A method for preparing primary retinal cell cultures for evaluating the neuroprotective and neuritogenic effect of factors on axotomized mature CNS neurons.

Retinal ganglion cells (RGCs) are central nervous system neurons with a very limited ability for axon regeneration. This unit details a cell culture technique, which can be used to functionally screen factors/compounds for their neuritogenic and neuroprotective effects on RGCs. In this protocol, the retina is isolated, digested in a papain solution, and after trituration, the RGCs are cultured. The neuritogenic effect of applied factors/compounds on RGCs in the medium is functionally determined by measuring the average neurite length of βIII-tubulin-positive RGCs in culture after 3 days. This protocol takes 3 to 7 days to perform depending on the application to complete, and is suitable to reliably test pharmacological and genetic approaches for their axon growth-promoting and neuroprotective potential on mature RGCs.