TITLE: Standard Cell Culture Practices
AUTHORS: Allan JW Lui

[DESCRIPTION]
Basic cell culture techniques

[BEFORE_START]
Cell culture numbers:
6-well1406759.60.3 x 1061.2 x 106111 to 312-well1506283.50.1 x 1060.5 x 1060.4 to 10.4 to 11 to 224-well1424751.90.05 x 1060.24 x 1060.2 to 0.30.2 to 0.30.5 to 1.048-well1506871.10.03 x 1060.12 x 1060.1 to 0.20.1 to 0.20.2 to 0.496-well1670080.320.01 1060.04 x 1060.05 to 0.10.05 to 0.10.1 to 0.2FlasksT-25156367;156340250.7 x 1062.8 x 106333–5T-75156499;156472752.1 x 1068.4 x 106558–15T-175159910;1599201754.9 x 10623.3 x 106171735–53T-225159934;1599332256.3 x 10630 x 106222245–68
 







[STEPS]
SECTION: Thaw cells
4. Request cells from RICS on or before 9am on the day of planned thaw
SECTION: Thaw cells
5. Confirm items are in the hood pre-thaw:
Warm media
15ml centrifuge tube
1ml pipette & tips
Waste bin
Aspiration pipettes
Motorised pipette controller
SECTION: Cell culture prep
1. Pre-warm media (and reagents to make complete media) +/- trypsin in 37 °C 
 

SECTION: Thaw cells
6. Prepare covered bucket of dry ice
Retrieve vials of frozen cells from -80C freezer and place into dry ice bucket and cover
SECTION: Thaw cells
7. Thaw vial in 37 °C , observing closely until only a small crystal is left, then transfer to hood

SECTION: Cell culture prep
2. While reagents are warming up:
Wipe down microscope table using paper towels and 70% ethanol
Wipe down microscope stage using Kimwipes and 70% ethanol
View cells under microscope
Switch on biosafety cabinet, wipe down using paper towels and 1% Chemgene, then with 70% ethanol
Fill a 1L plastic beaker with ~100-200ml 2% Virkon
Add a tablet of Virkon into a aspirator collection bottle and fill with 250-500ml tap water, then fit into vacuum aspirator and turn on system
 

SECTION: Cell culture prep
2.1. Any time before placing into the hood:
Pipettes: Wipe down using paper towels and 70% ethanol
All racks and containers (of liquids, pipette tips, etc but not cells): Spray down with 70% ethanol
All items in water bath: Wipe down using paper towels and 70% ethanol, then spray down with 70% ethanol

Spray hands with 70% ethanol frequently
SECTION: Thaw cells
8. Using 1ml pipette, transfer contents of vial into a 15ml centrifuge tube
Add 9 mL dropwise
Gently mix by inversion

Pelleting:
Centrifuge at 200 rcf, 5 min  
Aspirate supernatant using aspiration pipette connected to vacuum aspirator
SECTION: Thaw cells
9. Flick pellet to loosen



SECTION: Thaw cells
8.1. During centrifugation, transfer cell culture flasks to be used to the hood

 



SECTION: Thaw cells
8.2. Using alcohol-resistant markers, label with cell line name, passage number, date & initials

 

SECTION: Subculturing cells
13. Wash cells wiith PBS 1-2 times
Add the appropriate volume of trypsin for the flasks used.
Tilt plates to ensure trypsin evenly coats all cells
Incubate in 37 °C for up to 10 min 
 


SECTION: Cell culture prep
3. Wipe down flasks containing cells with 70% ethanol before handling, at least once per week
SECTION: Thaw cells
10. 1-2 days later, Check on cells to assess recovery/attachment and change media 
SECTION: Subculturing cells
14. Once the vast majority of cells have detached, inactivate trypsin w/ media, using at least double the volume of trypsin.
Pellet cells: 200 rcf, 5 min 
SECTION: Subculturing cells
13.1. Check under microscope whether cells have detached 3, 5, 8 & 10 minutes after placing into incubator, particularly the edges.
Tapping the sides of the flask may help to detach cells but also cause clumping.
SECTION: Freezing cells
19. Flick pellet to loosen
Resuspend cells in media 
Transfer 550 µL  to a 1.5ml centrifuge tube
Count cells on Vi-cell

SECTION: Subculturing cells
14.1. During centrifugation, transfer cell culture flasks to be used to the hood & label 

SECTION: Subculturing cells
15. Flick pellet to loosen
Resuspend cells in media 
SECTION: Thaw cells
9.2. Alternatively, resuspend cells in final volume required for culture vessel(s)
Transfer into culture vessel 

SECTION: Thaw cells
9.3. Check cells under microscope, observing for dead cells or large clumps
Transfer to 37 °C 
SECTION: Thaw cells
9.1. (Optional) Resuspend cells in 5 mL 
Count cells and assess viability. 


 

SECTION: Subculturing cells
11. Inspect cells under the microscope .
Cells are best trypsinised while in the exponential phase of growth, generally between 40-90% confluence.
Usually, cells are trypsinised near the end of the exponential growth phase, at 70-90% confluence.
Cells may also be subcultured to remove debris not detached during media changes.
 

SECTION: Freezing cells
17. Inspect cells under the microscope .
Cells should be frozen while in the exponential phase of growth, generally between 40-90% confluence.
Trypsinise and pellet cells (steps 11-13) 200 rcf, 5 min 

SECTION: Freezing cells
20. Calculate the total number of cells remaining after count.
Determine volume of 5*10^5 cells for Mycoplasma testing , or 1*10^6 cellsfor STR + Mycoplasma testing
Determine volume of cells to subculture, if any. 
Determine volume of cell suspension and freezing media to use
 

SECTION: Freezing cells
16. Prepare freezing media: 10 % (v/v) on ice 
  

 


SECTION: Freezing cells
18. Prepare items:
Warm media
Warm trypsin   or   (for firmly adherent cells)
15ml / 50ml centrifuge tubes
1.5ml centrifuge tubes
Cryovials
1ml pipette & tips
Waste bin
Aspiration pipettes
Motorised pipette controller
Freezing containers (cooling rate of <= 1 C/min) at Room temperature 

SECTION: Subculturing cells
12. Prepare items:
Warm media
Warm trypsin   or   (for firmly adherent cells)
15ml / 50ml centrifuge tube
1ml pipette & tips
Waste bin
Aspiration pipettes
Motorised pipette controller


SECTION: Freezing cells
21. Mix cell suspension well



SECTION: Freezing cells
20.1. Create labels for vials 
cell line name, 
current passage, 
cell number/vessel to thaw cells into
date, 
initials/name
SECTION: Freezing cells
21.3. Pellet cells to be frozen 200 rcf, 5 min 
Label cryovials during centrifugation
Resuspend cells in freezing media
Transfer cells in freezing media to cryovials; screw cap tightly
 

SECTION: Freezing cells
21.2. Replate cells as needed
SECTION: Freezing cells
21.1. Transfer cells needed for mycoplasma +/- STR testing into a 1.5ml centrifuge tube
Label tubes 
Place on ice 
SECTION: Freezing cells
22. Transfer cryovials into freezing container and freeze at -80C
SECTION: Freezing cells
23. Pellet cells 200 rcf, 5 min and submit for mycoplasma +/- STR testing, keep extracted DNA if possible

SECTION: Freezing cells
25. Transfer vials to liquid nitrogen storage at least 2 hours after freezing and when mycoplasma-negative.

Keep vials on dry ice during transfer.

SECTION: Subculturing cells
15.1. (Optional) Count cells on Vi-cell
 

SECTION: Subculturing cells
15.2. Dilute cells as necessary to achieve the desired subcultivation ratio and volume, and transfer to culture vessels
For culture plates: shake back and forth, then side to side to distribute cells evenly across wells. Do not swirl.

Transfer to 37 °C 
 

SECTION: Freezing cells
24. If establishing cell stock, thaw a vial of cells (steps 4-10) after >1 day of freezing to confirm good recovery and absence of bacterial and fungal contamination. 

If recovery is poor or contamination+, discard frozen vials and freeze new batch of cells.