Fish cell spheroids are promising 3D culture models for vertebrate replacement in ecotoxicology. However, new alternative ecotoxicological methods must be adapted for applications in industry and for regulatory purposes; such methods must be cost-effective, simple to manipulate and provide rapid results. Therefore, we compared the effectiveness of the traditional hanging drop (HD), orbital shaking (OS), and HD combined with OS (HD+OS) methods on the formation of zebrafish cell line spheroids (ZFL and ZEM2S). Time in HD (3–5 days) and different 96-well plates [flat-bottom or ultra-low attachment of round-bottom (ULA-plates)] in OS were evaluated. Easy handling, rapid spheroid formation, uniform-sized spheroids, and circularity were assessed to identify the best spheroid protocol. Traditional HD alone did not result in ZFL spheroid formation, whereas HD (5 days)+OS did. When using the OS, spheroids only formed on the ULA-plate. Both HD+OS and OS were reproducible in size (177.50 ± 2.81 µm and 225.62 ± 19.20 µm, respectively) and circularity (0.83 ± 0.02 and 0.80 ± 0.01, respectively) of ZFL spheroids. Nevertheless, HD+OS required a considerable time to completely form spheroids (10 days) and intensive handling, whereas the OS was fast (5 days of incubation) and simple. OS also yielded reproducible ZEM2S spheroids in 1 day (226.23 ± 0.57 µm diameter and 0.80 ± 0.01 circularity). In conclusion, OS in ULA-plate is an effective and simple spheroid protocol for high-throughput ecotoxicity testing. This study contributes to identify a fast, reproducible, and simple protocol of single piscine spheroid formation in 96-well plates and supports the application of fish 3D model in industry and academia.

鱼细胞球体是生态毒理学中脊椎动物替代的有前途的 3D 培养模型。然而，新的替代生态毒理学方法必须适用于工业应用和监管目的；此类方法必须具有成本效益、易于操作并能迅速产生结果。因此，我们比较了传统悬滴 (HD)、轨道振动 (OS) 和 HD 结合 OS (HD+OS) 方法对斑马鱼细胞系球体（ZFL 和 ZEM2S）形成的有效性。评估了 OS 中 HD（3-5 天）和不同 96 孔板 [平底或超低圆底附着（ULA 板）] 的时间。对易于处理、快速球体形成、均匀大小的球体和圆度进行了评估，以确定最佳的球体方案。单独的传统 HD 不会导致 ZFL 球体形成，而 HD（5 天）+OS 做到了。使用操作系统时，球体仅在 ULA 板上形成。HD+OS 和 OS 在 ZFL 球体的大小（分别为 177.50 ± 2.81 µm 和 225.62 ± 19.20 µm）和圆度（分别为 0.83 ± 0.02 和 0.80 ± 0.01）方面均具有可重现性。然而，HD+OS 需要相当长的时间才能完全形成球体（10 天）和密集处理，而 OS 快速（孵化 5 天）且简单。OS 还在 1 天内产生了可重复的 ZEM2S 球体（226.23 ± 0.57 µm 直径和 0.80 ± 0.01 圆度）。总之，ULA 板中的 OS 是用于高通量生态毒性测试的有效且简单的球体协议。这项研究有助于确定一种快速、可重复、