A detailed methodology for developing and applying a 3D polyHIPE scaffold to model myeloma cell dormancy in vitro

Background

Multiple myeloma is characterised by the outgrowth of malignant plasma cells in the bone marrow (BM). Disease relapse is driven by the reactivation of dormant myeloma cells (DMCs) that interact with osteoblastic lineage cells within the BM to evade therapy. Current two-dimensional (2D) in vitro culture systems lack the physiological and spatial complexity to replicate the BM microenvironment, while animal models offer limited control over microenvironmental components for systematic investigation of cell-to-cell interactions relevant to myeloma dormancy and are resource-intensive. Therefore, a more physiologically relevant and controllable in vitro model is needed to study dormancy mechanisms and microenvironmental regulation of drug responses.

Methods

We developed a reproducible and scalable three-dimensional (3D) polymerised High Internal Phase Emulsion (polyHIPE) scaffold model that mimics key features of the osteoblastic bone niche, where myeloma dormancy occurs. The osteoblast-supported model enables the investigation of dormancy induction, maintenance, and targeting within an endosteal-like environment. This protocol involves scaffold synthesis, sterilisation, osteoblast and myeloma cell seeding, and dormancy tracking using membrane dye retention.

Results

Using multiple osteoblastic (MG63, hFOB 1.19, and MC3T3) and myeloma (U266, JJN3, and 5TGM1) cell lines, we demonstrated the interchangeability of the model. PolyHIPE scaffolds facilitated osteoblast and myeloma cell infiltration and interaction over time and altered the drug sensitivity of cells in the 3D niche compared to conventional 2D cultures. Troubleshooting tips and time considerations were established to enhance the reproducibility.

Conclusions

While primarily intended for researchers studying myeloma dormancy, this 3Rs-aligned approach is adaptable and has the potential for broader use across the myeloma and cancer dormancy research. The model provides a controllable in vitro alternative for early-stage discovery and hypothesis-generating studies to minimise reliance on in vivo myeloma models, aligns with the 3Rs principles, and provides a platform to investigate dormancy-associated drug resistance and microenvironmental control of myeloma behaviour prior to targeted in vivo validation.