Validation of an in vitro muscle platform to evaluate myogenesis and calcium handling in control and dystrophic human myotubes

Validation of an in vitro muscle platform to evaluate myogenesis and calcium handling in control and dystrophic human myotubes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Validation of an in vitro muscle platform to evaluate myogenesis and calcium handling in control and dystrophic human myotubes Laura Mosqueira-Martín, Carolina Prendes-García, Pablo Marco-Moreno, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7137486/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Electrical impedance has emerged as a powerful tool for real-time, label-free, and non-invasive monitoring of cellular processes. Here, we employed an impedance-based assay to characterize the myogenic process of control and dystrophic human myoblasts. First, we conducted a comprehensive analysis of control myoblast differentiation, assessing the effects of initial seeding density and various extracellular matrix coatings. We also evaluated the influence of electrode presence and current application, both of which improved myoblast alignment. Immortalized myoblasts from Duchenne and Becker muscular dystrophy patients exhibited marked alterations in early differentiation and maturation, which were readily detected via impedance measurements. We further compared two differentiation protocols using one control and one dystrophic representative cell line. While both protocols supported the formation of mature myotubes, impedance profiles differed depending on the culture medium. Notably, we identified the protocol with superior impedance profile reproducibility over the culture lifespan. Finally, we successfully assessed calcium homeostasis in control and dystrophic myotubes differentiated on 96-well impedance plates. Our findings underscore the potential of impedance-based assays for monitoring myogenesis and identifying disease-associated phenotypes. Moreover, 96-well impedance plates represent a robust tool for high-throughput and high-content functional analysis in muscle disease modeling and therapeutic screening. Biological sciences/Biological techniques Biological sciences/Biotechnology Biological sciences/Cell biology Biological sciences/Stem cells Full Text Additional Declarations No competing interests reported. Supplementary Files MosqueiraMartn2025supplementaryinformation.pdf Cite Share Download PDF Status: Published Journal Publication published 17 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 08 Sep, 2025 Reviews received at journal 07 Sep, 2025 Reviews received at journal 19 Aug, 2025 Reviewers agreed at journal 31 Jul, 2025 Reviewers agreed at journal 30 Jul, 2025 Reviewers invited by journal 29 Jul, 2025 Editor assigned by journal 29 Jul, 2025 Submission checks completed at journal 22 Jul, 2025 First submitted to journal 22 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7137486","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":494010459,"identity":"862d532c-e062-45a0-b286-942b65bcb317","order_by":0,"name":"Laura Mosqueira-Martín","email":"","orcid":"","institution":"Group of Neuroscience, Departments of Pediatrics and Neuroscience, Faculty of Medicine and Nursing, UPV/EHU","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Mosqueira-Martín","suffix":""},{"id":494010462,"identity":"ab68e6e7-a9bb-4576-85d3-e08166b5a1ba","order_by":1,"name":"Carolina Prendes-García","email":"","orcid":"","institution":"Neuromuscular Diseases Group, Biogipuzkoa HRI","correspondingAuthor":false,"prefix":"","firstName":"Carolina","middleName":"","lastName":"Prendes-García","suffix":""},{"id":494010465,"identity":"af74ac6b-5d5c-4aa2-833b-9815c46709d0","order_by":2,"name":"Pablo Marco-Moreno","email":"","orcid":"","institution":"Neuromuscular Diseases Group, Biogipuzkoa HRI","correspondingAuthor":false,"prefix":"","firstName":"Pablo","middleName":"","lastName":"Marco-Moreno","suffix":""},{"id":494010469,"identity":"12c45cfd-bd7e-4786-b75a-65dc25d39d8f","order_by":3,"name":"Camila Vesga-Castro","email":"","orcid":"","institution":"Tecnun School of Engineering, University of Navarra","correspondingAuthor":false,"prefix":"","firstName":"Camila","middleName":"","lastName":"Vesga-Castro","suffix":""},{"id":494010471,"identity":"d20cc08e-e0f7-4e68-a7b5-6e31c8a23e76","order_by":4,"name":"Ainhoa Irastorza","email":"","orcid":"","institution":"Stem Cells and Aging Group, Biogipuzkoa HRI","correspondingAuthor":false,"prefix":"","firstName":"Ainhoa","middleName":"","lastName":"Irastorza","suffix":""},{"id":494010473,"identity":"477ce029-c0b3-4288-8dfc-2ddb95ba507f","order_by":5,"name":"Jacobo Paredes","email":"","orcid":"","institution":"Tecnun School of Engineering, University of Navarra","correspondingAuthor":false,"prefix":"","firstName":"Jacobo","middleName":"","lastName":"Paredes","suffix":""},{"id":494010475,"identity":"f2031fb0-c131-4601-8fa3-a0ef0f88ff58","order_by":6,"name":"Ander Izeta","email":"","orcid":"","institution":"Stem Cells and Aging Group, Biogipuzkoa HRI","correspondingAuthor":false,"prefix":"","firstName":"Ander","middleName":"","lastName":"Izeta","suffix":""},{"id":494010476,"identity":"79f3927f-69dd-49c2-ae95-01bc8381b419","order_by":7,"name":"Iratxe Madarieta","email":"","orcid":"","institution":"Tecnalia, Basque Research and Technology Alliance (BRTA)","correspondingAuthor":false,"prefix":"","firstName":"Iratxe","middleName":"","lastName":"Madarieta","suffix":""},{"id":494010477,"identity":"db71f1d3-c30d-4f41-bf68-2ae801a1e5cf","order_by":8,"name":"Itxaso Martí-Carrera","email":"","orcid":"","institution":"Pediatrics Group, Biogipuzkoa HRI","correspondingAuthor":false,"prefix":"","firstName":"Itxaso","middleName":"","lastName":"Martí-Carrera","suffix":""},{"id":494010478,"identity":"f6c2173a-fd5a-4d3b-9198-a789fd56cd84","order_by":9,"name":"Adolfo López de Munain","email":"","orcid":"","institution":"Neurology Department, Hospital Universitario Donostia, Osakidetza","correspondingAuthor":false,"prefix":"","firstName":"Adolfo","middleName":"López","lastName":"de Munain","suffix":""},{"id":494010479,"identity":"83faceeb-e925-4f73-ae9c-b6b0334afaf3","order_by":10,"name":"Ainara Vallejo-Illarramendi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAklEQVRIiWNgGAWjYDACZhiDnbmBQYLBhsGAeC3MjCAtaURoQegFamFgOExYC38778HPFTUMif3NjI0PLCrOy5lLJDA+5vmDW4vEYb5kyTPHGBJnHGZsNpA4c9vYckYCszFvGx5rDvMYSDawMRgzHGZsk5Bsu5244UYCmzRvA24d8od5jH82/GMwlj/M2P5Dsu1cPVAL+298DjM4zGMm2djGIGcAtIVBsu1AggHQFmYeNtxaDIFaLBv7JOQMgX6RkDiTbLjhzMNmybl4/CJ3/ozxzYZvNjxyx5sPfpaosJM3OJ588MMbPA6DAgkwyQyhwBFEJGD8QLzaUTAKRsEoGEEAALrrS0MVilklAAAAAElFTkSuQmCC","orcid":"","institution":"Group of Neuroscience, Departments of Pediatrics and Neuroscience, Faculty of Medicine and Nursing, UPV/EHU","correspondingAuthor":true,"prefix":"","firstName":"Ainara","middleName":"","lastName":"Vallejo-Illarramendi","suffix":""}],"badges":[],"createdAt":"2025-07-16 08:23:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7137486/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7137486/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-31522-z","type":"published","date":"2025-12-17T15:58:04+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":98813992,"identity":"b7af03ae-722a-46cf-b338-1068f5f62ff4","added_by":"auto","created_at":"2025-12-22 16:09:09","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6205986,"visible":true,"origin":"","legend":"","description":"","filename":"MosqueiraMartn2025Manuscriptfileamendment2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7137486/v1_covered_d7cb8b8c-40b1-4a25-a476-c5c66f0bdc05.pdf"},{"id":88211003,"identity":"7aea691a-1095-4982-a055-323b9c115169","added_by":"auto","created_at":"2025-08-04 05:12:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":6043764,"visible":true,"origin":"","legend":"","description":"","filename":"MosqueiraMartn2025supplementaryinformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7137486/v1/f31af6229f5488a5ccae8713.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Validation of an in vitro muscle platform to evaluate myogenesis and calcium handling in control and dystrophic human myotubes","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7137486/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7137486/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Electrical impedance has emerged as a powerful tool for real-time, label-free, and non-invasive monitoring of cellular processes. Here, we employed an impedance-based assay to characterize the myogenic process of control and dystrophic human myoblasts. First, we conducted a comprehensive analysis of control myoblast differentiation, assessing the effects of initial seeding density and various extracellular matrix coatings. We also evaluated the influence of electrode presence and current application, both of which improved myoblast alignment. Immortalized myoblasts from Duchenne and Becker muscular dystrophy patients exhibited marked alterations in early differentiation and maturation, which were readily detected via impedance measurements. We further compared two differentiation protocols using one control and one dystrophic representative cell line. While both protocols supported the formation of mature myotubes, impedance profiles differed depending on the culture medium. Notably, we identified the protocol with superior impedance profile reproducibility over the culture lifespan. Finally, we successfully assessed calcium homeostasis in control and dystrophic myotubes differentiated on 96-well impedance plates. Our findings underscore the potential of impedance-based assays for monitoring myogenesis and identifying disease-associated phenotypes. Moreover, 96-well impedance plates represent a robust tool for high-throughput and high-content functional analysis in muscle disease modeling and therapeutic screening.","manuscriptTitle":"Validation of an in vitro muscle platform to evaluate myogenesis and calcium handling in control and dystrophic human myotubes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-04 05:04:15","doi":"10.21203/rs.3.rs-7137486/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-08T10:33:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-07T17:19:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-19T23:03:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"136175817013359982706169040141532483769","date":"2025-07-31T21:11:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"29638778792638681985075157708704848192","date":"2025-07-30T19:15:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-29T19:33:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-29T16:13:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-22T08:37:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-07-22T08:36:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ff411d8a-7050-47a6-a9f3-085129acf5b3","owner":[],"postedDate":"August 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":52465510,"name":"Biological sciences/Biological techniques"},{"id":52465511,"name":"Biological sciences/Biotechnology"},{"id":52465512,"name":"Biological sciences/Cell biology"},{"id":52465513,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2025-12-22T16:02:26+00:00","versionOfRecord":{"articleIdentity":"rs-7137486","link":"https://doi.org/10.1038/s41598-025-31522-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-12-17 15:58:04","publishedOnDateReadable":"December 17th, 2025"},"versionCreatedAt":"2025-08-04 05:04:15","video":"","vorDoi":"10.1038/s41598-025-31522-z","vorDoiUrl":"https://doi.org/10.1038/s41598-025-31522-z","workflowStages":[]},"version":"v1","identity":"rs-7137486","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7137486","identity":"rs-7137486","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}