Figure 3.

Two days postinjury (DPI) in vivo recovered epithelial cells grown in culture demonstrate increased size and proliferation compared with control and 1 DPI cells. A: representative gating strategy for FACS-sorted cells isolated from control, 1 DPI, and 2 DPI jejunum based on live/dead (7 AAD) and EpCAM-Alexa647. B: representative time course images of enteroid growth in culture from FACS-sorted EpCAM⁺ cells from control, 1 DPI, and 2 DPI out to 168 h in culture. Scale bar 20 µm. C: box plots of log transformed daily enteroid area measurements from enteroids in B, showing increased growth of 2 DPI enteroids compared with control and 1 DPI, n = 4 pigs for each recovery day, 15,000 cells plated per 50 µL Matrigel, 15–20 enteroids measured per pig. D: means ± SD of bud counts per enteroid, showing significantly increased proliferative capacity for 2 DPI enteroids at 96 and 144 h in culture compared with control and 1 DPI. Analysis by two-way ANOVA with post hoc Tukey’s test, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data collected from same enteroids in C. E: culture overview images at 168 h, demonstrating increased size and bud count for 2 DPI enteroids. Scale bar 100 µm. FACS, fluorescence-activated cell sorting.