Bioprinting of a multi-composition array to mimic intra-tumor heterogeneity of glioblastoma for drug evaluation.

Microextrusion printing is widely used to precisely manufacture microdevices, microphysiological systems, and biological constructs that feature micropatterns and microstructures consisting of various materials. This method is particularly useful for creating biological models that recapitulate in vivo-like cellular microenvironments. Although there is a recent demand for high-throughput data from a single in vitro system, it remains challenging to fabricate multiple models with a small volume of bioinks in a stable and precise manner due to the spreading and evaporation issues of the extruded hydrogel. As printing time increases, the extruded bioink spreads and evaporates, leading to technical problems that decrease printing resolution and stability, as well as biological problems that affect 3D culture space and cell viability. In this study, we describe a novel microextrusion bioprinting technique to stably fabricate a multi-composition array consisting of massive and nanoliter-scale hydrogel dots by using multi-bioink printing and aerosol-based crosslinking techniques to prevent spreading and evaporation issues. We confirmed that the crosslinking aerosol effectively prevented spreading and evaporation by analyzing the morphological changes of the extruded hydrogel. By adjusting the extruding ratio of the bioinks, we were able to print a multi-composition array. This stable and massive array printing technique allowed us to improve the replicates of biological models and provide various data from a single culture system. The array printing technique was applied to recapitulate the intra-tumor heterogeneity of glioblastoma and assess temozolomide efficacy on the array model.