Development of a biohybrid lung - Preventing rejection of allogenic endothelial cells by HLA-class I silencing

由于终末期肺部疾病目前没有植入式长期辅助装置，肺移植仍然是目前唯一的治疗选择。该项目的目的是开发一种可植入式生物人工肺，作为移植的潜在长期替代品。用于体外膜氧合器的气体交换膜将被内皮化以改善血液相容性。生物人工肺可作为治疗选择的患者通常没有足够的自体内皮细胞（EC）用于细胞播种，因此异体内皮细胞的应用是必要的。因此，该项目将侧重于分析hla沉默。通过所谓的rnai技术，负责移植排斥反应的人白细胞抗原（HLA）的表面特征将被抑制，因此被沉默的异体EC将不会被免疫系统检测到。本文将研究这些沉默EC在气体交换膜上融合内皮单层的体外发育中的应用。除了优化hla沉默外，还将分析沉默EC对气体交换膜内皮化的适用性。为此，将结合膜的不同表面改性进行静态和动态条件下的分析，并验证各自的气体交换能力。功能分析将包括研究hla沉默的有效性（血液相容性分析，细胞毒性T和NK细胞效应分析，免疫细胞的粘附和迁移）和鉴定所谓的脱靶效应。为准备在小动物模型（大鼠）的体内应用，本项目的重点工作是分离EC，建立用于PCR和facs分析的内皮特异性引物和抗体，以及大鼠特异性EC的hla沉默，以及建立合适的生物反应器。

Due to the fact that there is currently no implantable long-term assist device for end-stage lung disease, lung transplantation remains the only curative option at present. The aim of the proposed project is the development of an implantable bioartifical lung as a potential long-term alternative to transplantation. Gas exchange membranes, which are used in the extracorporeal membrane oxygenators, will be endothelialized to improve hemocompatability. Patients for whom the bioartificial lung could be considered as a treatment option will typically not have enough autologous endothelial cells (EC) for cell seeding, rendering the application of allogenous EC necessary. This project will therefore focus on the analysis of HLA-silencing. By means of so-called RNAi-technology, the surface characteristics of the human leukocyte antigen (HLA), which are responsible for transplant rejection, will be suppressed, and therefore the silenced allogenous EC will not be detected by the immune system. The application of these silenced EC in the in vitro development of a confluent endothelial monolayer on the gas exchange membranes will be investigated. In addition to the optimization of the HLA-silencing, the suitability of silenced EC to endothelialize the gas exchange membranes will be analysed. For this purpose, analyses under static and dynamic conditions in combination with different surface modifications of the membranes, as well as the verification of the respective gas exchange capabilities, will be conducted. Functional analyses will include the investigation of the effectiveness of the HLA-silencing (hemocompatibility assays, analysis of cytotoxic T- and NK cell effects, adherence and migration of immunological cells) and the identification of the so-called off-target effects. In preparation for the in vivo application in a small animal model (rat), the isolation of EC, the establishment of endothelial specific primers and antibodies for PCR and FACS-analysis and the HLA-silencing of rat-specific EC, as well as the establishment of a suitable bioreactor, constitute the focal tasks in this project.