Establishing an effective repopulation of the endothelialized liver matrix with primary hepatocytes

背景：器官工程是一种解决供体器官短缺的实验性方法。第一步是脱细胞制备脱细胞支架，第二步是细胞再增殖，第三步是功能器官移植，表面活性剂灌注脱细胞已成为广泛接受的方法。到目前为止，肝细胞系、内皮细胞系、原代肝细胞和间充质基质细胞已用于再接种程序。到目前为止，再增殖的最大成功之一是脱细胞猪肝支架的完全再内皮化，尽管没有使用实质细胞。再内皮化的猪肝支架异位移植后，证明了体内门静脉灌注的维持长达20天。然而，没有肝支架repopulated与内皮细胞，以及实质细胞已经transplanted.Objective：本项目的目的是repopulationof脱细胞猪肝支架与实质细胞和内皮细胞和证明门静脉长期灌注后异位transplantation.Hypotheses：我们假设，通过门静脉和肝静脉的肝细胞和内皮细胞的顺序双向应用相比，单向应用的优势。双向应用模式促进了应用的肝细胞在脱细胞支架中的更高粘附性，增加了体外细胞特异性功能的增殖和恢复，并允许在vivo.Experimental design中长期灌注重新填充的器官：为了实现这一目标，计划了三个工作包（WP）。在WP 1和WP 2中，将在体外研究细胞应用途径对基质中细胞粘附以及增殖和肝细胞特异性功能恢复的影响。在WP 3中，将评估异位移植后内皮和实质再增殖对器官基质门静脉灌注的影响。在WP 1中，通过计算灌注液中应用的细胞数量与非粘附细胞数量之间的差异，间接确定基质中粘附细胞的数量。在WP 2中，在细胞培养基中重复测定肝细胞特异性功能（例如白蛋白合成）的恢复。在4周培养期结束时，使用组织学和免疫组织化学方法分析细胞分布、增殖率以及沿窦状隙的带状酶表达沿着。在WP 3中，在完成长期灌注培养后，将再增殖的器官异位移植，并每72小时通过计算机断层扫描评估门静脉灌注。前景：这些实验是下一步计划的胆道树再增殖和异种器官原位移植的先决条件。

Background:Organ engineering is an experimental approach to address the shortage of donor organs. The first step is to generate an acellular scaffold by decellularization, followed by the second step of repopulation with cells and the third step: transplantation of a functional organ.Perfusion with tensides has become the widely accepted method for decellularization. Up to now, liver cell lines, endothelial cell lines, primary hepatocytes, and mesenchymal stromal cells have been used for the reseeding procedure. One of the greatest successes in repopulation so far is the complete re-endothelialization of a decellularized pig liver scaffold, although without using parenchymal cells. After heterotopic transplantation of the re-endothelialized pig liver scaffold, the maintenance of portal venous perfusion in vivo for up to 20 days was demonstrated. However, no liver scaffold repopulated with endothelial as well as parenchymal cells has been transplanted so far.Objective:The aim of this project is the repopulation of the acellular pig liver scaffold with parenchymal and endothelial cells and the proof of portal venous long-term perfusion after heterotopic transplantation.Hypotheses:We hypothesize that the sequential bidirectional application of hepatocytes and endothelial cells via portal and hepatic vein is of advantage compared to unidirectional application. The bidirectional application mode promotes a higher adherence of the applied hepatocytes in the acellular scaffold, increases proliferation and resumption of cell-specific function in vitro and allows long-term perfusion of the repopulated organ in vivo.Experimental design:To achieve this goal, three work packages (WP) are planned. In WP 1 and WP 2, the effect of the cell application route on cell adherence as well as proliferation and resumption of hepatocyte-specific function in the matrix will be investigated in vitro. In WP 3, the influence of endothelial and parenchymal repopulation on portal venous perfusion of the organ matrix after heterotopic transplantation will be assessed. In WP 1, the number of adherent cells in the matrix is determined indirectly by calculating the difference between the number of applied cells and the number of non-adherent cells in the perfusate. In WP 2, resumption of hepatocyte-specific function (e.g. albumin synthesis) is determined repeatedly in cell culture medium. Cell distribution, proliferation rate as well as the zonal enzyme expression along the sinusoid, is analyzed using histological and immunohistochemical methods at the end of the 4-week culture period. In WP 3, after completion of the long-term perfusion culture, the repopulated organ is transplanted in heterotopic position and the portal venous perfusion is evaluated by computer tomography every 72 hours.Perspective:These experiments are the prerequisite for the repopulation of the biliary tree planned in the next step and for the orthotopic transplantation of the xenogeneic organ.