As the holiday season creeps painfully slowly towards us it’s not only the lab staff who need a little TLC over the festive break. Cell cultures will also be looking to take a much needed respite, but that doesn’t mean they can simply be abandoned.
If (like the writer of this article) you’re aiming for a gentle transition into the working week post New Year and won’t be requiring cells for experimental purposes, it may be advisable to freeze down your cultures and start afresh. The benefit of this is that your current stocks could already be a little long in the tooth and consequently their capacity for virus and protein generation diminished. You can find information on successfully freezing and thawing cells in previous blogs. However, if you’re eager to start off 2020 with a fresh round of protein expression then you’ll need to sustain your cell cultures over the coming period – which is no easy feat considering most insect cells must be sub-cultured at least twice a week. Fear not though for OET are here to help provide a solution!
First of all, do not assume that over diluting your cells in the hope this will prevent them from overgrowing is going to be a viable option. Insect cells can quickly become too dense or worse, die off completely if they are seeded too thinly. This is especially relevant for those in serum-free media as it lacks the necessary growth conditions to promote robust cell replication.
Secondly, it’s best not to try and increase the time between passages. Even if you are able to access your lab during the holidays to sporadically split your cultures (I’m not judging how you spend your free time), it may not be the wisest decision in the long run should you be looking to achieve healthy viable cells upon your return to work. Leaving extended periods between sub-culturing and allowing cells to overgrow too heavily before splitting can induce a stress response. When this occurs it can often be difficult for the culture to return to a healthy growth phase and in some cases, may give up entirely. It’s simply not worth it.
In our experience the optimal solution is to transfer your existing suspension cultures to monolayer and grow at a reduced temperature (we recommend 20-21°C). Through doing this you can achieve a slower rate of growth without impeding on cell viability, allowing the culture to survive for up to 10 days before the need to passage again. We find that leaving flasks at room temperature on a benchtop in the lab is sufficient. Alternatively some incubators can also be adjusted to maintain this temperature, which is particularly helpful in avoiding the risk of fatalities should your lab’s heating system be switched off over the holidays! The size and number of flasks will be dependent on how many cells you require upon return. Cells can be seeded with various densities following your standard cell culture procedure for monolayers (see OET’s Insect Cell Culture Guide for advice on how to do this). As an example, you may have a 175cm2 flask seeded with either 1 x 106 , 2.5 x 106 or 5 x 106 cells in 15ml medium. Including all three concentrations will increase the chances that at least one of your cultures will survive.
Once you are ready to begin passaging cells full-time you should transfer the monolayer cultures back to suspension following standard cell culture procedures. As your cells will be sufficiently confluent at this point they should easily adapt into the new shake flasks.
From all of us at OET we wish you a very merry Christmas and happy New Year!