Culturing HMEC, vHMEC and HME lines (PJ Keller)

Culturing HMECs ( Click to Download )

HME/C lines are slower to trypsinize than most cells. Use care when passaging to avoid over-trypsinizing and damaging cells.


  1. Grow cells to 70-80% confluence in MEGM media.
  2. Aspirate media from the plate; rinse with 10 ml PBS to remove cellular debris and traces of serum (though most HMEC lines are grown in serum-free conditions, this step is still done to wash the cells).
  3. Pipet 2 ml of trysin-EDTA  (we use 0.05% Trypsin) onto the cells in the dish (full coverage is essential for trypsinizing the HMECs, use up to 3 ml for 10 cm plate).  Incubate at 37oC for 6 minutes to dislodge most cells. Firmly tap the plate on the side of your hand to assist dislodging. Pipet the trypsin forcefully over the cells several times with a p1000 pipet tip to further dislodge any stubbornly stuck cells. If cells are still not dislodging do not look balled up at all under the microscope, return the cells to the incubator for 1-2 minutes more and repeat.
  4. For a 10 cm plate, pipet 8-10 ml of OM media onto the plate while tilting the plate.  Rinse the cells off the dish moving the pipet from the top to the bottom of the tilted plate.  Repeat several times.  Transfer cells to a 15 ml conical tube.
    1. If passaging cells for growth curves, it is useful to remove the cells from the plate with 5 mls and then rinse the plate with an additional 5 mls of media to make sure all cells are collected for counting.
  5. Pellet cells at 1000-1200 rpm for 5 minutes.
  6. Prepare the plate(s) you are passaging the cells onto by labeling them with the new passage number (P1, P2 etc.). Pipet 9 ml of MEGM media onto each plate.
  7. Resuspend the pellet in the amount of growth media that corresponds to the ‘split’ you are doing.  For a 1:4 split, for example, resuspend the cells in 4 mls of media. Pipet up and down until the pellet disappears; transfer 1 ml of the cells to the prepared plate(s).  Swirl and rock the plates to evenly distribute the cells and place in the incubator. In general, HMECs should be split 1:3 to 1:5, about 1X per week.
    1. If you are passaging the cells for growth curves, resuspend the cells in 2 mls of MEGM and count. Plate a defined number of cells for your passage. To calculate population doublings use the formula: PD = log(A/B)/log2 where A is the cells collected and B is the number of cells plated initially (Holst, CR et al. 2003 Cancer Research)
  8. To freeze cells, follow protocol through step 5. Resuspend at 1-2 million cells per ml in freezing media (OM media + 10 % DMSO and extra 5-10% CS). Aliquot 1 ml per vial and freeze in isopropanol freezing container at -80oC for up to 3 weeks. Transfer to liquid nitrogen for long term storage.

MEGM (MEBM + Bullet kit)

Purchased from Lonza (Cat# CC-3150)

MEBM basal media +

Bullet kit: Insulin (5 µg/ml), Hydrocortisone (0.5 µg/ml), hEGF (10 ng/ml), Pituitary Extract (52 µg/ml), Gentamycin (optional)

1% Antibiotic/Antimycotic (Invitrogen)

Organoid Media (OM)

DMEM/F12 1:1 formulation (Invitrogen)

5% calf serum

Insulin (5 µg/ml), Hydrocortisone (0.5 µg/ml), hEGF (10 ng/ml)

1% Antibiotic/Antimycotic (Invitrogen)