Generation of human fetal brain organoids and their CRISPR engineering for brain tumor modeling

The developing human brain displays unique features that are difficult to study in animal models. Current in vitro models based on human brain tissue face several challenges, including the limited cellular heterogeneity in two- or three-dimensional neural stem cell cultures, while tissue slice cultures suffer from short survival. We recently established culture conditions to derive organoid cultures directly from human fetal brain tissue by preserving tissue integrity, which can be long-term expanded and display cellular heterogeneity and complex organization. In this Protocol, we describe detailed procedures to establish human fetal brain organoids (FeBOs) that broadly retain regional characteristics, along with procedures for their passaging and characterization. In addition, we describe genome engineering approaches applied to FeBOs to generate mutant FeBO lines that serve as versatile bottom-up brain cancer models. Lastly, we exemplify various downstream applications applicable to both healthy and mutant FeBOs. Scientists with experience in tissue culture can expect the establishment of human FeBO cultures to take 2-3 weeks, while genome engineering of FeBOs takes 2-4 months.