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Detection of Hypoxia in 2D and 3D Cell Culture Systems Using Genetically Encoded Fluorescent Hypoxia Sensors

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Hypoxia

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2755))

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Abstract

In vivo oxygen availability varies widely between cellular microenvironments, depending on the tissue of origin and its cellular niche. It has long been known that too high or too low oxygen concentrations can act as a biological stressor. Thus, the precise control of oxygen availability should be a consideration for cell culture optimization, especially in the field of three-dimensional (3D) cell culture. In this chapter, we describe a system for visualizing oxygen limitations at a cellular level using human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) that were genetically modified to express a fluorescent hypoxia sensor. This sensor can detect the activation of hypoxia-induced factors (HIF) transcription factors that lead to the expression of the oxygen-independent fluorescent protein, UnaG, at low oxygen concentrations. The response of these hypoxia reporter cells can be evaluated in two-dimensional (2D) and 3D cultivation platforms during exposure to hypoxia (1% O2) and normoxia (21% O2) using fluorescence microscopy and flow cytometry. We show that hypoxia reporter MSCs exhibit a hypoxia-induced fluorescence signal in both 2D and 3D cultivation platforms with fast decay kinetics after reoxygenation, rendering it a valuable tool for studying the cellular microenvironment and regenerative potential of hAD-MSCs in an in vivo-like setting.

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Acknowledgments

This study was funded by the German Research Foundation, DFG Project 398007461 “Biomolecular Sensor Platform for Elucidating Hypoxic Signatures in 2D and 3D in vitro culture Systems.”

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Authors and Affiliations

  1. Institute of Technical Chemistry, Leibniz University of Hanover, Hanover, Germany

    Tabea Marie Fleischhammer, Sandra Dienemann, Nico Ulber, Iliyana Pepelanova & Antonina Lavrentieva

Authors
  1. Tabea Marie Fleischhammer
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  2. Sandra Dienemann
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  3. Nico Ulber
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  4. Iliyana Pepelanova
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  5. Antonina Lavrentieva
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Corresponding author

Correspondence to Antonina Lavrentieva .

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Editors and Affiliations

  1. Department of Oncology Johns Hopkins, University School of Medicine, Baltimore, MD, USA

    Daniele M. Gilkes

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© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Fleischhammer, T.M., Dienemann, S., Ulber, N., Pepelanova, I., Lavrentieva, A. (2024). Detection of Hypoxia in 2D and 3D Cell Culture Systems Using Genetically Encoded Fluorescent Hypoxia Sensors. In: Gilkes, D.M. (eds) Hypoxia. Methods in Molecular Biology, vol 2755. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3633-6_2

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  • DOI: https://doi.org/10.1007/978-1-0716-3633-6_2

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3632-9

  • Online ISBN: 978-1-0716-3633-6

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