Maintenance tips from donors (DRSC)
Maintenance: Cells grow at room temperature to 30C in vented flasks. High humidity in the incubator is advisable, though not necessary. Split every 4-5 days depending on density. When the culture becomes overgrown, it will start to cluster and detach progressively as they die. Cells can slowly recover from low densities but generally do not grow well at low densities.
Subculturing: Cells are very adherent. When confluent, cells are washed multiple times in PBS and treated for several minutes with Accumax (Innovative Cell Technologies #AM105). Any remaining attached cells, if present, are removed with a cell scraper and resuspended by pipetting in culture media. Cells can be subcultured with a ratio of 1:3-1:6. It is not necessary but recommended to use 10% of the previous growth media (conditioned media) while splitting to ensure prompt growth. Storage: Sub-confluent cells can be washed multiple times in PBS, centrifuged, and resuspended in freezing solution (FBS + 10% DMSO), aliquoted in cryovials, and slow frozen to -80C (using Styrofoam or Isopropanol filled containers) for at least 4h and transferred in liquid nitrogen for long term storage. When recovering the cells from a frozen stock use conditioned media if available and increase FBS concentration in the medium to 20%. Penicillin/Streptomycin This line has been cultured in the presence of antibiotics (Pen/Strep 1X), and we continued to culture it as such at the DGRC.