Fabrication of a Vertically Stacked Dentin-Pulp Complex Organ-on-a-Chip Device Using a Human Dentin Disc.

Conventional preclinical models used in dental research, including animal models and static cell cultures, present important limitations in reproducing the physiological conditions of the dentin-pulp interface and predicting human biological responses to dental biomaterials. Organ-on-a-chip (OoC) technology provides an alternative approach by enabling the recreation of tissue-specific microenvironments within controlled microfluidic systems. This manuscript describes a step-by-step protocol for the fabrication and assembly of a dentin-pulp complex OoC device incorporating a human dentin disc within a three-layer polydimethylsiloxane (PDMS) microfluidic platform. The protocol includes dentin disc preparation, fabrication of microstructured molds using photolithography, PDMS casting and curing, plasma-assisted layer bonding, device assembly, leak testing, and dental pulp stem cell seeding under static culture conditions. Critical fabrication parameters influencing device performance are discussed, including control of PDMS layer thickness, plasma surface treatment, and integration of the dentin disc within the device to minimize leakage. Cell adhesion and viability within the device were evaluated using scanning electron microscopy and confocal live/dead staining. Representative results demonstrate successful integration of the dentin disc within the microfluidic assembly, maintenance of leak-free conditions, and adhesion of viable dental pulp stem cells to the dentin surface. This protocol provides a reproducible approach for the fabrication of a dentin-pulp complex OoC platform that may support future studies investigating cellular responses to dental biomaterials and microenvironmental stimuli.