
Cell-based assays can now be performed on a versatile and adaptable platform that provides more information with less effort. It is time to welcome calorimetry to the cell-based assay world.
References
- Manneck, T.; Braissant, O. et al. J. Clin. Microbiol. 2011 Apr, 49(4), 1217–25; doi: 10.1128/JCM.02382-10; Epub 2011 Jan 26.
- Kirchhofer, C.; Vargas, M. Acta Trop. 2011 Apr, 118(1), 56–62; doi: 10.1016/j.actatropica.2011.02.003; Epub 2011 Feb 21.
- Global tuberculosis report 2013. WHO Library Cataloguing-in-Publication Data: 1. Tuberculosis—epidemiology. 2. Tuberculosis, Pulmonary—prevention and control. 3. Tuberculosis—economics. 4. Tuberculosis, Multidrug-Resistant. 5. Annual reports. I. World Health Organization. ISBN 978 92 4 156465 6.
- Howell, M.; Wirz, D. et al. J. Clin. Microbiol. 2012 Jan, 50(1):16–20; doi: 10.1128/ JCM.05556-11; Epub 2011 Nov 16.
- http://en.wikipedia.org/wiki/Isothermal_microcalorimetry
- Braissant, O.; Wirz, D. et al. Sensors (Basel) 2010, 10(10), 9369–83; doi: 10.3390/ s101009369; Epub 2010 Oct 18.
Magnus Jansson, Ph.D., is CSO, SymCel Sverige AB, Isafjordsgatan 39B, SE-164 40 Kista, Sweden; tel.: +46 8 5000 49 26; e-mail: magnus. [email protected]; www.symcel.se