Figure 5.

A: Photomicrographs depicting the graded increase in picrosirius red staining for collagen seen under bright-field microscopy (red staining) in representative histological sections from 3D organotypic gel cultures in which TDE_CC cholangiocarcinoma cells (initially plated at 2 × 10⁵ viable cells) were co-cultured with TDF_SM myofibroblastic cells at increasing initial viable cell plating densities ranging from 5 × 10⁴ to 8 × 10⁵ cells/gel. Note that the picrosirius staining reaction is proportionally enhanced in sections prepared from gel cultures in which the TDE_CC cells were co-cultured with TDF_SM cells as a function of increasing initial cell plating densities. When viewed microscopically under polarized light, the obvious differences in the intensities of the picrosirius red staining reaction for dense collagen fiber production and deposition into the gel matrix that distinguishes TDE_CC + TDF_SM co-cultures at the low versus the high TDF_SM proportions is clearly visualized. B: Graphic representation further showing gel matrix shrinkage (contraction) to also be a function of increasing initial TDF_SM myofibroblastic cell proportion when co-cultured with a constant number of TDE_CC cholangiocarcinoma cells at an initial cell plating density of 2 × 10⁵ cells/gel. Each value indicates the mean gel diameter determined from individual measurements made on two separate gel cultures per time point. C: Quantitative imaging data demonstrating picrosirius red staining intensity under polarized light for fibrous collagen to be most prominently increased in 3D gel co-cultures of TDE_CC + TDF_SM cells over staining intensity values measured in histological sections from TDF_SM, and TDE_CC mono-cell cultures, respectively. n = 3 cultures analyzed per data point, with staining intensity measurements made on 3 random sections/culture. ^∗∗∗∗P < 0.0001. Scale bars: 20 μm (A, top row); 100 μm (A, bottom row, and C).