Reconfigurable Hanging Drop Microarray Platform for On‐Demand Preparation and Analysis of Spheroid Array

In response to the increasing demand for spheroid‐based cancer research, the importance of developing integrated platforms that can simultaneously facilitate high‐throughput spheroid production and multiplexed analysis is emphasized. In addition, the understanding of how the size and cellular composition of tumors directly influence their internal structures and functionalities underlines the critical need to produce spheroids of diverse sizes and compositions on a large scale. To address this rising demand, this work presents a configurable and linkable in vitro three‐dimensional (3D) cell culture kit (CLiCK) for spheroids, termed CLiCK‐Spheroid. This platform consists of three primary components: a hanging drop microarray (HDMA), a concave pillar microarray (CPMA), and gradient blocks. The HDMA alone produces a homogeneous spheroid array, while its combination with the gradient block enables one‐step generation of a size‐gradient spheroid array. Using the size‐gradient spheroid arrays, the occurrence of necrotic cores based on spheroid size is demonstrated. Additionally, spheroids in a single batch can be conveniently compartmentalized and regrouped using a CPMA, enhancing the versatility of spheroid arrays and enabling multiplexed drug treatments. By combining the different assembly methods, this work has achieved high‐throughput production of cell composition‐gradient spheroid arrays, with noticeable variations in morphology and vascularization based on cell compositions. A configurable and linkable in vitro 3D cell culture kit for spheroids (CLiCK‐Spheroid) enables high‐throughput production of homogeneous, size‐gradient, and cell composition‐gradient spheroid arrays using a hanging drop microarray and gradient block. The generated spheroid array can be retrieved and transferred on demand using a concave pillar microarray. Multiplexed, sequential drug treatment and spheroid penetration and vascularization assay are demonstrated.