Thawing Cells: Cell Culture Basics

Always wear basic PPE and review the
MSDS for safety information. DMSO and cell culture freezing medium is
toxic. Thawing cells is a very stressful process for them to endure. To aid your
cells’ survival, perform each step quickly and under optimal conditions, such as
temperature, medium formulation, and seating density. Clean and set up the
cell culture hood before you remove the cells from liquid nitrogen storage. You
will need 10 milliliters of pre-warmed medium and a cell culture flask. This
protocol is the same for cells thawed into a suspension flask but the flask
used will be different. Be careful working with liquid nitrogen.
Ask the safety team at your Institute if you’re unsure what your PPE requirements are, or how to safely work with liquid nitrogen. Working quickly so that no
cells thaw while they’re out of the liquid nitrogen storage tank, pull the
vial of cells you need and place in a cup of dry ice to keep them cold in
transit to the water bath. The vial of cells should be thawed in a 37 degrees Celsius water bath quickly. Be careful not to submerge the vial as this
increases the risk for contamination. Remove the vial from the water bath when a small chunk of ice remains inside. The ice chunk will melt as you move it to
the cell culture hood. Wipe the vial with ethanol before placing it in the hood.
Working quickly, use a small pipette to transfer the contents of the vial to a
15 milliliter centrifuge tube. Very carefully, add 10 milliliters
pre-warmed cell culture medium to the tube. Start drop by drop, then gradually
increase your rate in order to avoid osmotic shock. Spinning the cells in the centrifuge
allows removal of the freezing medium that contains DMSO. The speed and time
may vary based on the cell type. Some cell lines are too delicate to spin, so
this step could be skipped in certain cases. Follow the instruction of your
laboratory manager if your cells are very delicate. Discard the medium by pouring, pipetting
or using a vacuum. Be careful not to disturb the cell
pellet. Re-suspend cells in appropriate volume,
usually ten milliliters of complete pre-warmed medium. Medium volume, flask size and number of flasks plated will depend on the cell number frozen in the vial and the
optimal seeding density for the cells. Sometimes a viable cell count is
required in order to determine the number of flasks and media volume to be
used and media volume to be used. With a north-south, west-west rocking motion, we ensure even distribution of
the cells and solution. Usually, cells are checked a few hours later or the next day for good adhesion, if adherent, and proper morphology.

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