Details
| Original language | English |
|---|---|
| Title of host publication | Hypoxia |
| Subtitle of host publication | Methods and Protocols |
| Editors | Daniele M. Gilkes |
| Publisher | Humana Press |
| Pages | 31-48 |
| Number of pages | 18 |
| Volume | 2755 |
| ISBN (electronic) | 978-1-0716-3633-6 |
| ISBN (print) | 978-1-0716-3632-9 |
| Publication status | Published - 7 Feb 2024 |
Publication series
| Name | Methods in molecular biology (Clifton, N.J.) |
|---|---|
| Publisher | Humana Press |
| ISSN (Print) | 1064-3745 |
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
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Molecular Biology
- Biochemistry, Genetics and Molecular Biology(all)
- Genetics
Cite this
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Hypoxia: Methods and Protocols. ed. / Daniele M. Gilkes. Vol. 2755 Humana Press, 2024. p. 31-48 (Methods in molecular biology (Clifton, N.J.)).
Research output: Chapter in book/report/conference proceeding › Contribution to book/anthology › Research › peer review
}
TY - CHAP
T1 - Detection of Hypoxia in 2D and 3D Cell Culture Systems Using Genetically Encoded Fluorescent Hypoxia Sensors
AU - Fleischhammer, Tabea Marie
AU - Dienemann, Sandra
AU - Ulber, Nico
AU - Pepelanova, Iliyana
AU - Lavrentieva, Antonina
N1 - 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.”
PY - 2024/2/7
Y1 - 2024/2/7
N2 - 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.
AB - 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.
KW - 3D cell culture
KW - Adipose tissue-derived mesenchymal stem cells
KW - GelMA
KW - Genetically encoded hypoxia sensors
KW - HIF-1
KW - Hydrogel
KW - Hypoxia reporter cells
KW - MSC
KW - UnaG
UR - http://www.scopus.com/inward/record.url?scp=85184440265&partnerID=8YFLogxK
U2 - 10.1007/978-1-0716-3633-6_2
DO - 10.1007/978-1-0716-3633-6_2
M3 - Contribution to book/anthology
C2 - 38319567
AN - SCOPUS:85184440265
SN - 978-1-0716-3632-9
VL - 2755
T3 - Methods in molecular biology (Clifton, N.J.)
SP - 31
EP - 48
BT - Hypoxia
A2 - Gilkes, Daniele M.
PB - Humana Press
ER -