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Generation of ‘semi-guided’ cortical organoids with complex neural oscillations


Temporal development of neural electrophysiology follows genetic programming, similar to cellular maturation and organization during development. The emergent properties of this electrophysiological development, namely neural oscillations, can be used to characterize brain development. Recently, we utilized the innate programming encoded in the human genome to generate functionally mature cortical organoids. In brief, stem cells are suspended in culture via continuous shaking and naturally aggregate into embryoid bodies before being exposed to media formulations for neural induction, differentiation and maturation. The specific culture format, media composition and duration of exposure to these media distinguish organoid protocols and determine whether a protocol is guided or unguided toward specific neural fate. The ‘semi-guided’ protocol presented here has shorter induction and differentiation steps with less-specific patterning molecules than most guided protocols but maintains the use of neurotrophic factors such as brain-derived growth factor and neurotrophin-3, unlike unguided approaches. This approach yields the cell type diversity of unguided approaches while maintaining reproducibility for disease modeling. Importantly, we characterized the electrophysiology of these organoids and found that they recapitulate the maturation of neural oscillations observed in the developing human brain, a feature not shown with other approaches. This protocol represents the potential first steps toward bridging molecular and cellular biology to human cognition, and it has already been used to discover underlying features of human brain development, evolution and neurological conditions. Experienced cell culture technicians can expect the protocol to take 1 month, with extended maturation, electrophysiology recording, and adeno-associated virus transduction procedure options.

Key points

  • This protocol describes the generation of cortical organoids with complex neural oscillations using a ‘semi-guided’ approach, as well as their functional characterization through microelectrode array measurements, calcium imaging and adeno-associated virus transduction.

  • The ‘semi-guided’ nature of this approach allows an intermediate between the cellular heterogeneity of unguided approaches and the predictability of guided approaches, which we speculate underlies the emergence of complex oscillations.

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Fig. 1: Overview of generation of cortical organoids and resulting features.
Fig. 2: Electrophysiology overview and characterization.
Fig. 3: Calcium imaging overview and AAV7m8–GCaMP transduction characterization in cortical organoids.

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Data availability

Further information for Figs. 1d,e and 2c–f can be found in Trujillo et al.7. Sequencing from Trujillo et al. for Fig. 1 can be found at National Center for Biotechnology Information Gene Expression Omnibus: GSE130238. Further information for Extended Data Fig. 3d–f can be found in Tanaka et al.8.

Code availability

Code for MEA processing can be found at


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The authors thank I.-H. Park and colleagues for approving the inclusion of published data (Supplementary Fig. 3) adapted from their figure list in Tanaka et al.8. Additionally, we acknowledge the Muotri Lab for their discussion on best organoid culture practices. This work was supported by US National Institutes of Health grants (R01MH100175, R01NS105969, MH123828, R01NS123642, R01MH127077, R01ES033636, R21MH128827, R01AG078959, R01DA056908, R01HD107788, R01HG012351, R21HD109616, R01MH107367), a California Institute for Regenerative Medicine grant (DISC2-13515) and a grant from the Department of Defense (W81XWH2110306).

Author information

Authors and Affiliations



M.Q.F., T.C. and A.R.M. updated existing protocol documents to match current best practices and devised a protocol manuscript outline. M.Q.F. wrote the first full draft of the protocol document, generated Figs. 1–2 and Extended Data Fig. 2 from existing laboratory documents and original additions, and formatted the document for submission. M.Q.F., T.C., F.P. and A.R.M. edited the manuscript and devised content for additional experimental procedures following the main protocol. M.Q.F. and T.C. wrote the comparison between protocols in the introduction and troubleshooting tips for organoid culture and quality control. T.C. wrote the AggreWell forced aggregation protocol in the Supplementary Methods, Procedure 2: MEA protocol, the MEA section of the ‘Anticipated results’ and Extended Data Fig. 3. F.P. wrote Procedure 3: calcium imaging, troubleshooting for Options B and C, and provided Supplementary Videos 1, 2 and 3. F.P. and R.B. wrote Procedure 4: AAV transduction, and provided Fig. 3. A.R.M., S.S. and M.C. oversaw generation of the protocol. All authors were involved in final editing of the protocol.

Corresponding author

Correspondence to Alysson R. Muotri.

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Competing interests

A.R.M. is a cofounder of and has an equity interest in TISMOO, a company dedicated to genetic analysis and brain organoid modeling focusing on therapeutic applications customized for autism spectrum disorder and other neurological disorders with genetic origins. The terms of this arrangement have been reviewed and approved by the University of California San Diego in accordance with its conflict-of-interest policies. A.R.M. is an inventor of several patents related to human functional brain organogenesis, including the protocol described here.

Peer review

Peer review information

Nature Protocols thanks Wei Niu, Ranmal Samarasinghe, Tjitse Vandermolen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Trujillo, C. et al. Cell Stem Cell. 25, 558–569.e7 (2019):

Trujillo, C. et al. Science 371, eaax2537 (2021):

Papes, F. et al. Nat. Commun. 13, 2387 (2022):

Extended data

Extended Data Fig. 1 Third-party comparison of different brain organoid protocols and the fetal human brain.

(a) Schematic View of the Culture System Generating human cortical organoids (hCOs). Guided protocols originated from Eiraku et al.5 while non-guided protocols are from Lancaster et al.19. Timeline of neural induction, differentiation, and maturation step is shown across protocols. Note that, while we have use the term ‘semi-guided’ here to describe our protocol and distinguish it from other Eiraku et al.5 -derived directed protocols, all panels in this figure are adapted from ref. 8, which only utilized the terms ‘guided’ and ‘unguided’ and thus correctly classified our protocol7 as guided. (b–d) Shared-nearest-neighbors (SNN) graph visualization for differentiation trajectory. (b) Differentiation directions (arrows) were determined by pseudotime. (c) Estimated trajectory backbone from the SNN graph. (d) Comparison of differentiation trajectory among different protocols. (e) The presence of cell types in each organoid protocol and human fetal brain. Cell types with >0.25% of cells are denoted with a plus sign. F, Fiddes et al.17; V, Velasco et al.6; B, Birey et al.14; M, Madhavan et al.18; T, Trujillo et al.7; X, Xiang et al.15; Q, Quadrato et al.20; G, Giandomenico et al.50. (f) Enrichment of disease-related genes in each organoid protocol. The red boxes indicate data generated from the protocol presented here. Figure adapted with permission from ref. 8, Elsevier.

Extended Data Fig. 2 Molecular and functional reproducibility of cortical organoids.

(a) Schematic showing the single-cell approach performed to assess the reproducibility of organoid generation using different iPSC lines (WT1 and WT2). (b) tSNE plot of single-cell mRNA sequencing data from two 6-month-old organoids. (c) Expression of gene markers for various cell types both batches. (d) Population ratio of each cluster by replicate. (e) Consistent and reproducible development of electrical activity in organoids over time across four cell lines, bars represent mean ± s.e.m (n = 8, independent experiments performed in duplicates using two clones of an iPSC line).

Extended Data Fig. 3 Selection of organoids for plating.

(a) Good-quality 1-month-old organoids with visible spatial arrangement of neural rosette structures. Scale bar, 1,000 μm. Mixed quality organoids, where there is a mixed population of fully differentiated organoids with visible rosette structures and incomplete differentiated organoids—select rosetted organoids only for downstream assays and discard incompletely differentiated organoids. Poor-quality spheroids that did not efficiently neuralize and differentiate into rosetted organoids—discard and start over. (b) Distinguish proper spatial organization and structural development in organoids; orange arrows indicate holes and green arrows indicate rosettes. Scale bar, 1,000 μm. (c) Use of immunohistochemistry to distinguish between good-quality organoids with spatial neural rosette arrangement (smaller circles within the organoid) and incompletely differentiated organoids that contain neurons but lack neural rosette structures and spatial organization. Immunostainings showing nuclei (DAPI), neuron microtubules (MAP2), and proliferating NPCs (Ki67 and Nestin). Scale bar, 50 μm.

Supplementary information

Supplementary Information

Supplementary Methods.

Supplementary Video 1

Representative movies of calcium activity in GCaMP-expressing cortical organoids

Supplementary Video 2

Representative movies of calcium activity in OGB1-labeled 2D cultures of iPSC-derived neurons. 2D cortical neurons are 4 and 5 months old, respectively.

Supplementary Video 3

Representative movies of calcium activity from neurons from OGB1- labeled organoids plated on imaging plates.

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Fitzgerald, M.Q., Chu, T., Puppo, F. et al. Generation of ‘semi-guided’ cortical organoids with complex neural oscillations. Nat Protoc (2024).

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