TITLE: CMT-93 Cell Culture Protocol
AUTHORS: Laura Gómez

[DESCRIPTION]
CMT-93 is a cell line exhibiting epithelial morphology that was isolated from the rectum of a mouse with polyploid carcinoma. 


[BEFORE_START]
Clean and prepare the laminar flow cabinet, turn on the water bath and warm up the culture media.

[GUIDELINES]
Working with cell cultures requires a laminar flow cabinet. It has to be radiated with UV light, cleaned with any highly effective terminal disinfectant (such as Tego‱ 2000 or Suredis‱) and 70% ethanol. All material introduced into the cabinet must also be sprayed with ethanol. 
Once the work is finished, we must clean the cabinet with the detergent, then with 70% etOH and turn on the UV light for 30 min.

[STEPS]
SECTION: Preparation of complete growth medium (DMEM+)
1. Add 445 mL 1X DMEM, 50 mL FBS and 5 mLglutamine 200 millimolar (mM) to a sterile 500 mL bottle and homogenize

SECTION: Preparation of complete growth medium (DMEM+)
2. Label the bottle with name, group, phone number, date and additions. 

SECTION: Preparation of complete growth medium (DMEM+)
3. Close with parafilm and store at 4 °C.

SECTION: Cell thawing procedure
4. Remove one vial of cell stock from the liquid nitrogen tank with gloves and forceps. Transfer them to the cell culture laboratory in an appropiate container or a box with ice.
SECTION: Cell thawing procedure
5. Thaw the vial by gently shaking it in a 37 °C water bath. Thawing should be rapid (approximately 2 min).

SECTION: Cell thawing procedure
6. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol
SECTION: Cell thawing procedure
7. Transfer the contents of the vial to a centrifuge tube containing 9 mL of complete culture medium and1200 rpm, 5 min

SECTION: Cell thawing procedure
8. Resuspend with 10 mL DMEM+ and distribute on 2 P60 plates.

SECTION: Cell thawing procedure
9. Incubate cultures at 37 °C, 5% CO2 5 min 

SECTION: Subculturing procedure
10. Remove and discard culture medium.
SECTION: Subculturing procedure
12. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53mM EDTA solution to remove all traces of serum which contains trypsin inhibitor. Discard.
SECTION: Subculturing procedure
13. Add 2 mL of Trypsin-EDTA solution to flask and incubate 10 min at 37 °C to facilitate detachment from the plate.

 

SECTION: Subculturing procedure
15. Add 5 mL of DMEM+ and aspirate cells by gently pipetting. Pour the existing volume down the walls of the flask in order to drag and collect as many cells as possible.

SECTION: Subculturing procedure
19. Add 1 mL of the cell supension to new culture vessels containing 14 mL DMEM+. 

SECTION: Subculturing procedure
20. Incubate cultures at 37 °C, 5% CO2 5 min 

SECTION: Subculturing procedure
11. Rinse with PBS 1X solution and discard
SECTION: Subculturing procedure
14. Observe cells under an inverted microscope until cell layer is dispersed.
 

SECTION: Subculturing procedure
16. Collect the cell suspension in a centrifugue tube and 1200 rpm, 5 min.

SECTION: Subculturing procedure
18. Resuspend in medium according to the dilution to be made.

 

SECTION: Subculturing procedure
17. Discard the supernatant into a beaker with 70% EtOH or 10% bleach.
SECTION: Cryopreservation and storage procedure
21. Repeat steps of Subculturing procedure until the "Resuspend in medium according to the dilution to be made" step.
SECTION: Cryopreservation and storage procedure
22. Resuspend in medium taking into account that for every p100 we can storage up to 2 cryovials of cells, containing 1 mL. 


SECTION: Cryopreservation and storage procedure
23. Prepare the cryovials with 50 µL DMSO. 

SECTION: Cryopreservation and storage procedure
25. Label the cryovials with cell line, passage, date and lab number or phone number.
SECTION: Cryopreservation and storage procedure
26. Store the cryovials in a slow freezing container at -80 °C for 1440 min.

SECTION: Cryopreservation and storage procedure
27. Transfer the cryovials to the liquid nitrogen tank.
SECTION: Cryopreservation and storage procedure
24. Add 950 µL of the cell suspension to every cryovial.