(C-120) Differential Effects of 2D and 3D Environments on Hydrogen Peroxide-induced Cellular Senescence

Introduction:: Cellular senescence, a state of irreversible growth arrest, is a fundamental process influencing various aspects of cellular biology. Hydrogen peroxide (H­­2O­2), a reactive oxygen species (ROS), is a key mediator of oxidative stress and has been known to trigger cellular senescence. Notably, the cellular microenvironment, specifically whether cells are grown in two-dimensional (2D) monolayer or three-dimensional (3D) tissue, can substantially influence cellular behaviors. In this study, we investigate the effects of 2D and 3D environments on hydrogen peroxide-induced cellular senescence by quantifying biochemical, structural, and physical properties.

Materials and Methods:: In this study, we employed human foreskin fibroblasts in both 2D monolayers and three-dimensional 3D tissues. H­­2O­2 solutions at concentrations ranging from 25 μM to 200 μM were administered to the cells cultured in a 24-well plate to examine the concentration-dependent effects of hydrogen peroxide (H­­2O­2) on cellular senescence. Rat tail collagen was adopted as the primary component to mimic the 3D environment. The experimental procedure involved adding H­­2O­2 to the cells and incubating them for 1 hour, followed by a 23-hour culturing period in the medium. To quantify the diverse biochemical and morphological properties of cells and organelles, we captured and used fluorescence images.

Results, Conclusions, and Discussions:: In this study, the control cells in 2D, which were not subjected to any amount of hydrogen peroxide (H­­2O­2), maintained a circular shape with small nuclei. However, even at the lowest concentration tested (25 μM), a noticeable enlargement of the nuclei was observed. Concentrations of 25, 50, and 100 μM displayed similar morphology, with no significant changes in cell appearance. As the concentration of H2O2 increased to 150 and 200 μM, a substantial increase in nucleus size and elongation of cell bodies were evident. Furthermore, cells treated with 100 μM and higher concentrations were observed to be densely packed together, indicating a possible impact on cell-cell interactions.
These findings demonstrate a concentration-dependent effect of H­­2O­2 on cellular morphology, with lower concentrations inducing subtle changes and higher concentrations causing more pronounced alterations in both nucleus size and cell shape. The observed changes in cell packing also suggest that higher concentrations of H­­2O­2 may influence cell behavior beyond mere morphological alterations, possibly affecting cell signaling and intercellular communication.


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