Cvetkovic Caroline, Basu Nupur, Krencik Robert
Center for Neuroregeneration, Department of Neurosurgery, Houston Methodist Research Institute.
Center for Neuroregeneration, Department of Neurosurgery, Houston Methodist Research Institute;
J Vis Exp. 2018 Aug 16(138):58034. doi: 10.3791/58034.
A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed 'organoids' or 'spheroids', for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures ('asteroids') are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.
我们在理解各种细胞类型和信号如何对突触回路功能产生影响方面面临的一个障碍是缺乏用于研究人类大脑的相关模型。解决这一问题的一项新兴技术是使用三维（3D）神经细胞培养物，即所谓的“类器官”或“球体”，以长期保存包括细胞外粘附分子在内的细胞间相互作用。然而，这些培养系统耗时且并非系统生成。在此，我们详细介绍一种从人类多能干细胞快速且一致地生成神经元和星形胶质细胞的3D共培养物的方法。首先，将预分化的星形胶质细胞和神经祖细胞解离并计数。接下来，将细胞与Rho激酶抑制剂以特定比例混合在成球培养皿中，以产生大小可重复的球体。作为漂浮球体培养数周后，最终将共培养物（“小行星体”）切片用于免疫染色，或接种到多电极阵列上以测量突触密度和强度。一般来说，预计该方案将产生显示成熟细胞类型特异性标记物、形成功能性突触并表现出自发性突触网络爆发活动的3D神经球体。总之，与单层培养相比，该系统允许在更合适的模型中进行药物筛选和疾病机制研究。
