Protocol – Subculturing suspension cells

Protocol – Subculturing suspension cells


  • Suspension cell cultures are passaged by diluting the existing culture.
  • Since cells float in the medium in suspension culture, they are not treated with trypsin-EDTA solution unless there are some special requirements. For example, if you need to plate estimated number of cells from a culture which forms tight clumps in suspension. In this case, cell clumps are treated with trypsin-EDTA to obtain single cell suspension to count the cell number accurately.
  • To subculture suspension cells, a small amount of cell suspension from the existing culture is transferred to a culture dish containing fresh growth medium.


  • Reagents and solutions:
    • Complete growth medium (room temperature / 37°C)
  • Equipment and disposables
    • T25 flask/Tissue culture dishes
    • Pipettes and pipette aid
    • Laminar flow hood
    • Beaker to discard the waste
Starting material:

Suspension cell culture (high density) ready to subculture

Prior to start:
  • Place complete medium in 37°C water bath for warming.
  • Clean and wipe workspace in the laminar flow hood with 70% ethanol, turn on UV light for 20 – 30 min. After 20 min, turn off the UV light and start the air flow. Let it flow for 10 min.


Subculturing of suspension culture growing

  • Check cells under the microscope to make sure cells are healthy and are not contaminated.
  • Use aseptic techniques while operating cell culture.


Step 1: Transfer all reagent bottles and disposables to the laminar flow hood
  • Spray and wipe all bottles and packets containing disposables (culture dish packets) with 70% ethanol and place them in the laminar flow hood.
  • Take out the estimated number of culture dishes from its packet and label them with the date of subculture, passage number, cell name and your name.
Step 2: Dilute cell suspension at recommended cell density (or split ratio)
  • Take out culture dish from the incubator and place inside laminar flow hood.
  • Pipet suspensension culture to suspend cells homogeneously and transfer an aliquot of cells into fresh culture flask/dish.
  • Add appropriate amount of growth medium (for T25 flask/ 60 mm dish, 4 – 5 ml medium is recommended).
  • Pipette up and down 2 – 3 times to suspend cells homogeneously.
  • Wright all details on culture dish including your name, cell line name, passage number and subculture date.
  • Discard remaining cell suspension if you don’t need them.
  • Cell counting is not necessary for the regular maintenance of cell in culture. Most researchers use split ratio to seed cells in a fresh culture dish.
  • Split ratio is a rough estimation of how many culture dishes can be prepared from the existing cell culture. For e.g., you can prepare 5 to 10 flask/dish from the cell culture (90% confluent) if the recommended split ratio is 1:5 to 1:10.
  • If recommended split ratio is 1:5, transfer ⅕th culture volume to the fresh culture dish. Alternatively, cells can be counted using haemocytometer or any other appropriate method and recommended number of cells can be transferred to the fresh dish. Often counting is performed to seed exact number of cells as needed for the experiment.
  • To count cells accurately, cell clumps should to broken into the single cells. To make single cell suspension, try several rounds of gentle pipetting. If still cell clumps are seen, collect cells by centrifugation, wash the pellet with PBS and briefly treat cells with trypsin-EDTA.
  • If you need to make many culture dishes/flasks, prepare master mix before transferring suspension to the fresh flask.

Step 3: Place the flask in the incubator. Open the lid of the flask slightly for air exchange if you are using sodium bicarbonate containing culture medium. Tighten the lid if you are using vented cap flask.