The bulk output of a biological response represents the combined behaviors of highly diverse cells ensemble in physiologic and pathologic conditions. Measuring biological phenomena in bulk populations blends and potentially masks the unique contributions of individual cells, particularly when behaviors are highly heterogeneous or driven by rare cell type. The ability to isolate and analyze cells based on the expression of specific markers has increased our understanding of cell biology and produced numerous applications for biomedicine.
With this in mind, we here propose to dissect the diversity of cell subpopulations through an innovative approach of highly-multidimensional cell separation and analysis, based on spectral cell sorting. To this aim, we are requesting the spectral cell sorter Cytek Aurora CS: this is a recently released instrument that combines the full-spectrum based flow cytometry with high-efficiency cell sorting. This will allow the sorting of human and mouse samples and of different cells and particles, including immune cells, cancer and stem cells, platelets, cell lines, and fluorescent beads. The configuration requested is suitable for both basic and advanced panel design and cell sorting. Importantly, it complies with biosafety level II, allowing the safe separation and the analysis of human cells.
This project stems from a consortium of scientists and physicians from 12 departments, 4 faculties and 3 macro-areas working in different biomedical research fields, who propose to exploit this instrumentation for multiple research lines. The main applications of this technological platform will be the analysis of the heterogeneity of complex immune cell populations and discovery of new immune subsets in several immune/endocrine-related disorders, in viral infections, in cardiovascular and metabolic diseases, in hematological disorders, and in cancer.
Spectral flow cytometry is a novel technology that is going to revolutionize the approach to cell analysis. J. P. Robinson, one of the greatest flow cytometry experts, has recently published an editorial supporting the need of moving from conventional to spectral flow cytometry (Robinson JP, Cytometry A 2019).
The instrument that we request, the Cytek Aurora CS, is a spectral cell sorter that combines spectral profiling with high efficiency cell sorting. Sapienza would be the first Italian institution to have this technology. It has been released in the market in the latest months and represents an absolute innovation in the national and international scenario. This instrument has just been released and the first instrument in Europe is going to be installed in France in August 2021.
The Cytek Aurora CS was designed to leverage the paradigm-shifting technology onboard the Cytek Aurora system and deliver the sorting capabilities you have come to expect in a high-end sorter. Like Aurora, the CS provides the benefits of Full Spectrum Profiling (FSP¿) technology. Its optical design and unmixing algorithm provide scientists remarkable flexibility, enabling the use of a wide array of new fluorochrome combinations without reconfiguring the system for each application. The state-of-the-art optics and low-noise electronics provide high-resolution, high-content and high sensitivity. The result is a system that delivers high resolution at the single cell level to resolve the most challenging cell populations, such as cells with high autofluorescence or low levels of expression of key biomarkers, regardless of assay complexity, and to isolate live cells for downstream studies.
We strongly believe that it would be a unique technological platform in Sapienza Research Infrastructures. At present, several flow cytometry-based analyzers are present in Sapienza Research Infrastructure, however none of them display the following characteristics which can be found instead in the requested instrumentation:
- none of them is a cell sorter able to separate populations: the Aurora CS is able to separate up to 6 different populations with >99% purity and even sort single cells into 96-well plates;
- most of them have a multi-parametricity of around 4-12 colors (obtained with 1-3 lasers): the requested Aurora CS has a configuration of 38 parameters obtained with only 3 lasers thanks to the revolutionary optics based on spectral profiling;
- all of them are based on conventional flow cytometry, using filters and photomultipliers: the Aurora CS will be able to resolve highly overlapping fluorescence signals with cannot be otherwise distinguished (as an example, FITC and GFP), and to subtract cellular autofluorescence which, in complex cells, can hinder the performance of reliable analyses.
- most of them are most suitable to the analysis of intermediate-size cells, like leukocytes: thanks to its optical properties, the Aurora CS allows discriminating at high resolution very small cells and particles as well as big and complex cell types,
- most of them are compatible with a relatively limited set of fluorochromes, according to the optical bank that they accommodate: the spectral analysis allows the detection of an extremely long list of fluorochromes and fluorescent proteins, thus significantly increasing the options for panel design.
This highly innovative system opens incredibly new perspectives for the study of multiple cells ex vivo and in vitro: we envisage that the acquisition of this new instrument will widen and improve the experimental possibilities of our groups and our consortium, possibly leading to discoveries and advances in several fields. Indeed, the scientific literature is full of examples of significant advances made possible through the use of flow cytometry, and in particular of spectral profiling.