This user guide provides a practical starting framework for transitioning adherent human pluripotent stem cells (hPSCs), including ESCs and iPSCs, from conventional high-protein maintenance media such as mTeSR™ Plus to the fully defined, animal-free HiDef® B8 medium. The guide outlines a recommended direct adaptation workflow in which cells are switched to 100% HiDef-B8 at passage or thaw, with Ready-CEPT® used during inoculation or immediately after passage to support recovery. It also discusses a more gradual adaptation option for sensitive lines, while noting that transition requirements can vary by cell line and that several passages may be needed for growth rates to normalize.

Beyond the initial transition, this guide provides practical recommendations for establishing reliable, weekend-free adherent hPSC culture, including optimal confluence, aggregate passaging with HiDef PBS/EDTA, skip-day feeding, seeding considerations for fragile lines, and the advantages of low-oxygen culture during adaptation. It also highlights the expected morphological shift from the tighter, “island-like” colonies commonly observed in protein-rich media to the looser, “continent-like” morphology characteristic of cells maintained in HiDef-B8, without loss of pluripotency. Together with published evidence supporting long-term pluripotency, genomic stability, and performance across numerous PSC lines, this guide gives researchers a straightforward path for moving toward a lower-protein, chemically defined, animal-free culture system that is compatible with both routine research and future cGMP-aligned workflows.