Culturing primary murine cardiac fibroblasts on elastic silicone substrata (5 kPa) with low nutrient media significantly reduced the spontaneous activation of myofibroblast markers and limited proliferation compared to conventional rigid plastic culture plates.
Eliminating mechanical stimulus via elastic substrata and limiting nutrient content extends the quiescent nature of primary cardiac fibroblasts in vitro, enabling more physiological studies of cardiac fibrosis.
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.
Landry et al. (Mon,) conducted a other in Healthy primary murine cardiac fibroblasts. Elastic silicone substrata (5 kPa) and low nutrient media (F10 with 2% FBS) vs. Conventional polystyrene tissue culture plates (rigid substrate) and high nutrient media was evaluated on Expression of myofibroblast markers (ED-A fibronectin, SMemb, alpha-SMA, PDGFR-alpha) and cell proliferation. Culturing primary murine cardiac fibroblasts on elastic silicone substrata (5 kPa) with low nutrient media significantly reduced the spontaneous activation of myofibroblast markers and limited proliferation compared to conventional rigid plastic culture plates.