Abstract
Purpose :
The culture of purified retinal ganglion cell (RGC) cultures has been a vital tool in the study of glaucoma and neural regeneration. Traditionally, RGCs are isolated from early postnatal animals using a two-step immunopanning (IP) technique, a procedure limited to RGCs of a specific maturity. This study aimed to develop a method using a low-pressure cell sorting protocol that will allow for the sorting of RGCs and their subtypes using optogenetic markers while maintaining the RGCs survival and electrophysiological properties, which can be affected using traditional fluorescence associated cell sorting (FACS).
Methods :
Using a combined IP-FACS protocol that initially depletes macrophage and photoreceptors using IP before enriching for RGCs using low-pressure FACS against Thy-1 expression. RGC viability was compared against a two-step IP using a live/dead analysis. Cell phenotype was compared using immunohistochemistry for RBPMS and β-III-Tubulin, while properties such as axon outgrowth and neurite branching were also measured. Further, RT-qPCR was carried out to verify the purity of isolated cells. Functionality was compared using calcium imaging to determine isolated RGC response to both potassium chloride (KCl) and glutamate.
Results :
RGCs purified by the IP-FACS method remained viable with no evident cytotoxicity and similarly extended their neurites to those purified by the IP method. RGCs isolated from both methods showed similar expression of RGC-specific proteins and similar gene expression levels. IP and IP-FACS methods resulted in no significant difference (p > 0.05) in a total number of neurites of 2.14 ± 0.3 and 2.47 ± 0.5, longest neurite length of 1098.14 ± 205.5 μm and 1184.15 ± 111.3 μm, total neurite length of 1765.5 ±367.7 μm and 2117.6 ± 23.3 μm and the average neurite length of 890.9 ± 148.9 μm and 882.2 ± 157.1 μm, respectively. Calcium imaging revealed that both populations respond to KCl and glutamate stimuli, confirming that cells are functional to standard calcium stimulus.
Conclusions :
Our results showed that low-pressure FACS can isolate RGCs from rodents with comparable viability, phenotype, and function compared to the traditional method of immunopanning. This method opens the possibility of isolating embryonic RGCs or subtype populations by combining FACS isolation with optogenetically labeled transgenic rodents.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.