Subculture of Suspension Cell Lines

Cook Book Sept 2010 Volume 12


Aim

In general terms cultures derived from blood (e.g. lymphocytes) grow in suspension. Cells may grow as single cells or in clumps (e.g. EBV transformed lymphoblastoid cell lines). For these types of cell lines subculture by dilution is relatively easy. However, for cell lines that grow in clumps it may be necessary to bring the cells into a single cell suspension by centrifugation and re-suspension by pipetting in a smaller volume before counting.

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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
  • Trypan blue (vital stain)

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Equipment

  • Personal protective equipment (sterile gloves, laboratory coat, safety visor)
  • Waterbath set to 37°C
  • Microbiological safety cabinet at appropriate containment level
  • Centrifuge
  • Incubator
  • Inverted phase contrast microscope
  • Haemocytometer
  • Pre-labelled fl asks
  • Marker Pen
  • Pipettes

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Procedure

  1. View cultures using an inverted phase contrast microscope. Cells growing in exponential growth phase should be bright, round and refractile. Hybridomas may be very sticky and require a gentle knock to the fl ask to detach the cells. EBV transformed cells can grow in very large clumps that are very diffi cult to count and the centre of the large clumps may be non-viable.
  2. Do not centrifuge to subculture unless the pH of the medium is acidic (phenol red = yellow) which indicates the cells have overgrown and may not recover. If this is so, centrifuge at 150 x g for 5 minutes, reseed at a slightly higher cell density and add 10-20% of conditioned medium (supernatant) to the fresh media.
  3. Take a small sample (100-200μl) of the cells from the cell suspension and count the cells (Protocol 6 - Cell Quantifi cation). Calculate cells/ ml and re-seed the desired number of cells into freshly prepared fl asks, without centrifugation, just by diluting the cells. Refer to the data sheet supplied with the cell line for the recommended seeding density.
  4. Repeat this every 2-3 days.

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Key Points

  1. If the cell line is a hybridoma or another cell line that produces a substance (e.g. recombinant protein or growth factor) of interest retain the spent media for analysis.

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Materials

     
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