title: Cryopreservation and quality control of mouse embryonic feeder cells.
abstract: Stem cell research is a highly promising and rapidly progressing field inside regenerative medicine. Embryonic stem cells (ESCs), reprogrammed "induced pluripotent" cells (iPS), or lately protein induced pluripotent cells (piPS) share one inevitable factor: mouse embryonic feeder cells (MEFs), which are commonly used for ESC long term culture procedures and colony regeneration. These MEFs originate from different mouse strains, are inactivated by different methods and are differently cryopreserved. Incomprehensibly, there are to date no established quality control parameters for MEFs to insure consistency of ESC experiments and culture. Hence, in this work, we developed a bench-top quality control for embryonic feeder cells. According to our findings, MEFs should be inactivated by irradiation (30Gy) and cryopreserved with optimal 10% DMSO at 1K/min freezing velocity. Thawed cells should be free of mycoplasma and should have above 85 ± 13.1% viability. Values for the metabolic activity should be above 150 ± 10.5% and for the combined gene expression of selected marker genes above 225 ± 43.8% compared to non-irradiated, cryopreserved controls. Cells matching these criteria can be utilized for at least 12 days for ESC culture without detaching from the culture dish or disruption of the cell layer.
authors.affiliation: Institute for Transfusion Medicine, Hannover Medical School, Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany.
authors.forename: Ulf
authors.initials: U
authors.lastname: Diekmann
authors.forename: Ralf
authors.initials: R
authors.lastname: Spindler
authors.forename: Willem F
authors.initials: WF
authors.lastname: Wolkers
authors.forename: Birgit
authors.initials: B
authors.lastname: Glasmacher
authors.forename: Thomas
authors.initials: T
authors.lastname: Müller
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D018920:Coculture Techniques; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000166:cytology*; D061252:Feeder Cells / Q000528:radiation effects; D005720:Gamma Rays; D057026:Induced Pluripotent Stem Cells; D051379:Mice; D009174:Mycoplasma / Q000302:isolation & purification; D011786:Quality Control
chemical_list: D003451:Cryoprotective Agents; D004121:Dimethyl Sulfoxide