Internal Delivery and Transport within Cellular Aggregates via Perfusable Glass-Sheathed Hydrogel Microtubes

Measuring the transport dynamics of soluble molecules such as nutrients, growth factors, and therapeutics within cell aggregates is essential to understand the transport-limiting effects of 3D cell culture models. Traditional methods to study molecular transport within engineered tissues often face challenges related to access for delivery and sampling and require sacrificing the culture. Here, we introduce an accessible, device-innovation platform that allows spatially defined delivery into a living cell aggregate. By integrating a highly perfusable, hollow-core agarose hydrogel microtube into a polyacrylamide microwell, our system allows local access to the aggregate's interior without advanced microfabrication or complex vascularization. Transport out of the hydrogel tube is limited to a specific spatial region within the aggregate by the deliberate placement and fracturing of a glass capillary sheath. We demonstrate an application of this system in an engineered model of placental trophoblast plugs, which arise temporarily during the first trimester of pregnancy to protect the placenta during development. Partial cell fusion occurs within these plugs, which would alter local transport characteristics, whether this morphological feature affects transport in a 3D tissue remains undefined. To address this, we created cylindrical plug-like aggregates and applied quantitative fluorescence measurements coupled with finite element modeling to determine that patterns of diffusivity are heterogeneous, with enhanced transport at the aggregate core compared to the periphery. This specific application highlights the platform's potential to study molecular transport dynamics within cell aggregates and provides a foundation for further exploration of diffusion-limited processes in 3D culture systems.