TITLE: Fallopian Tube Epithelial Cell Culture
AUTHORS: Ramlogan  Sowamber, Melissa Nicole Castillo, Iru  Paudel, Sophia HL HL George

[DESCRIPTION]
The purpose of this protocol is to grow primary fallopian tube epithelial cells as 2D culture.

This protocol explains the method of culturing and maintaining fallopian tube epithelial cells derived from processing human fallopian tube tissue.


[BEFORE_START]
All cell culture should be performed under sterile conditions in a biological safety cabinet (BSC)
All personal protective equipment (PPE) should be used
Use a solution of 70% EtOH (70% ethanol + 30% MilliQ H2O) to disinfect surface and hands prior to working with cells
All reagents should be at room temperature (20-22°C, 69-72°F). Do not warm using water bath. 
Obtain patient consent and approval from Research Ethics Board (REB) or Institutional Review Board (IRB) prior to commencing cell culture.

[GUIDELINES]
Confluent cells are contact growth inhibited. Ideal conditions for performing cell culture work (ie. Drug treatments, cell cycle analysis, growth analysis, etc) should be ~70-80% confluence. 

1.Freshly isolated FTE cells should be plated on Primaria plates at the following densities:

     Plate Size      Cell Number        12 well      2000        6 well      5000        60 mm      2.5-4x105        100 mm      0.6-1x106   

2. Primaria plates increase adhesion of cells to plate and promote an optimal substrate. The plate should be coated with collagen before plating the cells.
3. Cells should be cultured for 3-5 days with USG media, full media change every 48hrs, until the density is 70-80%.

     Plate Size      Tissue Culture Media Volume (mL)        24 well      0.5        12 well      1        6 well      2        60 mm      4        100 mm      8   


[STEPS]
SECTION: Dissociating cells from cell culture plate
2. Dissociate FTE cells from tissue culture plates
SECTION: Dissociating cells from cell culture plate
2.1. Wash cells with wash solution (PBS +1% P/S)
SECTION: Dissociating cells from cell culture plate
2.2. Aspirate wash solution with an autoclaved borosilicate glass pipette
SECTION: Dissociating cells from cell culture plate
2.3. Add 0.25% Trypsin EDTA to each plate

    Plate Size   Trypsin Volume (mL)     12 well   0.25     6 well   0.5     60 mm   1     100 mm   2    
 
SECTION: Dissociating cells from cell culture plate
2.4. Place plate in incubator (37°C, 5% CO2) for 5 minutes or until cells detach (visualize under inverted lab microscope to confirm detachment)
SECTION: Dissociating cells from cell culture plate
2.5. Remove plate from incubator and add double the volume of TNS to plate (ie 2 mL TNS : 1mL Trypsin). Gently pipette media up and down to collect all cells in pipette.
SECTION: Dissociating cells from cell culture plate
2.6. Transfer to 15 mL conical tube of choice
SECTION: Dissociating cells from cell culture plate
2.7. Centrifuge at 1100 rpm for 5 min
SECTION: Dissociating cells from cell culture plate
2.8. Aspirate supernatant and discard without disturbing pellet
SECTION: Dissociating cells from cell culture plate
2.9. Cells are now ready for: Passaging onto new plate; Freezing for future use or DNA/RNA/Protein extraction.
SECTION: Plating cells from pellets (ie. Passaging cells)
1. Cells can be grown after being pelleted in Step 1.8. This is called passaging the cells. The purpose of this step is expand cells onto a larger surface area for growth.
SECTION: Plating cells from pellets (ie. Passaging cells)
1.1. Prepare cell culture plate by treating cell culture plate with collagen (1mL of collagen for a 100 mm plate)
SECTION: Plating cells from pellets (ie. Passaging cells)
1.2. Coat entire surface of plate with collagen and aspirate remaining collagen
SECTION: Plating cells from pellets (ie. Passaging cells)
1.3. Add cell culture media to plate and let sit for 5 mins or more in BSC or until ready to use.
 
     Plate Size      Tissue Culture Media Volume (mL)        24 well      0.5        12 well      1        6 well      2        60 mm      4        100 mm      8   

 
 
SECTION: Plating cells from pellets (ie. Passaging cells)
1.4. Add 1 mL of cell culture media to 15mL conical tube with pellet (there should be no supernatant at this point)
SECTION: Plating cells from pellets (ie. Passaging cells)
1.5. Gently resuspend pellet by pipetting up and down until pellet has dissociated
SECTION: Plating cells from pellets (ie. Passaging cells)
1.6. Take entire volume and dispense into centre of plate while gently rocking the plate back and forth (this helps to evenly distribute cells onto plate)
SECTION: Plate previously frozen cells
3. Frozen cells should be stored at -80C for short term storage (no more than 1 month) or in Liquid N2 for long term storage). 
SECTION: Plating cells from pellets (ie. Passaging cells)
1.7. Place plate into incubator (37°C, 5% CO2) 
SECTION: Plate previously frozen cells
3.1. Take cells out of storage and warm to liquid by holding in the palm of hand (this process should be as quick as possible)
SECTION: Plate previously frozen cells
3.2. During thaw, add TNS to 15 mL labelled conical tube
SECTION: Plate previously frozen cells
3.3. Add thawed cells using pipette to 15 mL conical tube containing TNS. *For primary fallopian tube cells previously frozen, thaw cryo-vial rapidly into 5ml pre-warmed USG media. 
SECTION: Plate previously frozen cells
3.4. Centrifuge cells in centrifuge at 1100 rpm for 5 minutes
SECTION: Plate previously frozen cells
3.5. Aspirate supernatant
SECTION: Plate previously frozen cells
3.6. Re-suspend cells in cell culture media and add to prepared cell culture plate
SECTION: Freezing cells
4. Cells that have been pelleted can be resuspended in freezing media and placed directly at -80°C or Liquid N2