Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, United States.
George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, United States.
J Neurosci Methods. 2023 Jul 1;394:109898. doi: 10.1016/j.jneumeth.2023.109898. Epub 2023 May 24.
Brain organoids represent a new model system for studying developmental human neurophysiology. Methods for studying the electrophysiology and morphology of single neurons in organoids require acute slices or dissociated cultures. While these methods have advantages (e.g., visual access, ease of experimentation), they risk damaging cells and circuits present in the intact organoid. To access single cells within intact organoid circuits, we have demonstrated a method for fixturing and performing whole cell patch clamp recording from intact brain organoids using both manual and automated tools. We demonstrate applied electrophysiology methods development followed by an integration of electrophysiology with reconstructing the morphology of the neurons within the brain organoid using dye filling and tissue clearing. We found that whole cell patch clamp recordings could be achieved both on the surface and within the interior of intact human brain organoids using both manual and automated methods. Manual experiments were higher yield (53 % whole cell success rate manual, 9 % whole cell success rate automated), but automated experiments were more efficient (30 patch attempts per day automated, 10 patch attempts per day manual). Using these methods, we performed an unbiased survey of cells within human brain organoids between 90 and 120 days in vitro (DIV) and present preliminary data on morphological and electrical diversity in human brain organoids. The further development of intact brain organoid patch clamp methods could be broadly applicable to studies of cellular, synaptic, and circuit-level function in the developing human brain.
脑类器官代表了研究人类发育神经生理学的一种新模型系统。研究类器官中单神经元电生理和形态的方法需要使用急性切片或分离培养物。虽然这些方法具有优势（例如，可视化访问、易于实验），但它们有损伤完整类器官中细胞和回路的风险。为了在完整的类器官回路中获取单个细胞，我们展示了一种使用手动和自动化工具从完整的大脑类器官中进行固定和全细胞膜片钳记录的方法。我们展示了应用电生理学方法的开发，然后通过使用染料填充和组织透明化将电生理学与重建类器官内神经元的形态相结合。我们发现，使用手动和自动化方法都可以在完整的人类大脑类器官的表面和内部实现全细胞膜片钳记录。手动实验的成功率更高（手动全细胞成功率为 53%，自动全细胞成功率为 9%），但自动化实验效率更高（每天自动进行 30 次贴片尝试，每天手动进行 10 次贴片尝试）。使用这些方法，我们对体外培养 90 至 120 天的人类大脑类器官中的细胞进行了无偏倚调查，并展示了人类大脑类器官中形态和电生理多样性的初步数据。进一步开发完整的大脑类器官贴片钳方法可能广泛适用于研究人类发育大脑中的细胞、突触和电路水平功能。
