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.

Figure 1 |

Building a SpinΩ bioreactor. (a,b) CAD drawing (a) and photo (b) of a fully assembled SpinΩ bioreactor. (c) Enlarged view of individual parts of a SpinΩ bioreactor. (d) Photo of all parts needed to build one SpinΩ bioreactor. Please refer to Table 1 for corresponding part codes. The 3D PDF files for a and c allow the user to view the model from any angle (Supplementary Data 2 and 3). To open the 3D PDF file in Adobe Reader, click ‘Options’ on the pop-up notification (yellow bar) and agree to trust this document always.

Figure 2 |

Schematic diagram of brain region–specific organoid protocols. The protocols start with enzymatically detaching hiPSC colonies cultured on MEF at Day 0 to generate EBs (Steps 1–7 of the PROCEDURE). From here, the protocols diverge for the procedures to differentiate forebrain (Steps 9–32of the PROCEDURE), midbrain (Box 2) and hypothalamus (Box 3) organoids, respectively. The forebrain organoid protocol involves two unique procedures for embedding EBs in Matrigel at Day 7 and dissociating from Matrigel at Day 14, detailed in Steps 11–25. Finally, the procedures converge with culturing of organoids in the SpinΩ bioreactor, as described in Steps 26–30.

Figure 3 |

Progression and quality control of forebrain organoid development. (a) Example of an optimal hiPSC colony cultured on MEF, displaying sharp boundaries and homogeneous texture, with a diameter of ~1.5 mm. (b) Example of an optimal EB at Day 7 before embedding into Matrigel, showing a round and smooth morphology and a translucent surface. (c,d) Optimal neuroepithelium formation at Day 14, with a smooth surface and no sign of neuronal differentiation. The number of structures within each cluster may vary, but this does not substantially affect the outcome. (e) Optimal organoid formation at Day 35. The VZ appears more optically translucent than the surrounding immature neuronal layer. Note that the apical surface and ventricular lumen are also clearly visible. (f,g) At Days 56 (f) and 63 (g), the SVZ and CP layers outside the VZ should have expanded, whereas the VZ still appears to be bright. (h) At Day 84, the outer layers should further thicken, and the organoid overall should appear darker, whereas the VZ remains vaguely visible. The forebrain organoid should always exhibit bulges, because individual cortical structures expand independently. (i) Example of a suboptimal EB at Day 7. Note the uneven surface withdead or unhealthy cells attached. (j) Example of suboptimal EB forming underdeveloped neuroepithelial buds at Day 14. (k) Example of a failureto form neuroepithelium at Day 14. Note that cells extending neuronal processes or migrating into Matrigel both indicate premature differentiation. (l) Example of cystic organoid formed from failed neuroepithelium at Day 35. Scale bars, 100 μm (a–d, i–k), 500 μm (e–h, l).

Figure 4 |

Immunofluorescence characterization of brain region–specific organoids. (a) Forebrain organoid at Day 14, showing expression of dorsal forebrain NPC marker Pax6 (red). (b) A forebrain organoid with two cortical structures at Day 52. The ventricular surface is delineated by staining for adherens junction marker PKCλ (green). (c) Forebrain organoid at Day 84. The distribution of NPCs (SOX2, red), IPCs (TBR2, blue) and neurons (CTIP2, green) shows distinct VZ, SVZ and CP layers. (d) A forebrain organoid with multiple cortical structures coated by an ECM layer (laminin, green) from Matrigel treatment. (e,f) RGCs in forebrain organoids exhibit straight and radially oriented basal processes (P-Vimentin, red; Nestin, green) at Day45 (e) and Day 70 (f). (g) Midbrain organoid at Day 14, exhibiting radially organized neuroepithelium in the outer region positive for floor plate progenitor marker FOXA2 (green). (h, i) Midbrain organoid at Day 40, with clusters of FOXA2⁺ progenitor regions (red) and TH⁺ dopaminergic neurons (green). These panels show the same image, but i is a magnified view of a section of h. (j) Hypothalamus organoid at Day 21, consisting purely of RAX1⁺ progenitors (red). (k) Hypothalamus organoid at Day 40, containing clusters of OTP⁺ hypothalamic neurons (red) and POMC⁺ peptidergic neurons (green). Scale bars, 100 μm.