An Improved Method of Maintaining Primary Murine Cardiac Fibroblasts in Two-Dimensional Cell Culture

Figures

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  Figure 1. Myofibroblast marker expression in rat primary cardiac fibroblasts after >72 hours in culture. (a) Rat cardiac fibroblasts were plated on elastic plates with characteristic elastic moduli of 5 kPa, 10 kPa, and 100 kPa, with the indicated culture medium. Cells plated on conventional polystyrene tissue culture plates (rigid substrate – ranging from 30 to 100 MPa) were used as a comparative control. Protein from unpassaged primary cardiac fibroblasts was harvested ~3 days after plating. Vimentin expression was used as a pan-phenotypic control, and protein expression was normalized to total protein loading. (b) Graphical representation of data in (A). Data shown as the mean ± SD and is representative of n = 3–6 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001 when compared to cells cultured in F10 medium with 2% FBS on the same substrate.

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  Figure 2. αSMA is excluded from F-actin stress fibers when cultured on 5 kPa culture surfaces. P0 rat cardiac fibroblasts were seeded at low confluency (<10%) on either glass (rigid substrate) or elastic coverslips and probed for αSMA (green) and F-actin (red) 3 days after plating. Images are representative of n = 3 independent biological replicates. Scale bar = 50 μm.

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  Figure 3. Fibroblast and myofibroblast gene expression in primary cardiac fibroblasts after >72 hours in culture. RNA was harvested from P0 rat cardiac fibroblasts 3 days after plating and used for qPCR. Samples from cells cultured on conventional plastic tissue (rigid substrate) culture surfaces were used as comparative controls. All reactions were performed in technical triplicates and were normalized to HPRT. Data shown as the mean ± SD and is representative of n = 3 biological replicates. *P < 0.05, **P < 0.01, when compared to cells cultured on plastic in the same culture medium.

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  Figure 4. Nutrient restriction and decreased biomechanical input inhibits proliferation in primary cardiac fibroblasts. (a–c) P0 rat cardiac fibroblasts were plated on elastic plates with various elastic moduli, with the indicated culture medium, and stained with proliferation staining agent 24 hours after seeding. Images were captured immediately after staining and every 24 hours subsequently. Cells plated on conventional plastic tissue culture plates (rigid substrate) were used as a comparative control. Images are representative of n = 3 biological replicates. (d) Histrographical representation of cell counts 18 hours after plating and attachment (e) Histrographical representation of cell counts obtained after 3 days in culture (t > 72 hours). Data displayed as mean ± SD and is representative of n = 6 biological replicates. **P < 0.01, ***P < 0.005, ****P < 0.001 when compared to cells cultured on plastic in the same medium.

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  Figure 5. Assessment of cell viability in restricted nutrient conditions after >72 hours in culture. P0 rat cardiac fibroblasts were seeded onto substrata of various elastic moduli and cultured in F10 medium supplemented with 2% FBS for 4 days. Cell viability was assessed with Calcein-AM (green) staining, and cell death by ethidium homodimer (red). Each biological replicate was assessed by capturing 3 random fields at 10X magnification. Images are representative of n = 3 biological replicates, with 9 technical replicates each.

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  Figure 6. A schematic depicting the various factors affecting the fibroblast phenotype in vitro. Herein we summarize our main findings along with other known elements which influence primary cardiac fibroblast activation in vitro. We suggest that α-SMA is an ever-present marker on the cardiac fibroblast-myofibroblast spectrum, and that its incorporation in myofibroblast stress fibers is a key component of defining the activated phenotype. Previous studies on conventional plastic tissue culture plates have confirmed that passaging45, hyperglycemic-like conditions67, and treatment with ROS and hypertrophic agents indeed activate the myofibroblast phenotype and we conclude that excess serum also contributes to this activation.