We want to talk about how to grow cells over the holiday period spanning Christmas and New Year. (OK, so we have covered this before but we think it is worth repetition for new users of the baculovirus-insect cell system). Most labs, academic and industry, will have some time off over the next few weeks. It is often a problem keeping your cell cultures in good condition so that when you return in 2019 you can pick up that important protein expression project without too much delay. Given that most insect cells (usually Spodoptera frugiperda or Trichoplusia ni cell lines) need to be sub cultured at least twice a week if they are grown as suspension cultures, how will they survive for this protracted period? (more…)
While OET Ltd has built its reputation on baculovirus-insect cell expression systems, we also undertake work that requires growing mammalian cells successfully too. Much of this work involves the transduction of these cells with BacMam vectors to express genes placed under the control of mammalian-specific promoter elements. Some of our work in this area was recently published and involved the generation of improved BacMam vectors for transducing both mammalian cells in culture and also human pancreatic islet cells for pre-clinical tests of gene therapy. (more…)
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.
With Autumn, or Fall as some of you may call it, upon us everyone seems to back in action in the lab again and hopefully growing insect cells successfully. Regular readers of these blogs will know that we frequently return to the question of how to propagate insect cells. The reason for doing so is because growing insect cells successfully is fundamental to producing recombinant proteins using the baculovirus system. (more…)
Every so often we are asked if storing insect cells at -80°C is a viable option if you haven’t got access to liquid nitrogen cryogenic facilities. Our standard response is that this is not a good idea for long term storage of viable cells. This is largely based on historical dogma that says that cells do not preserve their viability for long at these temperatures. However, since one of our protocols for freezing insect cells involves an overnight cooling step in propanol in the -80°C we thought we would test the longevity of Sf21 cells left at this temperature for a little while longer. (more…)
An important factor in baculovirus expression is assessing when to passage Sf9 cells, which are commonly used to make recombinant viruses or for protein production. In our last blog we talked about how to make the transition from monolayer to suspension cultures and how maintain cells in a healthy state during this time. To define what is meant by ‘healthy’ cells, Figure 1 shows a sample of healthy Sf9 cells which appear largely uniform in size and round. What doesn’t come across in a web-based image is how the cells have a ‘shiny’ appearance. Although hard to define, when you see it you will recognize it! (more…)
The transition to suspension Sf9 cells from monolayer cultures is not difficult but does depend on harvesting the attached cells at the right time. Too early and it is extremely hard to detach the cells from the culture flask. Too late and the cells will be in such poor condition that they may not recover when in suspension. Trying to describe in words how the monolayer culture should look prior to harvest is difficult. Therefore, we have prepared a series of images depicting Sf9 cells from just after they were dispensed into a monolayer culture to the point of harvest for transition to suspension. (more…)
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. (more…)
Have you ever had the experience of adapting insect cells to a new growth medium? We recently had to do this for a client project because they had used a particular medium previously and were keen to maintain the same conditions to continue the work with us. This process made us realise that switching to a new serum-free insect cell culture medium can result in short term stress responses in the insect cells, which in this case were Sf9. (more…)
Transferring Sf9 cells from monolayers to suspension cultures can sometimes be difficult. These insect cells, commonly employed for baculovirus-mediated expression, are usually kept growing in suspension culture. However, sometimes monolayer cultures are used to keep them going during periods of low demand. These cultures are also useful as a reserve or back up for the main suspension cultures, which even in the best laboratories can sometimes become contaminated with unwanted microbial visitors! (more…)