Building multiple microenvironmental niches using a customizable 3D printed well insert

The increasing demand for advanced <i>in vitro</i> models that replicate physiological crosstalk between cell types within and between organs requires customized cellular microenvironments arranged within a single platform. Hydrogels are biomaterials that mimic the physicochemical properties of tissue niches to optimally support diverse cell types. However, they require integration with cell patterning platforms like bioprinting or microfluidics to create organized multi-niche environments. There is currently a gap between bioprinting, which is scalable but limited by availability of printable hydrogels, and microfluidic patterning, which is compatible with diverse biomaterials but is challenging to multiplex. Here, we developed the Localized Microenvironment Well-Insert (LM-Well), a 3D-printed device designed to pattern multiple hydrogel niches with customizable physicochemical properties in multi-well plates. The LM-Well features patterning structures that enable capillary force-driven patterning of various hydrogel formulations, including natural, photo-crosslinkable and synthetic click hydrogels. Functional materials, exemplified by oxygen-scavenging microcapsules, can be patterned within the LM-Well offering an additional layer of control over local oxygen levels in individual cell niches, which modulated tumor growth and zonation of hepatic activities. Micro-architectural supports, such as micropillars or scaffolds, can be integrated into the LM-Well to optimally support mechanically-active cells like myocytes. The LM-Well's multi-niche patterning capability enabled the establishment of a liver-tumor co-culture in a single well, recapitulating altered drug efficacy on MCF-7 tumor cells following activation of tamoxifen and deactivation of doxorubicin by HepaRG-derived hepatocytes. As a versatile and accessible platform, the LM-Well facilitates physiologically relevant co-cultures with customizable niches and diverse biomaterials.