Figure 6.

Peripheral blood mononuclear cell (PBMC) interact with adipocyte derived stem cell (ADSC)-derived fibroblasts in 3D collagen spheroid co-cultures. (A) Images demonstrating the culture techniques used to generate 3D collagen spheroid co-cultures. Day 3 ADSC, PBMC monocyte-derived macrophage (MDM) 2D co cultures (first panel), 12 µl droplets of cells suspended in Type-I collagen (second panel), 3D collagen spheroid co-cultures in six well-plate (third panel). The final panel depicts a phase contrast image of a 3D collagen spheroid co-culture at day 8 of culture, scale bar 200 µm. (B) Left-hand panel: Whole-mount staining of a 3D collagen spheroid co-culture containing ADSC (ICAM-1, cyan) and PBMC [B cells, CD20 (green), T cells, CD3 (red)], counterstained with DAPI nuclear stain (blue). White arrows indicate B cells in close association with ADSC-derived lymphoid-like fibroblasts, scale bar, 50 µm. Right-hand panel; representative double immunohistochemistry (IHC) staining of formaldehyde fixed paraffin embedded (FFPE) sections of collagen spheroid co-cultures containing ADSC-derived lymphoid fibroblasts (podoplanin and VCAM-1, red), PBMC, and MDM (CD68, brown), scale bars 100 µm and 50 µm for upper and lower images, respectively. (C) Representative histogram overlays of podoplanin and ICAM-1 staining of untreated and cytokine treated ADSC alone or cytokine treated ADSC co-cultured with PBMC +/− 10⁵ autologous MDM recovered from 3D collagen spheroid cultures after 8 days of culture, isotype controls are shown as filled silver histograms. Graphs show surface expression of podoplanin and ICAM-1 on lymphoid fibroblasts for each culture condition at day 8 of culture. Each closed circle represents data from an independent experiment, bars represent the mean. Group comparisons were made using the one-way ANOVA test with Sidak correction for multiple comparisons; p values < 0.05 were considered significant, *p < 0.05.