Robust protocol for feeder-free adaptation of cryopreserved human pluripotent stem cells
Robust protocol for feeder-free adaptation of cryopreserved human pluripotent stem cells
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DOI：
10.1007/s11626-019-00413-9
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发表时间：
2019-10-29
影响因子：
2.1
通讯作者：
van Hengel, Jolanda
中科院分区：
文献类型：
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作者：
Aalders, Jeffrey;Van den Vreken, Natasja;van Hengel, Jolanda
人多能干细胞（hPSC）通常维持在小鼠胚胎成纤维细胞（MEF）饲养层上。然而，下游应用，如定向分化方案，主要针对无饲养层培养物进行优化。因此，hPSC必须经常适应无饲养层的条件。在这里，我们提出了一种使用StemFlex培养基的新型无饲养层适应方案，该方案可直接应用于解冻的hPSC细胞系。在Geltrex涂层上使用StemFlex培养基的直接无饲养层适应方案在约2周内产生稳健的hPSC培养物。用三种人胚胎干细胞（hESC）系测试了这种方法。证实所有细胞系都是多能的，表达POU5F1、SOX 2和NANOG。无饲养层适应没有引起染色体不平衡。StemFlex培养基使hPSC能够在解冻后直接有效适应无饲养层条件。该方案易于在进行无饲养层培养的实验室中实施，即使在样品质量低或未知的情况下，也允许更方便地适应和更稳健地扩增冷冻保存的hPSC。
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.