Culturing of Cardiac Fibroblasts in Engineered Heart Matrix Reduces Myofibroblast Differentiation but Maintains Their Response to Cyclic Stretch and Transforming Growth Factor β1

Figure 1. Silicone mold design. Custom made 3D-printed casts to provide the shape for the silicone mold (A); The silicone mold had a diameter of 22 mm (B); and a centrally placed post 2 mm (C). The initial gel volume was 250 μL and after gelation 500 μL of the medium was added on top.

Figure 2. Images of EHM fibers in Flexcell Tissue Train culture plates. Image (A) shows the (pink) gel after pipetting in the 6 well Flexcell Tissue Train culture plate, still in the Trough Loader (white bottom) providing the mold for the gel; Image (B) shows the EHM fiber between the two anchors after polymerization of the gel. The black and white grid underneath represents 3 cm by 3 cm. The diameter of the well was 3.5 cm.

Figure 3. Images of EHM rings taken 24 h after casting the gel using different cell densities (A–D): 100; 200; 400 and 2000 CF cells/μL using a 1 mg/mL collagen concentration. The images show the top view of the EHM-rings within the clear silicone mold on a black background with white grid (1 cm by 1 cm). Surrounding the central pole is a light pink circle, most left vaguely showing, becoming clearer with increasing the cell density; The final image (E) shows the EHM-ring (8000 cells/µL, 1 mg/mL collagen concentration) after 7 days of culturing, next to the central pole. The EHM ring has been removed from the central pole manually to enhance visibility. Bars represent 2 mm.

Figure 4. Histological and immunohistological analysis of EHM-ring sections. HE staining of an EHM ring section showing even distribution of the cells throughout the EHM ring structure. EHM ring was formed using 1.5 mg/mL collagen concentration and 1000 CF cells/μL gel and was fixed and stained 24 h after casting. Darker dots implicate CF. Magnification 5× (A); or 20× (B). Bars represent 200 µm. Vimentin (green) and DAPI (blue) staining (C); and Z-stack image of CNA35 (red) and Vimentin (green) staining nuclei are blue (DAPI) (D) (bar represents 29.4 μm).

Figure 5. Stiffness measurements of the EHM ring (1.5 mg/mL collagen concentration; 1.000 cells/μL gel) at two different timepoints (24 h and 48 h). Images of tensile analysis at 0% elongation (A) and at elongation at break; (B). Bar represents 10 mm. Tensile stiffness displayed as Young’s Modulus (n = 2); (C); Stress–strain (σ/ε) curve of the moving average (20 points/strain value) (n = 1) after 24 h and 48 h (D).

Figure 6. Baseline gene expression levels of Nppb, Tgfβ1, Ctgf and Acta2 in CF cultured on the different substrates: 2D monolayer on hard plastic cell culture plates (Hard), 2D monolayer on Bioflex cell culture plates (Flex) and 3D EHM fibers (EHM). Data are presented as relative mRNA levels normalized to house-keeping gene cyclophylin (n = 15–40). *** p < 0.001; **** p < 0.0001. Bar indicates mean.

Figure 7. The effect of 4 h (A); and 24 h (B) cyclic stretch (10%, 1 Hz) on gene expression in CF cultured on 2D monolayer Bioflex plates (Flex) (data from [32]) or 3D EHM fibers (EHM) conditions. Presented are the relative gene expression levels of Nppb, Tgfβ1, Ctgf and Acta2 in 2D (n = 5–12) and 3D (n = 6–9) in stretch (S) and non-stretch (NS) conditions. * p < 0.05; ** p < 0.01; *** p < 0.001. Bar indicates mean.

Figure 8. The effect of TGFβ1 stimulation of rat CF for 24 h cultured on different substrates. Presented are the relative gene expression levels of Nppb, Tgfβ1, Ctgf and Acta2 after stimulation with TGFβ1 (TGFβ1) or control (C) conditions for 24 h in 2D hard plastic cell culture plates (Hard, n = 11), 2D Bioflex plates (Flex, n = 5) and 3D EHM (EHM, n = 6) (A); Graphs of the relative gene expression levels of Ctgf and Acta2 in EHM were enhanced to improve readability (B). * p < 0.05; ** p < 0.01; *** p < 0.001. Bar indicates mean.