Freezing Cells: Gibco® Cell Culture Basics

Uploaded by LifeTechnologiesCorp on 21.12.2011

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 a 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
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 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 will need based on the number of cells
and concentration for each vial.
Once the centrifuge completely stops, remove the tubes, return to the hood and clean with
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 1 ml cell solution into each 1 ml cryovial. If you have a large
volume, resuspend the cells frequently so they are homogenous.
Use the Mr. Frosty container, or similar device, to ensure the cell solution decreases in temperature
by 1 degree Celsius each minute. Slow, regulated freezing minimizes cell damage.
After freezing overnight at -80 degrees Celsius, the cells are frozen and ready for permanent
storage in a 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