Protocol – Feeding Suspension Culture (Complete Replacement of Medium)

Overview

  • 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 as cells float in the medium. There are two commonly used methods to feed suspension culture: 
    • Complete replacement of medium which involves cell harvesting by centrifugation
    • Partially replacing the culture medium with fresh medium
  • Complete replacement of culture medium is ideally the best way to feed suspension culture. Since it takes time and involves an additional step of harvesting cells, often researchers partially replace the medium that takes just a few minutes. However, partial medium replacement is associated with the loss of cells from the culture.
  • Complete replacement of culture medium although takes time and effort, it results in almost no loss of cells if the optimal centrifuge speed and time are used for cell harvesting. 
  • This procedure is required when 
    • You observe dead or sick cells/suspended particles.
    • Medium color (phenol red-containing medium) has turned yellow very quickly.
    • You can not afford cell loss.

Requirements:

Equipment and disposables
> Laminar flow hood (Class II)
> Sterile pipettes (5 ml, 10 ml) (glass  or disposable plastic pipettes)
> Pipette aid

> Pasteur pipettes and vacuum source (for aspiration) (optional)
> Centrifuge tubes (15 ml)
> Benchtop centrifuge with 45° fixed-angle or swinging-bucket rotor (e.g., Eppendorf™ 5804 Series)

Reagents and solutions
> Complete cell culture medium  (warm up to 37°C or temperature at which cells are maintained)
> PBS (room temperature/37°C or temperature at which cells are maintained)

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 for 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 (basal medium + serum + other supplements) 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.

Objective
Feeding suspension culture, growing in a T25 flask.

Procedure:

Step 1: Examine the flask under a phase-contrast microscope. 
> Check whether the cells are healthy and the 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: Harvest the cells from the culture by centrifugation
> Shake the flask and transfer all medium from the flask to a 15 ml centrifuge tube using a 5 ml sterile pipette/pipette controller.
> Harvest cells from cell suspension by centrifugation at room temperature for 5 – 10 min at 250 × g (1000 – 1500 rpm for Eppendorf™ 5804 Series benchtop centrifuge) *. 
> Remove the supernatant carefully without disturbing the pellet..

Tip:
> 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): Wash the cells with PBS
> Flick the centrifuge tube using your fingers to dislodge the pellet and add 5 ml PBS. Resuspend the cells by pipetting a few times. 
> Harvest cells from cell suspension by centrifugation again at room temperature for 5 – 10 min at 100 – 250 × g (1000 – 1500 rpm for Eppendorf™ 5804 Series benchtop centrifuge) *. 
> Remove the supernatant carefully without disturbing the pellet.

Step 4: Resuspend the cells pellet in 5 ml medium and transfer all the content to a T25 flask. 
> Flick the centrifuge tube using your fingers to dislodge the pellet and add 5 ml complete growth medium. Resuspend the cells by pipetting a few times.
> Transfer all the content in a T25 flask.

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

* You must optimize the centrifuge speed and time for each cell line.

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