Hana Hakami
2011
Culture of Breast Cancer Cell Lines
Introduction
Breast cell is adherent cell. So, we need to a good substrate for
cell adhesion when they are growing in vitro.
Breast Cancer Cell Lines
There are different types of breast cancer cell lines. The most
famous are MDA-MB-231 , MCF-7 and T-47D.
Materials
Regular Medium – Freezing Media – Fetal Bovine Serum –
Penicillin/Streptomycin – 1x PBS (Phosphate Buffer Slain) – 0.25 %
Trypsin EDTA - Flasks or Petri Dishes – Centrifuge Tubes – Cryovials –
Serological Pipette – Pipettes (different sizes) – 70% Ethanol – Clorex –
Kim Wipes Tissue – Hood – Vacuum Pump - Centrifuge
Media Preparation
Breast Cancer Cell Line
Complete Media Contents
MDA-MB-231
RPMI 1640 + 10 % FBS + 1% AB
MDA-MB-468
DMEM + 10 % FBS + 1% AB
MCF-7
RPMI 1640 + 10 % FBS + 1% AB
T-47D
RPMI 1640 + 10 % FBS + 1% AB
BT-20
DMEM + 1% L-Glutamine + 10 % FBS + 1% AB
SK-BR-3
McCoy's + 10 % FBS + 1% AB
FBS: fetal bovine serum
AB: antibiotic/antimycotic
Cell Culture Thawing
1. Ware gloves and open the liquid nitrogen tank.
Hana Hakami
2011
2. Take the cryovial from the box and place it in the incubator for 2-3
minutes to thaw.
3. Open the cryovail and transfer the cells to 15 ml tube.
4. Add 4 ml media and pipette up and down.
5. Centrifuge it for 5 minutes 2000 rpm.
6. Discard the supernatant and wash the cells by adding 4ml media.
7. Centrifuge it for 5 minutes 2000 rpm.
8. Discard the supernatant and add 4 ml media and pipette up and
down.
9. Transfer the cells to 25T flask, label it and place it in the incubator.
Cell Culture Feeding
1. Check the cells under the inverted microscope to confirm that the
cells are healthy.
2. Check the color of the media re-feed when the media goes yellow
3. Discard from old media.
4. Wash briefly with PBS.
5. Add new complete media to the cells.
6. Replace the flask in the incubator.
Subculture of Adherent Cells (Splitting)
1. Remove media, wash with 5ml PBS and leave it 4 minutes, then
remove it.
2. Add 1 ml Trypsin, incubate 2-5 minutes at 37˚C until the cell can
be seen to be detaching ( check under microscope).
3. Add 5 ml media containing (FBS) and pipette around the flask to
remove the cells.
4. Transfer all the harvested cells to 15 ml tube.
5. Add another 5ml media to the flask to make sure all the cells are
taken.
Hana Hakami
2011
6. Centrifuge the tube for 5 minutes at 3000 rpm.
7. Discard the supernatant.
8. Add 8ml media and pipette up and down and divide it to 2 flasks
9. Replace the flask in the incubator.
Cell Culture Freezing
1. Label vials with a permanent marking pen. List the cell name,
number of cells in vials, and freezing date including passage
number if known.
2. Make fresh freezing medium. In many cases it is the best to have
the freezing medium chilled to 4˚C. The freezing media formula is:
 50% Regular Growth media.
 40% FBS.
 10% DMSO (Dimethyl Sulfoxide).
3. Trysinize the cells
4. Pipette cells of flask into a sterile 15 ml conical tube.
5. Centrifuge on 1000-2000 rpm for 5 minutes.
6. Discard the supernatant.
7. Re-suspend in freezing Mix and transfer to labeled cryovials.
8. Freeze in -80˚C overnight in isopropanol container.
9. Transfer the vials on the next day to liquid nitrogen tank.