Revisiting ISO 10993-5 In Vitro Cytotoxicity Standard Tests for Evaluation of Extracellular Matrix-Based Biomaterials.

Cytotoxicity testing is a critical step in the preclinical evaluation of biomaterials and medical devices, with extracellular matrix (ECM)-based biomaterials constituting a significant category. The current ISO 10993-5:2009 standard, Biological Evaluation of Medical Devices-Part 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-positive results, contributing to interlaboratory discrepancies. This study systematically evaluated ISO 10993-5-recommended assays for ECM-based 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-ECM attachment (actin cytoskeleton), proliferation (Ki67), and apoptosis (annexin V) were analyzed. Notably, despite observing increased metabolic activity (100%-150%) under serum-free 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 ∼15% 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 10993-5 revisions for more reliable assessment of biomaterials and medical devices.