Image: The microfluidic control instrument performing a Drop-seq run (Photo courtesy of the New York Genome Center).
The complex architecture and associated higher-order function of human tissues relies on functionally and molecularly diverse cell populations. Defining the cellular subsets found in pathologic tissues provides insights into disease etiology and treatment options.
Traditional methods such as flow cytometry, which require a priori knowledge of cell type-specific markers, have begun to define this landscape, but fall short in comprehensively identifying cellular states in a tissue, with particular difficulty detecting extremely rare subpopulations.
Scientists at the New York Genome Center (New York, NY, USA) and their colleagues have facilitated broad access to single-cell sequencing by developing a 3D-printed, portable and low-cost microfluidic controller. They adapted this device to perform massively parallel single-cell RNA-seq (Drop-seq), observing metrics and performance that are indistinguishable from a study level Drop-seq setup.
The group used the instrument to profile joint synovial tissue from rheumatoid arthritis (RA) patients. RA is an autoimmune disease that affects 1% of the population and is associated with painful swelling in the joints. The precise cause of RA is undetermined and muddled by the diversity of cells found in the swollen joints of patients. The portability of the controller permitted patient samples to be processed on-site and immediately after surgery, minimizing handling and transport to optimize sample quality. The team collected samples from five RA patients totaling 20,387 cells and looked at the individual gene expression patterns for each cell.
By analyzing the complete dataset and searching for clusters of similar cells, the scientists identified 13 groups, representing both infiltrating immune and inflamed stromal populations. Of particular interest were distinct groups of fibroblasts with strikingly different gene expression patterns. They were able to validate the presence of these multiple groups using BD FACSAria II flow cytometry sorter, and discovered that they exhibited distinct localization patterns with the joint tissue as well. The instrument processes 1 mL of cells at a concentration of 150–200 cells/µL in about 30 minutes, generating over one million droplets at a generation rate of approximately 700 Hz.
Instructions and assembly manuals for the instrument can be found online at the popular microfluidics repository Metafluidics. The 3D-printed custom device, which, along with its electronic and pneumatic components, can be easily obtained and assembled for a total cost of about USD 600, a fraction of the cost of comparable commercial systems. The device occupies a small footprint as well, not much larger than a tissue box.
William Stephenson PhD, a Senior Research Engineer, and lead author of the study, said, “Most commercial microfluidic instruments are very costly; as a result, not every lab has access to exciting technology for single-cell analysis. We designed the instrument to perform droplet microfluidics and in particular Drop-seq, a massively parallel technology for single cell RNA-sequencing.” The study was published on February 23, 2018, in the journal Nature Communications.
New York Genome Center