Generation of Individualized, Standardized, and Electrically Synchronized Human Midbrain Organoids

Article Article scientifique Article Generation of Individualized, Standardized, and Electrically Synchronized Human Midbrain Organoids eng 2025-08-06 author El Harane Sanae 0000-0002-9738-7226 919289 Département de médecine (DEMED) author Nazari Bahareh 966202 author El Harane Nadia 956447 author Locatelli Manon 870687 author Zidi Bochra 965938 author Durual Stéphane 0000-0002-9772-8406 592156 CUMD - Biomatériaux (BMAT) author Karmime Abderrahim 29133 author Ravier Florence 969445 author Roux Adrien 0000-0001-7618-6129 author Stoppini Luc 0000-0002-2648-7051 author Preynat-Seauve Olivier 0000-0001-5364-967X 561776 Département de médecine (DEMED) author Krause Karlheinz 0000-0002-9033-6768 23022 Décanat et administration de la faculté de médecine (ADMED) Karl-Heinz Krause 1211 10.3390/cells14151211 40801645 2073-4409 PMC12346388 https://www.mdpi.com/2073-4409/14/15/1211 <p>Organoids allow to model healthy and diseased human tissues. and have applications in developmental biology, drug discovery, and cell therapy. Traditionally cultured in immersion/suspension, organoids face issues like lack of standardization, fusion, hypoxia-induced necrosis, continuous agitation, and high media volume requirements. To address these issues, we developed an air–liquid interface (ALi) technology for culturing organoids, termed AirLiwell. It uses non-adhesive microwells for generating and maintaining individualized organoids on an air–liquid interface. This method ensures high standardization, prevents organoid fusion, eliminates the need for agitation, simplifies media changes, reduces media volume, and is compatible with Good Manufacturing Practices. We compared the ALi method to standard immersion culture for midbrain organoids, detailing the process from human pluripotent stem cell (hPSC) culture to organoid maturation and analysis. Air–liquid interface organoids (3D-ALi) showed optimized size and shape standardization. RNA sequencing and immunostaining confirmed neural/dopaminergic specification. Single-cell RNA sequencing revealed that immersion organoids (3D-i) contained 16% fibroblast-like, 23% myeloid-like, and 61% neural cells (49% neurons), whereas 3D-ALi organoids comprised 99% neural cells (86% neurons). Functionally, 3D-ALi organoids showed a striking electrophysiological synchronization, unlike the heterogeneous activity of 3D-i organoids. This standardized organoid platform improves reproducibility and scalability, demonstrated here with midbrain organoids. The use of midbrain organoids is particularly relevant for neuroscience and neurodegenerative diseases, such as Parkinson’s disease, due to their high incidence, opening new perspectives in disease modeling and cell therapy. In addition to hPSC-derived organoids, the method’s versatility extends to cancer organoids and 3D cultures from primary human cells.</p> Cell Culture Techniques / methods Cell Differentiation Humans Mesencephalon / cytology Organoids / cytology Organoids / metabolism Pluripotent Stem Cells / cytology Dewey 617.6 Dewey 616.07 Dewey 616 211 Biomatériaux 24Q22 137 Département de pathologie et immunologie 21F 56 Département de médecine 221 false 239 Groupe Susanne Scherrer : Unité des biomatériaux 315 298 Immunologie de transplantation, immunogénétique et thérapie cellulaire 561 38 Radicaux libres et cellules souches embryonnaires 60 Cells 14 15 c8f7d821-1252-4c83-ad93-362c44248608 Article (Published version) Public 2025-_cells-.pdf 7060257 application/pdf CC BY-4.0 368a0f3c7933e0bd76bb061948019c90db8e6cd0ec83990febd1d64685398b15 d3065721-8336-4606-90ae-380619c5ba71 Supplemental data Public cells-3676337-Supplementaryfigures-.pdf 763996 application/pdf 7583ed24b2851ae53d71726be7a2cb34e7341653cde84423a570d14e75bfa9ba 4a2d9c5f-cc29-4112-87c3-381d50de30a7 Supplemental data Public Copie_de_Table_S1.xlsx 11697 application/vnd.openxmlformats-officedocument.spreadsheetml.sheet 2b82a8258075246fdf1fcd4fd35e4de04f80357986a60810faa357b426e47613 openAccess Faculté de médecine / Section de médecine dentaire / Département de réhabilitation oro-faciale / Division de prothèse fixe et biomatériaux / Biomatériaux Faculté de médecine / Section de médecine fondamentale / Département de pathologie et immunologie Faculté de médecine / Section de médecine clinique / Département de médecine

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Generation of Individualized, Standardized, and Electrically Synchronized Human Midbrain Organoids	Cells			2025	22	95

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Nazari, Bahareh