我们描述了一种使用悬浮生物反应器从人类多能干细胞中稳健生成 3D 胰岛样类器官的可扩展方法。我们的方案涉及 6 个阶段、20 天的定向分化过程，从而产生 104-105 个类器官。这些类器官包含具有葡萄糖反应性胰岛素和胰高血糖素分泌的 α 和 β 样细胞。我们详细介绍了将干细胞培养、传代和冷冻保存为悬浮簇的方法，以及通过特定的生长培养基和逐步添加的外源因子来分化它们的方法。此外，我们还致力于研究应用的质量控制措施、故障排除策略和功能测定。© 2024。编辑（如果适用）和作者获得 Springer Science Business Media, LLC（该公司的一部分）的独家许可。施普林格自然。

We describe a scalable method for the robust generation of 3D pancreatic islet-like organoids from human pluripotent stem cells using suspension bioreactors. Our protocol involves a 6-stage, 20-day directed differentiation process, resulting in the production of 104-105 organoids. These organoids comprise α- and β-like cells that exhibit glucose-responsive insulin and glucagon secretion. We detail methods for culturing, passaging, and cryopreserving stem cells as suspended clusters and for differentiating them through specific growth media and exogenous factors added in a stepwise manner. Additionally, we address quality control measures, troubleshooting strategies, and functional assays for research applications.© 2024. The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.