Adapting Cells to a New Medium

Most of the media that are currently available on the market are based on formulations that are quite similar (e.g. Weiss, et al.). This makes it fairly easy to adapt cells to different formulations. Here I will detail a plan that is quite conservative and can be shortened considerably in many cases. In the simplest, shortest case, cells can simply be split into the new medium and then observed for several passages to be certain that growth parameters are acceptable. This procedure assumes that you are culturing in suspension, but can be easily adapted to attached cultures.

Growth Parameters

Doubling Time

Cell doubling time is the easiest measurement to use to assess the health of insect cells. Doubling times should be determined during the period the cells are in Log Phase replication. Doubling times between 16 and 24 hours are typical of Sf21 and Sf9 lines, though most are around 20 to 22 hours. [dt = t*ln2/ln(Ct/Co) where dt= doubling time, t=time between cell counts Ct and Co, Co=initial count, and Ct=count after time t. All times in hours.]

Cell Size

Cell size is a very useful parameter to monitor the health of your culture. If the cells are healthy they will maintain a consistent size on the low end of what is normal for that particular line. This can be quite variable from line to line. With Sf9 and Sf21 lines that I have used, diameters in the 16-18 µm [2100-3000 fl (femptoliters=µm^3)] range are quite typical, though I know of lines that are as small as 14 µm (1400 fl) and as large as 19 µm (3500 fl) and are still quite happy.

Duration of Lag

The duration of the lag at low density is dependent upon the split density that you employ. In general, the lower the split density that you use, the longer the lag. However, above a certain density threshold, with a given cell line, there is little density dependence, and below a threshold density there is a prohibitively long lag and cells shouldn't be routinely subjected to this stress.

Low density split tolerance

Cells that are capable of recovering with a short (< 12 h) lag phase at low density (ca. 2E5 cells/mL) are in good shape. This is, however, a cell line characteristic, and a little investigation is necessary to find the density tolerance of your line. I have lines that can be split to 5E4 cells/mL and recover nicely, and lines that won't recover well if split below 5E5 cells/mL.

High density maximum

This is the maximum density to which your cells will grow if left to proceed into stationary phase. This is cell line-medium combination dependent. I advise strongly against allowing your lines to grow into stationary phase, but after splitting some into fresh medium, you can allow the remainder to proceed into stationary phase and monitor them for the information. Typically, I avoid culturing cells above about 80% of their characteristic maximum which has varied in my experience from 2E6 to over 12E6 cells/mL.

Rigorous Adaptation Protocol

• Stage 1

  1. Prepare an appropriate amount of medium that is 25% New Medium (NM) and 75% Old Medium (OM) where OM is the medium your cells have been cultured in and NM is the medium to which you would like to adapt them.
  2. Split them into this 25% NM plus 75% OM at a density that is twice your usual low end split density.
  3. Expand them to your usual high density and monitor growth parameters.
  4. Continue splitting at this density in this combination medium until the growth parameters are within an acceptable range. This may be as little as a single split, that is there may be almost no change in growth parameters.
  5. Once they've stablized, split them to once your usual low end split density, and again monitor growth parameters.
  6. Again, continue splitting at this density in this combination medium until the growth parameters are within and acceptable range. This may be as little as a single split; that is, there may be almost no change in growth parameters.
  7. If the cells return to normal behavior, proceed to the next stage.
  8. If the cells fail to achieve their normal behavior, the new medium may just not be appropriate.
  9. If the growth parameters obtain a new equilibrium different from what is normal, but still acceptable, proceed with the next stage.
  
  

• Stage 2

  1. Prepare an appropriate amount of medium that is 50% NM plus 50% OM.
  2. Split them into this 50% NM plus 50% OM at a density that is twice your usual low end split density.
  3. Proceed as in Stage 1 to monitor growth parameters and stablize the cells. Then drop to once your usual low split density and stabilize again.
  
  

• Stage 3

  1. Proceed as in Stage 1 and 2 with 75% NM plus 25% OM
  
  

• Stage 4

  1. Proceed as in Stages 1, 2 and 3 with 100% NM.
  2. After the cells have stabilized in the new medium, characterize all the growth parameters to see if they've shifted substantially in the new medium.

Media Sources (see vendor page)

• Biowhitaker
• Expression Systems
• HyClone
• JRH Biosciences
• Life Technologies

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jlitts@baculovirus.com
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