Aseptic technique & passaging
Cells that grow attached to the plastic are rinsed free of serum and divalent cations, released with trypsin-EDTA, neutralised with serum-containing medium and reseeded at a ratio chosen so they return to 70–90% confluence by the next split.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Adherent (trypsinization)
Cells that grow attached to the plastic are rinsed free of serum and divalent cations, released with trypsin-EDTA, neutralised with serum-containing medium and reseeded at a ratio chosen so they return to 70–90% confluence by the next split.
How it differs from the standard method
- Rinse with Ca²⁺/Mg²⁺-free PBS before the trypsin.
- Watch the cells, not the clock: most lines round up and lift within 2–5 min at 37 °C.
- Stop the trypsin with at least twice its volume of complete medium (or a trypsin inhibitor in serum-free work).
- Gentler alternatives exist for sensitive cells: recombinant proteases or non-enzymatic EDTA-based dissociation buffers.
1Keeping a line alive without letting anything else in
A cell line in culture is a population that keeps dividing for as long as it has room and food. Left alone, an adherent line covers the flask, stops dividing, acidifies its medium and starts to die; a suspension line climbs until it exhausts the medium and then crashes.
When to split
Adherent cells are split while still sub-confluent, before contact inhibition stops them dividing.
Split ratio
The fraction carried forward. It has to match the doubling time and the interval, or the culture drifts up into confluence or down into sparseness.
Passage number
Every split adds one. It is the culture's age, written on the flask and in the record.
2Adherent versus suspension cells
Adherent cells attach through integrins to proteins adsorbed on the plastic and to each other through cadherins; both need calcium and magnesium. To detach them you rinse those ions away with PBS that lacks them, then add trypsin-EDTA: the protease cuts the adhesion proteins and EDTA chelates what calcium remains.
3How it goes wrong
- Splitting too rarely or at too low a ratio: the flask sits confluent, cells pile up and die, and each split starts with a long lag.
- Over-trypsinising: surface proteins are stripped, fewer cells reattach, and the survivors are not a random sample.
- Too little trypsin or a serum film left on the cells: the most adherent cells stay behind, so the line is selected at every passage.
- Harsh pipetting shears cells; too little leaves clumps that grow as islands.
- A lapse in technique lets bacteria or yeast in; the medium clouds and yellows a day or two later. Mycoplasma gives no visible sign at all.
- Long continuous culture: high passage numbers select for fast-growing variants and the line drifts away from the one characterised in the literature.
Key terms
Practise these as flashcards →How many times a culture has been subcultured since it was established or thawed; written on every flask.
The fraction of the growth surface covered by adherent cells, judged under a phase-contrast microscope.
The dilution applied at a passage, written 1:N: one part of the harvested cells goes into N parts of new culture.
The slowing and arrest of division in many adherent lines once cells touch on all sides.
The period after seeding before cells divide again, lengthened by rough handling or very low density.
Protease plus chelator used to detach adherent cells; neutralised by serum or a soybean trypsin inhibitor.
Cabinet with HEPA-filtered downflow and front inflow that protects both the work and the operator.
Cumulative number of doublings since a culture began; a more exact measure of age than passage number.
When to use it
- Routine maintenance of any continuous cell line between experiments.
- Expanding cells to the number an experiment, a transfection or a freeze needs.
- Moving a culture into a new vessel or medium, or recovering it after a thaw.
Compared with alternatives
Counting is how you seed a defined number of cells rather than splitting by ratio, and the only way to keep a suspension line in range.
When the passage number nears the lab's limit, the answer is a fresh vial from the bank rather than more passaging.
Aseptic technique prevents contamination; routine mycoplasma tests and STR profiling are how you find out whether it worked.
Primary cells have a finite lifespan and need gentler, more frequent handling than an immortal line.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Cells that grow attached to the plastic are rinsed free of serum and divalent cations, released with trypsin-EDTA, neutralised with serum-containing medium and reseeded at a ratio chosen so they return to 70–90% confluence by the next split.
- Rinse with Ca²⁺/Mg²⁺-free PBS before the trypsin.
- Watch the cells, not the clock: most lines round up and lift within 2–5 min at 37 °C.
- Stop the trypsin with at least twice its volume of complete medium (or a trypsin inhibitor in serum-free work).
- Gentler alternatives exist for sensitive cells: recombinant proteases or non-enzymatic EDTA-based dissociation buffers.