Three-Dimensional Scaffold-Free Spheroids with Fibroblast/Macrophage Co-Culture for in vitro Fibrosis Modeling
Yu Tan, Ph.D, Matthew Garza, BS, Wilmina Landford, MD, Devin Coon, MD MSE.
Johns Hopkins University, Baltimore, MD, USA.
PURPOSE: An in vitro model of fibrosis is critical for designing surgical implants with better biocompatibility or new anti-fibrotic drug development. However, traditional 2D monolayer culture-based models of fibrosis show little resemblance to the in vivo fibrogenesis process. To enable screening of exponentially larger numbers of materials and conditions than can be achieved by sub-acute animal testing, we have developed a 3D scaffold-free spheroid system with human fibroblasts (spherical micro-tissue), which is more physiologically similar to real tissue. In addition, hybrid spheroids consisted of human fibroblasts and macrophages have been fabricated to investigate direct fibroblast-macrophage interaction and communication during fibrogenesis.
METHODS: Spheroids were fabricated of 100% human fibroblasts with difference sizes (100, 200, 300,400, 500Ám in diameter) respectively. Immunofluorescent staining with collagen-1 and apoptosis marker (Caspase-3) as well as qPCR with fibrosis genes (collagen-1 and aSMA) was performed to compare with the 2D control to identify the optimal size of the spheroids for an effective fibrosis model. In addition, we isolated human peripheral blood derived monocytes to differentiate into macrophages that could be used to populate the spheroids. Macrophages were co-cultured with fibroblasts under different ratios (1:2, 1:4, 1:8, and 1:16) to identify the ideal ratio in hybrid spheroids. We performed immunofluorescent staining with macrophages marker (MAC387) and fibroblasts marker (ER-TR7) and qPCR to optimize the ratio of macrophages in the hybrid spheroids to optimize similarity to physiologic in vivo fibrogenesis.
RESULTS: Fibroblasts in spheroid had higher expression levels of fibrosis genes compared to the 2D monolayer control. The spheroid size of 200um showed the highest viability, more homogenous collagen deposition and the highest expression level of fibrosis genes due to a lower expression level of proteinase-related genes such as MMP1, MMP2 and MMP7 (Figures 1-2). Interestingly, hybrid spheroids at a ratio of 1:16 (macrophage:fibroblast) showed higher survival rate of macrophages and a remarkably higher expression level of fibrosis genes (collagen-1, collagen-3 and TGFβ) with a lower expression level of anti-fibrotic MMP7 (Figure3-4).
CONCLUSIONS: We have developed a 3D scaffold-free spheroid system as a more physiologically relevant fibrosis model in vitro for implant material and drug development and screening as compared to current methods. The inclusion of macrophages led to a significant change in fibroblast behavior. The size of the spheroids and the ratio of macrophages have been optimized regarding the resulting fibrosis profile. Future work will involve correlation of our findings with the in vivo tissue response.
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