Abstract
Purpose :
Single cell resolution provides insight into a cell’s biology. Current methods that provide high throughput isolation of cells are limited to dissociated cells, often at the cost of important morphological features. Thus, methods that allow for the rapid isolation of single cells according to their morphological phenotype are valuable. Previously, our lab successfully isolated and cultured adult retinal ganglion cells (RGCs) from mice and found a wide range of neurite outgrowth capacity. To understand the molecular mechanisms by which certain RGCs inherently regenerate, we have implemented a magnetic microraft array system (QIAscout) to efficiently isolate cells according to morphology.
Methods :
Retinas from 6-12-week old transgenic mice expressing tdTomato in Vglut2-Cre+ cells were dissociated and incubated with CD90.2-APC antibody. Cells were sorted using Fluorescence-Activated Cell Sorting (FACS) to isolate RGCs. Following FACS, cells were stained with RBPMS, an RGC-specific marker, to determine purity. Sorted RGCs were seeded onto 96-well plates and onto 200μm2 microraft arrays, where they were cultured in RGC medium for 7 days. Prior to collection of RGCs, viable cells were stained with Calcein-AM and imaged to identify their distinct morphologies. Each microraft, carrying a single Calcein-AM+/tdTomato+ cell, was extracted using the QIAscout system and subsequently placed in a lysis buffer and stored at -20°C. Images were captured of each raft before, after, and during collection.
Results :
FACS collected cells were highly enriched for RGCs, and 99.3% of tdTomato+/APC+ cells were immunolabeled with RBPMS. Additionally, these cells were successfully cultured and showed neurite outgrowth. Using the QIAscout system, viable RGCs were collected at a rate of 1 cell per 3 minutes. Morphologically distinct groups of RGCs were collected according to four general patterns of neurite extension: 1. No neurite outgrowth; 2. Single neurite; 3. Multiple neurites; 4. Complex/branching neurites.
Conclusions :
Using the QIAscout system, our lab has successfully collected individually cultured RGCs by their morphological phenotype at an efficient and reproducible rate. Further single cell transcriptomic profiling can be performed on our morphologically distinct populations to provide insight as to why certain cells are more resilient and capable of higher levels of innate neurite outgrowth.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.