CultureofHumanEmbryonicStemCells(hESC)

All cell lines are initially grown according to the supplier's protocols but we are adapting them to one simple protocol outlined below:

• 6-well plates (Falcon Cat #353046) are coated for 20 to 60 minutes at room temperature with 0.1% gelatin (Sigma Cat #G1890) in dH₂O.

• Mouse embryonic fibroblasts (CF1 strain), cultured in MEF medium, are mitotically inactivated by treatment with 10μg/ml mitomycin C (Roche Cat #107 409) for 2 to 3 hours at 37°C. Cells are washed three to four times with PBS, trypsinized (Invitrogen Cat #25300-054), and plated at a density of 0.75 x 10⁵/ml with 2.5ml per well of a gelatin-coated 6-well dish. Alternatively, cells may be inactivated by exposure to 8000rads of X-irradiation and plated at the same density.

• Immediately before plating hESC, MEFs are rinsed once or twice with PBS. hESC are plated onto MEFs as small clumps in 2.5ml per well of hESC medium containing 4ng/ml bFGF (R&D Systems Cat #233-FB). Cells are fed every day until ready to passage which is determined by the size of colonies, the age of MEFs (should not be older than 2 weeks) or differentiation status of the cells.

• Colonies which appear to be differentiating, are manually removed before passaging.

• To passage hESC, cells are washed once or twice with PBS and incubated with filter-sterilized 1mg/ml collagenase IV (Invitrogen Cat #17104-019) in DMEM/F12 for 10 to 30 minutes. Plates should be agitated every 10 minutes until colonies begin to detach. When moderate tapping of the plate causes the colonies to dislodge, they are collected and the wells washed with hESC medium to collect any remaining hESC. Alternatively, colonies may be removed using a cell scraper and collected.

• Colonies are allowed to sediment for 5 to 10 minutes. The supernatant, containing residual MEFs, is aspirated, and the colonies are washed with 5ml hESC medium and allowed to sediment again. This is repeated once more.

• After the final sedimentation, the colonies are resuspended in 1ml of hES medium and triturated gently to break up the colonies to approximately 100-cell size. Generally, cell lines are passaged at a ratio of between 1:3 and 1:6 every four to seven days.