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

Intervertebral disc (IVD) degeneration (IVDD) is a major contributor to chronic low back pain, representing a substantial burden on the spinal healthcare system and serving as a leading cause of long-term disability worldwide. Biomimetic<i>in vitro</i>models that accurately replicate histological characteristics, three-dimensional structures, and multicellular interactions are lacking. Consequently, monocultures of cell lines and two-dimensional culture models are still used to study the pathomechanisms of IVDD. We established functional multicellular IVD spheroid cultures using primary human annulus fibrosus and nucleus pulposus cells. The spheroids maintained the IVD-specific phenotype, including hypoxic conditions and lamellar structures. Additionally, the spheroid markedly increased the expression level of inflammatory mediators and chemokines in the presence of the pro-inflammatory cytokine IL-1<i>β</i>, 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 the expression levels of chemotactic factors and cytokines including CCL-2, -3, -4, -5, IL-8 and IL-6 (<i>p</i>< 0.05). Moreover, we observed considerable activation and infiltration of microglia induced by soluble factors derived from IVDD spheroids, which are expected to occur during IVDD. The chemotactic effects on microglia were reduced upon the neutralization of CCL-2 or IL-8 or inhibition of NF-<i>κ</i>B signaling. These robust<i>in vitro</i>IVD spheroids can be used to model IVDD and provide a valuable platform for the assessment and development of IVDD therapeutics.