Generation of a Sandwich Based 3D Hydrogel to Support Human Mammary Fibroblast Viability and Proliferation

Date of Award


Degree Type


Degree Name

Master of Science in Biomedical Sciences

First Advisor

Abigail Hielscher, PhD

Second Advisor

Xinyu Wang, PhD

Third Advisor

Harold Komiskey, PhD


Carcinoma associated fibroblasts (CAFs) or myofibroblastsare activated fibroblasts which play a role in breast tumorigenesis. Matrix stiffness promotes fibroblast transition into myofibroblasts. In this study, we prepared a biologically relevant 30 mechanically tuned model in which to investigate the fibroblast to myofibroblast transition. To accomplish this, we cultured human mammary fibroblasts (HMFs) between two mechanically tuned hydrogels. The bottom hydrogel consists of gelatin cross~linked with microbial transglutaminase (mTG) to generate three conditions: compliant (30μg mTG), moderate (lOOμg mTG) and stiff (200μg mTG) hydrogels. HMFs were seeded oh these hydrogels and then a compliant hydrogel was plated atop the HMFs. To determine viability in each of the hydrogels, HMFs were stained with EtBr/Calcein. At days 2 and 4, results showed significantly more live cells than dead in all three hydro gel conditions. Next, HMF proliferation was measured using a WST-1 assay. Proliferation was similar at day 2 for all hydrogel conditions and increased significantly in the compliant sandwich at day 4 when compared to the moderate and stiff hydrogels. To investigate changes in morphology, HMFs in each hydrogel condition were analyzed for circularity using Image J. HMFs in the compliant hydrogel were more circular overall in comparison to HMFs in moderate and stiffhydrogels which were more elongated. Overall, the results from this study indicate that HMFs remain viable and proliferative over the culture period in the 30 hydrogel, work which supports the use of this platform to investigate the role of mechanical stiffness on fibroblast morphology and behavior.

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