In Vitro Hypoxia Studies

Hypoxia, where the body or localized regions of the body are deprived of adequate oxygen supply, occurs during numerous acute and chronic disease states.  Examples include reduced blood flow during heart attack or stroke, and the oxygen-restricted microenvironment within a tumor.  Though cells can be studied in vitro under similar oxygen-poor conditions using a hypoxia chamber glove box, the inefficiencies of traditional endpoint assays are exacerbated by the constraints of working inside the chamber.  In contrast, an xCELLigence instrument run within a hypoxia chamber will continuously provide a real-time assessment of cell number, cell size/morphology, cell attachment quality, and cell migration/invasion without the need for intermittent manipulation/intervention by the researcher.

In the below example ACEA’s xCELLigence® RTCA DP instrument and electronic cell invasion and migration plate (CIM-Plate®) are used to monitor the ability of Ewing sarcoma cells to undergo chemotactic migration under normoxic vs. hypoxic conditions. Using the ligand SDF-1a as chemoattractant, it is first shown that CXCR4 (the most commonly expressed chemokine receptor in human cancer) promotes migration: chemical inhibition of CXCR4 with the small molecule AMD3100 causes significant reduction in the rate of migration (Figure A).  Next, the effect of exposing Ewing sarcoma cells to multiple stresses simultaneously is evaluated.  Moving serum-starved cells from a normoxic to a hypoxic atmosphere dramatically increased their rate of migration towards SDF-1a chemoattractant (Figure B).

Role of CXCR4 and Hypoxia on Chemotactic Migration of Ewing Sarcoma Cells. (A) Real-time analysis of Ewing sarcoma cell line migration. Cell index correlates linearly with the number of cells that have migrated through the pores of the CIM-Plate® toward the chemoattractant SDF-1a in the lower chamber. AMD3100 is a small molecule inhibitor of the chemokine receptor protein CXCR4. (B) Hypoxia increases the rate of migration of serum-deprived Ewing sarcoma cell line. Figures adapted from Molecular Cancer Research 2014 June, 12(6), 953-964.

Importantly, the above study highlights the flexibility and power of real-time cell analysis using xCELLigence®.  The quality and quantity of data generated in this study would be extremely difficult to match using traditional end point analyses conducted within a hypoxia chamber.

Key Benefits of Using xCELLigence for Studying In Vitro Hypoxia:
  • Data quantity: Automatic real-time data acquisition makes it possible to collect, with ease, significantly more data points than what is possible using traditional end point assays. This is especially true for hypoxia studies because manual acquisition of end points inside a hypoxia chamber is difficult and laborious.
  • Data quality and reproducibility are significantly better than what is possible using end point assays.
  • Time savings: Once cells are plated, data can be acquired continuously for anywhere from minutes to days/weeks without the need for hands-on time by the researcher.

All seven xCELLigence instruments are compatible with in vitro hypoxia studies.  To learn more about them, click here.

xCELLigence Research Grant

The research grant winner will be provided access to the xCELLigence Real Time Cell Analysis (RTCA) DP instrument, consumables, and consultation for up to 6 months.  Apply by December 15, 2019.

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In Vitro Hypoxia Studies Supporting Information:

  • In Vitro Hypoxia Studies Publications:
  1. Bach1 differentially regulates distinct Nrf2-dependent genes in human venous and coronary artery endothelial cells adapted to physiological oxygen levels. Chapple SJ, Keeley TP, Mastronicola D, Arno M, Vizcay-Barrena G, Fleck R, Siow RC, Mann GE. 2015 Dec 15. pii: S0891-5849(15)01166-1.
  2. In vitro oxygen availability modulates the effect of artesunate on HeLa cells. Murray J, Gannon S, Rawe S, Murphy JE. Anticancer Res. 2014 Dec;34(12):7055-60.
  3. Stress-induced CXCR4 promotes migration and invasion of ewing sarcoma. Krook MA, Nicholls LA, Scannell CA, Chugh R, Thomas DG, Lawlor ER.  Mol Cancer Res. 2014 Jun;12(6):953-64.
  4. Hypoxia-driven cell motility reflects the interplay between JMY and HIF-1α. Coutts AS, Pires IM, Weston L, Buffa FM, Milani M, Li JL, Harris AL, Hammond EM, La Thangue NB.  2011 Dec 1;30(48):4835-42.