The expression of alpha-smooth muscle actin (αSMA), an indicator of fibroblast-to-myofibroblast transition, and filamentous actin (F-actin) were quantified by fluorescence staining and confocal imaging of samples stretched for 24 h and compared to static controls. Intensity values were extracted from the images through background subtraction and thresholding (details on the image processing steps are presented in Supplementary Information, Figure ). The 3T3 fibroblasts showed a statistically significant increase in αSMA expression (~2.8-fold) after 24 h of stretching compared to the static control (Figures ). These results indicate that the 3D printed GelMA-CBP constructs subjected to cyclic mechanical stress can effectively induce a fibroblast-tomyofibroblast transition, a hallmark of fibrosis in organs like the liver or the lung. Contrary to αSMA, we observed a statistically significant decrease in the expression of F-actin after 24 h of stretching (Figure ,C,E), in agreement with previous reported data. For example, the work from Zhang and coworkers reported a time dependent reduction of F-actin induced by uniaxial cyclic stretching (10%, 1Hz) in rat bone marrow mesenchymal stem cells. The F-actin cytoskeleton plays a relevant role in various cellular processes, and there is significant literature reporting that stretching can lead to either reinforcement or fluidization of the cell body. Reinforcement results from enhanced actin polymerization and a concomitant increase in cytoskeletal stiffness. On the other hand, fluidization is a cellular response intended to relieve stress and is characterized by a decrease of F-actin expression and the corresponding softening of the cell body. Under our experimental conditions, the cyclic stretching of 3T3 fibroblasts for cycles from 0 to 20% strain at 0.33 Hz. (A) Schematic representation of the seeding, stretching, and imaging process. (B) Fluorescence labelling of the nucleus (DAPI -cyan), αSMA (primary antibody -green), F-actin (phalloidin -magenta), and merged image of all channels for 3T3 fibroblasts cultured under static and cyclic stretch conditions. (C) Zoomed in merged image of static and stretched conditions. Quantification of (D) αSMA expression and (E) F-actin for 3T3 fibroblasts cultured under static and cyclic stretch conditions. ** indicates a statistically significant difference with p < 0.01. A two-tailed t-test was done for comparisons between two groups. Data is presented as the mean and standard deviation for n ≥ 3 independently prepared samples.