Quality Assessment of Cardiovascular Cells and Tissues by Raman Microspectroscopy and Imaging

Abstract

The increasing lifespan of the human population has been accompanied by a higher prevalence of cardiovascular diseases. It has been more than 50 years since the first heart valve was transplanted in a human patient and many new approaches in cardiovascular transplantation and tissue engineering (TE) have been evolving ever since. However, the availability of human donor tissues is limited. Ideal, vital, durable, nonimmunogenic heart valve or cardiovascular replacements are not yet commercially available. Thus, a better understanding of developmental and regulating mechanisms of cardiovascular tissues is essential to develop new implant materials. Moreover, cardiovascular tissue transplants or tissue-engineered grafts need to be monitored before transplantation. This thesis aimed to establish Raman microspectroscopy and Raman imaging as marker-independent, non-destructive technique for quality assessment of cardiovascular transplants and tissue-engineered products. Towards this aim, the influence of an ice-free cryopreservation technique (IFC) on tissue integrity and immunogenicity of heart valves was analyzed. The extracellular matrix (ECM) structures of standard cryopreserved (FC) and IFC allograft leaflets were compared to native leaflets after longterm implantation in sheep. Moreover, the mid-term immunogenic effects on IFC treated xenografts were assessed. Quantitative monitoring of interstitial cryoprotectant (CPA) concentrations was performed for quality control of cryopreserved heart valves. Furthermore, phenotype and tissue origin of human smooth muscle cells (SMCs) that are applied in cardiovascular TE, were analyzed. The ECM remodeling of SMC ring constructs under different culture conditions was monitored. In addition to Raman measurements, routine techniques such as immunocytochemistry, quantitative polymerase chain reaction and histological staining were performed. The results demonstrate the superiority of Raman microspectroscopy and Raman imaging as marker-independent, non-destructive and sensitive method, which is also time- and cost efficient when compared to routine techniques. Raman analysis combined with multivariate data analysis tools allowed for the determination and characterization of structural ECM changes in FC heart valves and real-time quantification of residual CPAs. These techniques enabled the identification and discrimination of single human SMCs based on their tissue origin and phenotype. Moreover, ECM remodeling in tissue-engineered SMC rings was non-invasively monitored. This work affirms the potential of Raman techniques for future applications in in situ quality assessment in cardiovascular research.