Engineering and Evaluating Vascularized Organotypic Spheroids On-Chip.

Organotypic spheroids are evolving as a mainstream in vitro modeling platform, but it is crucial to integrate vascular tissue and perfusion for maintaining their longevity, stability, and physiological relevance. Current vascularization methods remain underdeveloped, and several protocols are poorly reproducible and are limited to use by a few select groups who have designed these methods. To achieve standardization, we offer a step-by-step guide to vascularize organotypic spheroids in case studies of pancreatic islets and cancer spheroids. Our systematic approach spans microfluidic chip design, spheroid fabrication, and vascularization techniques (vasculogenesis and angiogenesis) while describing critical tissue engineering methods. We also include additional insights and operating guidelines within our protocols that characterize and quantitate these models with molecular assays as well as our integrated computational algorithms of mass transport through formed capillary vessels. These protocols contribute to establishing reproducibility, standardization, and enhanced adoption by other contemporary organ-chip researchers, who want to engineer vascularized organoid-based microphysiological platforms. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Design and fabrication of microfluidic chips for vascularized spheroids Basic Protocol 2: Organotypic spheroid fabrication Basic Protocol 3: Vascularized spheroids on-chip Basic Protocol 4: Functionality assays Support Protocol 1: Cell Culture Support Protocol 2: Immunocytochemistry.