Govindan Subashika, Batti Laura, Osterop Samira F, Stoppini Luc, Roux Adrien
Tissue Engineering Laboratory, Haute école du paysage, d'ingénierie et d'architecture de Genève, Haute école spécialisée de Suisse occidentale (HEPIA HES-SO), University of Applied Sciences and Arts Western Switzerland, Geneva, Switzerland.
ARIMA Lifesciences PVT Ltd., Chennai, India.
Front Bioeng Biotechnol. 2021 Jan 7;8:582650. doi: 10.3389/fbioe.2020.582650. eCollection 2020.
Minibrain is a spheroid model, composed of a mixed population of neurons and glial cells, generated from human iPSC derived neural stem cells. Despite the advances in human 3D models such as aggregates, spheroids and organoids, there is a lack of labeling and imaging methodologies to characterize these models. In this study, we present a step-by-step methodology to generate human minibrain nurseries and novel strategies to subsequently label projection neurons, perform immunohistochemistry and 3D imaging of the minibrains at large multiplexable scales. To visualize projection neurons, we adapt viral transduction and to visualize the organization of cell types we implement immunohistochemistry. To facilitate 3D imaging of minibrains, we present here pipelines and accessories for one step mounting and clearing suitable for confocal microscopy. The pipelines are specifically designed in such a way that the assays can be multiplexed with ease for large-scale screenings using minibrains and other organoid models. Using the pipeline, we present (i) dendrite morphometric properties obtained from 3D neuron morphology reconstructions, (ii) diversity in neuron morphology, and (iii) quantified distribution of progenitors and POU3F2 positive neurons in human minibrains.
微型脑是一种球体模型，由源自人诱导多能干细胞的神经干细胞产生的神经元和神经胶质细胞混合群体组成。尽管诸如聚集体、球体和类器官等人类三维模型取得了进展，但仍缺乏用于表征这些模型的标记和成像方法。在本研究中，我们提出了一种生成人类微型脑培养物的分步方法，以及随后标记投射神经元、进行免疫组织化学和在大的可多重化规模下对微型脑进行三维成像的新策略。为了可视化投射神经元，我们采用病毒转导；为了可视化细胞类型的组织，我们进行免疫组织化学。为了便于对微型脑进行三维成像，我们在此展示了适用于共聚焦显微镜的一步式安装和清除的流程及配件。这些流程经过专门设计，使得这些检测可以轻松地进行多重化，以便使用微型脑和其他类器官模型进行大规模筛选。使用该流程，我们展示了（i）从三维神经元形态重建获得的树突形态计量学特性，（ii）神经元形态的多样性，以及（iii）人类微型脑中祖细胞和POU3F2阳性神经元的定量分布。
