✍🏼 Kiran J. Mcloughlin, Afnan M. Aladdad, Afnan M. Aladdad, Andrew J. Payne, Andrew J. Payne, Anna I. Boda, Sayra Nieto-Gomez, Karl E. Kador

🏠 Department of Ophthalmology, School of Medicine, University of Missouri-Kansas City, Kansas City, MO, United States

📑 frontiers in Molecular Neuroscience (2023)

Abstract

Purified Retinal Ganglion Cells (RGCs) for in vitro study have been a valuable tool in the study of neural regeneration and in the development of therapies to treat glaucoma. Traditionally, RGCs have been isolated from early postnatal rats and mice, and more recently from human in vitro derived retinal organoids using a two-step immunopanning technique based upon the expression of Thy-1. This technique, however, limits the time periods from which RGCs can be isolated, missing the earliest born RGCs at which time the greatest stage of axon growth occurs, as well as being limited in its use with models of retinal degeneration as Thy-1 is downregulated following injury. While fluorescence associated cell sorting (FACS) in combination with new optogenetically labeled RGCs would be able to overcome this limitation, the use of traditional FACS sorters has been limited to genomic and proteomic studies, as RGCs have little to no survival post-sorting. Here we describe a new method for RGC isolation utilizing a combined immunopanning-fluorescence associated cell sorting (IP-FACS) protocol that initially depletes macrophages and photoreceptors, using immunopanning to enrich for RGCs before using low-pressure FACS to isolate these cells. We demonstrate that RGCs isolated via IP-FACS when compared to RGCs isolated via immunopanning at the same age have similar purity as measured by antibody staining and qRT-PCR; survival as measured by live dead staining; neurite outgrowth; and electrophysiological properties as measured by calcium release response to glutamate. Finally, we demonstrate the ability to isolate RGCs from early embryonic mice prior to the expression of Thy-1 using Brn3b-eGFP optogenetically labeled cells. This method provides a new approach for the isolation of RGCs for the study of early developed RGCs, the study of RGC subtypes and the isolation of RGCs for cell transplantation studies.

How the WOLF was used in this study
The authors used the WOLF  Cell Sorter as a low-pressure fluorescence-activated cell sorting platform to isolate retinal ganglion cells (RGCs) from dissociated retinal tissue. After immunopanning to remove macrophages and other unwanted cells, the retinal cell suspension was labeled with a fluorescently conjugated anti-Thy-1 antibody to mark RGCs. The WOLF sorter was then used to sort these labeled cells at a gentle, low pressure (~300 cells per second) using panning buffer as both the running buffer and sheath fluid, calibrated with unstained control cells to set appropriate gates. This approach enriched viable RGCs for subsequent culture and analysis, enabling comparisons of cell survival, neurite outgrowth, gene expression, and functional characteristics against traditional immunopanning methods. Additionally, the sorter was used to isolate embryonic RGCs based on endogenous GFP expression, demonstrating its utility for enriching fragile neuronal populations that are difficult to purify with high-pressure traditional FACS systems.