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Biotechnol."],"abstract":"<jats:p>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%\u20137%) and crosslinker (3\u20139\u00a0mM) concentrations via a response surface. This enabled precise tuning of hydrogel stiffness between 0.5 and 3.5\u00a0kPa, closely mimicking the mechanical properties of brain tissue. The optimized GelNB hydrogel formulation (5% GelNB, 8\u00a0mM 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 \u00d7 10<jats:sup>6<\/jats:sup> cells\/mL, hypoxic response was detected only after 7\u00a0days of cultivation, whereas at 8 \u00d7 10<jats:sup>6<\/jats:sup> cells\/mL, hypoxic response was already observed within 24\u00a0h, illustrating the importance for using adequate cell numbers to avoid or achieve <jats:italic>in situ<\/jats:italic> 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.<\/jats:p>","DOI":"10.3389\/fbioe.2025.1579580","type":"journal-article","created":{"date-parts":[[2025,5,30]],"date-time":"2025-05-30T08:18:26Z","timestamp":1748593106000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":6,"title":["3D culture of neural progenitor cells in gelatin norbornene (GelNB) hydrogels: mechanical tuning and hypoxia characterization"],"prefix":"10.3389","volume":"13","author":[{"given":"Sandra","family":"Dienemann","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ole Jacob","family":"Wohlenberg","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jan Georg","family":"Gerstenberger","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Antonina","family":"Lavrentieva","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Iliyana","family":"Pepelanova","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,5,30]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"4141","DOI":"10.1021\/acs.biomac.2c00585","article-title":"Heparinized gelatin-based hydrogels for differentiation of induced pluripotent stem cells","volume":"23","author":"Arkenberg","year":"2022","journal-title":"Biomacromolecules"},{"key":"B2","doi-asserted-by":"publisher","first-page":"1303","DOI":"10.1089\/scd.2017.0291","article-title":"The role of dissolved oxygen levels on human mesenchymal stem cell culture success, regulatory compliance, and therapeutic potential","volume":"27","author":"Bahsoun","year":"2018","journal-title":"Stem Cells Dev."},{"key":"B3","doi-asserted-by":"publisher","first-page":"3015","DOI":"10.1242\/jcs.079509","article-title":"Deconstructing the third dimension-how 3D culture microenvironments alter cellular cues","volume":"125","author":"Baker","year":"2012","journal-title":"J. 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