A Hybrid Bioprinting–Microfabrication Approach to Controllable Multitype Spheroid Arrays for Modeling Tumor Heterogeneity

Abstract

The intrinsic heterogeneity of solid tumors remains a crucial barrier to understanding cancer biology and predicting therapeutic outcomes. Here, we introduce a scalable and versatile biofabrication platform that enables the controlled assembly of multitype spheroid arrays (MSA) within standard culture microplates. By performing programmable bioprinting within soft, microstructured culture devices, we achieve the precise spatial deposition of multiple cell types into defined microwells, yielding reproducible arrays of heterogeneous 3D tumor spheroids that collectively recapitulate the macro- and micro-heterogeneity of solid cancers. We demonstrate that this approach permits the fine-tuning of spheroid size, composition, and arrangement. It also supports the delayed secondary programmed seeding of cells on pre-grown spheroids, mimicking biological processes such as the invasion of immune cells. These methods enable quantitative drug-response profiling within a single assay. They overcome some critical limitations of current 3D cancer models, introducing the concept of the “cancer-in-a-well” technology. This technology provides a simple-to-use, cost-effective, high-throughput, and highly reproducible platform to recreate the complexity of tumors in vitro using methods and instruments that can be potentially used by any researcher in any cell culture lab. We can foresee a positive impact on cancer biology studies, drug screening, and personalized oncology.