Researchers from Columbia University Medical Center have developed an important technical advance that marries the simplicity of microwells and the early technique of barcoding of droplets with the parallelizability of microfluidics to enable many single cells from multiple different samples or perturbations to be profiled in parallel. The researchers system consists of five parallel microfluidic channels, each of which contains over 2000 microwells for cell capture and processing. To operate the system, the authors load single cells into microwells by simply flushing in a cell suspension. They then profile single-cell gene expression using either a RNA-printing-based approach or a bead-based capture modality.
The RNA-printing approach is conceptually akin to microengraving: lysis buffer is added and the microwells are quickly pressed against a slide that contains covalently grafted oligo-dT primers. Mature cellular mRNA hybridizes to these oligos and then, after washing, is reverse-transcribed on-chip by flowing appropriate reagents through the device. Expression can then be quantified by hybridizing gene-specific probes to these slide-delimited cDNAs. Importantly, this mode of operation maintains spatial correspondence between cell and well, potentially enabling additional information collected before lysis (e.g. cytokine secretion) to be used in downstream analyses. In the bead-based capture approach, uniquely barcoded oligo-dT beads are co-loaded into the microwells before performing lysis. After mRNA capture, a modified variant of CEL-Seq is performed in which reverse transcription and second-strand synthesis take place on-chip, and other steps are performed off-chip after bead harvest. (read more…)
Review – Wadsworth MH 2nd, Hughes TK, Shalek AK. (2015) Marrying microfluidics and microwells for parallel, high-throughput single-cell genomics. Genome Biol 16(1):129. [article]