Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Via Balzaretti, 9, 20133 Milano, Italy.
Mario Negri Insitute for Pharmacolgical Research-IRCCS, Via Mario Negri, 2, 20156 Milano, Italy.
Cells. 2022 Jun 23;11(13):2008. doi: 10.3390/cells11132008.
The human brain is the most complex organ in biology. This complexity is due to the number and the intricate connections of brain cells and has so far limited the development of in vitro models for basic and applied brain research. We decided to create a new, reliable, and cost-effective in vitro system based on the Nichoid, a 3D microscaffold microfabricated by two-photon laser polymerization technology. We investigated whether these 3D microscaffold devices can create an environment allowing the manipulation, monitoring, and functional assessment of a mixed population of brain cells in vitro. With this aim, we set up a new model of hippocampal neurons and astrocytes co-cultured in the Nichoid microscaffold to generate brain micro-tissues of 30 μm thickness. After 21 days in culture, we morphologically characterized the 3D spatial organization of the hippocampal astrocytes and neurons within the microscaffold, and we compared our observations to those made using the classical 2D co-culture system. We found that the co-cultured cells colonized the entire volume of the 3D devices. Using confocal microscopy, we observed that within this period the different cell types had become well-differentiated. This was further elaborated with the use of drebrin, PSD-95, and synaptophysin antibodies that labeled the majority of neurons, both in the 2D as well as in the 3D co-cultures. Using scanning electron microscopy, we found that neurons in the 3D co-culture displayed a significantly larger amount of dendritic protrusions compared to neurons in the 2D co-culture. This latter observation indicates that neurons growing in a 3D environment may be more prone to form connections than those co-cultured in a 2D condition. Our results show that the Nichoid can be used as a 3D device to investigate the structure and morphology of neurons and astrocytes in vitro. In the future, this model can be used as a tool to study brain cell interactions in the discovery of important mechanisms governing neuronal plasticity and to determine the factors that form the basis of different human brain diseases. This system may potentially be further used for drug screening in the context of various brain diseases.
人类大脑是生物学中最复杂的器官。这种复杂性归因于脑细胞的数量和复杂的连接，迄今为止，这限制了用于基础和应用脑研究的体外模型的发展。我们决定基于 Nichoid 创建一种新的、可靠且具有成本效益的体外系统，该系统是通过双光子激光聚合技术微制造的 3D 微支架。我们研究了这些 3D 微支架设备是否可以创建一个允许体外操作、监测和功能评估混合脑细胞群体的环境。为此，我们建立了一种新的海马神经元和星形胶质细胞共培养模型，在 Nichoid 微支架中生成厚度为 30μm 的脑微组织。培养 21 天后，我们对微支架内海马星形胶质细胞和神经元的 3D 空间组织进行了形态学表征，并将我们的观察结果与经典的 2D 共培养系统进行了比较。我们发现共培养的细胞定植在 3D 设备的整个体积内。使用共聚焦显微镜，我们观察到在这段时间内，不同的细胞类型已经变得高度分化。使用 drebrin、PSD-95 和突触小体蛋白抗体进一步阐述了这一点，这些抗体标记了大部分神经元，无论是在 2D 还是 3D 共培养中。使用扫描电子显微镜，我们发现与 2D 共培养中的神经元相比，3D 共培养中的神经元具有明显更多的树突突起。后一种观察结果表明，在 3D 环境中生长的神经元比在 2D 条件下共培养的神经元更有可能形成连接。我们的结果表明，Nichoid 可作为一种 3D 设备用于体外研究神经元和星形胶质细胞的结构和形态。在未来，该模型可用作研究脑内细胞相互作用的工具，以发现控制神经元可塑性的重要机制，并确定构成不同人类脑疾病基础的因素。该系统可能在各种脑疾病的背景下用于药物筛选。
