Abstract Matrix stiffness is associated with breast tumor progression. This in part is due to changes in extracellular matrix composition, abundance and modification, a phenomenon which is largely accomplished by myofibroblasts, or carcinoma associated fibroblasts. While it has been shown that fibroblasts acquire a myofibroblast phenotype following culture atop mechanically stiff 2 and 3 dimensional (3D) matrices, such a phenomenon has yet to be tested for cells encapsulated in 3D matrices. In this study, we generated gelatin hydrogels cross-linked with different concentrations of microbial transglutaminase (mTg) to yield compliant and stiff hydrogels. These hydrogels were utilized for encapsulation of human mammary fibroblasts (HMFs) to investigate their effects on HMF viability and proliferation over a 7 day culture period. To ensure that mTg was non-toxic to HMFs, HMFs were first cultured in 2D with various concentrations of mTg (range from 6.3 to 500μg/ml). Viability results indicated that mTg was relatively non-toxic to HMFs at the tested concentrations. To investigate thermal stability, hydrogels were cross-linked with low (6.3 μg/ml) and high (500 μg/ml) concentrations of mTg and were cultured in PBS for up to 5 hours at 37°C. Results demonstrated minimal loss of gel weight in 500 μg/ml mTg hydrogels and increased weight (e.g. swelling) from 6.3 μg/ml mTg hydrogels, suggesting that hydrogels cross-linked with the lowest and highest possible concentrations of mTg exhibit thermal stability. Next, rheology was used to assess the bulk mechanical stiffness of mTg cross-linked hydrogels. mTg at 20 μg/ml resulted in gels with a stiffness of 300 Pascals (Pa) while mTg at 30 μg/ml resulted in gels with a stiffness of 1200 Pa. Although hydrogels with a mechanical stiffness of 1800 Pa were evaluated, it was found that these conditions did not support cell viability or proliferation. As such, mTg concentrations yielding 300 and 1200 Pa were elected to represent compliant and stiff hydrogels, respectively. HMFs were encapsulated in compliant and stiff hydrogels and their viability and proliferation were monitored over the 7 day culture period. Results demonstrated that both compliant and stiff hydrogels supported HMF cell viability throughout the culture period, evaluated using the Live/Dead assay. Moreover, compliant and stiff hydrogels facilitated cell proliferation, assessed using the WST assay. Not only did proliferation significantly increase for HMFs in both compliant and stiff gels during the 7 day culture period, but was significantly greater for HMFs in stiff gels in comparison to HMFs in compliant gels at days 1, 3 and 5. Overall, these results indicate that different concentrations of mTg generate gelatin hydrogels with unique stiffnesses, which support HMF viability and proliferation over the culture period. These results will serve as a platform from which to investigate matrix stiffness effects on a myofibroblast phenotype. Citation Format: Kathryn Woods, Catlyn Thigpen, Kyung Min Park, Abby Hielscher. Mechanically tuned 3 dimensional gelatin hydrogels support mammary fibroblast viability and growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 345. doi:10.1158/1538-7445.AM2015-345