ORGANOTYPIC MODELS TO STUDY THE DISCOGENIC PAIN-ASSOCIATED CELL-TO-CELL CROSSTALK

Chronic low back pain (LBP) represents the leading cause of disability. There are limited non-surgical options in treating LBP. Developing high-throughput in vitro models is important to screen for novel targets. LBP is associated with a complex cell-to-cell crosstalk. Particularly, LBP is initiated by the interaction between the intervertebral disc (IVD) and nociceptive neurons in the dorsal root ganglion (DRG). Furthermore, pain is influenced by the functional crosstalk among DRG neurons. Cells correctly crosstalk only when they are spatially arranged close. We demonstrate that the in vitro spatial organization of cells is essential to study their communication. Bovine DRG and tail IVD were collected from a local abattoir. The enzymatically dissociated DRG micro-tissue was fast assembled to recapitulate the densely packed multicellular architecture of DRG naïve tissue. (Figure1A∼B) This was achieved by the hydrodynamic forces generated from a mild vertical vibration (60 Hz and 0.5 g). Using this approach, DRG neurons could be assembled around a specimen of annulus fibrosus (AF) in a collagen-matrix-based hydrogel. AF primed with IL-1β and TNF-a (both 10 ng/mL) was compared with AF not treated in affecting the sprouting of nociceptors' axons. The nociceptors were labeled using immunofluorescence of calcitonin gene-related peptide (CGRP). In the assembled multicellular system, the YAP nuclei translocation in glia was reduced by a fold change of 3.4 compared to monolayer culture, indicating their distinct mechanobiological response. (Figure1C) The cellular calcium signal was highly correlated when they were assembled into a cluster. (Figure1D) In the AF∼DRG multi-organotypic system, when the neurons were close to the AF, the CGRP(+) axons' length was increased (∼50%) in the cytokine-primed-AF group compared to non-primed control. When the neurons were far from the AF, no such difference could be detected between groups. (Figure2B∼D) Notably, the CGRP(+) axon length was negatively correlated with the AF∼neuron distance in the cytokine-primed-AF group (Rho = −0.55, p < 0.001, Figure 2E). The hydrodynamic bio-assembly achieved an enrichment of neurons that were close to the AF. The spatial distance among organotypic compartments, and their accurate structural organization, impact cell-to-cell crosstalk and should be controlled to assure the robustness of in vitro models. Apart from the crosstalk between IVD and DRG neurons, other types of cells, e.g. , vessel cells, glia and inflammatory cells, need to be investigated as they are also key players in LBP. The hydrodynamic approaches empower a precise control of proper cellular spatial organization.