Hillebrandt Karl, Polenz Dietrich, Butter Antje, Tang Peter, Reutzel-Selke Anja, Andreou Andreas, Napierala Hendrik, Raschzok Nathanael, Pratschke Johann, Sauer Igor M, Struecker Benjamin
General, Visceral, and Transplantation Surgery, Charité - Universitätsmedizin Berlin.
General, Visceral, and Transplantation Surgery, Charité - Universitätsmedizin Berlin;
J Vis Exp. 2015 Aug 10(102):e53029. doi: 10.3791/53029.
Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.
实质器官的去细胞化和再细胞化或许能够在体外生成功能性器官，并且已经发表了几种用于啮齿动物肝脏去细胞化的方案。我们旨在通过构建一种能够经门静脉和/或肝动脉进行选择性灌注的专利灌注装置来改进去细胞化过程。此外，我们试图在振荡周围压力条件下进行灌注，以提高去细胞化的均匀性。迄今为止，灌注去细胞化的均匀性一直是一个被低估的因素。在去细胞化过程中，灌注不良的器官区域可能仍含有细胞，而灌注良好区域的细胞外基质（ECM）可能已受到碱性洗涤剂的影响。振荡压力变化可以模拟呼吸过程中发生的腹腔内压力变化，以优化肝脏内部的微灌注。在本文所呈现的研究中，通过组织学染色、DNA含量分析和铸型腐蚀对去细胞化的大鼠肝脏基质进行了分析。经肝动脉灌注比经门静脉灌注显示出更均匀的结果。振荡压力条件的应用提高了灌注去细胞化的效果。经肝动脉并在振荡压力条件下灌注的肝脏显示出最佳结果。本文所介绍的使用我们的专利装置进行肝脏采集、插管和灌注的技术，能够实现复杂的灌注设置，以改进大鼠肝脏的去细胞化和再细胞化实验。
