TY - JOUR

T1 - Live reporting for hypoxia: Hypoxia sensor–modified mesenchymal stem cells as in vitro reporters

AU - Schmitz, Carola

AU - Pepelanova, Iliyana

AU - Seliktar, Dror

AU - Potekhina, Ekaterina

AU - Belousov, Vsevolod V.

AU - Scheper, Thomas

AU - Lavrentieva, Antonina

N1 - Funding information: This study was supported by the German Research Foundation (DFG Project 398007461 488 “3D Dual?Gradient Systems for Functional Cell Screening”) and Grant # 075?15?2019?1789 from the Ministry of Science and Higher Education of the Russian Federation. The publication of this article was funded by the Open Access Fund of Leibniz Universität Hannover. The authors also want to acknowledge support by the SMART BIOTECS initiative, financially supported by the Ministry of Science and Culture (MWK) of Lower Saxony, Germany. Open access funding enabled and organized by Projekt DEAL. This study was supported by the German Research Foundation (DFG Project 398007461 488 ?3D Dual-Gradient Systems for Functional Cell Screening?) and Grant # 075-15-2019-1789 from the Ministry of Science and Higher Education of the Russian Federation. The publication of this article was funded by the Open Access Fund of Leibniz Universit?t Hannover. The authors also want to acknowledge support by the SMART BIOTECS initiative, financially supported by the Ministry of Science and Culture (MWK) of Lower Saxony, Germany. Open access funding enabled and organized by Projekt DEAL.

PY - 2020/10/15

Y1 - 2020/10/15

N2 - Natural oxygen gradients occur in tissues of biological organisms and also in the context of three-dimensional (3D) in vitro cultivation. Oxygen diffusion limitation and metabolic oxygen consumption by embedded cells produce areas of hypoxia in the tissue/matrix. However, reliable systems to detect oxygen gradients and cellular response to hypoxia in 3D cell culture systems are still missing. In this study, we developed a system for visualization of oxygen gradients in 3D using human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) modified to stably express a fluorescent genetically engineered hypoxia sensor HRE-dUnaG. Modified cells retained their stem cell characteristics in terms of proliferation and differentiation capacity. The hypoxia-reporter cells were evaluated by fluorescence microscopy and flow cytometry under variable oxygen levels (2.5%, 5%, and 7.5% O 2 ). We demonstrated that reporter hAD-MSCs output is sensitive to different oxygen levels and displays fast decay kinetics after reoxygenation. Additionally, the reporter cells were encapsulated in bulk hydrogels with a variable cell number, to investigate the sensor response in model 3D cell culture applications. The use of hypoxia-reporting cells based on MSCs represents a valuable tool for approaching the genuine in vivo cellular microenvironment and will allow a better understanding of the regenerative potential of AD-MSCs.

AB - Natural oxygen gradients occur in tissues of biological organisms and also in the context of three-dimensional (3D) in vitro cultivation. Oxygen diffusion limitation and metabolic oxygen consumption by embedded cells produce areas of hypoxia in the tissue/matrix. However, reliable systems to detect oxygen gradients and cellular response to hypoxia in 3D cell culture systems are still missing. In this study, we developed a system for visualization of oxygen gradients in 3D using human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) modified to stably express a fluorescent genetically engineered hypoxia sensor HRE-dUnaG. Modified cells retained their stem cell characteristics in terms of proliferation and differentiation capacity. The hypoxia-reporter cells were evaluated by fluorescence microscopy and flow cytometry under variable oxygen levels (2.5%, 5%, and 7.5% O 2 ). We demonstrated that reporter hAD-MSCs output is sensitive to different oxygen levels and displays fast decay kinetics after reoxygenation. Additionally, the reporter cells were encapsulated in bulk hydrogels with a variable cell number, to investigate the sensor response in model 3D cell culture applications. The use of hypoxia-reporting cells based on MSCs represents a valuable tool for approaching the genuine in vivo cellular microenvironment and will allow a better understanding of the regenerative potential of AD-MSCs.

KW - 3D cell culture

KW - AD-MSCs

KW - hydrogels

KW - hypoxia sensor

KW - reporter cells

UR - http://www.scopus.com/inward/record.url?scp=85088565064&partnerID=8YFLogxK

U2 - 10.1002/bit.27503

DO - 10.1002/bit.27503

M3 - Article

C2 - 32667700

AN - SCOPUS:85088565064

VL - 117

SP - 3265

EP - 3276

JO - Biotechnology and bioengineering

JF - Biotechnology and bioengineering

SN - 0006-3592

IS - 11

ER -