In our previous blog we talked about the early stages of growing insect cells when you might just have revived a frozen stock and set up either monolayer or suspension cultures (or both). If you have established a viable suspension culture – and here we are talking primarily about insect cells such as Sf21 or Sf9 – you will probably monitor them fairly closely for their rate of growth. The ideal scenario is that your cells will double in number every 24 hours. However, frequently this isn’t the case. Why not?
A lot can depend on the seeding density of your cells after dilution from a previous culture. Another factor is the cell count attained by a culture before you sub culture or passage them to a fresh flask.
Let’s consider the first factor, seeding density. Most insect cells are grown in serum-free medium. This has permitted greatly increased cell culture densities to be attained compared to the days of using medium that contained serum. Anti-foam additives have also reduced shear forces, which develop from frothing and bubbles, so vigorous shaking can be used to increase the aeration of cultures. A slight disadvantage to this is that some cells, particularly Sf9s, are not happy if they are diluted too much on sub culture. We recommend a seeding density of no lower than 4-5 x 105 cells/ml. This can be particularly important in the first few passages of cells after they have been established from a frozen stock. It is very hard to define, but we have noted that cells freshly thawed can take a few sub cultures before they attain their maximum growth rate.
The second factor to consider is the density of your cells at the point of sub culture. Manufacturers of insect cell culture medium proudly claim that your cells can reach 107 or greater cells/ml in their product. This is great marketing material, but in day to day use you probably don’t want to force your cells to these densities on a routine basis. At the higher cell densities your culture will be approaching nutrient exhaustion and accumulation of waste by products. These can cause stress to your cells and result in the development of vacuoles and a grainy appearance. Your cells may still be close to 100% viable, but their growth rate will slow and the stationary phase of a culture is reached. This has the consequence that when you eventually sub culture your cells they may not start dividing immediately they are placed into fresh medium. If you monitor their growth you may see a lag before they begin to increase in number.
A further consequence of setting up a new culture of cells from one that had attained a very high density is that your dilution factor will be quite high. We have noted that dilution factors greater than 10 tend to result in slower initial growth of cells. The reasons for this are unclear but are probably a combination of the cells being in the stationary phase at passage and the low concentration of “conditioned” medium in the new culture. Conditioned or used medium is thought to contain factors generated by cells grown in the absence of serum that help other cells divide efficiently.
So ideally you should never grow insect cells in suspension culture to very high densities. We think it is best to let them reach about 3-6 x 106 cells/ml and then passage them to a new seeding density of 4-5 x 105 cells/ml. However, if your cells are only recently established from a frozen stock it may be wise to increase this seeding density slightly to 106 cells/ml to ensure they start dividing quickly.
More information of growing insect cells may be found in our baculoCOMPLETE manual: A Complete Laboratory Guide to the Baculovirus Expression System and Insect Cell Culture. If you do experience difficulties growing insect cells please contact us via firstname.lastname@example.org and we will try to help you.