Protocol: Primary Embryonic Fibroblast Culture
Thawing Cells
| 1. | Thaw a vial of P0 primary embryonic fibroblast cells, as prepared above, by holding at 37°C in a water bath. |
| 2. | Prepare media by adding 4mL fibroblast media to 4mL FBS in a 15mL tube. Mix well. |
| 3. | Layer 1mL of thawed fibroblasts onto media. There is no need to mix. |
| 4. | Centrifuge at 850rpm for 5 minutes to pellet cells. |
| 5. | Aspirate the media and discard. |
| 6. | Resuspend the pellet in 10mL fibroblast media. Transfer to a 10cm tissue culture plate and incubate 37°C, 5% CO2. Label these cells as P1. |
Passaging Cells
| 1. | When the PMEF cells are fully confluent (about 3 days) passage the cells 1 in 3. |
| 2. | Aspirate the media and discard (tilt plate to ensure complete removal). |
| 3. | Wash the plate with 10mL PBS. Do this by pipetting onto the side of the plate, not the base so as not to dislodge cells. Swirl gently, then aspirate. Repeat. |
| 4. | Add 1mL Trypsin/EDTA solution and swirl to cover. Incubate at 37°C for 2 minutes. |
| 5. | Check plates under a microscope to ensure that cells are fully trypsinised. Dislodge any adherent cells by flicking the plate. Cells should be rounded and float freely. |
| 6. | Add 8mL Fibroblast media to each plate. Pipette up and down 5 times, washing each plate thoroughly. |
| 7. | Add 3mL of the cell suspension to a fresh plate containing 7mL media (3 plates in total). Pipette up and down 3-5 times to ensure cells are fully mixed. Incubate. Label these cells as P2. |
| 8. | Cells can be passaged 1 in 2 or 1 in 4, depending on if you require fewer or more cells. Adjust volumes accordingly: i.e. for 1 in 2, add 9mL media to cells, then 5mL to 5mL fresh media in 2 plates; for 1 in 4, add 7mL media to cells, then 2mL to 8mL fresh media in 4 plates. |
| 9. | Continue growing and passaging until cells reach P4. |
PMEFs grown to confluency at 6 days in culture (x10)
Irradiation and Freezing
| 1. | When P4 cells reach confluency they are ready for freezing. |
| 2. | For ease of handling process plates in 3-4 batches of about 8 plates at a time. |
| 3. | Aspirate media, wash twice with 10mL PBS and trypsinise as usual. |
| 4. | Add 2-3mL media to each plate to stop Trypsin. |
| 5. | Add ~5mL media to one plate and wash down well to collect cells. Transfer the media to another plate and wash down. Continue transferring and washing down each plate. If the media becomes too thick or frothy, transfer the cells to a 50mL tube and continue with fresh media. |
| 6. | Once all the plates are washed and cells harvested transfer the contents to a 50mL tube. Wash down all plates a second time using 5-8mL media to ensure all cells are harvested. |
| 7. | Continue for next batch of cells. When all plates are harvested mix the cells well before counting. |
| 8. | Count cells by placing 10mL onto a hemocytometer, and counting the number of cells on a 25-grid square. The concentration of cells = number counted x104/mL. Multiply by the volume you have for the total number of cells. Freeze cells at 2x106 per vial. |
| 9. | Prior to freezing, g -irradiate cells, 25 GRAY. |
| 10. | After irradiation, spin down cells, as usual. |
| 11. | Aspirate the media and resuspend cells in the appropriate volume of freezing media to obtain 2x106/mL. Finally, aliquot 1mL of cells into cryovials and freeze immediately at -80°C. |
| 12. | Transfer vials to liquid nitrogen storage next day. |