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Reviving Cells from Liquid Nitrogen

Updated: Nov 21, 2023

The process of reviving cells from liquid nitrogen, or indeed from transport on dry ice, can be a little difficult on occasion.  Two events at OET recently have highlighted this. 

Firstly, we received a new cell line that came with a very complicated procedure for its culture – it is a mammalian species, quite different from your run of the mill HeLa’s and HEK293’s.  Its culture is known to be problematic, so the instructions we received from the supplier are very detailed.  We have yet to thaw the vial and initiate the culture but the information we have will hopefully make the process very smooth.

Secondly, we had feedback from a customer who had purchased some insect cells from us.  They were having difficulties as the cells did not look very healthy.  Fortunately, with some exchange of images we were able to diagnose that they were sub culturing the cells a little too soon while they were at a low density.  They were not quite following our instructions to the letter.  We actually have very few customers who experience such problems, but it does show how even minor deviations from a protocol can have unintended consequences.

So why should there be problems culturing insect cells?  Mostly, we find that these arise because the cells are grown in serum-free medium.  The absence of serum and addition of other ingredients has meant that insect cells can be amplified to much higher densities.  The minor disadvantage of this system is that the cells are not happy at low densities, particularly when they are freshly revived from cryostorage.  This problem can be made worse if the medium in a thinly populated cell culture is replaced with fresh.  It might seem better to give a flask a complete medium change, but you might be better off just adding a little more medium to the old.  We recommend this because those mysterious factors produced by cells that allow them to grow in serum-free medium need to be retained.  Once your cells are almost confluent they seem to tolerate a complete medium change more readily.

Those of you experienced in growing any type of cells, mammalian or insect, will know that problems can arise from time to time.  These may involve reviving cells from liquid nitrogen or from dry ice shipment, adapting cells to a new medium when your regular supply literally dries up, changes in plasticware that affect growth and worst of all, microbial contamination!  The latter might seem entirely avoidable but it happens to the best of us.

If you need any tips in growing insect cells then please get in touch with us.  We market Expression Systems ESF 921 and other serum-free media, which we also use in house for our own work.  We also sell Sf9 cells and Super Sf9 cells that are particularly useful for enhancing expression of some recombinant proteins.

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