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

authored by: Tabea Marie Fleischhammer, Sandra Dienemann, Nico Ulber, Iliyana Pepelanova, Antonina Lavrentieva
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.
Organisation(s): Institute of Technical Chemistry
Type: Contribution to book/anthology
Volume: 2755
Pages: 31-48
No. of pages: 18
Publication date: 07.02.2024
Publication status: Published
Peer reviewed: Yes
ASJC Scopus subject areas: Molecular Biology, Genetics
Electronic version(s): https://doi.org/10.1007/978-1-0716-3633-6_2 (Access: Closed)

BibTeX

@inbook{9599a812fea14e5c8e693c8097454093,
title = "Detection of Hypoxia in 2D and 3D Cell Culture Systems Using Genetically Encoded Fluorescent Hypoxia Sensors",
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.",
keywords = "3D cell culture, Adipose tissue-derived mesenchymal stem cells, GelMA, Genetically encoded hypoxia sensors, HIF-1, Hydrogel, Hypoxia reporter cells, MSC, UnaG",
author = "Fleischhammer, {Tabea Marie} and Sandra Dienemann and Nico Ulber and Iliyana Pepelanova and Antonina Lavrentieva",
note = "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.”",
year = "2024",
month = feb,
day = "7",
doi = "10.1007/978-1-0716-3633-6_2",
language = "English",
isbn = "978-1-0716-3632-9",
volume = "2755",
series = "Methods in molecular biology (Clifton, N.J.)",
publisher = "Humana Press",
pages = "31--48",
editor = "Gilkes, {Daniele M.}",
booktitle = "Hypoxia",
address = "United States",
}