Shell microelectrode arrays (MEAs) for brain organoids

Qi Huang¹, Bohao Tang², July Carolina Romero³, Yuqian Yang¹, Saifeldeen Khalil Elsayed⁴, Gayatri Pahapale¹, Tien-Jung Lee¹, Itzy E Morales Pantoja³, Fang Han⁵, Cynthia Berlinicke⁶, Terry Xiang¹, Mallory Solazzo¹, Thomas Hartung^{3

7

8

9}, Zhao Qin⁴, Brian S Caffo², Lena Smirnova^{3

8}, David H Gracias^{1

10

11

12

13

14}

Affiliations

¹ Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
² Department of Biostatistics, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21287, USA.
³ Center for Alternatives to Animal Testing, Department of Environmental Health and Engineering, Bloomberg School of Public Health and Whiting School of Engineering, Johns Hopkins University, Baltimore, MD 21205, USA.
⁴ Department of Civil and Environmental Engineering, Syracuse University, Syracuse, NY 13244, USA.
⁵ Department of Statistics, University of Washington, Seattle, WA 98195, USA.
⁶ Department of Ophthalmology, Johns Hopkins University School of Medicine, Wilmer Eye Institute, Baltimore, MD 21287, USA.
⁷ CAAT-Europe, University of Konstanz, 78464 Konstanz, Germany.
⁸ Environmental Metrology & Policy Program, Georgetown University, Washington, DC, 20057, USA.
⁹ Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, 21205, USA.
¹⁰ Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
¹¹ Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA.
¹² Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.
¹³ Laboratory for Computational Sensing and Robotics (LCSR), Johns Hopkins University, Baltimore, MD 21218, USA.
¹⁴ Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

PMID: 35977026
PMCID: PMC9385157
DOI: 10.1126/sciadv.abq5031

Shell microelectrode arrays (MEAs) for brain organoids

Qi Huang et al. Sci Adv. 2022.

. 2022 Aug 19;8(33):eabq5031.

doi: 10.1126/sciadv.abq5031. Epub 2022 Aug 17.

Authors

Affiliations

¹ Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
² Department of Biostatistics, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21287, USA.
³ Center for Alternatives to Animal Testing, Department of Environmental Health and Engineering, Bloomberg School of Public Health and Whiting School of Engineering, Johns Hopkins University, Baltimore, MD 21205, USA.
⁴ Department of Civil and Environmental Engineering, Syracuse University, Syracuse, NY 13244, USA.
⁵ Department of Statistics, University of Washington, Seattle, WA 98195, USA.
⁶ Department of Ophthalmology, Johns Hopkins University School of Medicine, Wilmer Eye Institute, Baltimore, MD 21287, USA.
⁷ CAAT-Europe, University of Konstanz, 78464 Konstanz, Germany.
⁸ Environmental Metrology & Policy Program, Georgetown University, Washington, DC, 20057, USA.
⁹ Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, 21205, USA.
¹⁰ Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
¹¹ Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA.
¹² Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.
¹³ Laboratory for Computational Sensing and Robotics (LCSR), Johns Hopkins University, Baltimore, MD 21218, USA.
¹⁴ Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

PMID: 35977026
PMCID: PMC9385157
DOI: 10.1126/sciadv.abq5031

Abstract

Brain organoids are important models for mimicking some three-dimensional (3D) cytoarchitectural and functional aspects of the brain. Multielectrode arrays (MEAs) that enable recording and stimulation of activity from electrogenic cells offer notable potential for interrogating brain organoids. However, conventional MEAs, initially designed for monolayer cultures, offer limited recording contact area restricted to the bottom of the 3D organoids. Inspired by the shape of electroencephalography caps, we developed miniaturized wafer-integrated MEA caps for organoids. The optically transparent shells are composed of self-folding polymer leaflets with conductive polymer-coated metal electrodes. Tunable folding of the minicaps' polymer leaflets guided by mechanics simulations enables versatile recording from organoids of different sizes, and we validate the feasibility of electrophysiology recording from 400- to 600-μm-sized organoids for up to 4 weeks and in response to glutamate stimulation. Our studies suggest that 3D shell MEAs offer great potential for high signal-to-noise ratio and 3D spatiotemporal brain organoid recording.

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Figures

**Fig. 1.. Fabrication process flow and experimental images of the 3D shell MEAs.**
(A) Fabrication process flow of 3D shell MEAs. (B) An optical image of the fabricated shell MEAs. (C) A zoomed-in optical image of the shell electrodes in the flat state. Scale bar, 200 μm. (D) Scanning electron microscopy (SEM) image of the shell MEAs after actuation. Scale bar, 100 μm. (E) The recording electrodes on the leaflet before (left) and after (right) conductive polymer PEDOT:PSS electroplating. Scale bar, 50 μm.

**Fig. 2.. Brain organoid model.**
(A) Eight-week brain organoid stained with neuronal marker MAP2 (green) and neuroprogenitor marker, Nestin (red). (B) Eight-week brain organoid stained with neuronal marker β-III-tubulin (green) and astrocyte marker, GFAP (red). Nuclei are stained with Hoechst (blue). Scale bar, 100 μm. (C) RT-PCR showing expression of neuroprogenitor (*Nestin*), neuron (*NeuN*), astrocyte (*GFAP*), and oligodendrocyte (*MBP*) genes over the course of 10 weeks of differentiation. Data are shown as means ± SEM, n = 3. MAP2, microtubule-associated protein 2; GFAP, glial fibrillary acidic protein; MBP, myelin basic protein; NeuN, neuronal nuclei; NPC, neuroprogenitors; 2w, 4w, 8w, and 10w indicate 2, 4, 8, and 10 weeks of differentiation starting from NPC stage, respectively.

**Fig. 3.. FEM simulation of the programmable folding of 3D MEAs and optical images of brain organoid encapsulation.**
(A) Plot depicting the simulated radius of curvature (ROC) as a function of the SU8 bilayer thickness and the top layer exposure energy. The top left image shows the size of the shell in flat state. The tip-to-tip distance from left to right is 1450 μm. (B) FEM snapshots showing organoids of different sizes (400 to 600 μm) fitting in tailored shell electrodes. (C) Corresponding SEM images of 3D shell electrodes with different levels of folding. The images are false-colored, with blue indicating the SU8 shell and yellow indicating the electrodes. Scale bar, 100 μm. (D) Bright-field image of the organoid in a 3D shell MEA, and (E) confocal image showing the top view (projected confocal stack) of a brain organoid (green, Fluo-4 calcium dye) with a diameter around 500 μm encapsulated in the 3D shell (blue) electrodes. Scale bar, 100 μm.

**Fig. 4.. 3D shell MEA recordings from encapsulated brain organoids.**
(A) Image of a quartz wafer–integrated 3D shell MEA. (B) Electrode distribution of the 3D shell MEA around the brain organoid. (C) Typical field potentials recorded from a brain organoid encapsulated within a 3D shell MEA. CH, channel. (D) Representative raster plot of the spontaneous firing of the brain organoid. (E) Representative overlaid spike waveform from channel 1. (F) Comparison of spike distribution from the recorded brain organoid before and after glutamate treatment.

**Fig. 5.. Investigation of different electrode configurations.**
(A) Optical images of the 3D shell electrodes and 2D electrodes in flat (left) and self-folded (right) state. Scale bar, 200 μm. (B) Optical image of the 3D shell electrodes (nos. 1, 2, and 3) encapsulating the brain organoid, along with the 2D electrodes (nos. 4, 5, 6, and 7). Scale bar, 200 μm. (C) Raster plot of the 3D shell and 2D recordings. (D) Histograms of spike counts having different signal-to-noise ratios (SNRs), comparing 3D and 2D channels. The graph displays that the 3D channels have a greater capacity to capture signals with different SNRs. (E) The SNR of the paired spikes recorded by 3D and 2D electrodes. (F) Trend statistics of the 2D and 3D channels with glutamate stimulation.

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Shell microelectrode arrays (MEAs) for brain organoids

Affiliations

Shell microelectrode arrays (MEAs) for brain organoids

Authors

Affiliations

Abstract

Figures

References

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Miscellaneous