Cells can be grown in serum-free MEGM media (LONZA, CC-3051, $81/bottle) or DME/F12 with supplements (However the latter will not expand the same types of cells). More detailed methods on culturing the cells can be found in the Clonetics instruction manual included with the MEGM media.
- Wash cells with PBS and add high concentration trypsin (0.15%) to the plate. Incubate the plate at 37oC until the cells detach (usually about 3-5 minutes).
- Add 4 volumes of DME + 10% fetal bovine serum or Trypsin Neutralizing Solution (Clonetics) to inactivate the trypsin.
- Place cells in an appropriate sized tube and spin as usual. Remove supe, resuspend cell pellet in MEGM media and plate. The serum can inhibit the growth of these HMECs so it needs to be removed before replating in serum-free media.
These cells grow very slowly if split too sparsely. Split them 1:4 or 1:5 at most.
Refeed cells every 2-3 days with fresh media.
These cells can undergo an epithelial-to-mesenchymal transition in culture if not handled properly. We have used immunofluorescence staining of cytokeratins 14 and 18 or immunobloting for E-cadherin to confirm their epithelial character.
I typically freeze the cells in a solution of (75% MEGM media, 15% calf serum, 10% DMSO). I thaw the cells in the morning by placing them directly onto a 10 cm plate containing 10 ml warm media. After 6-8 hour, remove the media containing dead cells and add fresh media.
We typically grow the cells with drug co-expressed with Ras (so below) to help maintain it’s expression levels. However, there is apparently a strong biological selection pressure to maintain expression of LT and TERT so we do not need to add drugs for their co-expressed drug-resistance markers.
200 mg/ml G418 for neomycin (neo), 50 mg/ml hygromycin (hygro), 0.5 mg/ml puromycin (puro) or 500 mg/ml zeocin (zeo).
We have found that it is difficult to maintain high levels of H-rasV12 protein in the cells as they are passaged in culture. Initially after infection of a population of HMECs with an H-rasV12-expressing retrovirus, the levels of expression are high (typically 12-15 fold over the endogenous H-Ras protein in the cells. However, as you continue to grow and passage the cells over several weeks, the levels decrease slowly, suggesting that the high-level H-rasV12 expression is not well tolerated in the cells during in vitro passaging. However, this high-level of expression is critical for the tumorigenicity of the cells when injected s.c. into irradiated nude mice. Therefore, it is important to check the levels or Ras protein by western in a given population of cells for experiments relating to the levels of Ras. Also, we typically grow the cells in the presence of drug for the resistance gene associated with the Ras to help maintain the levels of H-rasV12 protein in a given polyclonal population.
