How to Create a Hypoxic Environment for Cell Culture

How to assemble and purge the Hypoxia Chamber, and how to assess the resulting low oxygen environment

Researchers try to mimic physiological conditions in vitro to expand our knowledge about the regenerative properties of stem cells. One physiological condition that can be replicated is the hypoxic conditions that can be found in tissue. The effects of low oxygen tension are species- and tissue source-dependent. For example, although low oxygen tension has been shown not to affect human mesenchymal stromal cell (MSC, also known as mesenchymal stem cell) phenotype1, it has been reported that it influences proliferation kinetics and metabolism2. In contrast, low oxygen tension has been shown to affect both the cell surface marker expression rate and expansion capability of mouse MSCs2. These findings show that mimicking physiological conditions is key. A low oxygen environment for cell culture can be achieved with the Hypoxia Incubator Chamber. This protocol describes how to assemble and purge the Hypoxia Chamber, and how to assess the resulting low oxygen environment.


  • Hypoxia Incubator Chamber (Catalog #27310)
  • Single Flow Meter (Catalog #27311)
  • Mixed gas tank with a regulator valve
    • Certified medical grade pre-mixed gas is required. For culturing in 5% oxygen, we recommend ordering a nitrogen tank, with the following hypoxic mixture: 5% O2, 10% CO2, 85% N2. If a lower oxygen percentage is required (e.g. 2% oxygen), adjust the percentage N2 to make up the balance (e.g. for culturing in 2% oxygen, increase the N2 to 88%)
  • 5/16" i.d. Tygon® tubing
  • Two adapters (to connect the flow meter to the gas chamber; optional depending on the version of the Hypoxia Incubator Chamber)


Part I: Opening the Hypoxia Chamber

  1. With your left hand, grasp the chamber and ring clamp at the opposite end of the clamp handle.
  2. Place your right hand firmly over the clamp handle and open slowly.
  3. Remove the clamp by extending it to its full diameter.
  4. Remove the lid and trays using both hands.

Part II: Stacking and Reassembling the Hypoxia Chamber

  1. To prevent excessive evaporation of cultures, the chamber must be humidified. Place one or two 10 mm uncovered Petri dishes containing 10 - 20 mL of sterile water in the chamber.
  2. Check all O-ring surfaces, making sure they are free of any particles that would cause leaks.
  3. Properly seat and position the trays on the base.
  4. Arrange your cultures in the chamber.
  5. Place the lid on the base.
  6. Grasp the clamp with your left hand and center the clamp at the position where the base and lid join together. The left hand should be held firmly against the clamp and the chamber.
  7. Slowly close the clamp handle with your right hand, ensuring that the clamp is centered.

Part III: Purging the Hypoxia Chamber

  1. Open both ports for the inlet and outlet tubing.

    Note: Both clamps must be open to avoid rupturing the chamber.
  2. Open the tank valve control all the way counterclockwise.
  3. Slowly open the regulator valve control clockwise to approximately 8 - 10 psi.
  4. Attach the bottom connection of the flowmeter to the inlet port tubing of the tank.
  5. Attach the tubing coming from the top of the flow meter to the chamber.
  6. Adjust the regulator valve control to 20 L/min by using the flow meter. A flow rate of 20 L/min completely purges the chamber in 4 minutes.
  7. After purging, disconnect the chamber from the gas source and close the two tubing clamps. This seals the mixed gases in the chamber.
  8. Turn off the valve control clockwise. Allow the gas to purge from the regulator until the pressure reads zero.
  9. Close the regulator by turning the regulator valve control counterclockwise.
  10. Put the chamber in an incubator at the appropriate temperature.

Additional Tips

  • Checking the Seal: For chambers that are filled with large quantities of culture plates, re-flush after 1 hour of incubating to remove any gases trapped in the plasticware.
  • Adjusting the Ring Clamp if Chamber Fails to Seal: Loosen the locking nut and turn the bolt clockwise to tighten or counterclockwise to loosen the clamp. After adjusting, tighten the locking nut.
  • Ensuring the Chamber is Hypoxic: Take the chamber out and place the tubing close to your ear. Open the tubing clamp and listen for gas escaping. If you hear a puff, rather than a continuous flow, it's a good seal.
  • Cleaning: The chamber can be sterilized with 70% ethanol, 5% formalin, or sterilizing gas mixtures.
  • For additional information, see the Product Information Sheet for the Hypoxia Incubator Chamber.
  • Document #PR00012
  • Version 1.0.0
  • May 2020

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    1. Wagey et al 2008. STEMCELL Poster presented at TERMIS NA 2008
    2. Ex vivo expansion of human mesenchymal stem cells: A more effective cell proliferation kinetics and metabolism under hypoxia. Cabral et al 2010 J Cell Physiol