Protocol – Passaging/Subculturing Suspension Culture


  • Suspension cell lines are passaged by diluting the existing culture.
  • Since cells float in the medium in suspension culture, they are not treated with a trypsin-EDTA solution unless there are some special requirements. For example, if you need to plate an estimated number of cells from a culture that 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 a suspension cell line, 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
1. Place the complete growth medium in a 37°C water bath for warming up.
2. 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 airflow. Let it flow for 10 min.

Subculturing suspension cell line

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

1. Do all operations aseptically.
2. All transfer of medium should be done inside the laminar flow hood.
3. Wear a lab coat and disposable latex gloves at all times.
4. Do all steps which involve changing the medium from cells quickly to avoid any risk of drying of cells.
5. Don’t decant medium from the flask/dish. This can increase the risk of contamination.


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 and label them with the date of the subculture, passage number, cell line name, and your name.

Step 2: Dilute cell suspension at recommended cell density (or follow recommended split ratio)
> Take out culture dishes from the incubator and place inside the laminar flow hood.

> Pipet cell culture to suspend cells homogeneously and transfer an aliquot of cells into a fresh culture flask/dish.
> Add an 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.
> Write all details on the 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 cells in culture. Most researchers use a 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 flasks/dishes from the cell culture (90% confluent) if the recommended split ratio is 1:5 to 1:10.

> If the recommended split ratio is 1:5, transfer ⅕th culture volume to the fresh culture dish. Alternatively, cells can be counted using a hemocytometer or any other appropriate method and a recommended number of cells can be transferred to the fresh dishes. Often counting is performed to seed the exact number of cells as needed for the experiment.

> A master suspension culture can also be prepared if you need to prepare more than 1 dish for experiments. Master suspension of cells ensures an equal number of cells among all dishes.
> To count cells accurately, cell clumps should be broken into single cells. To make the 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 a 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 a vented cap flask.

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