Co-culture of Human Tenon Fibroblast and macrophages as a novel in-vitro model for conjunctival scarring

Abstract

 

Purpose

 

Glaucoma filtration surgery is one of the most effective treatments for lowering intraocular pressure. However, conjunctival contraction and scarring can lead to failure of this surgery. It is established that persistent inflammation after surgery can cause aggressive scarring. The fibroblast populated collagen gel is a well-established assay to study contraction in vitro, but it lacks the complex interaction between inflammatory cells and fibroblasts, which mediate wound-healing process in vivo. To provide a more realistic model of conjunctival scarring and develop more effective treatments, we developed a novel in-vitro model with co-culture of fibroblasts and inflammatory cells.

 

Methods

 

The U937 monocyte-like human cell line was differentiated into macrophage-like cells using phorbol 12-myristate 13- acetate (PMA). An antibody against CD 68 was used to confirm macrophage differentiation. Macrophages were co-cultured with human Tenon’s capsule fibroblasts (HTFs) in three-dimensional collagen gels and contraction was measured over 7 days using digital photography.

 

Results

 

PMA promoted the differentiation of monocytes into CD68-positive macrophages (Figure 1). While neither U937 cells, nor differentiated macrophages alone contracted collagen gels, co-culture of macrophages with HTFs at a 2:1 ratio led to a 50% increase in the contraction potential of HTFs for the first 5 days compared to HTFs alone (Figure 2).

 

Conclusions

 

Inflammatory cells significantly increased the contractibility of HTF as measured in collagen contraction assay. We propose that this new model, recapitulating both aspects of scarring - inflammation and contraction may prove useful in increasing our understanding of the complex wound environment for the development of combination therapies against scarring.

 

 

CD 68 staining of U937 cells following PMA - induced differentiation to macrophages.

 

 

Co-culture of HTF with macrophage increases contraction. Collagen gel contraction for HTF co-cultured with or without macrophage (1:2).

 

Keywords: 765 wound healing • 557 inflammation • 474 conjunctiva