Human pluripotent stem cells (hPSCs) are conventionally maintained on mouse embryonic fibroblast (MEF) feeder layers. However, downstream applications, such as directed differentiation protocols, are primarily optimized for feeder-free cultures. Therefore, hPSCs must often be adapted to feeder-free conditions. Here we propose a novel feeder-free adaptation protocol using StemFlex medium, which can be directly applied to thawed hPSC lines.The direct feeder-free adaptation protocol using StemFlex culture medium on Geltrex coating led to robust hPSC cultures in approximately 2 weeks. This approach was tested with three human embryonic stem cell (hESC) lines. All lines were confirmed to be pluripotent, expressing POU5F1, SOX2, and NANOG. No chromosomal imbalances were induced by the feeder-free adaptation.StemFlex medium enabled the efficient adaptation of hPSCs to feeder-free conditions directly after thawing. This protocol is easy to implement in laboratories that perform feeder-free cultures, allowing more convenient adaptation and more robust expansion of cryopreserved hPSCs, even in cases when sample quality is low or unknown.

中文翻译：

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用于冷冻保存的人多能干细胞的无饲养适应的鲁棒方案。

人多能干细胞（hPSC）通常保持在小鼠胚胎成纤维细胞（MEF）饲养层上。但是，下游应用（例如定向分化方案）主要针对无饲养层培养进行了优化。因此，hPSC必须经常适应无饲养者的条件。在这里，我们提出了一种使用StemFlex培养基的无饲养者适应新方案，该方案可以直接应用于解冻的hPSC系。在StereFlex涂层上使用StemFlex培养基进行的无饲养者直接适应方案可以在约2周内培养出稳定的hPSC培养物。该方法已通过三种人类胚胎干细胞（hESC）系进行了测试。确认所有品系均具有多能性，表达POU5F1，SOX2和NANOG。无饲养者适应不会引起染色体失衡。StemFlex培养基可在融化后直接使hPSC有效适应无饲养者的条件。该协议易于在进行无饲养员培养的实验室中实施，即使在样品质量较低或未知的情况下，也可以更方便地适应并更有效地扩展冷冻保存的hPSC。