In Vitro Development of Human iPSC-Derived Functional Neuronal Networks on Laser-Fabricated 3D Scaffolds
In Vitro Development of Human iPSC-Derived Functional Neuronal Networks on Laser-Fabricated 3D Scaffolds
复制标题
DOI：
10.1021/acsami.0c16616
复制
发表时间：
2021-02-09
影响因子：
9.5
通讯作者：
Chichkov, Boris
中科院分区：
文献类型：
--
作者：
Koroleva, Anastasia;Deiwick, Andrea;Chichkov, Boris
由人类诱导多能干细胞（hiPSC）产生的神经祖细胞是“脑芯片”的最前沿。调查事务所可行的和功能性的hiPSC衍生的神经元网络正在形成强大的体外模型，用于评估皮质回路的正常和异常形成，了解潜在的疾病机制，并研究对药物的反应。因此，它们代表了科学界和制药业的理想工具。然而，个别神经元和网络的完整功能成熟所需的培养条件仍然是未知的。已经认识到，与2D培养物相比，三维（3D）培养条件可以更好地模拟体内神经元组织发育，从而提供更理想的体外方法。在本文中，我们提出了一个三维支架平台，支持和促进复杂的神经元网络的发展的设计和实施。3D支架是通过双光子聚合（2PP），一种高分辨率的3D激光微结构技术，使用生物相容性和不可降解的光反应性树脂Dental LT Clear（DClear），通过直接激光写入生产的。神经元在由垂直堆叠的支架层形成的3D环境中发育和互连。开发的网络可以支持不同的细胞类型。从3D培养的第50天开始，神经元祖细胞可以发育成所有六层的皮质投射神经元（CNP）、不同类型的抑制性神经元和胶质细胞。此外，与2D条件相反，3D支架支持在120天的过程中长期培养神经元网络。通过钙成像探测网络健康和功能，这揭示了结合个体和集体事件的强烈自发神经元活动。总之，我们的研究结果突出了先进的微结构3D支架作为神经元功能的3D体外建模的可靠平台。
Neural progenitor cells generated from human induced pluripotent stem cells (hiPSCs) are the forefront of "brain-on-chip". investigations. Viable and functional hiPSC-derived neuronal networks are shaping powerful in vitro models for evaluating the normal and abnormal formation of cortical circuits, understanding the underlying disease mechanisms, and investigating the response to drugs. They therefore represent a desirable instrument for both the scientific community and the pharmacological industry. However, culture conditions required for the full functional maturation of individual neurons and networks are still unidentified. It has been recognized that three-dimensional (3D) culture conditions can better emulate in vivo neuronal tissue development compared to 2D cultures and thus provide a more desirable in vitro approach. In this paper, we present the design and implementation of a 3D scaffold platform that supports and promotes intricate neuronal network development. 3D scaffolds were produced through direct laser writing by two-photon polymerization (2PP), a high-resolution 3D laser microstructuring technology, using the biocompatible and nondegradable photoreactive resin Dental LT Clear (DClear). Neurons developed and interconnected on a 3D environment shaped by vertically stacked scaffold layers. The developed networks could support different cell types. Starting at the day 50 of 3D culture, neuronal progenitor cells could develop into cortical projection neurons (CNPs) of all six layers, different types of inhibitory neurons, and glia. Additionally and in contrast to 2D conditions, 3D scaffolds supported the long-term culturing of neuronal networks over the course of 120 days. Network health and functionality were probed through calcium imaging, which revealed a strong spontaneous neuronal activity that combined individual and collective events. Taken together, our results highlight advanced microstructured 3D scaffolds as a reliable platform for the 3D in vitro modeling of neuronal functions.