Tissue engineering of the human corneal endothelium: From primary cell culture to transplantation of highly functional cell sheets

拟议的研究项目旨在体外培养并随后转移用于组织工程目的的人角膜内皮细胞(HCEC)片材。为了能够直接转化为临床前环境，对原代、非永生化HCEC培养进行了研究。分离方案和培养条件被调整，以确保适当的细胞增殖和维护。通过符合后来临床应用的基本要求(例如，动物复合无培养条件)的方法生成功能强大和可行的HCEC片材。采用了申请人先前开发的并且允许满足HCEC的特定要求的可调谐温度响应细胞培养载体。在刺激分离后，细胞膜需要保留完整的细胞间接触和细胞外基质，以稳定地在转移、储存和移植中存活。为了实现这一目标，应用了一系列全面的细胞片形态、活性和生理功能的表征技术。温和的操作技术以及专用的移植工具是利用体外移植模型开发的。除了单一的内皮细胞密度外，还有更重要的移植质量标准被识别和评估。

The proposed research project aims at in vitro cultivation and subsequent transfer of human corneal endothelial cell (HCEC) sheets for tissue engineering purposes. To allow a straightforward translation into a pre-clinical setting, primary, non-immortalized HCEC cultures are investigated. Isolation protocols and culture conditions are adapted to ensure an appropriate cell proliferation and maintenance. Highly functional and viable HCEC sheets are generated by methods which comply with essential requirements for later clinical application (e.g. animal compound free culture conditions). A tunable thermo-responsive cell culture carrier that was previously developed by the applicants and that allows to meet particular requirements of HCEC is employed. Upon stimulated detachment the cell sheets need to preserve intact intercellular contacts as well as extracellular matrix to stably survive transfer, storage and transplantation. Towards this goal a comprehensive range of characterization techniques for cell sheet morphology, viability, and physiological function is applied. Gentle manipulation techniques as well as dedicated transfer tools are developed using an in vitro transplantation model. More significant criteria for the transplant quality beyond the sole endothelial cell density are identified and evaluated.