Purification of retinal ganglion cells using low-pressure flow cytometry

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.

Keywords: FACS (fluorescence-activated cell sorting); cell isolation; immunopanning; neurite outgowth; retinal ganglion cell (RGC).

Figure 1

Summary of both immunopanning and low-pressure cell sorting protocols for isolation of RGCs from postnatal rodents. Diagrammatic representation of the isolation of primary RGCs from rodent litters. Both the protocol for immunopanning and protocol for cell sorting will include isolation of retinas from eyes via surgery (Step 1), followed by enzymatic digestion and centrifugation to obtain a single-cell suspension (Step 2). This suspension is then exposed to a series of antibody-coated plates to remove unwanted macrophage, and in some cases photoreceptors (Step 3). The protocol can then proceed either to selection for RGCs, through immunopanning (Step 4A) or via flow-based cell sorting (Step 4B). For purification by immunopanning (Step 4A), cells were immunopanned against Thy-1. Isolated RGCs are then removed via trypsinization in combination with pipetting to facilitate cell release. For RGCs isolated by flowcytometry (Step 4B), the retinal suspension can be incubated with a fluorescently labeled Thy-1 antibody before being sorted using a low-pressure cell sorter. Created with BioRender.com.

Figure 2

Representative fluorescence microscopy images showing attachment and neurite extension of RGCs purified by immunopanning method and sorting method using Cellstain™ staining to show green fluorescent viable cells (calcein AM-stained) and red fluorescent dead cells (propidium iodide, PI), over 3 days at 37°C. By day 3, cells had adopted a more spread RGCs morphology. Positive control images (PI stain on fixed cells from a separate sample cultured from the same experiment) are shown. RGC viability was calculated for both isolation methods after 3, 7, and 14 days. The data is reported as the mean ± SD, significance was determined by Student’s t-test p < 0.05. (Scale bar = 50 μm).

Figure 3

(A) Representative fluorescence microscopy images showing effects of immunopanning method and sorting method as RGCs purification techniques on neuronal and RGCs markers. Immunocytochemical staining was performed for βIII-tubulin (green), RPBMS (red), with DAPI nuclear counterstain (Blue), after 3 days of culture. Negative control images are shown (scale bar = 100 μm). (B) Graphs showing tracing data for RGCs obtained from the immunopanning method and sorting method with a minimum of 20 cells traced per well. The total number of neurites/cell, longest neurite length/cell, total neurite length/cell, and average neurite length/cell were calculated. The values were reported as the mean ± SD of three independent experiments: n = 3 in triplicates. Statistical significance was determined by Student’s t-test: (ns) p > 0.05.

Figure 4

Comparative effects of purification methods on gene expression of RGCs. ∆∆CT was determined by RT-qPCR for Brn3a, Brn3b, CRX, PKCA, STX1a, and GFAP. Expression is normalized to GAPDH and represented relative to total retinal digest (mean ± SEM of three independent experiments: n = 3 in duplicates). Statistical significance versus total retinal digest (one-way ANOVA): (ns) p > 0.05.

Figure 5

Percentage of living cells staining positive or negative for the RGC marker RBPMS for both immunopanning and IP-FACS sorted cells. The values were reported as the mean ± SD of three independent experiments: n = 3 in triplicates. Statistical significance RGCs versus non-RGCs (Student’s t-test): (****) p < 0.0001.

Figure 6

RGCs respond to calcium-associated stimuli regardless of isolation technique. (A,C) RGCs depolarize in response to 100 mM KCl stimuli and (B,D) 50 μM glutamate stimuli. Baseline activity was established over 60 s before the stimulus was applied to cells via a manual pipette (addition of stimulus indicated on traces via an arrow). The maximum response between conditions was non-significant. Original videos are included in Supplementary material.

Figure 7

RGCs were isolated from Brn3b-P2A-eGFP at embryonic day 14–15 and purified via low-pressure flowcytometry. (A) Gating conditions for the purification of RGCs. (B) Staining of isolated cells with βIII-tubulin following growth for 2 days in vitro. (Scale bar 250 μm).