**3. Results**

### *3.1. Attachment of the hiPSC-CMs to the PET Textiles*

Five PET textiles (Figure 1) were tested as a scaffold for the hiPS-CMs. None of the fiber-related parameters (thickness range of the fibers and straight vs. textured quality of the fibers) or the weave pattern changed the behavior of the cells, but all the studied PETs (1–5) supported the growth of the hiPS-CMs in a similar manner (data not shown). PET 5, with a plain weave derivative pattern, was chosen for the following experiments. A combination of plain weave derivative pattern and the reed density used produced the most variating topography for the studied PET textile samples (Figure 1). PET 5 was also blue and had slight autofluorescence, which made the fibers visible with fluorescent imaging.

Gelatin has been used as a basic coating material for hiPS-CMs culturing in our laboratory; therefore, it was used also in the above-mentioned PET textile screening study. However, the number of the attached hiPS-CMs remained relatively low. To improve the cell attachment on the PET textile, coating with commercial basement membrane matrix GeltrexTM was also tested. In addition, plasma treatment prior to gelatin coating and dopamine-bound gelatin were tested. There were no clear differences in the cell attachment (Figure S1) or structural maturation state of the hiPS-CMs (Table S1) with the coating material or plasma treatment. Thus, after testing multiple PET textile types and coatings, PET 5 with normal gelatin coating was chosen for further experiments.

### *3.2. hiPSC-CM Morphology, Sarcomere Orientation, and Sarcomere Length*

hiPSC-CMs cultured on PET 5 and glass coverslips were immunolabeled with Troponin T and Myocin binding protein C3 (MyBPC3) antibodies. Qualitative analysis revealed that the cells aligned according to the PET 5 textile fibers and exhibited clearly elongated structures and increased sarcomere orientation, as shown in Figure 2. The orientation of the CM sarcomeres was significantly higher on PET 5 (*n* = 98) compared to controls (*n* = 174), which was indicated by the lower circular variance (0.611 ± 0.162 and 0.882 ± 0.069, respectively; *p* < 0.05). Table 3 shows examples of the distribution of the sarcomeres in hiPSC-CM cultured on PET 5 and coverslip. Sarcomeres in CMs grown on PET 5 were more oriented than those in the controls. The difference in sarcomere length (Table 3) between PET 5 and control samples was not significant and was 1.736 ± 0.187 μm and 1.749 ± 0.122 μm on average, respectively. The shape of CMs was determined using the aspect ratio, and CMs grown on PET 5 had significantly higher aspect ratios than controls (4.915 ± 2.263 and 1.567 ± 0.455, respectively; *p* < 0.05), indicating that the cells exhibited a more rod-like structure essential for efficient contraction (Figure 2). However, confocal imaging revealed that the hiPS-CMs are still flat and wrap around single PET fibers (Figure 3).

**Figure 2.** Two representative examples of the structure of human-induced pluripotent stem cell- derived cardiomyocytes (hiPSC-CMs) cultured on gelatin-coated polyethylene terephthalate (PET)-based textiles (PET) textiles and coverslips (controls). The hiPS-CMs were immunostained with myosin binding protein C (MyBPC3) (green) and Troponin T (red). The nuclei of the cells were stained with DAPI (blue). Scale bar is 25 μm. On PET 5, the cells and their sarcomeres clearly aligned according to the fibers of the textile, whereas the control cells exhibited no longitudinal axis or sarcomere orientation to one direction. Orientations of the sarcomeres were analyzed with CytoSpectre. The analysis results of sarcomere orientation and length of sarcomeres confirmed that the orientation of the sarcomeres improved when the cells were cultured on PET 5 textiles, but the sarcomere length distribution in the cells did not differ significantly.

**Table 3.** The data of the CytoSpectre analysis of cells grown on PET 5 and glass coverslips (control). The orientation of the CM sarcomeres was significantly higher on PET 5 compared to control (*p* < 0.05) as indicated by the average circular variance. The difference in sarcomere length between PET 5 and control samples was not significant. However, the shape of CMs was determined using the aspect ratio and CMs grown on PET 5 had a significantly higher ratio than the control (*p* < 0.05).


**Figure 3.** (**<sup>a</sup>**–**<sup>c</sup>**) Confocal images from single human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) on the polyethylene terephthalate PET type 5 textiles, with Troponin T (red), myosin binding protein C, MYBPC (green), nuclear stain DAPI (blue) from different projections. Images reveal that the hiPS-CMs were aligned with the PET fibers. However, the cells wrapped around the single PET 5 fibers and exhibited a relatively flat structure. The sarcomeres of the cells were clearly oriented along the fibers. Scale bar is 25 μm.
