The brain is the most complex organ in the human body with billions of neurons and trillions of connections. I have always been intrigued by the brain’s mechanisms and by the pathologies that affect the “mind”. As I have learned of how little we understand about the mechanisms behind some of the treatments prescribed for those who suffer from mental illnesses and the unsystematic way in which some treatments have been developed, I have come to consider it my scientific duty to contribute to the world-wide effort to create a proper test platform for treatments for neurological disorders.
Three-dimensional (3D) neuronal models, such as brain organoids and assembloids (also known as “mini brains”), have provided pioneering platforms for understanding various aspects of brain development1–5 and brain pathologies6–14. However, despite this progress, building a manipulatable in-vitro model to study the altered or disrupted 3D functional interconnectivity in multiregional network pathologies such as a focal epileptic seizure remains a major challenge.
At the same time, microfluidic organ-on-chip platforms have been developed, aiming to study neuronal interactions. However, these approaches lack either the 3D connectivity or clinically relevant cellular diversity and complex functionality of organoid approaches.15,16
To fill this gap, in this study we introduce a novel method to develop 3D neuronal tissues which, while preserving the potential of organoids, opens a range of possibilities for engineering approaches to mechanistically analyze clinically relevant 3D functional network connectivity.
In this method, by promoting matrix-supported active cell reaggregation, we engineered multiregional cerebral tissues (Figure 1) with intact 3D neuronal networks and functional interconnectivity characteristic of brain networks. Furthermore, using a multi-chambered tissue-culture chip, we show that our separated but interconnected cerebral tissues can mimic neuropathological signatures of a seizure, such as the propagation of epileptiform discharges (Figure 2).
To our knowledge, this is the first method to produce “mini brains” that is not based on the SFEBq (or even SFEB) method, which uses (a quick) mechanically-enforced aggregation of the (dissociated) cells.
The combination of this culturing method and culturing platform holds the potential to mimic any pathology whereby the activity of one area of the brain is (pharmacologically) altered, which can in turn contribute to drug development.
Figure. 1 | Formation of human cerebral tissues via matrix-supported active reaggregation of cells (MARC).
a, Schematics of the MARC culture method, showing the different culture steps. Timeline and additives supplied in each step are indicated. b, Example phase-contrast images at the different 3D-culture phases, showing the matrix-supported active reaggregation of the cells into cerebral tissues. Single dissociated cells suspended in Matrigel grew into small spheroids (Day 1–7). During pre-terminal differentiation, neurite outgrowths extended from the spheroids (white arrows) and merged into neurite bundles (white arrowheads) between spheroids (Day 10, 15). The spheroids migrated and subsequently merged into large cerebral tissues (Day 20) (n = 120 samples across 6 independent experiments). Scale bar: 500 µm. c, Immunohistochemical co-staining of cryosections of MARC-produced cerebral tissues at Day 90 revealed the presence of markers of neural progenitor cells (NPCs; SOX2), early and mature neurons (Tuj1 and MAP2), mature excitatory Glutamatergic neurons (VGLUT1), inhibitory GABAergic neurons (VGAT), mature dopaminergic neurons (DAT), and astrocytes (GFAP), indicating the cellular diversity of the MARC-produced cerebral tissues (n = 5 samples across 2 independent experiments). Scale bar: 500 µm. d, Zoom-ins of the images in c as indicated by the white squares. Scale bar: 100 µm.
a-c, Schematics indicating the steps involved in the Penicillin-treatment experiments. Two cerebral tissues were generated in the two chambers of the PDMS-based iS3CC chip (see also Supplementary Fig. 2 for more details of the chip) and formed a connection through the porous membrane (a, see also Supplementary Fig. 3). To study the propagation of abnormal discharges from one tissue to the other, calcium imaging was performed on both tissues (b) and Penicillin G (“Pen”) was added to one of the chambers (c). d, Fluorescence pictures of intracellular calcium detected by fluo-4 direct in cerebral tissues at day 45 where one of the chambers (left, “treated”) was treated with Penicillin G, whereas the other (right, “untreated”) was not. The activity of 522 neurons was detected in the treated (blue circles) and untreated (red circles) tissues and analyzed by live calcium imaging (see also Supplementary Movie 3). Scale bar: 250 µm (n = 6 samples across 6 independent experiments). e-f, Time traces of 4 selected cells in the treated (blue) and untreated (red) cerebral tissues pre- (e) and post-treatment (f) with Penicillin G (see also Supplementary Fig. 4). The black vertical lines indicate instances where all 4 cells in the treated tissue showed synchronized transient peaks. This synchronicity propagated ~45% of the time to the cells in the untreated tissue. g-h, Quantification of the change in fluorescence intensity (g) and fold change in neuronal activity (h, log scale) induced by addition of Penicillin G in the treated (blue) and untreated (red) cerebral tissues. The symbols represent data for each cell; the boxes represent the median, 1st and 3rd quartiles; and the whiskers represent the 5th and 95th percentiles of the population data. Asterisk denotes statistically significant difference (Mann-Whitney U test, p < 10−11).
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