Deterministic Culturing of Single Cells in 3D

Models using 3D cell culture techniques are increasingly accepted as the most biofidelic in vitrorepresentations of tissues for research. These models are generated using biomatrices and bulk populations of cells derived from tissues or cell lines. This thesis study focuses on an alternate method to culture individually selected cells in relative isolation from the rest of the population under physiologically relevant matrix conditions. Matrix gel islands are spotted on a cell culture dish to act as support for receiving and culturing individual single cells; a glass capillary-based microfluidic setup is used to extract each desired single cell from a population and seed it on top of an island. Using examples of breast and colorectal cancers, we show that individual cells evolve into tumors or aspects of tumors displaying different characteristics of the initial cancer type and aggressiveness. By implementing a morphometry assay with luminal A breast cancer, we demonstrate the potential of the proposed approach to studying phenotypic heterogeneity. Results reveal that intertumor heterogeneity increases with time in culture and that varying degrees of intratumor heterogeneity may originate from individually seeded cells. Moreover, we observe a positive correlation between fast-growing tumors and the size and heterogeneity of their nuclei