Phenotypes of primary retinal macroglia: Implications for purification and culture conditions

• Published in: Experimental eye research 2019-05, Vol.182, p.85-92
• Main Authors: Backstrom, Jon R., Sheng, Jinsong, Fischer, Rachel A., Sappington, Rebecca M., Rex, Tonia S.
• Format: Article
• Language: English
• Subjects: Animals; Astrocyte-mesenchymal transition; Astrocytes; Astrocytes - physiology; Cell Communication - physiology; Cell Culture Techniques; Cell Differentiation; Culture Media - pharmacology; Intermediate filaments; Müller glia; Neuroglia - physiology; Phenotype; Primary culture; Rats; Rats, Sprague-Dawley; Retina; Retina - cytology; Retinal Neurons - physiology
• ISSN: 0014-4835; 1096-0007
• DOI: 10.1016/j.exer.2019.03.008

Many neurodegenerations, including those of the visual system, have complex etiologies that include roles for both neurons and glia. In the retina there is evidence that retinal astrocytes play an important role in neurodegeneration. There are several approaches for isolating and growing primary retinal astrocytes, however, they often lead to different results. In this study, we examined the influence of culture conditions on phenotypic maturation of primary, purified retinal glia. We compared retinal astrocytes and Müller glia purified by immunomagnetic separation, as differentiation between these astrocyte subtypes is critical and immuno-based methods are the standard practice of purification. We found that while time in culture impacts the health and phenotype of both astrocytes and Müller glia, the phenotypic maturation of retinal astrocytes was most impacted by serum factors. These factors appeared to actively regulate intermediate filament phenotypes in a manner consistent with the induction of astrocyte-mesenchymal transition (AMT). This propensity for retinal astrocytes to shift along an AMT continuum should be considered when interpreting resulting data. Our goal is that this study will help standardize the field so that studies are replicable, comparable, and as accurate as possible for subsequent interpretation of findings. •Some current purification methods may result in co-purification of both astrocytes and Müller cells.•Müller cells are initially reactive in culture after purification, but over time they return to a quiescent state.•Primary astrocytes convert to a phenotype consistent with induction of astrocyte-mesenchymal transition (AMT).•Factors in fetal bovine serum actively promote AMT in retinal astrocytes.•Primary astrocytes can convert back to an in vivo healthy phenotype by culturing in serum-free media.