Date of Thesis
Spring 2025
Description
In tissue engineering, 3-dimensional (3D) experiments are necessary for simulating histological and physiological conditions, and a standardized model is required to effectively consider the mechanobiological effects of various substrates throughout these experiments. Biomaterials can be difficult to integrate into the body without the presence of vasculature to facilitate integration. Endothelial cells are responsible for creating and propagating vasculature under appropriate native tissue or in vitro substrate conditions. This work aimed to develop and refine a 3D vascular endothelial culture on a porcine gelatin scaffold to conduct physiologically relevant in vitro studies utilizing a substrate that resembled the extracellular matrix (ECM) associated with human soft tissues. This model cultured human umbilical vein endothelial cells (HUVECs) within a porcine gelatin scaffold and evaluated various cell seeding techniques, resuspension concentrations, and incubation periods to develop a robust model for endothelial vasculature growth in 3D. Evaluated culture parameters included scaffold handling and saturation with vascular culture media, one-sided and two-sided seeding of HUVECs into the scaffold, cell resuspension concentrations of 400,000, 600,000, and 800,000 cells in 100 μL of vascular media, and incubation periods of 96, 120, 144, and 168 hours. Confocal microscopy was utilized to document vasculature throughout the scaffolds under each set of culture conditions. The ImageJ Angiogenesis Analyzer toolset was applied to each image and statistical analysis was performed on the results to determine an optimal protocol. The primary metrics of interest assessed in this study were the quantity of vascular junctions, total vascular length, and total master segment length in each representative sample. When analyzed in conjunction, these parameters provided a global overview of the density and interconnectedness of the vasculature associated with each testing condition.
Two-sided seeding with scaffold saturation and a resuspended cell concentration of 600,000 cells per 100 μL of vascular media provided the most uniform distribution of vasculature within the scaffold while maintaining an appropriate density that permitted imaging. The 120-hour period associated with the aforementioned parameters yielded the greatest ratio of developed master segment length relative to the quantity of junctions, with an average of 1070 μm of vascular segmentation connected by approximately 15 junctions. These data suggested that the most optimal seeding protocol for endothelial vasculature development and observation using confocal microscopy was seeding 600,000 cells per scaffold, seeding from both sides of the scaffold, and incubating for 120 hours after seeding. It is recommended that all researchers investigating development of a 3D cell culture protocol assess the effects of variation in substrate handling, cell concentration, and network incubation. This standardized protocol will enable global experimentation requiring a consistent vascularized 3D matrix for the investigation of the effects of various external stimuli on endothelial networks.
Keywords
3D Culture, Endothelial, Vascular Network, Angiogenesis, Scaffold, Tissue Engineering
Access Type
Honors Thesis (Bucknell Access Only)
Degree Type
Bachelor of Science in Biomedical Engineering
Major
Biomedical Engineering
Minor, Emphasis, or Concentration
Mathematics
First Advisor
Dr. Olivia Boerman
Second Advisor
Dr. Matthew Heintzelman
Third Advisor
Dr. Donna Ebenstein
Recommended Citation
Walker, Sophia F., "Optimization of Angiogenesis in 3D Human Umbilical Vein Endothelial Cell Networks: How Can We Build a More Physiologically Accurate Model?" (2025). Honors Theses. 700.
https://digitalcommons.bucknell.edu/honors_theses/700