As I was looking for a protocol for primary culture of human breast cancer cells, I found widely variable protocols concerning reagents and steps so I wanted to know the stable and common protocol for this culture and what are the reagents I could replace or not essential ?
Thanks
I have cultured HuMEC (human mammary epithelial cells) with success using HuMEC ready medium combined with HuMEC Supplement and Bovine Pituitary Extract. These cells should be cultured the same way as immortalized cell lines, just make sure to change the media every 2 days. Also be sure to not expose them to trypsin for too long as they are extremely sensitive. Good luck!
You can use the medium from Lonza (MEGM™ Mammary Epithelial Cell Growth Medium) in a bullet kit containing essential reagents as BPE, hydrocortisone, hEGF, insulin and gentamicin/ amphotericin-B, it is a medium optimized for HMEC.
Do not forget to replace medium every 2-3 days, remove Trypsin any time you split them, and do not split more than 1:4 because at lower seeding density this cell type could not survive.
You may refer to the book by Dr. R. Ian Freshney, Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, for any difficulty related to culture of mammalian cells. He has got answers to all difficulties encountered during culturing of mammalian cells.
Dear Toni and Angelo, thanks a lot for your valuable advice.
Dear Imran, thanks for this important book it is really useful.
Dear Mohamed, Using Humec medium and supplements such as BPE etc will result in you culturing normal myoepithelial cells from human breast tissue. A lot of people think these are breast cancer epithelial or even normal epithelial cells but they are not. Just try staining them for myoepithelial cell markers and you will see. They also have a classic myoepithelial morphology with a 'shiny' plasma membrane. In mixed 2D culture with normal epithelial cells they form 'walls' around the epithelial colonies. Normal breast epithelial cells need FBS and who knows what primary breast malignant epithelial cells need because, to my knowledge, no one has cultured them in sufficient numbers to be useful in experiments. I do wish people would not forget the humble myoepithelial cell. It does have a very useful role to play in maintaining normal breast architecture.
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