Society for Neuroscience (SfN) Annual Meeting Neuroscience 2022

Presenter: Megan Ciarlo

Megan Ciarlo, Evelyn Rodriguez-Mesa, Rachel Barhouma, Vuong Tran, Archita Gadkari and Joe Mansmucker

Recovering highly viable and pure populations of neural cell subtypes from brain tissue has been historically challenging. Neural cells often have complex morphologies that require careful dissociation and, once dissociated, can be especially sensitive to the harsh conditions of traditional cell sorting technologies. The high pressures, long fluidics pathways, electrostatic charges, and long processing times of conventional jet-in-air/droplet-based sorters are often not well-tolerated by neural cells and can have significant negative effects on the cells’ ability to perform well in downstream assays.

We were able to overcome these challenges by using a proven, automatic dissociation technique that utilizes mild enzymatic and mechanical mechanisms to dissociate the tissue in combination with a gentle microfluidic cartridge and microchip-based sorting technology to isolate cell subtypes. Here we show fast, efficient, and high purity sorting of neurons, microglia, and astrocytes from dissociated adult mouse brain while still preserving the viability and functionality of the sorted cells. Sort purities were greater than 90% and viability remained high. After sorting, we assessed the cells’ functionality by plating the sorted cells for seven days under the optimal conditions for each cell type. After seven days of culture, we used microscopy and immunocytochemistry techniques to confirm the morphology and cell surface/intracellular protein expression was consistent with the expected isolated cell type. Finally, single-cell RNA sequencing (scRNA-seq) of the sorted cells was performed using the Evercode whole transcriptome technology revealing no significant difference in gene expression or clustering between the sorted and unsorted cells. This data demonstrates a complete workflow for gentle and efficient isolation of neural cell subtypes from whole brain tissue that preserves cell viability, functionality, and transcriptome.