Robust protocol for feeder-free adaptation of cryopreserved human pluripotent stem cells. 
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.