Jul 23, 2020
Passaging adherent cancer cell lines
- Emily Souster1,
- Verity Goodwin1,
- Adam Jackson1,
- Charlotte Beaver1,
- Fiona Behan1,
- Rizwan Ansari1,
- Mathew Garnett1
- 1Wellcome Sanger Institute
Protocol Citation: Emily Souster, Verity Goodwin, Adam Jackson, Charlotte Beaver, Fiona Behan, Rizwan Ansari, Mathew Garnett 2020. Passaging adherent cancer cell lines. protocols.io https://dx.doi.org/10.17504/protocols.io.bgtbjwin
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 24, 2020
Last Modified: July 23, 2020
Protocol Integer ID: 37443
Abstract
This SOP is for the routine maintenance and expansion of adherent cancer cell lines.
It also details how cells are harvested and counted for use in downstream protocols.
This protocol is also used in expasion of Cas9 transduced cell lines, prior to banking.
Process diagram:
Guidelines
- Cancer cells should be passaged at ~80-90% confluent where possible. They should not be allowed to reach 100% confluence.
Figure 1. Adherent cancer cell line ready to be passaged (x5).
Materials
MATERIALS
Falcon™ 15mL Conical Centrifuge TubesFisher ScientificCatalog #14-959-53A
TrypLE™ Express Enzyme (1X), no phenol redThermo FisherCatalog #12604021
Cell culture treated T75 flasksCorningCatalog #430641
Cell Culture Treated T150 FlasksCorningCatalog #430825
DPBSInvitrogen - Thermo FisherCatalog #14190
10mg/ml BlasticidinInvivoGenCatalog #ant-bl-1
Select an appropriate culture media for your cell line. Common culture medias used for cancer cell lines are serum supplemented Advanced DMEM F-12 or RPMI, in the presence of pen-strep.
Equipment
Microbiological Safety Cabinet (MSC)
Light Microscope
Nucleocounter/Cell counter
Pipette Boy
Stripettes
Centrifuge
Pipettes and tips
37 °C , 5% CO2 incubator
Safety warnings
- Blasticidin is toxic if swallowed and harmful in contact with skin.
Before start
- Pre-warm complete culture media to room-temperature.
- Check the cells under a microscope and record percentage confluency. Cancer cells should be passaged when ~80-90% confluent.
Detaching and collecting cells
Detaching and collecting cells
Remove the old culture media using a sterile aspirator pipette.
Gently wash the cells with PBS (see Table 1, column B for recommended volume). Aspirate the PBS from the flask.
Note
Always aim to add reagents at the side/back of the flask initially to avoid dislodging any cells.
| Size of flask | Volume of PBS (ml) | Volume of TrypLE (ml) | Volume of media and cells (ml) | |
| T25 | 5 | 3 | 7 | |
| T75 | 7.5 | 4 | 12 | |
| T150 | 10 | 5 | 24 | |
| T525 (3 layer) | 30 | 15 | 60 | |
| T875 (5 layer) | 50 | 25 | 100 |
Table 1. Recommended volumes for different flask types.
Add sufficient TrypLE Express Enzyme to cover the bottom of the flask (see Table 1, column C for recommended volume). Rock the flask to allow the TrypLE to cover the surface of the cells.
Incubate the flask at 37 °C for 3-5 minutes. Use the microscope to check if the cells have detached. If necessary, tap the side of the flask, or return to incubator until all cells have detached.
Add fresh media to the flask at an equal volume to the TrypLE added in Step 3. Use the liquid in the flask to rinse the cells off the bottom of the flask. Transfer the entire volume to an appropriately sized sterile tube.
Spin the tube at 300 x g for 3 minutes.
Safety information
Tubes should be spun inside centrifuge buckets with sealed safety caps. After spinning, the sealed bucket should be transferred to a microbiology safety cabinet before removing the tubes.
Aspirate the supernatant, taking care to avoid disturbing the cell pellet. Resuspend the pellet in an appropriate volume of fresh media (around 5 mL -20 mL ) and mix well to ensure a single cell suspension.
If a cell count is not required, proceed to Step 9.
To take a cell count, aliquot 100 µL of cell suspension and count using your preferred method.
Re-seeding cells
Re-seeding cells
Add fresh media and an appropriate volume of cell suspension to a new flask, to achieve the desired split ratio or cell number (where possible, aim for the total volume of media and cells in the flask to follow the recommeded volumes in Table 1, column D).
For example, if splitting a T75 flask 1:2, either add the entire cell suspension to fresh media in a T150 flask (achieving a total volume of 24 mL ), OR add half the cell suspension to fresh media in a T75 (achieving a total volume of 12 mL ).
Note
The required split ratio will be dependend on the growth rate and behaviour of individual cell lines.
If maintaining a selected population of Cas9 cells, add blasticidin to achieve the desired concentration, as determined by: 'Blasticidin titration of cancer cell lines' protocol.
Add the required volume of 10mg/ml blasticidin depending on the flask size (Table 2), to achieve the desired final concentration.
| Size of flask | Volume of media (ml) | 10µg/ml Blasticidin (µl) | 25µg/ml Blasticidin (µl) | 50µg/ml Blasticidin (µl) | 75µg/ml Blasticidin (µl) | |
| T25 | 7 | 7 | 17.5 | 35 | 52.5 | |
| T75 | 12 | 12 | 30 | 60 | 90 | |
| T150 | 24 | 24 | 60 | 120 | 180 | |
| T525 (3 layer) | 60 | 60 | 150 | 300 | 450 | |
| T875 (5 layer) | 100 | 100 | 250 | 500 | 750 |
Table 2. Volume of 10mg/ml blasticidin stock required in different media volumes to achieve the final concentrations shown.
Safety information
Blasticidin is toxic if swallowed and harmful in contact with skin.
Transfer the flask to a 37 °C , 5% CO2 incubator. Agitate the flask gently back and forth and side to side to ensure even distribution of cells across the flask.
Inspect the cells daily and record confluency. Cancer cell lines should have a complete media change twice per week, or as needed if the media looks yellow.