The advent of human induced pluripotent stem cells (iPSCs) has accelerated the development of miniature in vitro 3D human brain models for the purpose of disease modeling, drug testing and molecular screening. In vitro culture models have been integral in studying aspects of brain development and function in real time. 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. 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. To facilitate 3D imaging of minibrains, we present here pipelines and accessories for one step mounting and clearing suitable for confocal microscopy. To visualize projection neurons, we adapt viral transduction and to visualize the organization of cell types we implement immunohistochemistry. 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. Despite the advances in human 3D in vitro models such as aggregates, spheroids and organoids, there is a lack of labeling and imaging methodologies to characterize these models. Minibrain is a 3D brain in vitro spheroid model, composed of a mixed population of neurons and glial cells, generated from human iPSC derived neural stem cells. 4Swiss Center for Applied Human Toxicology (SCAHT), Bern, Switzerland.3Wyss Center for Bio and Neuroengineering, Geneva, Switzerland.2ARIMA Lifesciences PVT Ltd., Chennai, India.1Tissue 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.Osterop 3†, Luc Stoppini 1,4 and Adrien Roux 1,4* Subashika Govindan 1,2, Laura Batti 3, Samira F.

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