Whether it's a stock you've expanded following it's arrival from the cell bank, or a clone you've carefully nurtured from the single cell, banking down your cells in the right way is crucial if you're going to be able to return to them time and again, and revive them quickly so that you can get on with your experiments.
Here's our protocol for banking human cell lines:
Reagents and equipment:
- TrypLE® or equivalent cell dissociation buffer (Life Technologies 12605010)
- 15 and 50 mL conical bottom tubes
- Complete cell culture media
- Fetal Bovine Serum (FBS)
- Microcentrifuge tubes
- Trypan blue
We use the following freezing medium depending on cell line type:
- Adherent lines: 50% serum, 45% Basal media, 5% DMSO
- Suspension lines: 50% serum, 40% Basal media, 10% DMSO
1. Pre warm the appropriate culture media for the cell line.
2. Obtain the culture flask(s) with cells to bank growing at up to 80-90% confluence.
3. Aspirate media from flask and place in waste bottle.
4. Wash cells with 10 mL PBS.
5. Add 5 mL of 1x TrypLE® to cells.
6. Incubate cells in a 37°C incubator for 5 minutes (or until cells have detached from surface).
7. Triturate cells 3-4 times with a 5 mL stripette to break up any cell clumps.
8. Transfer the cell suspension to a 50 ml conical bottom tube.
9. Rinse the flask with 10 mL of media and transfer to the 50 mL conical bottom tube and mix gently by pipetting.
10. Add 20 μL 1x Trypan Blue to a microcentrifuge tube.
11. Add 20 μL of the cell suspension to the tube and mix by gentle pipetting.
12. Transfer 20 μL of the cell suspension to a well of disposable hemocytometer.
13. Using an Inverted microscope, count the number of live cells in 3 fields of the hemocytometer (where 1 field is a 4x4 grid) and determine the mean number of cells per field.
14. Concentration (cells/mL) can be determined by the following calculation
Mean (from line 13) x 2 (dilution factor from cell count) x 104 = cells/mL
15. Calculate the number of cryovials needed :
(1 cryovial per 1 mL of cells at 1x106/mL), number = #cryovials required
16. Add labels to the cryovials. Horizon would recommend including the following information: Date, Cell line name, Description, Clone ID, Passage number. Concentration and volume of cells.
17. Calculate the volume of cells needed:
number of vials needed x 1x106 / concentration of cells/mL (from 14) = volume cells required
18. Transfer the calculated volume of cells to a new conical bottom tube.
19. Pellet cells by centrifuging for 3 minutes at 200 x g at room temperature.
20. Remove media from the cell pellet in the conical bottom tube and resuspend the cells in the freezing media (1 mL for every 106 cells pelleted).
21. Aliquot 1 mL of the cell suspension in freezing media into each cryovial.
22. Seal the cryovials and place in a room temperature Mr Frosty. Transfer the Mr Frosty to a -80°C freezer overnight.
23. Transfer the cryovials containing the frozen cells to liquid nitrogen.