Cell Culture

How to Grow Cells over the Holiday Period

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…)

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Growing Mammalian Cells Successfully

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…)

Growing insect cells successfully – Part II

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? (more…)

Growing Insect Cells Successfully

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…)

Storing Insect Cells at -80°C?

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…)

When to Passage Sf9 Cells

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…)

Suspension Sf9 Cells from Monolayer Cultures

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…)

Adapting Insect Cells to a New Growth Medium?

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…)

Sf9 Cells from Monolayers to Suspension Cultures

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…)