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Best Tips on Cell Culture Cryopreservation

Cryopreservation is a mandatory technique for cell culture researchers as it allows the storage of cells and tissues in the cryopreserved stage for future culturing. Cryopreservation can impact research efficacy owing to how the whole process is managed and what materials are used. For example, if cells are frozen too quickly, membrane damage and cell death can occur due to the formation of ice crystals. The successful strategy of cryopreservation requires thoughtful consideration of cryoprotective agents, storage vessels, cooling rate, and other factors. As KOSHEEKA is involved with primary cell culture and stem cell culture, we requested Ms. Shraddha Singh Gautam, Director-Lab Operations of Advancells Group, to give her valuable insight on some FAQs on cell culture cryopreservation.

  1. Choosing a cryovial is crucial. When to choose internal or external threaded cryovials?

These two options depend on the preference of the researcher. Some researchers choose external thread cryovials that may help in minimizing contamination. Some researchers choose internal thread cryovials as they fit better in freezer boxes. For researchers using automated machinery, they need to consult with the instrument manufacturers.

  1. Can insulated cardboard or polystyrene foam boxes be used as freezing chambers?

These materials or homemade devices do not always give controlled uniform cooling and therefore might affect the cells with serious viability differences among the vials upon thawing.

  1. Any alternatives to DMSO for cryopreservation applications?

Cryoprotective agents can be divided into intracellular and extracellular cryoprotectants. Intracellular agents are small molecules that can penetrate the cell membrane. Examples are DMSO, glycerol, ethylene glycol, propylene glycol, etc. Extracellular agents are large molecules that are added to the cryoprotectant solution. Examples are sucrose, dextrose, methylcellulose, etc.

  1. Are there any protocol tips for efficient cryopreservation of stem cells?

The success of the cryopreservation process can depend on 4 critical areas:

Good cell condition: Stem cells should be healthy before cryopreservation and should be frozen after being passaged for 2-3 days. Do not use overgrown or over-confluent cells as they might show poor viability after thawing. Ensure that cell clumps are not present as that will lead to inefficient cryoprotection to the cells. 1-2 x 106 cells/ml is the typical density of cryopreservation.

Correct cryoprotective agent use: DMSO is the most common cryoprotectant. Generally, 10% DMSO is the final concentration in the freezing media along with FBS.  For better efficiency, researchers should use freshly prepared cryoprotectant mix on the day of the experiment.

Controlled freezing rate: For cells, the ideal cooling rate is -1°C per minute. Electronic automated freezing units are best for controlling cooling rates.

Proper cryogenic storage: Liquid N2 freezers permit storage at a temperature between -140°C and -180°C in the vapor phase. Using the vapor phase storage greatly reduces the possibility of leaky or exploding vials during taking them out.

  1. Is it ok to transfer cells stored at -80 to liquid nitrogen directly?

In general, it is okay to transfer cryovials from -80°C to -196°C directly for long-term storage, without any other interim steps.

  1. Due to COVID-19, some labs had to shut down quickly and liquid N2 storage was not possible. How long can we expect cells to maintain themselves in -80 storage?

Chances of good viability after revival are poor if months have passed with cells stored at -80°C.
Researchers are trying to develop better protocols for improving long-term cryopreservation at -80°C. It has been reported in some studies that 10% Ficoll 70 in the freezing medium can extend cell storage duration at -80°C to around a year with comparable viability and plating efficiency versus liquid N2 storage.

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