Leveraging Design of Experiments and Extracellular Matrix Coatings to Modulate Intestinal Fibroblast Activation In Vitro

Introduction: Crohn's disease progression frequently manifests with intestinal fibrosis, characterized by excessive extracellular matrix (ECM) deposition. Despite its high incidence, the precise mechanisms initiating this fibrotic process remain unclear. Fibroblasts, the primary cell type responsible for ECM production within the intestinal stroma, are implicated in fibrosis development through their activation in Crohn’s disease. However, researchers seeking to understand intestinal fibroblast activation in vitro face a significant challenge because these cells spontaneously activate when cultured on traditional tissue culture polystyrene (TCPS) flasks. Consequently, conventional cell culture methodologies fail to accurately model fibroblast activation processes relevant to Crohn's disease pathogenesis. To address this limitation, our research focuses on developing a reproducible protocol that can induce quiescence in colonic fibroblasts in in vitro culture. Using Design of Experiment (DOE) analysis, we seek to identify the specific micro-environmental conditions that drive quiescence in CCD-18 colonic fibroblasts.
Specifically, we are investigating the potential of natural ECM proteins (Collagen I, III, and laminin) as cell culture coatings, alongside vitamin D (VD) supplementation as soluble stimuli. Since VD deficiency is linked to stricture formation and fibrosis in Crohn's. Therefore, incorporating VD-supplemented media into the protocol explores its potential as a treatment, influencing the transition of activated fibroblasts to a quiescent state. Additionally, literature suggests both ECM coatings and VD as potential factors to mitigate pro-fibrotic effects in fibroblasts in other tissues. Hence, by creating a more physiologically relevant microenvironment that mimics the lamina propria, rich in these collagens, we aim to better understand fibroblast activation in Crohn's Disease.

Materials and

Methods: After the initial passage on TCPS, CCD-18co fibroblasts were cultured on different ECM coatings (Collagen I, Collagen III and Laminin) at 2, 15 and 30 µg/cm2 or in VD-containing media at 1, 5 and 10 µM using EMEM media with 2% fetal bovine serum (FBS). Additionally, we assessed media supplementation with fibroblast growth factor (FGF) at 1, 5, 10 and 20 ng/mL. The Fibroblast phenotype was evaluated on day 5 by quantifying cell proliferation, and qualitatively assessing the expression of alpha smooth muscle actin (α-SMA) protein via immunofluorescence and fibronectin deposition via ELISA. After gathering preliminary data on individual treatments, we assessed the synergistic effects of these treatments using a full factorial DOE. We had 5 continuous factors including collagen I, collagen III, laminin, VD and FGF with 3 levels for each. Using a full factorial design, all possible combinations of these factor levels were investigated. This resulted in 243 unique experimental conditions. For all conditions, cells originating from one vial of CCD-18co fibroblasts were cultured on designated combined ECM protein coating and treated with the corresponding concentrations of VD and FGF. This approach enabled us to determine the optimal protocol, considering all concentrations, for inducing the transition of colonic fibroblasts to their quiescent phenotype. We measured α-SMA expression intensity, proliferation rate, viability and fibronectin deposition using ELISA to collect data followed by using JMP software to analyze collected data for DOE intention.

Results, Conclusions, and Discussions: Through our initial investigation, we sought to characterize fibroblasts' response to each treatment. The corresponding results revealed diverse effects of the individual-tested interventions on CCD-18co fibroblasts. Laminin coating displayed a marked reduction in α-SMA expression (Fig.1C-D) by increasing the concentration compared to other proteins. Conversely, collagen type III appeared to induce a more active phenotype, as evidenced by an up-regulation of α-SMA intensity (Fig.1B). Collagen type I elicited a significant reduction at 15 μg/cm2 in α-SMA expression (Fig.1A). Interestingly, FGF and VD supplementation demonstrated a dose-dependent suppressive effect on α-SMA expression, with the 10 ng/mL and 10 μM doses of FGF and VD exhibiting the most significant decrease compared to 1 ng/mL and 1 μM, respectively (Fig.1E-H). Our findings support existing literature suggesting ECM protein coatings and VD can mitigate pro-fibrotic effects in fibroblasts from various tissues. No significant difference was observed between different coatings and the control in proliferation rate and viability. The diverse activity phenotypes observed in response to laminin, collagen type I, FGF and VD (Fig.1A-H) underscore the complexity of establishing a standardized protocol for inducing fibroblast quiescence. While these treatments offer promising avenues, the lack of a uniform response suggests the need for a more nuanced approach. To overcome this challenge, we are performing a full-factorial DOE analysis as previously described. We have already measured α-SMA intensity, proliferation rate, viability and fibronectin deposition for all 243 conditions. Upcoming analysis of these data will lead to the identification of the treatment conditions that minimize α-SMA expression and ECM production while maximizing cell viability. This approach allows us to systematically evaluate the combined effects of ECM proteins, VD, and FGF on fibroblast quiescence.
In conclusion, these findings highlight the complexity of inducing fibroblast quiescence. To address this, we are developing a robust protocol using a full-factorial DOE to optimize the combined effects of ECM proteins, VD, and FGF. This systematic approach holds promise for not only elucidating the mechanisms of fibroblast quiescence but also establishing a standardized protocol that will empower further research into the triggers of intestinal fibrosis in Crohn's disease.

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