Figure 1.

Schema for freezing/thawing of hPSCs. Passage: One fourth of cell clumps after dissociation with GCDR were seeded onto rhVitronectin (rhVTN)-N-coated dishes and cultured with TeSR-E8 medium for four days before the next passage. Cells were dissociated into single cells with several dissociation reagents (Step 1) and frozen with several cryopreservation mediums (Step 2). All possible combinations of dissociation reagent and cryopreservation medium were tested. Cells were thawed (Step 3) either via cell clumping with AggreWell 400 plates for two days followed by seeding and culturing in TeSR-E8 medium on rhVTN-N-coated dishes for two days (Step 3A) or seeded on rhVTN-N-coated 24-well flat bottom dish at various cell concentrations (6 × 10⁴, 6 × 10³, 6 × 10² cells/well) as non-cell clump formation (single cell suspension) controls (Step 3B).