Fibroblast Gap-closure Assay-Microscopy-based in vitro Assay Measuring the Migration of Murine Fibroblasts

Background

Few treatments for fibrosing diseases exist because of an incompletely understood and complex etiology (Rockey et al., 2015). Current efforts to develop therapies for organ fibrosis have focused on pathologic fibroblasts, also known as myofibroblasts (Blackwell et al., 2014). In addition to secretion of matrix proteins such as collagen, which comprise scar, a hallmark of pathologic fibroblasts is their increased proliferative and migratory capacity (Kendall and Feghali-Bostwick, 2014). A number of studies have shown that innate immune cells interact with and mediate fibroblast activation (Desai et al., 2018). Thus, we investigated the effects of lung macrophages on fibroblasts in a recent study, where we found a novel population of macrophages that localizes to fibrotic scar in murine lung (Aran et al., 2019). These macrophages highly expressed PDGF-AA, a secreted factor known for promoting fibroblast migration and proliferation. Thus, we treated mouse 3T3 embryonic fibroblasts with conditioned media from cultured mouse lung macrophages, with and without PDGF-AA blocking antibody, and measured fibroblast gap closure.

Here we describe the protocol for the assay, which should be useful for other investigators studying paracrine signaling between adjacent cellular lineages. Since the method presented employs an established, adherent cell line, it may be easily adapted to other cell types and treatments in fields where cell migration is an important pathological characteristic, such as cancer or wound healing. Importantly, fibroblasts and the extracellular matrix have been recently recognized as fundamental players in the tumor microenvironment and as such are of the outmost interest in oncology research (Bu et al., 2019).

Compared with other methods for investigating chemotaxis (Justus et al., 2014), such as the scratch assay, our approach does not introduce mechanical stress to the cells, which can potentially activate fibroblasts and obscure results. It also does not require optimization of cell culture as required for the transwell/chamber invasion assay, and data from the same well may be collected at multiple timepoints while cells are visualized in real time. Our assay can be easily scaled up to a high throughput format for drug screening purposes. One limitation of the assay is that it does not address directional cell migration. Also, our protocol measures both migration and proliferation of fibroblast cells; a proliferation assay should be conducted if further distinction is needed.