Development and evaluation of a 3D-engineered neural co-culture system: Impacts on oxidative stress, pentose phosphate pathway, trace element and mineral metabolisms

Abstract

Background Co-culturing multiple cell types within three-dimensional (3D) systems enhances the capacity to investigate intricate cell-cell and cell-microenvironment interactions, offering deeper insights into multicellular dynamics. This study comprehensively investigates the role of cellular metabolism within 3D cell culture models, offering a detailed examination of the underlying metabolic processes. New method In this study, a three-dimensional co-culture system was developed by encapsulating human neuroblastoma (SH-SY5Y) and human umbilical vein endothelial (HUVEC) cells within photopolymerized gelatin methacrylate (GelMA) hydrogels using photomasks for studying multiplex neural co-cultures. The structural characteristics of the hydrogel were analyzed using Fourier-transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Cell proliferation and antioxidant enzyme activities, including glucose 6-phosphate dehydrogenase (G6PD), 6-phosphoglucanate dehydrogenase (6-PGD), glutathione reductase (GR), glutathione s-transferase (GST), and glutathione peroxidase (GPx) were measured. The levels of trace elements and minerals were also quantified. Results The 3D-co-culture system can be considered non-toxic based on ISO 10993–5 since cell viability did not reduce below 80 % on the 7th day compared to day 0. The 3D model did not adversely affect the indicated enzymes in the co-culture system for up to 7 days. Na, Ca, Cu, Zn, and Mg levels significantly increased in the first, 4th, and 7th days compared to day 0. Comparison with existing methods Although photomask-based patterning of GelMA scaffolds has been previously demonstrated, our approach is unique as it combines multiplex photomask fabrication with the co-culture of neuronal and endothelial cells. Additionally, we measure multiple metabolic pathways, including the pentose phosphate pathway (PPP) and antioxidant enzymes, as well as the dynamics of trace and mineral elements in spatially defined neurovascular co-cultures. This integration allows for the simultaneous production of numerous geometrically controlled replicates and provides the first comprehensive assessment of PPP activity alongside trace element dynamics in engineered neural co-cultures. Conclusion This study highlights the benefits of using GelMA-based constructs in supporting viable and metabolically active cells in a 3D environment and presents a robust framework of methodologies that can be employed in future research to elucidate the complex metabolic dynamics in 3D environments, in tissue engineering, disease modeling, and drug development. © © 2025. Published by Elsevier B.V.