Aspiration-assisted bioprinting of spheroids

Aspiration-assisted bioprinting (AAB) is a versatile biofabrication technique that enables the precise and selective patterning of biologics, such as tissue spheroids and organoids, addressing limitations of conventional bioprinting techniques. AAB facilitates the fabrication of (1) tissues with physiologically relevant cell densities using spheroids and (2) advanced tissue models that replicate three-dimensional microenvironments essential for studying cellular responses, disease development and drug testing. Here we provide reliable and reproducible guidelines for the precise positioning of abovementioned biologics, incorporating two operational modes: (1) a single-nozzle mode for precise, one-by-one bioprinting and (2) a high-throughput mode using a digitally controllable nozzle array, enabling the rapid and simultaneous placement of multiple spheroids for scalable tissue fabrication. Comprehensive instructions are included for setting up the AAB platform, operating software and key operational procedures, including optimization of bioprinting conditions. This Protocol enables users to build and operate their own AAB platform depending on target applications, achieving fine control over spheroid positioning through successful aspiration and their precise placement under optimized conditions. This Protocol enables the setup of the AAB platform within 1–2 d. Bioprinting time varies depending on the number of spheroids to bioprint: the single-nozzle mode requires ~30 s per spheroid, while the high-throughput mode can print 64 spheroids in 3–4 min. Designed for accessibility and adaptability, this Protocol is suitable for users from a variety of backgrounds, including engineering, biology, pharmacy and medical sciences, who require bioprinting of spheroids for creating microphysiological systems for drug testing and disease modeling and implantable grafts for regenerative medicine.