Abstract
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.
Highlights
In vitro culture of enriched neuronal populations allows for enhanced study of direct/intrinsic factors under a variety of conditions
To further deplete amacrine cells (ACs) in order to allow for improved binding of retinal ganglion cells (RGCs) to Thy1.2 antibody in the final step, we added a selection plate coated with anti-CD15 [43] just prior to the existing anti-CD57 plate
Immunostaining of Thy1.2+ cells with RBPMS revealed that these cells are highly enriched for RGCs, reaching 85.4 ± 1.7% purity
Summary
In vitro culture of enriched neuronal populations allows for enhanced study of direct/intrinsic factors under a variety of conditions. Such cultures are commonly derived from either embryonic/neonatal tissues [1,2,3,4,5] or induced pluripotent stem cells (iPSCs) [6,7,8]. Embryonic/neonatal tissues have weak neuronal connections which allow for their isolation and culture without severe injury [9] and are useful for the study of neuronal development [10,11,12,13].
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