Available at: https://digitalcommons.calpoly.edu/theses/3175
Date of Award
10-2025
Degree Name
MS in Biomedical Engineering
Department/Program
Biomedical Engineering
College
College of Engineering
Advisor
Chistopher Heylman
Advisor Department
Biomedical Engineering
Advisor College
College of Engineering
Abstract
Colorectal cancer is the second leading cause of cancer related deaths worldwide. It currently affects millions of people across the globe and is only expected to increase in impact over the coming years. The most common treatment for metastatic colon cancer is chemotherapy, however, the development of chemotherapeutic drugs is a long and expensive process. A large portion of this development process is spent in preclinical drug testing. However, the models used often lack enough physiological relevance to guarantee the drug’s success in a clinical trial. Due to this gap in testing, researchers seek to develop a more physiologically relevant in vitro tumor model to better represent the interactions of a drug with the tumor microenvironment. The aim of this research was to develop a tri-culture tumor spheroid model that improved upon previous models’ physiological relevance. This was accomplished through a series of experiments. The first aimed to determine the optimal cell density and number of days in culture to develop a baseline tumor spheroid within the targeted size range. The next investigated how the addition of HUVECs would affect tumor spheroid formation and endothelial network formation. The third explored how the addition of HDFs would affect the same parameters. The fourth investigated the viability of spheroids in dense Matrigel to pursue the idea of culturing the spheroids in microfluidic devices. It also investigated the impact of a new method of HDF inclusion, a shell-like layer, on the model’s physiological relevance. The fifth explored the impact of the number of days before transfer as well as the effect of the location of additional HUVECs and HDFs plated in the extracellular matrix on the development of the spheroid model. The first three experiments established the ideal plating density for the spheroid and demonstrated that a tri-culture spheroid best mimics the tumor microenvironment. The fourth experiment revealed that the shell-like plating method improved both tumor spheroid shape and formation of angiogenic sprouts. The fifth experiment further refined the tumor spheroid model and established a protocol for tri-culture tumor spheroids that demonstrated physiologically relevant size, shape, and angiogenic sprouting. However, further work is required to foster these angiogenic sprouts into a perfusable vascular network to further increase the physiological relevance of this model for accurate preclinical drug testing.