Abstract
Primary cardiac fibroblasts are notoriously difficult to maintain for extended periods of time in cell culture, due to the plasticity of their phenotype and sensitivity to mechanical input. In order to study cardiac fibroblast activation in vitro, we have developed cell culture conditions which promote the quiescent fibroblast phenotype in primary cells. Using elastic silicone substrata, both rat and mouse primary cardiac fibroblasts could be maintained in a quiescent state for more than 3 days after isolation and these cells showed low expression of myofibroblast markers, including fibronectin extracellular domain A, non-muscle myosin IIB, platelet-derived growth factor receptor-alpha and alpha-smooth muscle actin. Gene expression was also more fibroblast-like vs. that of myofibroblasts, as Tcf21 was significantly upregulated, while Fn1-EDA, Col1A1 and Col1A2 were markedly downregulated. Cell culture conditions (eg. serum, nutrient concentration) are critical for the control of temporal fibroblast proliferation. We propose that eliminating mechanical stimulus and limiting the nutrient content of cell culture media can extend the quiescent nature of primary cardiac fibroblasts for physiological analyses in vitro.
Highlights
Www.nature.com/scientificreports likened in vitro to seeding cells on stiff plastic surfaces
In order to determine the physiological effects of two-dimensional cell culture on primary cardiac fibroblasts, we examined the influence of cell culture medium and serum, and whether the compressibility of the culture surface was a greater factor in the spontaneous phenotype of the cells in vitro
After three days in culture, variable expression of myofibroblast markers ED-A fibronectin, non-muscle myosin heavy chain (SMemb or myosin IIB), and αSMA was observed in conditions of either low nutrient media (F10 with 2% fetal bovine serum (FBS)) or high nutrient media (DMEM/F12 with 10% FBS), plated on substrates that mimic the compressibility of healthy myocardium (5 kPa), or fibrosis-stiff substrate (100 kPa) (Fig. 1)
Summary
Www.nature.com/scientificreports likened in vitro to seeding cells on stiff plastic surfaces. Mechanical loading and stretching modulates fibroblast function and phenotype, promoting the deposition of ED-A fibronectin[25] and enhancing TGF-β signaling[43] While it has traditionally been viewed as a permanent event, the activation of myofibroblasts has recently been observed as a reversible process in resident fibroblasts in vivo[5,44]. While it is common practice to passage primary cells to promote homogeneity in the culture population, passaging further drives myofibroblast activation and prevents physiologicallypertinent studies[45]. This phenotypic plasticity presents a unique problem in that fibroblast physiology that is representative of healthy myocardium cannot be readily observed and manipulated in vitro. These data support an alternative means by which to investigate the molecular and cellular physiology of primary cardiac fibroblasts in vivo studies
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