Human ES cells (H1 and H9) were usually maintained in specific media on Matrigel-coated tissue culture plates32 (link). Cells were passaged routinely with EDTA as described previously13 (link). Briefly, cells were washed twice with PBS/EDTA medium (0.5 mM EDTA in PBS, osmolarity 340 mOsm), then incubated with PBS/EDTA for 5 minutes at 37°C. PBS/EDTA was removed, and cells were washed off swiftly with a small volume of corresponding media.
E8 media composition: Media contained DMEM/F12, L-ascorbic acid-2-phosphate magnesium (64 mg/l), sodium selenium (14 µg/l), FGF2 (100 µg/l), insulin (19.4 mg/l), NaHCO3 (543 mg/l) and transferrin (10.7 mg/l), TGFβ1(2 µg/l) or NODAL (100 µg/l). Osmolarity of all media was adjusted to 340 mOsm at pH7.4. All the media were stored at 4°C, and were used within 2 weeks of production. L-ascorbic acid-2-phosphate magnesium is the stable form of L-ascorbic acid in cell culture.
E8 media composition: Media contained DMEM/F12, L-ascorbic acid-2-phosphate magnesium (64 mg/l), sodium selenium (14 µg/l), FGF2 (100 µg/l), insulin (19.4 mg/l), NaHCO3 (543 mg/l) and transferrin (10.7 mg/l), TGFβ1(2 µg/l) or NODAL (100 µg/l). Osmolarity of all media was adjusted to 340 mOsm at pH7.4. All the media were stored at 4°C, and were used within 2 weeks of production. L-ascorbic acid-2-phosphate magnesium is the stable form of L-ascorbic acid in cell culture.
