|Cell Type:||Immortalized embryonic mouse fibroblasts with drug resistance to blasticidin and zeocin.|
|Subculturing:||Subculture the cells once they reach 60-80% confluency. NOTE: It is very important not to let the cells get confluent or their ability to support hES cells will be diminished.|
|Growth Conditions:||Cells grow in 10% CO2 at 37C in a humidified incubator. Media: DMEM supplemented with 10% bovine calf serum. Split the cells at a density of ~ 3.3 x 103cells/cm2. Feed every 2 to 3 days with DME supplemented with 10% bovine calf serum. DO NOT use fetal bovine serum.|
|Cryopreservation:||DMEM with 10% bovine calf serum containing 10% sterile DMSO|
|Source:||12-13 day mouse embryos|
MMM cells are an immortalized murine cell line developed from embryos of 12-13 days of gestation. The line is described in an article entitled "An Immortalized Drug-Resistant Cell Line Established From 12-13 Day Mouse Embryos For The Propagation Of Human Embryonic Stem Cells" by S. Iuchi, M. Marsch-Moreno, C. Velez-DelValle, K. Easley, W. Kuri-Harcuch and H. Green,published in Differentiation (2006) 74: 160-166.
For antibiotic selection: zeocin at 100µg/ml and blasticidin S at 5µg/ml in DME + 10% bovine calf serum.
To support hES cells, irradiate MMMbz cells with 6000 rads and plate the cells at 1.3 x 105/cm2 (this corresponds to 2.6 x 106 cells per 60 mm dish and to 1.2 x 106 per 35 mm dish) in DME + 10% bovine calf serum. Add hES cells on the following day in SR medium, which consists of Knockout DMEM, supplemented with 20% Knockout Serum Replacement (Knockout SR), 1mM L-glutamine, 1% MEM non-essential amino acids, 0.1 mM 2-mercaptoethanol (BME), and bFGF at 4 ng/ml.
- Iuchi S, Marsch-Moreno M, Velez-DelValle C, Easley K, Kuri-Harcuch W, Green H. An immortalized drug-resistant cell line established from 12-13-day mouse embryos for the propagation of human embryonic stem cells. Differentiation. 2006 Apr;74(4):160-6.
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