Description
- Abstract:
- Abstract This study presents a scaffold-free 3D ring-shaped co-culture model composed of human dermal fibroblasts and THP-1 monocyte-derived macrophages to recapitulate cellular interactions associated with tissue fibrogenic conditions more closely. We optimized the 50:50 culture medium and established a macrophage-to-fibroblast ratio of 1:16 as the most ring-structured, stable culture setting with M0 macrophages. With M1 and M2 co-culture in optimized settings, significant contraction in the x, y- plane from day 2 was observed onward compared with the fibroblast-only control. In contrast, the addition of M1 macrophages led to a reduction in x, y-thickness starting from 24 hours post-seeding. To examine variations in immunological roles in tissue morphology, proinflammatory M1 and profibrotic/anti-inflammatory M2a and M2c were observed in the x, y-, and z-dimensions until day 7. To assess biochemical changes, we measured P1CP levels in the supernatants of each co-culture model to evaluate relative collagen production over time. Profibrotic M2a macrophage rings secreted the highest level of P1CP, suggesting the most fibrogenesis effect on fibroblast cells at an early stage. Proinflammatory M1 macrophages increased collagen production at later time points, suggesting potential activation of the profibrotic process through a non-canonical pathway activation by the inflammatory cytokine. Due to macrophages' high plasticity, we visualized the M1, M2a, and M2c phenotypes by immunostaining for CD86, CD206, and CD163 using fluorescent immunohistochemistry. Myofibroblast activation was also assessed in different macrophage co-cultures using αSMA staining. The reduced activation of myofibroblasts was associated with a reduction in collagen production across later time points in M2 co-cultured rings and myofibroblast-only rings. In contrast, prolonged myofibroblast activation in M1 co-cultured rings demonstrated a correlation with increased collagen production towards later time points. These findings demonstrate the potential of this 3D in vitro platform as a physiologically relevant and quantifiable alternative to conventional 2D culture, 3D spheroids, and animal models for studying fibrosis induced by macrophage-fibroblast crosstalk and for evaluating therapeutic intervention.
- Notes:
- Thesis (Sc. M.)--Brown University, 2026
Citation
Kim, Yaunghyun,
"A 3D In Vitro Ring Model with Macrophage and Fibroblast Co-culture: A Platform for Preclinical Fibrosis Studies as an Alternative to Animal Testing"
(2026).
Biology and Medicine Theses and Dissertations, Biotechnology.
Brown Digital Repository. Brown University Library.
https://repository.library.brown.edu/studio/item/bdr:5uvk6p7b/
Relations
Collections:
-
Biology and Medicine Theses and Dissertations
Theses and Dissertations for the Biology and Medicine department....