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Frequently Asked Cell Culture Questions

1. If I receive a tube of frozen cells, can I put it directly into liquid nitrogen for storage?

In many cases, cells transported on dry ice (-80°C) can be put back into liquid nitrogen and then thawed quickly afterwards. However, cell viability may be reduced after such treatment. For some sensitive cell lines, this may make cell recovery more difficult. This phenomenon is thought to be due to a change in the structure of the ice crystals within the cells as a result of the temperature change. It is therefore recommended that cells should be thawed and cultured as soon as possible after receipt. Reduce the storage time at -80°C. This temperature is only used for transport.

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2. What safety measures should be taken when removing cells from liquid nitrogen for recovery?

Cell cryotubes in liquid nitrogen that are not completely sealed and have liquid nitrogen leaking into them can cause an explosion if the temperature of the cryotube rises sharply when thawing. It is therefore recommended that goggles and protective gloves are worn when removing cells from liquid nitrogen. For resuscitation, the freezing tube should be shaken continuously in a 37°C water bath to completely thaw the freezing solution within 1-2 minutes. Afterwards, wipe the outside of the tube with an alcohol wipe, then take it into the ultra-clean table and transfer the cells to a centrifuge tube with 10 ml of culture medium added, centrifuge at 1000 rpm for 5-10 minutes, discard the supernatant, add the appropriate amount of culture medium and inoculate the culture flask and incubate in a 5% CO2 incubator.

3. Why should cells be stored in the vapour phase of a liquid nitrogen tank rather than in the liquid phase?

Cells stored in the gas phase of liquid nitrogen are more likely to be revived. Whereas in the liquid phase of liquid nitrogen, if the lyophilisation tubes are not properly sealed or have leaks, direct contact between the cells and the liquid nitrogen can compromise the viability of the cells after thawing.

4. For suspension cells, how do I change the culture medium?

Culturing suspension cells can be done by simply adding fresh medium (if space allows) or by separating the cells from the old medium by centrifugation (100 x g for 5 minutes) and subsequently resuspending the precipitated cells in fresh medium. However, for most suspension cell lines, simply adding medium is a better method. Either way, the medium needs to be renewed before the cells reach their largest saturation density. The saturation density of cells varies between 3 x 10 5 and 2 x 10 6 depending on the cell line and culture conditions (resting or stirring, oxygenation levels, etc.). Cells must be diluted to a lower cell concentration to allow sufficient nutrient recovery to keep the cells growing logarithmically. If the medium is simply changed without reducing the cell density, the cells will rapidly deplete the medium and die. If the cells are diluted below their smallest density, they will enter a lag phase and grow very slowly or will die. Each suspension cell line has a different saturation density and passaging interval, so daily cell counts are the way to monitor suspension cell lines*.

5. What is the recommended CO2 level for cell culture?

Although CO2 levels in cell culture systems range from 0.03% to 40% (typically around 0.03% CO2 in the atmosphere), it is very common to have no CO2 added to the air or a CO2 concentration of 5% to 10%. It is important to adjust the concentration of sodium bicarbonate in the medium to balance with the CO2 level in the gas phase. Cells produce CO2 and require a small amount of carbonic acid for growth and survival. If no CO2 is added and the cells are multiplying, 4 mM (0.34 g/L) of anhydrous sodium bicarbonate can be used. However, the lid of the culture flask should be tightened at this point. If the culture system requires 5% or 10% CO2, use 23.5 mM (1.97 g/L) or 47 mM (3.95 g/L) sodium bicarbonate at 37°C, respectively, with an initial pH of approximately 7.6. Under these conditions, the flask should be left uncapped or a Petri dish should be used to maintain gas equilibrium.

6. Why do some cells need sodium pyruvate? How much sodium pyruvate should I add to the medium?

Pyruvate is an organic acid metabolite in the glycolytic pathway* that readily enters and leaves the cell. Therefore, the addition of sodium pyruvate to the medium provides both an energy source and a carbon source for anabolism, helps to maintain certain specific cells, helps with cell cloning or is needed when serum concentrations in the medium are reduced. Sodium pyruvate also helps to reduce fluorescence-induced cytotoxicity. Sodium pyruvate is usually added at a final concentration of 1 mM. commercially available sodium pyruvate solutions are usually 100 mM storage solution (100X).

Translated with www.DeepL.com/Translator (free version).


Post time: Jun-21-2022