Cell cultures represent a basic approach for multiple areas of biomedical research, due to the fast, cost-effective, versatile and easily reproducible methods.
Because of the complexity of cellular environment, analytical methods for cell studies need different requirements, including sensitivity, specificity and ability to measure different cell parameters. In this context, multianalyte testing appears the most desirable research approach, as it allows to achieve multiple measures from each sample with work simplification, high throughput, and reduction in overall cost per test.
In line with this concept, our proposal is to acquire a Cell Imaging Equipment combined with a Multi-Mode Plate Reader and an incubator-like system, in order to study multiple endpoints, in both fixed and live cells, through integrated phenotypic information and intensity data.
The instrument takes advantage of spectrophotometric tools, based on both UV-VIS absorbance, fluorescence and chemiluminescence, an incubator-like system for live-cell analysis and kinetic studies and a high contrast brightfield and fluorescence imaging module, all controlled by an intuitive and powerful software for data capture and analysis.
Applications of the instrument include concentration measurement for chemicals, phytochemicals, protein, DNA or RNA, free-label cell-counting, cell proliferation, apoptosis, migration, invasion, morphology profiling of cell structures, intracellular localization of antibodies, peptides and delivery systems, DNA damages, ELISA assays, kinetic measures and live-cell analysis.
These features support the main objective of present proposal, which is to provide researchers of different biomedical and nonbiomedical sciences with a new generation system, equipped with multiple, automated and well-integrated advanced services and also easy to use, for improving considerably the quality of their researches and the international performance of Sapienza University.
The proposed instrument, based on a Cell Imaging equipment and a Multi-Mode Reader, combines multiple advanced technologies, including high performance filter-based fluorescence detection, monochromator-based ultraviolet and visible absorbance, fluorescence, luminescence and high contrast brightfield and fluorescence imaging, and incubator-like environmental control (temperature, CO2/O2 control and shaking) for real-time live cell and longer-term kinetic studies. These features make the instrument of interest not only for biomedical sciences (including biochemistry, molecular biology, pharmacology, toxicology, pharmacognosy, microbiology, nutraceutical, pathology and experimental medicine) but also for nonbiological disciplines, particularly chemistry, phytochemistry, merceology, drug design and pharmaceutical technology.
Along with the wide application fields, a key point to be outlined is that this kind of instrument allows to perform rapid multi-sample analyses and simultaneous determinations in a short time, using low volumes and few manual manipulations, thus providing a powerful tool for obtaining reliable data with reduced both time and costs.
The possibility to work with small volumes also reduces reagent costs, cell and nutrient consumption, and optimizes the management of laboratory wastes (including handling, segregation in suitable containers, storing in suitable areas, disposal and removal by specialized personnel and processes), and the laboratory financial resources. This is also reflected in a reduced requirement of technical personnel for instrument control and maintenance.
At the moment, researchers involved in the present proposal project take advantages of basic instruments for cell analyses, mainly filter-based spectrophotometers, brightfield inverted microscopes for basic evaluation of cell morphology and manual dye-based counting systems, with possibility of errors and inter-operator variations.
The proposed instrument will allow to perform automated counting, with high reproducibility compared to manual counting, high throughput due to the counting of multiple samples, and availability of more detailed analysis reports (including graphical display of the cells counted) which can be storaged and overlooked later, thus allowing new and deeply interpretation of the results.
The advanced brightfield imaging system of the proposed instrument will allow to perform also label-free direct cell counting, with the considerable advantage of generating a bright point of light for each cell which that can easily identified and counted by the provided software.
This fully automated equipment, also supported by the incubator-like control system, enables accurate quantitative and phenotypic kinetic analysis of cell growth, without using stain, which alter the native cell properties and limit experiment duration due to cytotoxicity.
Also, confluence measurements, which are routinely determined prior to splitting cells in tissue culture by dye-based counting, can be obtained by label-free methods, taking advantage of the automated high contrast brightfield system of the proposed instrument.
This excellent feature, combined with the fluorescent imaging system, can be also applied to advanced 3D spheroidal tumor systems, which provide a more predictive model than the traditional two-dimensional (2D) cultures, thus giving proper microscopic and quantitative analysis of tumoroid size, proliferation and invasion. The advanced technologies of imaging tool allow researchers to study not only spheroid proliferation through brightfield imaging, but also phenotypic events such as hypoxia, apoptosis, or necrosis induction through the use of fluorescent probes and fluorescence imaging. At the moment, this kind of analysis is hindered for researchers, due to the limited available instruments, despite the well-recognized importance of culturing cells in three-dimensions as a more representative approach of the in vivo tissue.
A further advantage to be exploited, instead of the traditional slide fluorescent visualizations, is the new microplate-based approach for automated immunofluorescence analyses, which allows greater number of samples to be easily processed. This automated approach can be also applied to study intestinal drug absorption and the involvement of uptake and efflux transporters, by both directly measuring the passage of a compound across intestinal epithelium (bidirectional transport assay) and indirect measures (e.g. ATPase activity, uptake, accumulation and efflux assays), since it is a ready and throughput method respect to the more time-consuming chromatographic analysis.
Taken together, the automated, versatile and well-integrated features of the proposed instrument allow to overcome the available limited instrumental equipments and to strengthen the quality of both biomedical and nonbiomedical researches at Sapienza University, improving, as a consequence, its international performance.