本文介绍了一种利用人多能干细胞在悬浮生物反应器中产生三维胰样器官的方案。我们描述了一种可扩展的技术，可以生成10,000-100,000个器官，经过4-5周进一步分化为具有葡萄糖响应的胰岛素和胰高血糖素释放的α和β细胞。我们详细说明了培养、传代和冷冻保存干细胞悬浮团块的步骤，并指定了分化所需的培养基和分化因子。最后，我们讨论了用于研究应用的功能性评估方法。有关使用和执行该方案的完整细节，请参阅Alvarez-Dominguez 等1。版权所有© 2023 The Authors. Published by Elsevier Inc. All rights reserved.

Here, we present a protocol for producing 3D pancreatic-like organoids from human pluripotent stem cells in suspension bioreactors. We describe scalable techniques for generating 10,000-100,000 organoids that further mature in 4-5 weeks into α- and β-like cells with glucose-responsive insulin and glucagon release. We detail procedures for culturing, passaging, and cryopreserving stem cells as suspended clusters and specify growth media and differentiation factors for differentiation. Finally, we discuss functional assays for research applications. For complete details on the use and execution of this protocol, please refer to Alvarez-Dominguez et al.1.Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.