Cell Culture Basics – Freezing Cells A properly maintained frozen cell stock
is an important part of cell culture. It gives you the opportunity to go back to
early passage cells or thaw more cells as needed. In preparation for freezing, be
sure to label each vial with cell type, passage number and date using a
permanent marker. You will need pre-chilled freezing
medium and Mr. Frosty container filled with isopropanol. The general freezing method is the same for suspension and adherent cells except
that adherent cells need to be removed from the culture plates before starting
the freezing procedure. First, the conditioned media is aspirated off and discarded. The attached cells are washed with PBS. Then TrypLE is added to dissociate the culture and detach the cells from the flask. If needed, the flask can be tapped to dislodge and break up cell clumps. Cells from multiple plates should be collected into one or two 15 milliliter centrifuge tubes. Add medium to the cells. mix gently and transfer to a centrifuge tube No inactivation is necessary when using TrypLE. Take a small sample for a cell counting. Make sure your tubes are balanced in the centrifuge. spin-down the cells to form a pellet While the cells are spinning, count the sample for total cell number and percent viability. The Countess Automated Cell Counter can be used for a fast and accurate reading. Make sure the percent viability is greater than 90%. Only healthy cells should be frozen. Optimal cell concentration for freezing varies by cell type. Also, calculate the volume of freezing medium you’ll need based on the number of cells and concentration for each file. Once the centrifuge completely stops, remove the tubes, return to the hood and clean with ethanol. Remove and discard the medium, paying close attention not to disturb the cell pellet. Resuspend the pellet in the correct volume of cold freezing medium for optimal cell density for freezing. Triturate the cells for a single cell suspension. Transfer approximately one milliliter of cell solution into each cryovial. If you have a large volume, resuspend the cells frequently, so they’re homogeneous. Use the Mr. Frosty container or similar device to ensure the cell solution decreases in temperature by one degree Celsius each minute. Slow, regulated freezing minimizes cell damage. After freezing overnight at minus 80 degrees Celsius, the cells are frozen and ready for permanent storage in the liquid nitrogen tank. Always wear the appropriate protective equipment and handle liquid nitrogen carefully. Do not attempt to handle liquid nitrogen until you read and fully understand the potential hazards, their consequences and the related safety precautions. When transferring cryovials, don’t let the cell solution begin thawing. You risk decreasing cell viability. Freezing is a very stressful process for the cells. Make sure every step is performed quickly and carefully under optimal conditions of temperature, medium formulation and freezing density.