Animal Free Culturing of hESCs

1. 0.1% gelatin (type A, from porcine) (Sigma-Aldrich Inc.; cat. no. G-1890). All culture dishes should be covered with 0.1% gelatin at least 1 h before plating foreskin fibroblasts (see Note 4).

2. Foreskin fibroblast culture medium: 80% DMEM, 20% human serum, 2 mM L-glutamine (Invitrogen Corporation; cat. no. 25030-024) (see Notes 3 and 5). For the derivation of foreskin fibroblasts, supplement media with the addition of penicillin-streptomycin (Sigma-Aldrich Inc.; cat. no. P-3539) and kanamycin (Amresco, Solon, OH; cat. no. 0408-106).

3. hESC-medium: 80% DMEM or knockout DMEM (ko-DMEM; Invitrogen; cat. no. 10829018), 20% human serum (Sera Care Life Sciences, Inc., Oceanside, CA; cat. no. CC-5520), 1% nonessential amino acids (Invitrogen; cat. no. 11140-035), 1 mM L-glutamine, and 0.1 mM ß-mercaptoethanol (Invitrogen; cat. no. 31350010) (see Note 3).

4. hESCs serum-free medium: 85% ko-DMEM, 15% SR, 1% nonessential amino acids, 1 mM L-glutamine, 0.1 mM ß-mercaptoethanol, 4 ng/mL human recombinant bFGF (Invitrogen; cat. no. 13256-029). For the preparation of 500 mL of culture medium: add 75 mL SR, 5 mL nonessential amino acids, 2.5 mL L-glutamine, 1 mL ß-mercaptoethanol, 2000 ng bFGF, and 416.5 mL ko-DMEM into a filter unit and filter (see Note 3).

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