Subculture of Semi-Adherent Cell Lines

Cook Book Sept 2010 Volume 12

Fundamental Techniques in Cell Culture Laboratory Handbook-2nd Edition

12.5 Protocol 4 - Subculture of Semi-Adherent Cell Lines

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Aim

Some cultures grow as a mixed population (e.g. B95-8 - marmoset) where a proportion of cells do not attach to the tissue culture fl ask and remain in suspension. Therefore to maintain this heterogeneity both the attached cells and the cells in suspension must be subcultured.

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Materials

  • Media– pre-warmed to 37°C (refer to the ECACC Cell Line Data Sheet for the correct medium)
  • 70% (v/v) isopropanol in sterile water
  • PBS without Ca2+/Mg2+.
  • 0.25% trypsin/EDTA in HBSS, without Ca2+/Mg2+.
  • Soyabean Trypsin Inhibitor
  • Trypan blue (vital stain)

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Equipment

  • Personal protective equipment (sterile gloves, laboratory coat, safety visor)
  • Waterbath set to appropriate temperature
  • Microbiological safety cabinet at the appropriate containment level
  • Centrifuge
  • Inverted phase contrast microscope
  • Incubator
  • Haemocytometer
  • Pre-labelled fl asks
  • Marker Pen
  • Pipettes

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Procedure

  1. View cultures using an inverted phase contrast microscope to assess the degree of confl uency and confi rm the absence of bacterial and fungal contaminants. Give the fl ask a gentle knock fi rst, this may dislodge the cells from the fl ask and remove the need for a trypsinisation step with the subsequent loss of some cells due to the washes.
  2. Decant medium containing non-adherent cells into a sterile centrifuge tube and retain.
  3. Wash any remaining attached cells with PBS without Ca2+/Mg2+ using 1-2ml for each 25cm2 of surface area. Retain the washings.
  4. Pipette trypsin/EDTA onto the washed cell monolayer using 1ml per 25cm2 of surface area. Rotate fl ask to cover the monolayer with trypsin. Decant the excess trypsin.
  5. Return fl ask to incubator and leave for 2-10 minutes.
  6. Examine the cells using an inverted microscope to ensure that all the cells are detached and fl oating. The side of the fl asks may be gently tapped to release any remaining attached cells (see Protocol 6 - Cell Quantifi cation).
  7. Transfer the cells into the centrifuge tube containing the retained spent medium and cells.
  8. Centrifuge the entire cell suspension at 150 x g for 5 minutes.
  9. Remove the supernatant and re-suspend the cell pellet in a small volume (10-20ml) of fresh culture medium. Count the cells.
  10. Pipette the required number of cells into a new labelled fl ask and dilute to the required volume using fresh medium (refer to ECACC Cell Line Data Sheet for the required seeding density).
  11. Repeat this process every 2-3 days as necessary.

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Key Points

  1. Although most cells will detach in the presence of trypsin alone the inclusion of EDTA is used to enhance the activity of the enzyme.
  2. Trypsin is inactivated in the presence of serum. Therefore, it is essential to remove all traces of serum from the culture medium by washing the monolayer of cells with PBS without Ca2+/Mg2+. Repeated warming to 37°C also inactivates trypsin.
  3. Cells should only be exposed to trypsin/EDTA long enough to detach cells. Prolonged exposure could damage cell surface receptors. In general, a shorter time of exposure to trypsin is required for semiadherent cell lines in comparison to adherent cell lines.
  4. Trypsin should be neutralised with serum prior to seeding cells into new flasks otherwise cells will not attach.
  5. Trypsin may also be neutralised by the addition of Soyabean Trypsin Inhibitor, where an equal volume of inhibitor at a concentration of 1mg/ml is added to the trypsinised cells. The cells are then centrifuged, resuspended in fresh culture medium and counted as above.
  6. If a CO2 incubator is not available gas the flasks for 1-2 minutes with 5% CO2 in 95% air filtered through a 0.2μm filter.

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