Présentation musicale : [Partition d'orchestre

Abigail Hielscher (848582)

Catlyn Thigpen (4689742)

Hana Park (2827127)

Jennifer Wang (3804277)

Kathryn Woods (4689739)

BMC Cell Biology

Material 1, Circularity 20 μg, Circularity analyses on HMFs encapsulated in 20 μg hydrogels. (XLSX 19 kb

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Additional file 5: of Mechanically tuned 3 dimensional hydrogels support human mammary fibroblast growth and viability

Material 9, Proliferation 75 and 100 μg, Proliferation analyses for HMFs encapsulated in 75 and 100 μg hydrogels. (XLSX 12 kb

Additional file 13: of Mechanically tuned 3 dimensional hydrogels support human mammary fibroblast growth and viability

BACKGROUND: Carcinoma associated fibroblasts (CAFs or myofibroblasts) are activated fibroblasts which participate in breast tumor growth, angiogenesis, invasion, metastasis and therapy resistance. As such, recent efforts have been directed toward understanding the factors responsible for activation of the phenotype. In this study, we have investigated how changes in the mechanical stiffness of a 3D hydrogel alter the behavior and myofibroblast-like properties of human mammary fibroblasts (HMFs).
RESULTS: Here, we utilized microbial transglutaminase (mTG) to mechanically tune the stiffness of gelatin hydrogels and used rheology to show that increasing concentrations mTG resulted in hydrogels with greater elastic moduli (G\u27). Upon encapsulation of HMFs in 200 (compliant), 300 (moderate) and 1100 Pa (stiff) mTG hydrogels, it was found that the HMFs remained viable and proliferated over the 7 day culture period. Specifically, rates of proliferation were greatest for HMFs in moderate hydrogels. Regarding morphology, HMFs in compliant and moderate hydrogels exhibited a spindle-like morphology while HMFs in stiff hydrogels exhibited a rounded morphology with several large cellular protrusions. Quantification of cell morphology revealed that HMFs cultured in all mTG hydrogels overall assumed a more elongated phenotype over time in culture; however, few significant differences in morphology were observed between HMFs in each of the hydrogel conditions. To determine whether matrix stiffness upregulated expression of ECM and myofibroblast markers, western blot was performed on HMFs in compliant, moderate and stiff hydrogels. It was found that ECM and myofibroblast proteins varied in expression during both the culture period and according to matrix stiffness with no clear correlation between matrix stiffness and a myofibroblast phenotype. Finally, TGF-β levels were quantified in the conditioned media from HMFs in compliant, moderate and stiff hydrogels. TGF-β was significantly greater for HMFs encapsulated in stiff hydrogels.
CONCLUSIONS: Overall, these results show that while HMFs are viable and proliferate in mTG hydrogels, increasing matrix stiffness of mTG gelatin hydrogels doesn\u27t support a robust myofibroblast phenotype from HMFs. These results have important implications for further understanding how modulating 3D matrix stiffness affects fibroblast morphology and activation into a myofibroblast phenotype

Woods, Kathryn

Thigpen, Catlyn

Wang, Jennifer Peyling

Park, Hana

Hielscher, Abigail

Philadelphia College of Osteopathic Medicine: DigitalCommons@PCOM

English

Mechanically Tuned 3 Dimensional Hydrogels Support Human Mammary Fibroblast Growth and Viability

Material 3, Circularity 60 μg, Circularity analyses on HMFs encapsulated in 60 μg hydrogels. (XLSX 19 kb

Additional file 7: of Mechanically tuned 3 dimensional hydrogels support human mammary fibroblast growth and viability

Material 14, Thermal stability 20, 30 and 60Â Îźg, Analyses of gel encapsulated HMFs for changes in gel weights following culture in a 37C incubator. (XLSX 11Â kb

Additional file 18: of Mechanically tuned 3 dimensional hydrogels support human mammary fibroblast growth and viability

Material 11, Gel Weight Measurements, Gel weight of HMFs encapsulated in 20, 30 and 60Â Îźg hydrogels. Weights were taken shortly after polymerization and the end of the culture period. (XLSX 13Â kb

Additional file 15: of Mechanically tuned 3 dimensional hydrogels support human mammary fibroblast growth and viability

Material 2, Circularity 30 μg, Circularity analyses on HMFs encapsulated in 30 μg hydrogels. (XLSX 19 kb

Additional file 6: of Mechanically tuned 3 dimensional hydrogels support human mammary fibroblast growth and viability

Material 5, Cell Viability 60 μg, Cell viability analyses for HMFs encapsulated 60 μg hydrogels. (XLSX 12 kb

Additional file 9: of Mechanically tuned 3 dimensional hydrogels support human mammary fibroblast growth and viability

Abstract Background Carcinoma associated fibroblasts (CAFs or myofibroblasts) are activated fibroblasts which participate in breast tumor growth, angiogenesis, invasion, metastasis and therapy resistance. As such, recent efforts have been directed toward understanding the factors responsible for activation of the phenotype. In this study, we have investigated how changes in the mechanical stiffness of a 3D hydrogel alter the behavior and myofibroblast-like properties of human mammary fibroblasts (HMFs). Results Here, we utilized microbial transglutaminase (mTG) to mechanically tune the stiffness of gelatin hydrogels and used rheology to show that increasing concentrations mTG resulted in hydrogels with greater elastic moduli (G’). Upon encapsulation of HMFs in 200 (compliant), 300 (moderate) and 1100 Pa (stiff) mTG hydrogels, it was found that the HMFs remained viable and proliferated over the 7 day culture period. Specifically, rates of proliferation were greatest for HMFs in moderate hydrogels. Regarding morphology, HMFs in compliant and moderate hydrogels exhibited a spindle-like morphology while HMFs in stiff hydrogels exhibited a rounded morphology with several large cellular protrusions. Quantification of cell morphology revealed that HMFs cultured in all mTG hydrogels overall assumed a more elongated phenotype over time in culture; however, few significant differences in morphology were observed between HMFs in each of the hydrogel conditions. To determine whether matrix stiffness upregulated expression of ECM and myofibroblast markers, western blot was performed on HMFs in compliant, moderate and stiff hydrogels. It was found that ECM and myofibroblast proteins varied in expression during both the culture period and according to matrix stiffness with no clear correlation between matrix stiffness and a myofibroblast phenotype. Finally, TGF-β levels were quantified in the conditioned media from HMFs in compliant, moderate and stiff hydrogels. TGF-β was significantly greater for HMFs encapsulated in stiff hydrogels. Conclusions Overall, these results show that while HMFs are viable and proliferate in mTG hydrogels, increasing matrix stiffness of mTG gelatin hydrogels doesn’t support a robust myofibroblast phenotype from HMFs. These results have important implications for further understanding how modulating 3D matrix stiffness affects fibroblast morphology and activation into a myofibroblast phenotype

Kathryn Woods

Catlyn Thigpen

Jennifer Peyling Wang

Hana Park

Abigail Hielscher

Directory of Open Access Journals

Mechanically tuned 3 dimensional hydrogels support human mammary fibroblast growth and viability

Material 12, Proliferation 20, 30 and 60 μg, Proliferation analyses for HMFs encapsulated in 20, 30 and 60 μg hydrogels. (XLSX 9 kb

Additional file 16: of Mechanically tuned 3 dimensional hydrogels support human mammary fibroblast growth and viability

https://figshare.com/articles/Additional_file_16_of_Mechanically_tuned_3_dimensional_hydrogels_support_human_mammary_fibroblast_growth_and_viability/5709247