Details
| Original language | English |
|---|---|
| Article number | 1579580 |
| Journal | Frontiers in Bioengineering and Biotechnology |
| Volume | 13 |
| Publication status | Published - 30 May 2025 |
Abstract
The development of physiologically relevant three-dimensional (3D) culture platforms for neural stem cell (NSC) cultivation is essential for advancing neuroscience research, disease modelling, and regenerative medicine. In this study, we introduce norbornene-functionalized gelatin (GelNB) hydrogels crosslinked with a laminin-based peptide as a bioactive scaffold for NSC culture. A central composite design of experiments (DoE) approach was employed to systematically map hydrogel mechanical properties across varying macromer (4%–7%) and crosslinker (3–9 mM) concentrations via a response surface. This enabled precise tuning of hydrogel stiffness between 0.5 and 3.5 kPa, closely mimicking the mechanical properties of brain tissue. The optimized GelNB hydrogel formulation (5% GelNB, 8 mM crosslinker) supported NSC viability and enhanced NSC cluster formation demonstrating its suitability for 3D neural cell culture. Furthermore, we characterized the onset of hypoxia in 3D constructs using genetically encoded fluorescent hypoxia biosensors, revealing a cell density-dependent hypoxic response. At 3 × 106 cells/mL, hypoxic response was detected only after 7 days of cultivation, whereas at 8 × 106 cells/mL, hypoxic response was already observed within 24 h, illustrating the importance for using adequate cell numbers to avoid or achieve in situ physiological hypoxia. These findings highlight the importance of controlled ECM properties and oxygen microenvironments in NSC cultivation and provide valuable insights for the development of advanced biomimetic neural tissue models.
Keywords
- 3D cell culture, design of experiments, gelatin-norbornene, hydrogels, hypoxia, IKVAV, neural stem cells, response surface methodology
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biotechnology
- Chemical Engineering(all)
- Bioengineering
- Medicine(all)
- Histology
- Engineering(all)
- Biomedical Engineering
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In: Frontiers in Bioengineering and Biotechnology, Vol. 13, 1579580, 30.05.2025.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - 3D culture of neural progenitor cells in gelatin norbornene (GelNB) hydrogels
T2 - mechanical tuning and hypoxia characterization
AU - Dienemann, Sandra
AU - Wohlenberg, Ole Jacob
AU - Gerstenberger, Jan Georg
AU - Lavrentieva, Antonina
AU - Pepelanova, Iliyana
N1 - Publisher Copyright: Copyright © 2025 Dienemann, Wohlenberg, Gerstenberger, Lavrentieva and Pepelanova.
PY - 2025/5/30
Y1 - 2025/5/30
N2 - The development of physiologically relevant three-dimensional (3D) culture platforms for neural stem cell (NSC) cultivation is essential for advancing neuroscience research, disease modelling, and regenerative medicine. In this study, we introduce norbornene-functionalized gelatin (GelNB) hydrogels crosslinked with a laminin-based peptide as a bioactive scaffold for NSC culture. A central composite design of experiments (DoE) approach was employed to systematically map hydrogel mechanical properties across varying macromer (4%–7%) and crosslinker (3–9 mM) concentrations via a response surface. This enabled precise tuning of hydrogel stiffness between 0.5 and 3.5 kPa, closely mimicking the mechanical properties of brain tissue. The optimized GelNB hydrogel formulation (5% GelNB, 8 mM crosslinker) supported NSC viability and enhanced NSC cluster formation demonstrating its suitability for 3D neural cell culture. Furthermore, we characterized the onset of hypoxia in 3D constructs using genetically encoded fluorescent hypoxia biosensors, revealing a cell density-dependent hypoxic response. At 3 × 106 cells/mL, hypoxic response was detected only after 7 days of cultivation, whereas at 8 × 106 cells/mL, hypoxic response was already observed within 24 h, illustrating the importance for using adequate cell numbers to avoid or achieve in situ physiological hypoxia. These findings highlight the importance of controlled ECM properties and oxygen microenvironments in NSC cultivation and provide valuable insights for the development of advanced biomimetic neural tissue models.
AB - The development of physiologically relevant three-dimensional (3D) culture platforms for neural stem cell (NSC) cultivation is essential for advancing neuroscience research, disease modelling, and regenerative medicine. In this study, we introduce norbornene-functionalized gelatin (GelNB) hydrogels crosslinked with a laminin-based peptide as a bioactive scaffold for NSC culture. A central composite design of experiments (DoE) approach was employed to systematically map hydrogel mechanical properties across varying macromer (4%–7%) and crosslinker (3–9 mM) concentrations via a response surface. This enabled precise tuning of hydrogel stiffness between 0.5 and 3.5 kPa, closely mimicking the mechanical properties of brain tissue. The optimized GelNB hydrogel formulation (5% GelNB, 8 mM crosslinker) supported NSC viability and enhanced NSC cluster formation demonstrating its suitability for 3D neural cell culture. Furthermore, we characterized the onset of hypoxia in 3D constructs using genetically encoded fluorescent hypoxia biosensors, revealing a cell density-dependent hypoxic response. At 3 × 106 cells/mL, hypoxic response was detected only after 7 days of cultivation, whereas at 8 × 106 cells/mL, hypoxic response was already observed within 24 h, illustrating the importance for using adequate cell numbers to avoid or achieve in situ physiological hypoxia. These findings highlight the importance of controlled ECM properties and oxygen microenvironments in NSC cultivation and provide valuable insights for the development of advanced biomimetic neural tissue models.
KW - 3D cell culture
KW - design of experiments
KW - gelatin-norbornene
KW - hydrogels
KW - hypoxia
KW - IKVAV
KW - neural stem cells
KW - response surface methodology
UR - http://www.scopus.com/inward/record.url?scp=105007836582&partnerID=8YFLogxK
U2 - 10.3389/fbioe.2025.1579580
DO - 10.3389/fbioe.2025.1579580
M3 - Article
AN - SCOPUS:105007836582
VL - 13
JO - Frontiers in Bioengineering and Biotechnology
JF - Frontiers in Bioengineering and Biotechnology
SN - 2296-4185
M1 - 1579580
ER -