HMEC Cell Line

human mammary epithelial cells

HMEC Cell Culture

HMEC Cell Culture Information

HMEC cells should be cultured in Mammary Epithelial Growth Media (MEGM) or MCDB 170. This needs to be supplemented with the following:

HMEC Cell Culture
  • 5 ng/mL EGF (20,000X stock, 100ug/mL)
  • 0.5 ug/mL Hydrocortisone (2000X stock, 1 mg/mL)
  • 5 ug/mL Insulin (200X stock, 1 mg/mL)
  • 70 ug/mL BPE (200X stock, 14 mg/mL)
  • 5 ug/mL Transferrin (2000X stock, 10 mg/mL)
  • 10-5 M Isoproterenol (500X stock, 5 x 10-3 M )
  • 10-4 M Ethanolamine (1000X stock, 10-1 M)
  • 10-4 M O-Phosphothanolamine (1000X stock, 10-1 M)

Cells should be grown at 37°C in a low CO2 setting (0.1-2%), as opposed to the typical 5% CO2 cell culture setting. Media should be maintained at the appropriate pH. If the media is yellow, it is indicative of being acidic, whereas a red hue indicates it is too basic. The perfect media is a salmon orange. Cells can be grown in 12-15 mL of media in a T75 flask and should be seeded at 3-5 x 105 density. Note that cells can also be grown in plates for better gas exchange.

HMEC Subculture Protocol

Subculture cells when they are subconfluent, roughly 85-95% confluent. Cells will increase 6-10x in number before confluence if grown at the densities described in the cell culture instructions. Subculture roughly once a week and split cells at a ratio of 1:6 or 1:10.

To subculture:

  1. Make sure cells appear healthy under the microscope
  2. Aspirate media from the parent dish
  3. Wash cells once by adding 3 mL Trypsin (in T75 flask or 10 cm plate) and then aspirating reagent
  4. Detach cells by adding 3 mL of Trypsin and then incubate 2-5 minutes at 37°C
  5. Observe the cells under an inverted microscope to confirm cell detachment
  6. Add at least 9 mL complete media when most cells have detached
  7. Transfer the cell suspension to a 15 mL tube
  8. Centrifuge cells at 800-1000 rpm for ~5 minutes
  9. Aspirate supernatant from the tube being careful not to disturb the cell pellet
  10. Bring the cell pellet to desired density in flask with complete media or freeze cells for long-term storage

HMEC Cryo-Preservation

HMEC cells should be frozen in Glycerol I freezing media. This is a combination of MCDB 170 culture media with 10% glycerol and 15% FCS. Cells should be maintained at a density of 106 cells/mL. After slowly freezing the cells overnight, store in the vial in the vapor phase of liquid nitrogen.

Tips

  • To achieve optimal cell densities, be persistent on changing culture medium daily
  • For maximum proliferating subcultures, subculture HMEC before 90% confluency is reached
  • Damage to HMEC cells can occur during the trypsinization step; damage may result from excessive exposure to Trypsin or too harsh pipetting
  • The number of passages achieved are individual specific and vary with the methods employed by investigators
  • Freshly thawed aliquot: Thawed cells are fragile and should be thawed in less than 1 minute. Do not pellet the cells with centrifugation to remove freezing media.  Rather, the DMSO or Glycerol will be diluted to a low enough concentration by adding the thawed aliquot directly to a flask containing media

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Procedures for Culture of Human Mammary Epithelial Cells

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