Fallopian Tube Epithelial Cell Culture
Ramlogan Sowamber, Melissa Nicole Castillo, Iru Paudel, Sophia HL HL George
Abstract
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.
Steps
Plating cells from pellets (ie. Passaging cells)
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.
Prepare cell culture plate by treating cell culture plate with collagen (1mL of collagen for a 100 mm plate)
Coat entire surface of plate with collagen and aspirate remaining collagen
Add cell culture media to plate and let sit for 5 mins or more in BSC or until ready to use.
| A | B |
|---|---|
| Plate Size | Tissue Culture Media Volume (mL) |
| 24 well | 0.5 |
| 12 well | 1 |
| 6 well | 2 |
| 60 mm | 4 |
| 100 mm | 8 |
Add 1 mL of cell culture media to 15mL conical tube with pellet (there should be no supernatant at this point)
Gently resuspend pellet by pipetting up and down until pellet has dissociated
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)
Place plate into incubator (37°C, 5% CO2)
Dissociating cells from cell culture plate
Dissociate FTE cells from tissue culture plates
Wash cells with wash solution (PBS +1% P/S)
Aspirate wash solution with an autoclaved borosilicate glass pipette
Add 0.25% Trypsin EDTA to each plate
| A | B |
|---|---|
| Plate Size | Trypsin Volume (mL) |
| 12 well | 0.25 |
| 6 well | 0.5 |
| 60 mm | 1 |
| 100 mm | 2 |
Place plate in incubator (37°C, 5% CO2) for 5 minutes or until cells detach (visualize under inverted lab microscope to confirm detachment)
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.
Transfer to 15 mL conical tube of choice
Centrifuge at 1100 rpm for 5 min
Aspirate supernatant and discard without disturbing pellet
Cells are now ready for: Passaging onto new plate; Freezing for future use or DNA/RNA/Protein extraction.
Plate previously frozen cells
Frozen cells should be stored at -80C for short term storage (no more than 1 month) or in Liquid N2 for long term storage).
Take cells out of storage and warm to liquid by holding in the palm of hand (this process should be as quick as possible)
During thaw, add TNS to 15 mL labelled conical tube
Add thawed cells using pipette to 15 mL conical tube containing TNS. *For primary fallopian tube cells previously frozen, t haw cryo-vial rapidly into 5ml pre-warmed USG media.
Centrifuge cells in centrifuge at 1100 rpm for 5 minutes
Aspirate supernatant
Re-suspend cells in cell culture media and add to prepared cell culture plate
Freezing cells
Cells that have been pelleted can be resuspended in freezing media and placed directly at -80°C or Liquid N2
