Fig. 3.

Long-term culture of PLN FRCs in collagen scaffolds recapitulates LN-like reticula structure of donor mice and reticular alterations of T1D PLNs. (a) Representative maximum projection images of confocal z-stacks taken 1, 7, 14 or 21 days after seeding of 12-week old NOD (top row) or NOR (bottom row) PLN FRCs in collagen sponges with protocols shown in Supplemental Fig. 2. FRCs are identified by gp38 (green) and F-actin (red); nuclei are counterstained with DAPI (blue). Scalebars = 100 µm. (b) FRC remodeling of their reticular networks in 3D scaffolds as reticular pore size (Feret diameter) as a function of culture time (3–21 days) and of type of FRCs seeded (derived from NOD: red vs. NOR: black). For each condition, n = 30 pores per scaffolds were quantified blindly and n = 3 independent scaffolds were measured. (c) FRC proliferation in 3D scaffolds as total FRC numbers retrieved from the scaffolds at each time point per treatment (NOD: red vs. NOR: black derived FRCs). (d) Expression of the important FRC marker gp38, as mean fluorescence intensity (MFI) of NOD (red) and NOR (black) cells isolated from scaffolds 7–21 days after seeding and normalized to the MFI of cells at same passage but cultured in traditional 2D tissue culture flasks. Biological replicates were n = 3 scaffolds. Both the effects of culture time and of FRC origin (NOD vs. NOR) were evaluated. ns not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001