Supporting cells, such as short-term cultures of fibroblasts, are used to cultivate human embryonic stem (ES) cells, however they are susceptible to senescence after a number of passages. The Mouse Embryonic Blasticidin and Zeocin-resistant (MMMbz) Immortalized Feeder Cells eliminates the problems associated with the use of primary cell lines. This cell line has a doubling time of 16 hours and may be used indefinitely to propagate human embryonic stem cells. It has the ability to facilitate the growth of hES H9 and hES H1. MMMbz cells are resistant to blasticidin and zeocin due to transgene expression allowing for selection of hES cells transfected with genes conferring resistance.
Biosafety Level
BioSafety Level II
Growth Properties
Adherent
Quantity
106 cells/1.0 ml
Morphology
Fibroblast-like
Species
Mouse (M. musculus)
Propagation
Grow cells in T25 ECM-coated flasks (G299) with the following conditions. The base medium for this cell line is Prigrow III medium available at abm, Cat. No. TM003. To make the completed growth medium, add the following components to the base medium: Bovine Calf Serum (available from Hyclone, SH30072) to final concentration of 10% and Penicillin/Streptomycin Solution (G255) to a final concentration of 1%. Carbon dioxide (CO2): 5%, Temperature: 37.0°C. To freeze: suspend cells in PriGrow III (TM003) with 10% bovine calf serum and 10% sterile DMSO. To support the growth of hES cells: irradiate MMMbz cells with 6000 rads and seed at a density of 1.3x105/cm2 in propagation medium. The next day add hES in Knockout DMEM (Invitrogen), supplemented with 20% Knockout Serum Replacement (Invitrogen), 1mM L-glutamine (G275), 1% MEM non-essential amino acids (Invitrogen), 0.1mM 2-mercaptoethanol (CH045) and bFGF at 4 ng/ml (Z200015).
Source Organ
Reproductive
Quality Control
Selective growth with media containing blasticidin and zeocin to select for cells with transgene expression.
Shipping
Dry Ice
Storage
-180°C
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