Journal: APL Bioengineering
Article Title: Intercellular junction-driven stromal cell stacking in a confined 3D microcavity
doi: 10.1063/5.0197187
Figure Lengend Snippet: NIH 3T3 cells stacking on 3D microcavity. (a) The illustration shows the stages of stacking formation in NIH 3T3 mouse fibroblast cells. The cells crawl down from the reservoir, as indicated by the arrows. After 72 h, the stacking is fully formed within the microcavity. (b) 3D reconstructions of confocal images, displaying the z-projection and corresponding y–z and x–z sections of cells (along the white lines). The cells are stained for F-actin (red) and the nucleus (blue) at the initial (left) and final (final) stages of stacking cell formation. The yellow dotted boxes indicate the boundaries of the PDMS substrate. The scale bars represent 100 μ m. (c) The plot shows the height of the initial and final stages of stacking cell formation for different gaps ranging from L s = 100–400 μ m. The results demonstrate that the final stage of stacking cells has a significantly greater cell height compared to the initial stage. Statistical analysis was performed using Kolmogorov–Smirnov test, comparing the initial cell height of each group as the control, ** P < 0.01, n = 5, error bars show standard deviation. (d) The plot presents the number of cells at the initial and final stages of stacking formation for various microcavity side lengths. Statistical analysis was conducted using the Kolmogorov–Smirnov test, comparing the cell number at the initial stage as the control, * P < 0.05, n = 4, error bars show standard deviation. (e) Region of interest (ROI) for migration trajectory analysis around the microcavity represented by L s = 100 μ m. The scale bar is 100 μ m. (f) Determination of the angle during cell migration from the reservoir to the microcavity. The rose plot represents the migration direction from the initial point to the final point during the stacking formation from (e). (g) This is a 3D reconstruction of confocal images stained for F-actin, Fn, and Col I at the final stage of stacking formation along the white lines represented by L s = 100 μ m. Single-channel micrographs have been presented in grayscale to improve contrast and clarity. Scale bar: 100 μ m. h, These plots display gene expression of Col I and Fn with GAPDH as the housekeeping gene, indicating the upregulation of both genes during the final stage of stacking cell formation. Statistical analysis was performed using Wilcoxon test, comparing D1 (initial time) as the control, n = 3, error bars show standard deviation.
Article Snippet: NIH3T3 mouse fibroblast (ATCC CRL-1658) cells, human dermal fibroblast cells (ATCC PCS-201-012), and HaCaT cells were maintained in DMEM supplemented with 10% of fetal bovine serum (FBS) and antibiotics (100 U mL −1 penicillin and 100 μ g mL −1 streptomycin; Gibco, Grand Island, NY, USA).
Techniques: Staining, Control, Standard Deviation, Migration, Expressing