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hESC line (RCM1) was obtained from a failed-to-fertilize inseminated egg recovered by parthenogenetic activation. Standard in vitro and in vivo characterization demonstrated that this line to possesses all of the properties attributed to a normal euploid hESC line. Cells have a normal 46XX female karyotype.
For feeder-free culture, grow RCM1 in HDF-CM (human dermal fibroblast-conditioned medium) on a substrate of either 1:30 Matrigel or a mixture of four extracellular matrix proteins as described by Ludwig et al. (2006), namely laminin, fibronectin, vitronectin, and collagen IV. Preparation of HDF-CM involves mitotic inactivation of HDFs followed by exposure to and conditioning of fully supplemented KO DMEM/SR medium (80% Knockout-DMEM, Knockout serum replacement (Knockout SR, 20%), glutamine (1 mM), β-mercaptoethanol (0.1 mM), nonessential amino acids (1%), and human bFGF (4 ng/ml)) over 24-h intervals over 7 days. Just before usage, frozen HDF-CM can be thawed, filtered for sterility through a 0.2-μm filter, and supplemented with an additional 24 ng/ml human bFGF.