Three-dimensional (3D) culture platforms have been adopted in a high-throughput screening (HTS) system to mimic in vivo physiological microenvironments. The automated dispenser has been established commercially to enable spotting or distributing non-viscous or viscous biomaterials onto microplates. However, there are still challenges to the precise and accurate dispensation of cells embedded in hydrogels such as Alginate- and Matrigel-extracellular matrices. We developed and improved an automated contact-free dispensing machine, the ASFA SPOTTER (V5 and V6), which is compatible with 96- and 384-pillar/well plates and 330- and 532-micropillar/well chips for the support of 3D spheroid/organoid models using bioprinting techniques. This enables the distribution of non-viscous and viscous biosamples, including chemical drugs and cancer cells, for large-scale drug screening at high speed and small volumes (20 to 4000 nanoliters) with no damage to cells. The ASFA SPOTTER (V5 and V6) utilizes a contact-free method that minimizes cross-contamination for the dispensation of encapsulated tissue cells with highly viscous scaffolds (over 70%). In particular, the SPOTTER V6 does not require a washing process and offers the advantage of almost no dead volume (defined as additional required sample volume, including a pre-shot and flushing shot for dispensing). It can be successfully applied for the achievement of an organoid culture in automation, with rapid and easy operation, as well as miniaturization for high-throughput screening. In this study, we report the advantages of the ASFA SPOTTER, which distributes standard-sized cell spots with hydrogels onto a 384-pillar/well plate with a fast dispensing speed, small-scale volume, accuracy, and precision.

Proton beam therapy (PBT) is a critical treatment modality for head and neck squamous cell carcinoma (HNSCC). However, not much is known about drug combinations that may improve the efficacy of PBT. This study aimed to test the feasibility of a three-dimensional (3D) tumor-spheroid-based high-throughput screening platform that could assess cellular sensitivity against PBT. Spheroids of two HNSCC cell lines—Fadu and Cal27—cultured with a mixture of Matrigel were arrayed on a 384-pillar/well plate, followed by exposure to graded doses of protons or targeted drugs including olaparib at various concentrations. Calcein staining of HNSCC spheroids revealed a dose-dependent decrease in cell viability for proton irradiation or multiple targeted drugs, and provided quantitative data that discriminated the sensitivity between the two HNSCC cell lines. The combined effect of protons and olaparib was assessed by calculating the combination index from the survival rates of 4 × 4 matrices, showing that Cal27 spheroids had greater synergy with olaparib than Fadu spheroids. In contrast, adavosertib did not synergize with protons in both spheroids. Taken together, we demonstrated that the 3D pillar/well array platform was a useful tool that provided rapid, quantitative data for evaluating sensitivity to PBT and drug combinations. Our results further supported that administration of the combination of PBT and olaparib may be an effective treatment strategy for HNSCC patients.