Development of a Protocol to Induce Quiescence in In Vitro Culture of CCD-18 Colonic Fibroblasts
Background:
Intestinal fibrosis is a common complication that arises during progressive Crohn’s disease (CD). Despite its prevalence, the precise mechanisms responsible for initiating intestinal fibrosis in CD remain elusive. One of the most common cell populations residing in intestinal wall are fibroblasts which can become activated in CD and contribute to the development of intestinal fibrosis. However, studying these cells in their quiescent, resting state in vitro presents a challenge, as they tend to become activated when cultured on tissue culture-treated polystyrene (TCPS) culture flasks. Due to this phenomenon, standard culture methods are insufficient to study the process of fibroblast activation. To address this challenge, our focus is on developing a protocol to deactivate colonic fibroblasts in vitro.
Methods:
To investigate the process of transitioning activated CCD-18co fibroblasts into a resting state, we assessed the impact of Vitamin D (VD) and various extracellular matrix (ECM) coatings, including Collagen I, Collagen III, and Laminin, on these cells. According to the literature, both ECM coatings and VD may have the capacity to mitigate pro-fibrotic effects in fibroblasts in other tissues. In summary, after the initial passage on tissue culture-treated polystyrene (TCPS), the fibroblasts were cultured on different ECM coatings or in media containing VD. Additionally, we also assessed supplementation of the media with fibroblast growth factor (FGF). The Fibroblast phenotype was evaluated on day 5 by quantifying cell proliferation and apoptosis, and qualitatively assessing the expression of smooth muscle actin (α-SMA) protein via immunofluorescence.
Results:
As a result of treatment with 10 μM of VD in conjunction with FGF, our cells exhibited a more pronounced fibrotic phenotype, as opposed to becoming quiescent. Conversely, under the influence of 10 μM VD alone, cells exhibited reduced production of α-SMA a significant contrast compared to the other experimental groups. Furthermore, the type of coating used on our culture plates had varying effects on cell activation. Coatings of Collagen I and Collagen III appeared to drive the cells towards a more activated phenotype, characterized by increased cell projection and higher α-SMA intensity. In contrast, Laminin-coated plates induced a less active phenotype, with reduced cell projection and lower α-SMA intensity. There was no significant difference between different coatings and the control proliferation rate and apoptosis.
Conclusions:
These findings underscore the critical need for the establishment of a protocol aimed at achieving quiescent, physiologically relevant CCD-18co cells. Our data strongly suggests that various treatment options can elicit distinct activity phenotypes in these cells. The discovery of a protocol capable of transitioning myofibroblasts into normal intestinal fibroblasts holds the promise of unlocking new avenues for prospective research studies, providing deeper insights into the factors that must be monitored for colonic fibroblasts to attain a quiescent or activated state. Once we have access to quiescent fibroblasts, we can advance to the next stage in unraveling the underlying mechanisms and causes of intestinal fibrosis.
