Chronic back and joint pain has been rated as a top risk factor of disability worldwide. In vitro culture of peripheral sensory nerve, namely dorsal root ganglion (DRG) neurons, is a useful model to investigate pain-associated biology and to discover novel regenerative medicine in terms of pain alleviation. Typical monolayer culture of DRG cells, however, losses the multicellular shape, which may cause a disturbed intercellular communication. For example, the multicellular structure provides the basis of synchronized cross excitation commonly observed in vivo . We used the sound induced morphogenesis (SIM) method to aggregate DRG cells into a multicellular system . Viability and calcium signal synchronization of the DRG cells were evaluated in the sound induced multicellular system.
DRG cell line ND7/23 was aggregated using SIM in a 0.5 mg/mL collagen solution with a cell seeding density of 0.2 M cells/ml. Cells randomly distributed in collagen gel without SIM served as control (non-SIM control). The DRG cell viability was analysed using live dead staining. After 2 days of culture, the neuronal discharge was evaluated using calcium imaging (Fluo4) . In each culture, 5000 pairs of neurons were randomly sampled to investigate the synchronization of their calcium signalling which was quantified by the ratio dividing their synchronized calcium event number by total calcium event number (synchronization ratio).
Cells in the SIM-induced aggregates displayed higher viability comparing to those outside the aggregate in the same culture (viability 91.9% vs 50.6%) and cells in the non-SIM control (viability 91.9% vs 77.1%). Higher calcium synchronization was found in the SIM group compared to non-SIM control. The synchronization ratio was elevated from 20.4% to 28.1% comparing SIM to non-SIM control. Interestingly, in the SIM culture, cells outside the aggregate also displayed elevated synchronization ratio (29.9%), indicating that the SIM-induced intercellular communication did not depend on an intercellular apposition. Generation of multicellular systems by patterning primary DRG neurons from large animal is ongoing. Omics studies will be performed to unravel their physiological relevance.
The multicellular system allows a better inter-neuronal communication to form the synchronized neuronal discharge which has been formerly observed in vivo . Thus, our multicellular culture system not only reconstruct the complexity of in vivo morphology but is also important to recapitulate the in vivo function.
1. Kim, YS. et al., Neuron. 91(5), 1085-1096 (2016).
2. Petta, D. et al., Biofabrication. 13, 015004 (2020).
3. Ma, J. et al., Neurospine. 17(1), 42–59 (2020).