Human pluripotent stem cells (hPSCs) are inherently sensitive cells. Single-cell dissociation and the establishment of clonal cell lines have been long-standing challenges. This inefficiency of cell cloning represents a major obstacle for the standardization and streamlining of gene editing in induced pluripotent stem cells for basic and translational research. Here we describe a chemically defined protocol for robust single-cell cloning using microfluidics-based cell sorting in combination with the CEPT small-molecule cocktail. This advanced strategy promotes the viability and cell fitness of self-renewing stem cells. The use of low-pressure microfluidic cell dispensing ensures gentle and rapid dispensing of single cells into 96- and 384-well plates, while the fast-acting CEPT cocktail minimizes cellular stress and maintains cell structure and function immediately after cell dissociation. The protocol also facilitates clone picking and produces genetically stable clonal cell lines from hPSCs in a safe and cost-efficient fashion. Depending on the proliferation rate of the clone derived from a single cell, this protocol can be completed in 7–14 d and requires experience with aseptic cell culture techniques. Altogether, the relative ease, scalability and robustness of this workflow should boost gene editing in hPSCs and leverage a wide range of applications, including cell line development (e.g., reporter and isogenic cell lines), disease modeling and applications in regenerative medicine.

人类多能干细胞（hPSC）本质上是敏感细胞。单细胞解离和克隆细胞系的建立一直是长期存在的挑战。细胞克隆的低效率是基础和转化研究中诱导多能干细胞基因编辑标准化和简化的主要障碍。在这里，我们描述了一种化学定义的方案，使用基于微流体的细胞分选结合 CEPT 小分子混合物进行稳健的单细胞克隆。这种先进的策略可促进自我更新干细胞的活力和细胞适应性。使用低压微流体细胞分配可确保将单细胞轻柔、快速地分配到 96 孔板和 384 孔板中，而快速起效的 CEPT 混合物可最大程度地减少细胞应激，并在细胞解离后立即维持细胞结构和功能。该方案还有助于克隆挑选，并以安全且经济高效的方式从 hPSC 中产生遗传稳定的克隆细胞系。根据来自单个细胞的克隆的增殖率，该协议可以在 7-14 天内完成，并且需要无菌细胞培养技术的经验。总而言之，该工作流程的相对简易性、可扩展性和稳健性应该会促进 hPSC 中的基因编辑，并利用广泛的应用，包括细胞系开发（例如报告细胞系和同基因细胞系）、疾病建模和再生医学应用。