Developing a Biofunctional Xeno-free Hydrogel Platform for Long-term 3D Neuronal Differentiation and Maintenance

Abstract:

The development of in vitro systems for long-term neuronal differentiation and survival is crucial for understanding both physiological processes and various disease states. While traditional two-dimensional (2D) neuron models have been instrumental in neuroscience research, these systems fail to maintain neuron viability over extended periods, impeding prolonged neuronal maturation. One reason for this limitation is that 2D-based models misrepresent the in vivo microenvironment by lacking an extracellular matrix (ECM) and tissue-specific cells, negatively affecting cell survival, differentiation, and behavior. To address these challenges, advanced 2D models incorporating an ECM and three-dimensional (3D) culture systems have emerged as a superior approach for performing in vitro studies that are more physiologically relevant. However, many of these models use animal-based ECM derived from murine tumors, which are temperature-sensitive, contain over 2,000 undefined components, and, as a result, introduce experimental variability and affect reproducibility, hindering potential lab automation capabilities and clinical applications.

In this study, we developed VitroGel® NEURON, a novel xeno-free hydrogel platform with biofunctional modifications that resemble the native ECM to assess long-term neuronal maturation and survival. Human-induced pluripotent stem cell (iPSC)-derived neuronal stem cells (NSCs) and rat neuronal neuroblasts were embedded in a hydrogel matrix for 3D culture. In addition to the 3D hydrogel neuron culture system, we established 2D thin-coating and hydrogel-covering “blanket” methods for NSC and neuronal neuroblast culture and maintenance. VitroGel® NEURON supported robust NSC proliferation and maintenance in both 2D and 3D formats without compromising multipotency. The hydrogel facilitated extensive neurite and axonal outgrowth and sustained long-term neuronal differentiation and maturation, as evidenced by beta-III-tubulin expression. Collectively, these findings demonstrate that VitroGel® NEURON provides a defined, xeno-free cell culture platform that supports neuronal development and long-term maintenance, offering a reproducible and clinically relevant alternative to animal-based ECM systems. 

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VitroGel® NEURON

RocketCell™ NSC Xeno-Free
Complete Growth Kit

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