Advancing 3D Cell Cultures of Stem-Cell Derived Pancreatic Islets and Breast Cancer Cells Using Recombinant Functionalized Spider Silk

QC 2024-08-30

Abstract

The culture of cells in 3D creates a more physiologically relevant cell environment than conventional 2D cultures. Interactions of cells with the extracellular matrix induce important cellular signalling that regulates cell adhesion, migration, proliferation, differentiation, and survival. This is crucial for modelling cell development and disease. This thesis aims to develop and analyse improved 3D cell culture methods for stem cell-derived pancreatic islets (SC-islets) and breast cancer cell lines using a functionalized recombinant spider silk. Spider silk is a natural protein-based material with remarkable mechanical properties of high strength and elasticity. It is also biodegradable and cytocompatible. FN-silk, the recombinant spider silk protein utilized in this thesis, is functionalized with a cell binding motif (RGD) from fibronectin, to improve cell adhesion. Notably, FN-silk self-assembles at the liquid-air interface into a fibrillar structure, making it favourable as support for cell culture. In this thesis, bioinformatic methods were used to discover how the FN-silk supported environment affects the gene expression of cells and the cellular heterogeneity of SC-islets during a differentiation process. Additionally, bioinformatical analysis of the effect of 3D cell culture of breast cancer cell lines in FN-silk networks was performed. The first part of the thesis addresses serval challenges in pancreatic islet transplantation, by presenting an optimized protocol for pancreatic differentiation from human pluripotent stem cells, improving in vitro cultivation, and developing a cryopreservation method for SC-islets. The differentiation protocol presented in Paper 1 resulted in pure endocrine cell populations, avoiding unwanted proliferating and non-endocrine cells. It was also demonstrated that these SC-islets matured in vivo, and could effectively reverse diabetes in a diabetic mouse model. Single-cell RNA sequencing analysis provided new insights into the cellular composition and gene expression of the SC-islets before and after transplantation. In Paper 2, an innovative method for 3D in vitro cultivation of SC-islets using FN-silk networks mimicking the extracellular matrix was established. The FN-silk networks provided structural support for in vitro cultivation and handling during in vivo transplantation. The viability and functionality of free and FN-silk incorporated SC-islets were evaluated and compared. Single-cell RNA sequencing analyses confirmed maintenance of cellular composition, with a slightly improved beta cell maturation for SC-islets supported by FN-silk. In Paper 3, a novel strategy for cryopreservation of SC-islets was explored. The twisted vitrification method, previously employed for 2D cultures, was adapted for 3D cultures by utilizing integration into FN-silk networks to facilitate handling during the vitrification process. The second part of the thesis aimed to develop a method for 3D culture of breast cancer cells to better replicate the complexity of the tumour microenvironment. In Paper 4, FN-silk networks were used to generate a 3D environment for breast cancer cells where crucial cell-ECM interactions can be established. Proliferation rates and key marker expression of the cells cultured in 2D versus in the FN-silk network environment were investigated. Bioinformatic analysis of bulk RNA sequencing data was used to compare breast cancer cells in conventional 2D cell cultures with those cultured in 3D with the support of FNsilk. In conclusion, the work conducted in this thesis presents significant advancements in the development and analyses of 3D cell cultures of both SCislets and breast cancer cell lines, potentially enhancing therapeutic applications, disease modeling, and drug testing.

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