Deciding what goes into the medium and what it commits you to: why cell culture supplements and cell culture media supplements are the least controlled variable in most laboratories and why complete media cell culture preparation, and the complete cell culture media that results, should be a recorded step, what msc cell culture and other primary systems demand of supplement lots, where ypd media and other microbial formulations sit as a different discipline, what an hbss buffer and a citrate buffer are for and why trizma as a buffering base is a reagent with a grade, how pma and ionomycin are used as a stimulation control rather than as a supplement, what a shake flask, shake flask cell culture, a cell culture shake flask arrangement and a cell culture well plate impose on gas exchange and evaporation, and where cell culture services are the cheaper answer than doing it badly in house

Controlling the medium

1. Record every component and lot. Base medium, serum or replacement, growth factors, antibiotics and any additive, each with a lot number, in the experimental record. Without it a change in behaviour has no candidate explanation.
2. Reserve supplement lots for a study. Ask the supplier to hold a lot and buy enough to finish the work. A supplement lot change mid study is a variable that cannot be removed from the analysis afterwards.
3. Prepare complete medium as a documented step. Written composition, volume, date of preparation and a shelf life on the bottle. Growth factors and glutamine degrade, and an old bottle is a different medium.
4. Match the vessel to gas exchange. Shake flask fill volume, plate format and lid design decide oxygen transfer and evaporation. Edge effects in plates and oxygen limitation in over filled flasks are vessel problems, not biology.
5. Keep stimulation reagents separate from supplements. Reagents used to provoke a response are experimental treatments with their own controls, not medium components. Recording them in the same place as supplements confuses the two.
6. Consider outsourcing where scale exceeds capability. Expansion, banking and characterisation at scale are done better and cheaper by a service than by a research laboratory improvising, and the service returns documentation.

The uncontrolled variable

Most laboratories control the base medium carefully and treat supplements casually, which is the wrong way round: the supplements are the biologically active components and the ones that vary between lots.

Bringing supplements under the same lot discipline as the base is a small change that removes a large amount of unexplained variation.

Buffers are reagents too

Buffering bases and salt solutions come in grades, and the impurity profile of a cheap grade can matter for sensitive cells. Where a formulation is critical, the grade belongs in the recipe.

Write the buffer composition and the pH adjustment temperature into the protocol, and label prepared solutions with a date and a maker.

Common questions

Why did cells change behaviour after a medium change?

Almost always a supplement lot rather than the base medium. Recording lots and reserving them for the length of a study is what makes this investigable and usually preventable.

How long does complete medium last?

Shorter than the base medium, because glutamine and growth factors degrade. Set a shelf life per formulation, label the bottle with the preparation date, and do not extend it informally.

Do plate edge effects matter?

Considerably. Evaporation from outer wells changes concentration and osmolality over a multi day experiment. Use inner wells for samples and fill the outer ring with buffer.

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