Monday, May 7, 2012

How to Prepare Large-Scale Cell Culture Media

Cell culture media is to mammalian cells what a workout smoothie is to your human cells.

the matrix dozer cell culture media
Media's purpose is to make a stainless steel bioreactor hospitable to CHO cells by providing volume where temperature, dissolved oxygen (dO2), and pH can be controlled.

As well, it must provide nutrients intended for cellular uptake as well as a place to absorb metabolic waste. At the start of cell culture, the nutrient supply is defined; by the end of cell culture, the nutrients are depleted. Despite the added nutrients, media is still largely water and can thus be modeled at 1g/mL.

You make cell culture media the same way you'd make a smoothie, except at large-scale, you're making 100 or 500 or even 15,000 liters and so there are differences.
  1. Initial QS.
    This is where you add water-for-injection (WFI) into a clean media preparation tank.
  2. Add media powder.
    Once the powder touches the water, the media can promote growth. Since the bioreactor is only clean (and not sterile), if you don't proceed quickly, you may have a contamination on your hands.
  3. Add bicarbonate powder
    Bicarbonate is the buffer. This whole time, you are agitating, and pH control is OFF.
  4. Add peptones (optional).
    Over the past decade, we've seen movement away from bovine (cow) to porcine (pig) peptone. I've read that we now use veggie peptone, but have never seen it.
  5. Adjust pH
    Some will dispute the necessity of adjusting the pH in the media prep tank because once the media is transferred to the bioreactor the pH will get adjusted there.
  6. Final QS
    This is where you add the rest of the water. If you have an osmolality specification, you'd measure it here as an in-process test before transferring the media to the bioreactor
  7. Transfer/sterile filter the media
    While pumping the media over to the bioreactor, there will be sterile filters that remove 0.1 micron particles so that the media that ends up in the bioreactor is free of microbes. In some cases, the media is virally inactivated by passing through a "pasteurizer" that raises the temperature to 121 degC, holds it for 1 minute and cools it down.

It's a bit more than making a smoothie since mixing in a blender is forgiving. But in the preparation of cell culture media where you are making thousands of liters of this stuff at 7 bucks per gallon ($2/liter), the large-scale media preparation procedure has to be written to be highly reproducible.

Credits: Image above is from the greatest movie of all time - The Matrix (1999).

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