Between passages four to eight, selected feeder-dependent iPSC lines were transferred to feeder-free culture. The feeder-dependent iPSC lines were split and passaged onto both feeder-dependent and feeder-free conditions. The feeder dependent lines continued to be cultured on MEF-CF1 feeder plates in iPSC medium, whilst the feeder free lines were cultured in Essential 8 (E8) medium on tissue culture dishes coated with 10 µg/ml Vitronectin XF (StemCell Technologies, UK, 07180). E8 complete medium consists of basal medium DMEM/F-12(HAM) 1:1(Life technologies, UK, A1517001) supplemented with E8 supplement (50X) (Life technologies, UK, A1517001) and 1% Pen/Strep (Life technologies, UK, 15140122). Media was changed daily. To passage feeder-free iPSC lines, cells were washed with PBS and incubated with PBS-EDTA solution (0.5 mM) for 5-8 minutes. PBS-EDTA solution was removed, and cells were resuspended in E8 medium and seeded at split ratios ranging from 1:3 to 1:6 onto Vitronectin coated tissue culture dishes. Cells were passaged every four to seven days (depending on the confluence and morphology of the cells). Once the feeder-free iPSC lines were established in culture, the cells were expanded for banking and characterisation.
