Ruogang Zhao, PhD

Research update for year 1. Progress 1, established a co-culture of lung microtissues and human blood monocyte-derived macrophages (Mo-MFs) to model macrophage-regulated lung fibrosis. First, we established the protocol for efficient human monocyte-derived macrophage culture and differentiation. The profibrotic M2a macrophages were successfully induced from primary human monocyte purified from PBMC. We performed tests on the interaction between M2a macrophages and lung fibroblasts in both 2D culture and 3D culture. In 2D culture, compared to untreated lung fibroblasts, M2a MF treated lung fibroblasts expressed strong a-SMA, suggesting the differentiation of these fibroblasts to myofibroblasts. Next, we co-cultured M2a MFs with lung fibroblast-populated 3D microtissue. We show that M2a macrophages attached well to the microtissue surface in large numbers. Macrophages displayed an elongated morphology and formed obvious alignment on the microtissue surface. Quantitative analysis of fluorescence intensity shows much higher expression of a-SMA in co-cultured microtissue as compared to mono-cultured fibroblast-populated microtissue, confirming myofibroblast differentiation. Together, these results showed that the lung microtissue fibrosis can be induced by a human blood monocyte derived macrophages M2a. Progress 2, integrated the lung microtissues with a microfluidic device to study the effect of shear flow on the micromechanics of lung fibrogenesis. The lung microtissue array microfluidic system consists of a top PDMS layer containing microfluidic channels for cell medium flow and a bottom PDMS layer containing arrays of fibrotic microtissues. Media flow was provided through syringe pump draw. To test flow-mediated changes in microtissues, the microtissues were subjected to media perfusion at 3µl/min for 21 hours. The phase contrast images show that fibroblasts exhibited higher order of diagonal alignment and the microtissues generated higher contractile force under flow treatment as compared to the static condition. Together, these results show that shear flow has an important impact on the microtissue morphology and force generation. Plans for the coming year. Since we have developed monocyte derived macrophage (Mo-MF) co-cultured lung microtissue model in the past 10 months, the remaining task for Aim 1 is to develop a lung interstitial macrophage (TR-MFs) co-cultured microtissue. We originally proposed to use human TR-MFs that are isolated from donor lungs, but we tried this on human lung tissues obtained from the tissue bank and all the cells are dead. This is mainly because the tissue was no longer fresh after shipping to us (more than 12 hours). Since fresh human tissues are very hard to obtain from the tissue bank, we proposed to use mouse cells, which are easy to obtain and widely used by researchers in the field. We also plan to complete all the tasks in Aim 2, which is to test the clinical relevancy and power of the MF-fibrous tissue models for the screening of MF-targeting anti-fibrosis drugs, in the coming year.

Update: In year 1 of this project, we established a co-culture of lung microtissues and human blood monocyte-derived macrophages (Mo-MFs) to model macrophage-regulated lung fibrosis. The profibrotic M2a macrophages were successfully induced from primary human monocyte purified from PBMC. We performed tests on the interaction between M2a macrophages and lung fibroblasts in both 2D culture and 3D culture. Our results showed that the lung microtissue fibrosis can be induced by a human blood monocyte derived macrophages M2a. We also integrated the lung microtissues with a microfluidic device to study the effect of shear flow on the micromechanics of lung fibrogenesis. The lung microtissue array microfluidic system consists of a top PDMS layer containing microfluidic channels for cell medium flow and a bottom PDMS layer containing arrays of fibrotic microtissues. The results show that lung fibroblasts exhibited higher order of diagonal alignment and the microtissues generated higher contractile force under flow treatment as compared to the static condition.