Intervertebral disc spheroids as an in vitro multicellular platform for recapitulating the microenvironment of intervertebral disc degeneration

Intervertebral disc (IVD) degeneration (IVDD) is a major contributor to chronic low back pain (LBP), representing a significant burden on the spinal healthcare system and a leading cause of long-term disability worldwide. Reliable in vitro models are lacking and thus monocultures of cell lines and 2D-based culture models are still used to study the patho-mechanisms of IVDD. We established functional multicellular IVD spheroid cultures using primary human annulus fibrosus (AF), nucleus pulposus and microglial cells. The IVD spheroid culture was observed to facilitate the IVD-specific phenotype and function. The spheroid culture markedly increased the expression of inflammatory mediators and chemokines in the presence of pro-inflammatory cytokines IL-1b, a master regulator of IVDD. Furthermore, we implemented our microfluidic chemotaxis platform to investigate microglial neuroinflammation in response to our reconstituted IVD spheroid models. Transcriptome sequencing revealed that microglia stimulated by potential contributing factors derived from IVDD spheroids exhibited a significant upregulation of chemotactic factors and cytokines including CCL2, 3, 4, 5, CXCL8, IL6, and IL1b (p < 0.05). Moreover, we observed considerable activation and infiltration of microglia induced by soluble factors derived from IVDD spheroids, which is expected to occur during IVDD. The chemotactic effects on microglia were reduced upon neutralization of CCL-2, IL-8 or inhibition of NF-kB signaling. These robust in vitro IVD spheroids can be used to model IVDD and provide a valuable platform for the assessment and development of IVDD therapeutics. Overall design: Samples: SV40 human microglia cells were treated with DF12, SPCM, SPCM_IL for 48 hours. DF12: DMEM/F12 medium SPCM: A spheroid was cultured in DMEM/F12 for 48 hours, and the supernatant was collected. The spheroid consisted of 1.8 × 10^4 nucleus pulposus cells and 3.2 × 10^4 annulus fibrosus cells. SPCM_IL: A spheroid was cultured in DMEM/F12 with 1 ng/ml of IL-1ß for 48 hours, and the supernatant was collected. The spheroid consisted of 1.8 × 10^4 nucleus pulposus cells and 3.2 × 10^4 annulus fibrosus cells.