Generation of human brain region–specific organoids using a miniaturized spinning bioreactor

Abstract

Human brain organoids, 3D self-assembled neural tissues derived from pluripotent stem cells, are important tools for studying human brain development and related disorders. Suspension cultures maintained by spinning bioreactors allow for the growth of large organoids despite the lack of vasculature, but commercially available spinning bioreactors are bulky in size and have low throughput. Here, we describe the procedures for building the miniaturized multiwell spinning bioreactor SpinΩ from 3D-printed parts and commercially available hardware. We also describe how to use SpinΩ to generate forebrain, midbrain and hypothalamus organoids from human induced pluripotent stem cells (hiPSCs). These organoids recapitulate key dynamic features of the developing human brain at the molecular, cellular and structural levels. The reduction in culture volume, increase in throughput and reproducibility achieved using our bioreactor and region-specific differentiation protocols enable quantitative modeling of brain disorders and compound testing. This protocol takes 14–84 d to complete (depending on the type of brain region–specific organoids and desired developmental stages), and organoids can be further maintained over 200 d. Competence with hiPSC culture is required for optimal results.

Using brain organoids to study human neurodevelopment, evolution and disease

“…In these protocols, brain region specific patterning factors were supplemented shortly after the formation of embryoid bodies from the human iPSCs (Kadoshima et al, 2013;Paşca et al, 2015;Watanabe et al, 2017). This resulted in the generation of forebrain, midbrain or hypothalamic brain organoids in a more reproducible way (Qian et al, 2016;Qian et al, 2018), the preference towards gliogenesis (Paşca et al, 2015), or the generation of hippocampal-like brain organoids (Sakaguchi et al, 2015).…”

“…To overcome this problem, scientists are trying to use other model systems. Following the great advances in isolating and culturing embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), several protocols have been developed for generating brain specific organoids derived from human ESCs or iPSCs, giving us a novel and useful tool for studying human corticogenesis (Kadoshima et al, 2013;Lancaster et al, 2013;Lancaster & Knoblich, 2014;Qian et al, 2016;Qian et al, 2018;Sloan, Andersen, Paşca, Birey, & Paşca, 2018). Brain organoids are a valuable model and recently a large variety of techniques have been developed allowing us to understand human-specific features of cortical development.…”

Improved single-cell ATAC-seq reveals chromatin dynamics of in vitro corticogenesis

“…Differentiation of forebrain organoids from human iPSCs was carried out using a previously described protocol with certain modifications 4,40 . In this modified protocol, cortical neurogenesis is initiated in AggreWell800 plates (STEMCELLTechnologies; Cat.…”

“…On day 14, GFR Matrigel-embedded organoids were transferred to culture in either a nylon (laser sintered) or ULTEM 9085m 3D-printed Spin Omega 12-well miniature bioreactor 4,40 (refer to Qian et al 4 . for detailed instructions on bioreactor 3D-printing and assembly) in Forebrain Differentiation Medium II (FDM II) consisting of a 1:1 mix of DMEM/F12 with GlutaMax and Neurobasal (Thermo Fisher, Cat.…”

“…M3148) with medium changes every 2-3 days. Approximately every two weeks, organoids were transferred to different wells in a new 12-well bioreactor plate to avoid position effects 4,40 . On day 70, we began adding 0.2 mM L-Ascorbic Acid (Sigma, Cat.…”