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The current ISO 10993\u20105:2009 standard, Biological Evaluation of Medical Devices\u2010Part 5: Tests for In Vitro Cytotoxicity, delineates four metabolic assays that primarily rely on colorimetric methods and colony formation. However, relying solely on colorimetric assays or colony formation fails to provide precise insights into cell function\/activity and may yield false\u2010positive results, contributing to interlaboratory discrepancies. This study systematically evaluated ISO 10993\u20105\u2010recommended assays for ECM\u2010based commercial products, specifically assessing the impact of key assay variables including cell types, contact mode (test extracts versus test material itself), and media components (with or without serum) on biological outcomes. These evaluations support the development of more accurate and robust test methods. While all four assays indicated the noncytotoxic nature of the test samples, metabolic activity readings varied substantially depending on the serum presence, cell types, and assay method employed. To address these limitations and achieve more precise insights into cellular activity, cell membrane integrity (live\/dead staining), cell\u2010ECM attachment (actin cytoskeleton), proliferation (Ki67), and apoptosis (annexin V) were analyzed. Notably, despite observing increased metabolic activity (100%\u2013150%) under serum\u2010free conditions measured using MTT and XTT assays, live\/dead and actin staining showed no corresponding changes in cell viability or attachment, and Ki67 indicated only \u223c15% proliferation. Annexin V staining was detected only in human primary dermal fibroblasts, highlighting their greater reliability over L929 cells for detecting apoptosis. These findings provide a valuable reference for researchers, regulatory bodies, and industry stakeholders in refining cytotoxicity testing protocols and guiding future ISO 10993\u20105 revisions for more reliable assessment of biomaterials and medical devices.<\/jats:p>","DOI":"10.1155\/ijbm\/7395612","type":"journal-article","created":{"date-parts":[[2026,5,30]],"date-time":"2026-05-30T09:02:37Z","timestamp":1780131757000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Revisiting ISO 10993\u20105 In Vitro Cytotoxicity Standard Tests for Evaluation of Extracellular Matrix\u2010Based Biomaterials"],"prefix":"10.1155","volume":"2026","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5202-4897","authenticated-orcid":false,"given":"Rashmi","family":"Ramakrishnan","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6848-4476","authenticated-orcid":false,"given":"Andrew C.","family":"Daly","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,5,30]]},"reference":[{"key":"e_1_2_10_1_2","doi-asserted-by":"publisher","DOI":"10.3389\/ftox.2023.1337468"},{"key":"e_1_2_10_2_2","doi-asserted-by":"publisher","DOI":"10.1007\/10_2011_111"},{"key":"e_1_2_10_3_2","unstructured":"ISO 10993-5:2009-Biological Evaluation of Medical Devices-Part 5: Tests for in Vitro Cytotoxicity 2009 https:\/\/www.iso.org\/standard\/36406.html."},{"key":"e_1_2_10_4_2","unstructured":"ISO 10993-1:2025 Biological Evaluation of Medical Devices Part 1: Requirements and General Principles for the Evaluation of Biological Safety Within a Risk Management Process 2025 https:\/\/www.iso.org\/standard\/10993-1."},{"key":"e_1_2_10_5_2","unstructured":"ASTM F895-84 Standard Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity 2024 https:\/\/www.astm.org\/f0895-84r01.html."},{"key":"e_1_2_10_6_2","unstructured":"ISO 10993-12:2021 Biological Evaluation of Medical Devices Part 12: Sample Preparation and Reference Materials 2021 https:\/\/www.iso.org\/standard\/75769.html."},{"key":"e_1_2_10_7_2","doi-asserted-by":"publisher","DOI":"10.3892\/br.2015.481"},{"key":"e_1_2_10_8_2","volume-title":"Genotoxicity-A Predictable Risk to Our Actual World; IntechOpen","author":"Aslant\u00fcrk \u00d6. 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