The tumor microenvironment plays an important role in regulating cancer cell behavior. The tumor microenvironment describes the cancer cells, and the surrounding endothelial cells, fibroblasts, and mesenchymal stem cells, along with the extracellular matrix (ECM). The tumor microenvironment stiffens as cancer undergoes malignant progression, providing biophysical cues that promote invasive, metastatic cellular behaviors. This project investigated the influence of three dimensional (3D) chitosan-alginate (CA) scaffold stiffness on the morphology, growth, and migration of green fluorescent protein (GFP) – transfected MDA-MB-231 (231-GFP) breast cancer (BCa) cells. The CA scaffolds were produced by the freeze casting method at three concentrations, 2 wt%, 4 wt%, and 6 wt% to provide different stiffness culture substrates. The CA scaffold material properties were characterized using scanning electron microscopy imaging for pore structure and compression testing for Young's Modulus. The BCa cell cultures were characterized at day 1, 3, and 7 timepoints using Alamar Blue assay for cell number, fluorescence imaging for cell morphology, and single-cell tracking for cell migration. Pore size calculations using SEM imaging yielded pore sizes of 253.29 ± 52.45 µm, 209.55 ± 21.46 µm, and 216.83 ± 32.63 µm for 2 wt%, 4 wt%, and 6 wt%, respectively. Compression testing of the CA scaffolds yielded Young's Modulus values of 0.064 ± 0.008 kPa, 2.365 ± 0.32 kPa and 3.30 ± 0.415 kPa for 2 wt%, 4 wt%, and 6 wt% CA scaffolds, respectively. The results showed no significant difference in cell number among the 3D CA scaffold groups. However, the 231-GFP cells cultured in 2 wt% CA scaffolds possessed greater cellular size, area, perimeter, and lower cellular circularity compared to those in 4 wt% and 6 wt% CA scaffolds, suggesting a more prominent presence of cell clusters in softer substrates compared to stiffer substrates. The results also showed cells in 6 wt% CA having a higher average cell migration speed compared to those in 2 wt% and 4 wt% CA scaffolds, indicating a positive relationship between substrate stiffness and cell migration velocity. Findings from this experiment may contribute to the development of enhanced in vitro 3D breast tumor models for basic cancer research using 3D porous biomaterial scaffolds.

Thesis Completion




Thesis Chair/Advisor

Steward, Robert L.


Florczyk, Stephen J.


Bachelor of Science in Mechanical Engineering (B.S.M.E.)


College of Engineering and Computer Science


Mechanical and Aerospace Engineering

Degree Program

Mechanical Engineering



Access Status

Open Access

Length of Campus-only Access

1 year

Release Date