Culture of Primary Neurons from Dissociated and Cryopreserved Mouse Trigeminal Ganglion

Corneal nerves originate from the ophthalmic branch of the trigeminal nerve, which enters the cornea at the limbus radially from all directions toward the central cornea. The cell bodies of the sensory neurons of trigeminal nerve are located in the trigeminal ganglion (TG), while the axons are extended into the three divisions, including ophthalmic branch that supplies corneal nerves. Study of primary neuronal cultures established from the TG fibers can therefore provide a knowledge basis for corneal nerve biology and potentially be developed as an in vitro platform for drug testing. However, setting up primary neuron cultures from animal TG has been dubious with inconsistency among laboratories due to a lack of efficient isolation protocol, resulting in low yield and heterogenous cultures. In this study, we used a combined enzymatic digestion with collagenase and TrypLE to dissociate mouse TG while preserving nerve cell viability. A subsequent discontinuous Percoll density gradient followed by mitotic inhibitor treatment effectively diminished the contamination of non-neuronal cells. Using this method, we reproducibly generated high yield and homogenous primary TG neuron cultures. Similar efficiency of nerve cell isolation and culture was further obtained for TG tissue cryopreserved for short (1 week) and long duration (3 months), compared to freshly isolated tissues. In conclusion, this optimized protocol shows a promising potential to standardize TG nerve culture and generate a high-quality corneal nerve model for drug testing and neurotoxicity studies. IMPACT STATEMENT: Isolation of neurons from trigeminal ganglion (TG) has been used for deeper insights on corneal nerve biology. However, the lack of standardized isolation protocols may be the attributes of the heterogenous culture with inconsistent results among laboratories. In this study, we investigated an optimized protocol for TG neuron culture with good efficiency. Furthermore, compared with freshly prepared TG culture, the culture prepared with cryopreserved TG tissues or neurons showed similar morphology with preserved neuronal marker expression. This study highlights the promising potential of using optimized protocol as a standardized isolation method as in vitro corneal nerve model for corneal nerve studies.