Yan Mengying, Wang Lulu, Wu Yiyong, Wang Liping, Lu Yi
CAS Key Laboratory of Brain Connectome and Manipulation, the Brain Cognition and Brain Disease Institute, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions, Shenzhen 518055, China.
CAS Key Laboratory of Brain Connectome and Manipulation, the Brain Cognition and Brain Disease Institute, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions, Shenzhen 518055, China.
Acta Biomater. 2023 Feb;157:252-262. doi: 10.1016/j.actbio.2022.12.011. Epub 2022 Dec 12.
Biomimetic brain structures and artificial neural networks have provided a simplified strategy for quantitatively investigating the complex structural and functional characteristics of highly interconnected neural networks. To achieve this, three-dimensional (3D) cell culture approaches have attracted much attention, which can mimic cell-cell interactions at the organism level and help better understand the function of specific neurons and neuronal networks than traditional two-dimensional cell culture methods. However, 3D scaffolds similar to the natural extracellular matrix to support the culturing, recording, and manipulation of neurons have long been an unresolved challenge. To resolve this, 3D hydrogel scaffolds can be fabricated via an innovative thermal treatment followed by an esterification process. A highly porous microstructure was formed within the bulk hydrogel scaffold, which showed a high porosity of 91% and a low Young's modulus of 6.11 kPa. Due to the merits of the fabricated hydrogel scaffolds, we constructed 3D neural networks and detected spontaneous action potentials in vitro. We successfully induced seizure-like waveforms in 3D cultured neurons and suppressed hyperactivated discharges by selectively activating γ-aminobutyric acid-ergic (GABAergic) interneurons. These results prove the advantages of our hydrogel scaffolds and demonstrate their application potential in the accurate dissection of neural circuits, which may help develop effective treatments for various neurological disorders. STATEMENT OF SIGNIFICANCE: While 3D cell culture approaches have attracted much attention and offer more advantages than two-dimensional cell culture methods, 3D scaffolds similar to the natural extracellular matrix to support the culturing, recording, and manipulation of neurons have long been an unresolved challenge. Herein, we developed a simplified and low-cost strategy for fabricating highly porous and cytocompatible hydrogel scaffolds for the construction of three-dimensional (3D) neural networks in vitro. The cultured 3D neural networks can mimic the in vivo connection among different neuron subgroups and help accurately dissect and manipulate the structure and function of specific neural circuits.
仿生脑结构和人工神经网络为定量研究高度互连神经网络的复杂结构和功能特性提供了一种简化策略。为此，三维（3D）细胞培养方法备受关注，它可以在生物体水平模拟细胞间相互作用，比传统的二维细胞培养方法更有助于深入理解特定神经元和神经网络的功能。然而，长期以来，制备类似于天然细胞外基质的3D支架以支持神经元的培养、记录和操作一直是一个未解决的难题。为了解决这个问题，可以通过创新的热处理和酯化过程制备3D水凝胶支架。在块状水凝胶支架内部形成了高度多孔的微观结构，其孔隙率高达91%，杨氏模量低至6.11 kPa。由于所制备水凝胶支架的优点，我们构建了3D神经网络并在体外检测到自发动作电位。我们成功地在3D培养的神经元中诱导出癫痫样波形，并通过选择性激活γ-氨基丁酸能（GABA能）中间神经元抑制了过度激活的放电。这些结果证明了我们水凝胶支架的优势，并展示了其在精确剖析神经回路方面的应用潜力，这可能有助于开发针对各种神经疾病的有效治疗方法。重要性声明：虽然3D细胞培养方法备受关注且比二维细胞培养方法具有更多优势，但长期以来，制备类似于天然细胞外基质的3D支架以支持神经元的培养、记录和操作一直是一个未解决的难题。在此，我们开发了一种简单且低成本的策略，用于制备高度多孔且具有细胞相容性的水凝胶支架，以在体外构建三维（3D）神经网络。培养的3D神经网络可以模拟体内不同神经元亚群之间的连接，并有助于精确剖析和操纵特定神经回路的结构和功能。
