Protocol – Feeding Suspension Culture (Partial Replacement of Medium)


  • Feeding cell 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, and dying cells. 
  • Replacement of culture medium from suspension culture is a bit tricky job as cells float in the medium. Ideally, cells are harvested from the medium by centrifugation and then resuspended in a fresh culture medium. However, in practice, a part of the culture medium is replaced with a fresh culture medium without harvesting the cells. This procedure is associated with the loss of cells. 
  • This procedure is similar to the method used for the subculturing of suspension cells.

Note: Use this procedure if cells are perfectly healthy and you can afford the loss of cells.


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 or temperature at which cells are maintained)

Prior to start:
> Warm up the growth medium to 37°C or temperature at which cells are maintained 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).

The complete culture medium is usually stored at +4°C. It is recommended to warm up the culture medium to the cell’s physiological temperature (the temperature culture is maintained, 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 suspension culture, growing in a T25 flask.


Step 1: Examine the flask under a phase-contrast microscope. 
> Check whether the cells are healthy and culture is clean without suspended particles. Usually, healthy cells are shiny and rounded whereas sick/dead cells are dull and have an irregular shape (you must know the features of the cell to differentiate healthy cells from sick/dead cells and contamination).

Step 2: Tilt the flask and allow the cells to settle. Remove a portion of medium (half or 2/3) carefully.
> Keep the flask in a standing position for a few minutes. All the cells will be settled at the bottom. Now slightly title the flask towards you and aspirate the culture medium using pipette/pipette collector. Make sure that the pipette tip touches only the surface of the liquid (do not insert deep inside the liquid). This step is crucial as the loss of cells depends on this step and the amount of medium you have taken out.

You can store the medium in a centrifuge tube that you have taken out from the culture dish. Throw it after ensuring that there are enough cells in the culture dish at the end of the process (see step 4).

Step 3: Add a sufficient amount of fresh medium
> Suppose you have taken out 50% medium, you can add the same amount of medium or more, depending on the volume of the culture dish. 
> Agitate the culture dish a bit to resuspend the cells evenly.

Step 4: Examine the flask under a phase-contrast microscope again and place it back in the incubator.
It is important to examine cells under a phase-contrast microscope to ensure that there are enough cells. If there are not enough cells, collect the cells by centrifugation the medium you stored in a centrifuge tube in step 2 and add them in the culture dish.

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