Investigating Fibroblast-Induced Collagen Gel Contraction Using a Dynamic Microscale Platform

Abstract

Mechanical forces have long been recognized as fundamental drivers in biological processes, such as embryogenesis, tissue formation and disease regulation. The collagen gel contraction (CGC) assay has served as a classic tool in the field of mechanobiology to study cell-induced contraction of extracellular matrix (ECM), which plays an important role in inflammation and wound healing. In a conventional CGC assay, cell-laden collagen is loaded into a cell culture vessel (typically a well plate) and forms a disk-shaped gel adhering to the bottom of the vessel. The decrement in diameter or surface area of the gel is used as a parameter to quantify the degree of cell contractility. In this study, we developed a microscale CGC assay with an engineered well plate insert that uses surface tension forces to load and manipulate small volumes (14 µL) of cell-laden collagen. The system is easily operated with two pipetting steps and the microscale device moves dynamically as a result of cellular forces. We used a straightforward onedimensional measurement as the gel contraction readout. We adapted a conventional lung fibroblast CGC assay to demonstrate the functionality of the device, observing significantly more gel contraction when human lung fibroblasts were cultured in serum-containing media versus serum-free media (p≤0.01). We further cocultured eosinophils and fibroblasts in the system, two important cellular components that lead to fibrosis in asthma, and observed that soluble factors from eosinophils significantly increase fibroblast-mediated gel contraction (p≤0.01). Our microscale CGC device provides a new method for studying downstream ECM effects of intercellular cross talk using 7-35 fold less cell-laden gel than traditional CGC assays.

An in vitro 3D annulus fibrosus cell culture model with type I collagen: An examination of cell–matrix interactions

“…Bowles et al 39 and Takai et al 54 experiments demonstrate versatility of this model. While cells, 56,57 including IVD cells, 38,39 have been previously cultured in collagen in various forms, our model explains a relatively easy and versatile matrix that can be used for not only mechanical testing but also the interplay between inflammation and mechanics for AF cells, which then provides insight into the functional impact of structural changes. Another hypothesis could be that adding LPS to cells+collagen constructs has caused an increase in matrix metalloproteinases (MMPs) secretion which in turn has affected matrix molecules.…”

Targeted apoptosis of myofibroblasts by elesclomol inhibits hypertrophic scar formation

“…Treatment of human skin fibroblasts with TGF-b1 resulted in the cells gaining a myofibroblast-like phenotype, as indicated by the significant upregulation of the myofibroblast marker a-SMA and collagen gel contraction [31]. The collagen gel contraction assay has become a classic method for studying cell-induced extracellular matrix contraction in the field of mechanical biology; the extracellular matrix is crucial for wound healing [32]. In the collagen gel contraction test, 10 mM elesclomol significantly inhibited myofibroblast differentiation as indicated by a marked decrease in gel contraction at 24 h after drug administration.…”

Eozinofilų poveikis tarpląstelinio užpildo baltymų genų raiškai, bronchų lygiųjų raumenų ląstelių ir plaučių fibroblastų kontraktiliškumui ir migracijai sergant astma

“…Mūsų tyrimo duomenimis, astma sergančių asmenų eozinofilai skatina tarpląstelinio užpildo baltymų, tokių kaip kolagenas I, fibronektinas, versikanas, elastinas, tenaskinas C, dekorinas, periostinas ir vimentinas, genų raišką BLRL ir PF. Kitų autorių duomenys neprieštarauja mūsų tyrime gautiems duomenims: žmogaus astmos tyrimų metu (in vivo, ex vivo), gyvūnų modeliuose ir ląstelių kultūrų tyrimuose nustatytas kolageno I [39,40], fibronektino [40][41][42], versikano [43,44], elastino [45], tenaskino C, periostino [46][47][48][49][50][51][52][53][54] ir vimentino [55] genų ir (arba) baltymų raiškos padidėjimas BLRL ir PF. Taip pat, mūsų tyrimo metu nustatytas tarpląstelinio užpildo baltymus degraduojamųjų fermentų -užpildo metaloproteinazių -MMP-2, MMP-9, ADAM33 genų raiškos padidėjimas BLRL ir PF po inkubacijos astma sergančių asmenų eozinofilais.…”