A stress-reduced passaging technique improves the viability of human pluripotent cells.
A stress-reduced passaging technique improves the viability of human pluripotent cells.
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DOI：
10.1016/j.crmeth.2021.100155
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发表时间：
2022-02-28
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无异种培养系统扩大了人类多能干细胞（PSC）的临床和工业应用。然而，再现性问题，经常引起的变异性在传代步骤中，仍然存在。在这里，我们描述了一种改进的方法，用于人PSC的传代培养。改进后的方法通过降低DNA损伤和细胞凋亡显著提高了人PSC的活力，从而实现了更有效和可重复的下游应用，如基因编辑和定向分化。此外，该方法在长期培养后不改变PSC特征，并减弱异常亚群的生长优势。这种稳健的传代方法最大限度地减少了实验误差，降低了PSC在人PSC研究和应用中质量控制失败的比率。一种改进的传代方法显著增加了人PSC的活力。与常规方法相比，该方法引发较少的DNA损伤和凋亡。应力降低的方法改善了下游应用的结果。该方法减弱了高活力异常亚群的过度生长。尽管用于人多能干细胞（PSC）的化学限定的培养系统被广泛使用，PSC克隆间传代后的细胞活力通常是可变的，导致再现性的问题。为了提高先进PSC培养系统的效率和再现性，我们通过优化细胞分离和解离的程序开发了一种改进的传代方法。修改后的传代方法提高了细胞活力，使下游应用更有效和可重复。Takahashi等人修改在重组基质上培养的人多能干细胞的传代方法。修订后的方案通过减少DNA损伤和细胞凋亡来提高细胞活力，提高下游应用的效率和重现性，并减弱异常细胞群的过度生长。
Xeno-free culture systems have expanded the clinical and industrial application of human pluripotent stem cells (PSCs). However, reproducibility issues, often arising from variability during passaging steps, remain. Here, we describe an improved method for the subculture of human PSCs. The revised method significantly enhances the viability of human PSCs by lowering DNA damage and apoptosis, resulting in more efficient and reproducible downstream applications such as gene editing and directed differentiation. Furthermore, the method does not alter PSC characteristics after long-term culture and attenuates the growth advantage of abnormal subpopulations. This robust passaging method minimizes experimental error and reduces the rate of PSCs failing quality control of human PSC research and application. An improved passaging method significantly increases the viability of human PSCs The method triggers less DNA damage and apoptosis compared to the conventional method The stress-reduced method improves the results of downstream applications The method attenuates the overgrowth of highly viable abnormal subpopulations Although chemically defined culture systems for human pluripotent stem cells (PSCs) are in widespread use, cell viability after passaging among PSC clones is often variable, leading to issues with reproducibility. To improve the efficiency and reproducibility of advanced PSC culture systems, we have developed an improved passaging method by optimizing procedures for cell detachment and dissociation. The revised passaging method improves cell viability and makes downstream applications more efficient and reproducible. Takahashi et al. modify the passaging method for human pluripotent stem cells cultured on recombinant matrices. The revised protocol increases cell viability by reducing DNA damage and apoptosis, improves the efficiency and reproducibility of downstream applications, and attenuates the overgrowth of abnormal cell populations.