Protocol – Feeding Adherent Culture

  • Feeding adherent culture involves replacing the old consumed medium with the fresh growth medium. The purpose is to supply fresh nutrients and to remove the waste which keeps accumulating in the medium due to cell metabolism, degradation of medium components (for example ammonia is accumulated in the culture medium as a result of degradation of L-glutamine), and dying cells. 
  • Since adherent cells are attached to the substratum, medium replacement is an easy job. However, the process must be quick enough to protect the cell layer from drying.
  • The cell layer can also be washed with PBS if needed. Often PBS washing is included when you identify dead cells or floating particles in the medium during the inspection of the culture under a microscope.

Here we have taken an example of feeding C2C12 cells growing in a T25 flask. C2C12 cells adhere firmly to the substratum, therefore, pose no risk of cell loss during medium removal and PBS washing. These cells are maintained in DMEM medium supplemented with 10% FCS at 37°C in a humidified incubator.


Equipment and disposables:
> Laminar flow hood 
> Sterile pipettes (5 ml, 10 ml) and Pipetboy/Pipette Controller
> Pasteur pipettes and vacuum source (for aspiration) (optional)

Reagents and solution:
> Complete cell culture medium  (warm up to 37°C)
> PBS (room temperature/37°C)

Prior to start:
> Warm up the growth medium and PBS to 37°C by keeping them in a water bath (set at 37°C)
> Make the laminar flow hood ready cell culture work (wipe the work surface with 70% ethanol, turn the UV-light on, and after 20 min switch off the UV-light and turn on the laminar flow).

Note: Complete culture medium is usually stored at +4°C. It is recommended to warm up the culture medium as well as PBS to the cell’s physiological temperature (the temperature used to maintain the culture, e.g., 37°C for most mammalian cell lines and 28°C for most insect cell lines) in order to avoid unnecessary cold shock to cells.

Feeding adherent culture, growing in a T25 flask.


Step 1: Examine the flask under a phase-contrast microscope. 
> If you observe dead cells and suspended particles, you must include a PBS washing step.

Step 2: Remove the old medium from the flask quickly.
> Tilt the flask and aspirate out all the medium using a 5 ml pipette/pipette controller or Pasteur pipette connected with a vacuum source. 

> Remember to handle culture aseptically. Use one hand to operate the flask and the other hand to add or remove liquid. Usually, an experienced worker opens the lid of the culture dish and lifts it slightly with the help of two fingers of one hand, and uses the other hand to remove or add medium from the culture flask.

Step 3 (optional): Quickly give a PBS wash if necessary.
> Add 5 ml PBS to the flask. Now swirl the flask carefully to rinse the cell layer.  Tilt the flask to collect PBS at one side and aspirate out PBS completely.

> Make sure when you release the PBS from the pipette, the flow should not be toward the cell layer.

Often subculturing of adherent cells results in some cell death due to the harsh treatment (e.g., trypsin) used to detach cells from the substratum. Therefore, it is expected to see some dead cells and suspended particles in the dish. Washing cells with PBS removes these dead cells and suspended particles, and makes the observation under a microscope clear and easy without any confusion with microorganism contamination. 

Step 4: Quickly add 5 ml fresh medium to the flask.
Make sure the flow of liquid released from the pipette should not be towards the cell layer.

Step 5: Examine the flask under a phase-contrast microscope and place it back 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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