Klein Martin, Varga Ivan, Danišovič Ľuboš, Gálfiová Paulína, Kleinová Mária, Žiaran Stanislav, Novotná Oľga, Kuniaková Marcela
Institute of Histology and Embryology, Faculty of Medicine, Comenius University in Bratislava, Špitálska Street 24, Bratislava 813 72, Slovakia.
Institute of Medical Biology, Genetics and Clinical Genetics, Faculty of Medicine, Comenius University in Bratislava, Špitálska Street 24, Bratislava 813 72, Slovakia.
Tissue Cell. 2024 Dec;91:102623. doi: 10.1016/j.tice.2024.102623. Epub 2024 Nov 14.
Decellularization is a technique of cell removal that prepares vacant extracellular matrices (ECMs), which can be reseeded with patient-specific cells and used in regenerative medicine. Histological methods are vital for protocol standardization and efficacy assessment, which are necessary for preparing such bioscaffolds. Foreskins from male subjects (n=20) aged three to fifteen years were harvested and decellularized using detergent-based and enzyme-based protocols. To acquire a standardized protocol that would provide the optimal balance between effective cell removal and ECM preservation, a total of seven protocols were evaluated using the most basic formalin-fixed paraffin-embedded (FFPE) tissue blocks, which were sectioned and stained with HE, Masson's blue trichrome and orcein. After achieving the optimal result, we further morphologically characterized the scaffolds using scanning electron microscopy (SEM) and immunohistochemical (IHC) detection of fibronectin and collagen IV. Histology proved to be a quick, relatively easy, and cheap method to achieve protocol standardization and assess decellularization efficacy and ECM preservation. Morphological analysis via normal histology, SEM, and IHC showed that both definitive protocols are effective in cell removal but, at the same time, don't compromise the structural integrity of the ECM. Morphological characterization of foreskin-derived bioscaffolds via normal histology, IHC, and SEM is irreplaceable for standardizing decellularization protocols, assessing the efficacy of cell removal, and evaluating ECM preservation.
去细胞化是一种去除细胞的技术，可制备出空的细胞外基质（ECM），这些基质可重新接种患者特异性细胞并用于再生医学。组织学方法对于方案标准化和疗效评估至关重要，而这对于制备此类生物支架是必不可少的。收集了20名年龄在3至15岁男性受试者的包皮，并使用基于洗涤剂和基于酶的方案进行去细胞化处理。为了获得一种能在有效去除细胞和保留ECM之间实现最佳平衡的标准化方案，使用最基本的福尔马林固定石蜡包埋（FFPE）组织块对总共七种方案进行了评估，这些组织块被切片并用苏木精-伊红（HE）、马森三色蓝和orcein染色。在获得最佳结果后，我们使用扫描电子显微镜（SEM）以及对纤连蛋白和IV型胶原进行免疫组织化学（IHC）检测，进一步对支架进行形态学表征。组织学被证明是一种快速、相对简便且廉价的方法，可实现方案标准化并评估去细胞化效果和ECM保留情况。通过常规组织学、SEM和IHC进行的形态学分析表明，这两种确定的方案在去除细胞方面均有效，但同时不会损害ECM的结构完整性。通过常规组织学、IHC和SEM对包皮来源的生物支架进行形态学表征，对于标准化去细胞化方案、评估细胞去除效果以及评估ECM保留情况而言是不可替代的。
