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

用原代肝细胞建立内皮化肝基质的有效再增殖

• 批准号: 455452355
• 负责人: Dr. Philipp Felgendreff
• 金额: --
• 依托单位: Artificial Liver and Liver Transplantation Laboratory
• 依托单位国家: 德国
• 项目类别: WBP Fellowship
• 财政年份: 2021
• 资助国家: 德国
• 起止时间: 2020-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/455452355?language=en
• 关键词: Establishing; effective; repopulation; endothelialized; liver

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.

背景：器官工程学是解决供体器官短缺的一种实验性方法。第一步是通过脱细胞生成脱细胞支架，第二步是细胞再繁殖，第三步是功能器官的移植，肌腱苷灌流已成为被广泛接受的脱细胞方法。到目前为止，已有肝细胞系、内皮细胞系、原代肝细胞和间充质基质细胞用于补种。到目前为止，在再繁殖方面最大的成功之一是完全重新内皮化脱细胞的猪肝支架，尽管没有使用实质细胞。在异位移植重新内皮化的猪肝支架后，证明门静脉在体内的灌流可维持长达20天。目的：本课题的目的是实现脱细胞猪肝支架内皮细胞和内皮细胞的再生，并证实异位移植后门静脉的长期灌流。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，促进了体外细胞的增殖和细胞特异性功能的恢复，并允许在体内长期灌流再生的器官。实验设计：为实现这一目标，设计了三个工作包(WP)。在WP 1和WP 2中，将在体外观察细胞应用途径对细胞黏附以及基质中肝细胞特异性功能的增殖和恢复的影响。在WP 3中，将评估异位移植后血管内皮细胞和实质细胞再生对器官基质门静脉血流的影响。在WP 1中，基质中的贴壁细胞数量是通过计算应用的细胞数量与灌流液中非贴壁细胞数量之间的差值来间接确定的。在WP 2中，肝细胞特异性功能(如白蛋白合成)的恢复是在细胞培养液中反复测定的。培养4周后，用组织学和免疫组织化学方法分析细胞的分布、增殖率以及沿血窦的带状酶表达。在WP 3中，在完成长期灌流培养后，将再生的器官移植到异位位置，每72小时进行一次门静脉CT检查。视角：这些实验是下一步计划的胆道树再生和异种器官原位移植的先决条件。