Optimized culture of retinal ganglion cells and amacrine cells from adult mice

• Published in: PloS one 2020-12, Vol.15 (12), p.e0242426
• Main Authors: Park, Yong H, Snook, Joshua D, Zhuang, Iris, Shen, Guofu, Frankfort, Benjamin J
• Format: Article
• Language: English
• Subjects: Aging; Amacrine cells; Amacrine Cells - drug effects; Amacrine Cells - physiology; Animals; Antibodies; Axonogenesis; Biology and Life Sciences; Brain-derived neurotrophic factor; CD57 antigen; Cell culture; Cell survival; Cell Survival - drug effects; Cell Survival - physiology; Cells, Cultured; Cloning; Culture media; Development and progression; Diagnosis; Dose-Response Relationship, Drug; Embryos; Female; Health aspects; In vivo methods and tests; Inhibitory Concentration 50; Inhibitory postsynaptic potentials; Laboratories; Male; Medical schools; Medicine; Medicine and Health Sciences; Methods; Mice; Molecular biology; Neonates; Nervous system; Nervous system diseases; Neurological diseases; Neuronal Outgrowth; Neurons; Optimization; Physiological aspects; Pluripotency; Population studies; Populations; Primary Cell Culture - methods; Puromycin; Puromycin - pharmacology; Research and Analysis Methods; Retina; Retinal ganglion cells; Retinal Ganglion Cells - drug effects; Retinal Ganglion Cells - physiology; Stem cell transplantation; Stem cells
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0242426

Cell culture is widely utilized to study the cellular and molecular biology of different neuronal cell populations. Current techniques to study enriched neurons in vitro are primarily limited to embryonic/neonatal animals and induced pluripotent stem cells (iPSCs). Although the use of these cultures is valuable, the accessibility of purified primary adult neuronal cultures would allow for improved assessment of certain neurological diseases and pathways at the cellular level. Using a modified 7-step immunopanning technique to isolate for retinal ganglion cells (RGCs) and amacrine cells (ACs) from adult mouse retinas, we have successfully developed a model of neuronal culture that maintains for at least one week. Isolations of Thy1.2+ cells are enriched for RGCs, with the isolation cell yield being congruent to the theoretical yield of RGCs in a mouse retina. ACs of two different populations (CD15+ and CD57+) can also be isolated. The populations of these three adult neurons in culture are healthy, with neurite outgrowths in some cases greater than 500μm in length. Optimization of culture conditions for RGCs and CD15+ cells revealed that neuronal survival and the likelihood of neurite outgrowth respond inversely to different culture media. Serially diluted concentrations of puromycin decreased cultured adult RGCs in a dose-dependent manner, demonstrating the potential usefulness of these adult neuronal cultures in screening assays. This novel culture system can be used to model in vivo neuronal behaviors. Studies can now be expanded in conjunction with other methodologies to study the neurobiology of function, aging, and diseases.