Fig. 5: Vasculature supports immune cell trafficking and reveals disease-associated recruitment.

A Schematic illustrating addition of human PBMC-derived monocytes on Day 8 of device culture, after perfusable vasculature has been established, followed by 4 days of co-culture before endpoint fixation or tissue harvesting. Created in BioRender. Tevonian, E. (https://BioRender.com/e15y242). B Maximum intensity projection of devices 12 h after monocyte addition shows that monocytes (CellTracker Deep Red, false-colored yellow) localize within GFP-HUVEC vascular networks (green) and begin interacting with spheroids (CellTracker Red, magenta). Scale bars = 200 µm. C Representative maximum intensity projection images (insets of 5B) showing monocyte interactions with spheroids. Arrows indicate an example of a monocyte that has extravasated from the vascular networks near a hepatic spheroid (left) and a cluster of monocytes pausing in the networks at a spheroid (right). Scale bars = 200 µm. This experiment was repeated independently three times with similar results, where each experiment used a unique primary monocyte donor. D Imaging after 4 days of monocyte co-culture shows monocytes persist in the devices. Insets (right) show that most monocytes appear to localize with hepatic spheroids (visible in brightfield channel), and some monocytes appear to be differentiating based on their changed morphology. Scale bars = 200 µm (left), 50 µm (insets). This monocyte imaging experiment was repeated independently four times with similar results, where each experiment used a unique primary monocyte donor. E Flow cytometry gating strategy to quantify the frequency of monocytes (CellTracker Deep Red) in each device. F Monocytes were significantly enriched within insulin resistance (IR) devices compared to the physiological condition. Comparison to devices containing vascular networks alone (no hepatic spheroids) also demonstrates the presence of spheroids significantly increases monocyte frequency. n = 6 devices per condition across N = 3 experiments. Statistical analysis performed with Kruskal-Wallis test and post-hoc Dunn’s multiple comparison test. *p < 0.05 and **p < 0.01. G Representative histogram shows elevated CD163 on CellTracker+ monocytes cultured in MPS devices compared to naive monocytes (gray).