2. DMEM with 10% FBS
4. 1X PBS
5. Cryomedium (DMSO & FBS)
8. Centrifuge tubes (15 ml)
1. Remove vial from liquid nitrogen freezer and immediately place it in a 37°C water bath. Agitate vial continuously until medium is thawed. The medium usually thaws in <60 sec. Cells should be thawed as quickly as possible to prevent formation of ice crystals that can cause cell lysis. Try to avoid getting water around the cap of the vial.
2. Wipe top of vial with 70% ethanol before opening.
3. Transfer thawed cell suspension into a sterile centrifuge tube. Centrifuge for 10 min at 150 to 200 × g, room temperature. Discard supernatant. Cells are washed with fresh medium to remove residual DMSO.
4. Gently resuspend cell pellet in 3ml of DMEM with 10% FBS. Perform cell counting using hemocytometer. Determine how much cells you need to plate, based on that calculate the required volume of cell culture to be plated.
5. Transfer to properly labeled petriplate containing the appropriate amount of medium. Cultures are re-established at a higher cell density than that used for original cultures because there is some cell death associated with freezing.
6. Incubate in a humidified 37°C, 5% CO2 Incubator.
7. Check cultures after 24 hrs to ensure that cells have attached to the plate.
8. Change medium after 2 to 3 days or when pH indicator (e.g. phenol red) in medium changes color. Keep cultures in medium with 10% FBS until cell line is reestablished. If recovery rate is extremely low, only a subpopulation of the original culture may be growing; be especially careful of this when working with cell lines known to be mosaic.
Trypsinizing and Sub culturing Cells from a Monolayer (Adherent cells):
1. Remove all medium from primary culture with a sterile Pasteur pipette. Wash adhering cell monolayer once or twice with a small volume of 37°C PBS without Ca2+ and Mg2+ to remove any residual FBS that may inhibit the action of trypsin.
2. Add 1 ml of 37°C trypsin/EDTA solution to culture to cover adhering cell layer in a 10 cm dish.
3. Place plate in incubator for 1 to 2 min. Tap bottom of plate on the countertop to dislodge cells. Check culture with an inverted microscope to be sure that cells are rounded up and detached from the surface.
4. Add 3 ml 37°C complete medium to stop trypsinization.
5. Draw cell suspension into a Pasteur pipette and rinse cell layer two or three times to dissociate cells and to dislodge any remaining adherent cells. As soon as cells are detached, transfer to a 15 ml tube. (Cells can be counted using a hemocytometer or Coulter counter and diluted to the desired density so a specific number of cells can be added to each culture vessel. A final concentration of ∼5 × 104 cells/ml is appropriate for most subcultures. Cultures should be labeled with date of subculture and passage number.)
6. Add appropriate volume of fresh medium to each new cell culture plate.
7. Transfer cell suspension to the petriplate containing medium.
8. Incubate in a humidified 37°C, 5% CO2 Incubator.
9. If necessary, feed sub confluent cultures after 2 or 3 days by removing old medium and adding fresh 37°C medium.
Freezing the cell line:
1. Trypsinize cells from plate (this step only in case of adherent cells) as mentioned above in adherent cell culturing. It is best to use cells in log-phase growth for cryopreservation.
2. Transfer cell suspension to a sterile centrifuge tube and centrifuge 5 min at 300 to 350 × g (∼1500 rpm), room temperature. Cells from three or more dishes from the same subculture of the same source can be combined in one tube.
3. Remove supernatant. Prepare cryomedium (10% DMSO in FBS)
4. Add 10 ml of 4°C freezing medium. Resuspend pellet.
5. Pipette 1-ml aliquots of cell suspension into labeled 2-ml cryovials. Tighten caps on vials.
6. Place vials 1 hr to overnight in a -70°C freezer, and then transfer to liquid nitrogen storage freezer.
Difference Encountered in a Real Laboratory
In an actual laboratory setting, there are certain important steps that are not necessarily applicable in a virtual lab:
1. Discard all contaminated materials properly and return your supplies to the proper storage locations, and clean up your working area.
2. Make sure that all the condense in solution mixed well.
3. Always disinfect your work area when you are finished.