Abstract 3755: Generation of a sandwich based 3D hydrogel to support human mammary fibroblast viability and proliferation
Authors: Abbott Anna , Byrd Jana , Hielscher Abby 
Published: August 2019

Myofibroblasts are activated fibroblasts which play a role in breast tumor growth, metastasis and therapy resistance. One factor which has been shown to facilitate the transition of fibroblasts to myofibroblasts is increased matrix stiffness, a phenomenon which has been reported in 2D, but not widely investigated in 3D. One of the limitations associated with 3D cultures is that cells are unable to remodel an environment which is too rigid and fail to remain viable as a result. To this end, we sought to develop a mechanically tunable 3D hydrogel to investigate whether this platform was supportive for growth of human mammary fibroblasts (HMFs). To accomplish this, we generated a sandwich-based gelatin hydrogel in which we cultured HMFs between two mechanically tuned hydrogels. The bottom hydrogel consisted of gelatin cross-linked with microbial transglutaminase (mTG) to generate compliant (30µg), moderate (100µg) and stiff (200µg) mTG hydrogels. HMFs were seeded on these hydrogels and then a compliant hydrogel was plated atop the HMFs, generating the 3D hydrogel sandwich. As a control, HMFs were cultured in 2D atop compliant, moderate and stiff hydrogels. After a period of 2 and 4 days, HMFs cultured in the 2D and 3D hydrogels were investigated for changes in viability and growth using EtBr/Calcein and WST, respectively. At days 2 and 4, results showed significantly more live cells than dead in all 3D hydrogels, indicating that HMFs remained viable despite increases in mechanical stiffness of the bottom hydrogel. Differences in viability between HMFs grown in 2D vs 3D were negligible with the exception for HMFs in 2D compliant hydrogels which had a significantly greater proportion of live cells in comparison to HMFs in 3D compliant hydrogels. Analyzing changes in proliferation, we found that proliferation increased over the 4-day culture period for HMFs grown in all 3D hydrogels, but when compared to one another, HMFs were most proliferative in the compliant 3D hydrogels than in the moderate and stiff 3D hydrogels. Analyzing differences in 2D and 3D cultures, it was found that HMFs were more proliferative in 2D as opposed to 3D. To investigate changes in morphology, a property which is influenced in part by changes in the mechanical properties of the substrate, HMFs in 2D and 3D hydrogels were analyzed for changes in circularity using Image J. HMFs in the compliant 3D hydrogel were more circular at days 1 and 2 in comparison to HMFs in moderate and stiff hydrogels. At days 3 and 4, HMFs in all 3D hydrogels exhibited similar elongated morphologies. Comparing 2D and 3D hydrogels, HMFs were more elongated overall in 2D than in 3D in each of the hydrogels. Overall, the results indicate that HMFs remain viable and proliferate over the culture period in the mechanically tuned 3D hydrogels. This work supports the use of this platform to investigate the role of mechanical stiffness on fibroblast acquisition of a myofibroblast phenotype.

Citation Format: Anna Abbott, Jana Byrd, Abby Hielscher. Generation of a sandwich based 3D hydrogel to support human mammary fibroblast viability and proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3755.