You’re getting ready to finally take some time off and relax. I’m sharing ways to make sure you don’t have your frozen cells on your mind as you lay on the beaches of Bermuda. Taking time to organize your lab will allow you to leave the lab with piece of mind. Last time I began to discuss how you can freeze your cell lines correctly before leaving the lab or if you’ve isolated a fantastic clone that expressed your gene with GFP. I’ll continue on this topic today with tips for freezing and thawing your cell lines.
Before embarking on massive expansion for stock generation, you should check your source cell line for the following criteria:
- Viability - No use to freeze unhealthy cells, right? If your source has less than 90% viability, split the cells and postpone expansion.
- Possible contamination (Mycoplasma, fungus etc.); cells that were found to be infected should be discarded immediately.
- Morphology – Check the cells maintain their typical morphology and behavior. Look at high magnification for membrane-related changed (filopodia formation/deformation in anchorage-dependent) and for intracellular changes (granule formation). Make sure you take notes and update your lab notebook or your Labguru account under the specific entry of these cells within the collection module. While doing so, you can track the different morphological changes the cells under go over time.
Freezing Cell Lines
- Media replenishment – One day before freezing the cells media should be replenished with fresh media. Use the BioKM task feature to add that reminder!
- Harvest cells before confluence reaches ~70%. Count viable cells and calculate the volume of cryo-media needed for ~5 x106 cells/ml media (the optimal number can vary among different cell types).
- Cryo-media – Wash cells from media and resuspend them with cryo-media according to your calculation.There are various cryomedia and you should empirically define the appropriate cryo-media for your cell culture (base media can be regular media/Serum):
- 5-10% DMSO – it is important to ensure that the stock DMSO solution doesn’t contain residual toxic material.
- 5-15% Glycerol
- Cells grown in serum-free media should be frozen and thawed in 50% conditional medium (serum-free media in which cells were grown for 24 hours).
- Mark cryogenic vials with the name of cell line, date and your name and fill vials to no more than 1.5ml.
- Gradual decrease in temperature is crucial for the survivability of frozen cells when thawed in the future. Use Mr. Frosty for gradual temperature decrease of the samples (between 1°-3°C) and place at -80°C freezer for overnight incubation. If you don’t have a Mr. Frosty you can either use Styrofoam holders/polystyrene foam/cardboard/lab diapers to control the decrease of the temperature.
- Document the number of frozen vials prepared and the expected location in the liquid nitrogen storage.
- Transfer the tubes from -80°C to liquid nitrogen storage as quickly as possible. Thus, it is best to either use dry ice box (-80°C) or submerge the vials within liquid nitrogen (which is preferable) and then quietly plan where to place your frozen vials.
- Due to the fact that there is not much place on the vial to document the exact name of the cell type/clone, make sure you document the exact position of each vial within the cryobox and the exact position within the liquid nitrogen tank. In your lab notebook (or BioKM experiment module) document the history and special characteristics of each frozen cell batch.
- Don’t forget to keep a backup box of all cell lines in separate (colleague’s?) liquid nitrogen tank for any possible crisis with your storage tank.
Thawing Cell Lines
You’re back from vacation and you’re ready to revive your cells. Correct thawing of cells is as important as correct freezing. Keep these points in mind:
- When removing vials from liquid nitrogen storage there is a risk that vials can explode or their cap bursting; use face shield and related protecting clothing.
- Transferring tubes from liquid nitrogen to hot tub should be done in liquid nitrogen.
- Thawing should be fast and at 37°C while keeping the tube’s cap above the water line to prevent possible contamination. Once the smallest ice particle dissolves within the vial, dip or spray the vial with 70% ethanol and aseptically transfer the content onto fresh and pre-warmed media within a 10cm culture plate/25 cm2 flask.
- Monitor for morphological changes and growth rate and make sure you update your Labguru experiment page.