*2.6. Immunocytochemistry*

Immunocytochemistry was performed on day 10–11 after plating the cells on the PET 5 textile samples. The samples were fixed with 4% paraformaldehyde, blocked with 10% normal donkey serum (Biowest, Nuaille, France) solution, and stained with goa<sup>t</sup> anti-cardiac troponin T (1:1,000, Abcam) and mouse anti-MyBPC3 (1:400, Santa Cruz Biotechnology, Dallas, Texas, USA) at 4 ◦C overnight. Donkey anti-goat Alexa Fluor 568 and donkey anti-mouse Alexa Fluor 488 (1:800, Thermo Fisher Scientific, Waltham, Massachusetts, USA) were used as secondary antibodies. The cell nuclei were stained using Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, California, USA). Fluorescence was visualized with a Nikon A1R+ Laser Scanning Confocal Microscope (Nikon, Tokyo, Japan) using a Nikon Apo 60× 1.40NA oil objective and with Zeiss Axio Imager.M2 with ApoTome.2 and AxioCamHRm3 camera using a Zeiss EC Plan-Neofluar 40× 1.30NA oil objective.

### *2.7. Analysis of Cell Alignment and Sarcomere Orientation*

The orientation and sarcomere length of the hiPSC-CMs cultured on the PET textiles were analyzed from microscopy images using a spectral analysis tool, CytoSpectre [34]. CytoSpectre allows quantification of orientation and size distribution of cellular structures by using Fourier transform. In this study, the circular variance and wavelength of the detailed spectral component were used to determine the sarcomere orientation and modal sarcomere length of the hiPSC-CMs, respectively. Circular variance ranges from zero to one, with zero describing perfect anisotropy and one describing perfect isotropy. CytoSpectre determines the axes of the cell, which can be used to determine the aspect ratio. Prior to the analysis, the images were processed with ImageJ for masking.

### *2.8. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)*

hiPSC-CMs were prepared for qRT-PCR on day 1 and day 11 after plating the cells as previously described [35] to study the expression of several cardiac related genes. PET 5 and control samples (cells from glass coverslips) were collected from six independent experiments (*n* = 6). Two replicate samples from each independent experiment were collected. The cells were lysed with lysis solution of a CellsDirect One-Step qRT-PCR Kit (Invitrogen, Carlsbad, California, USA) following the manufacturer's protocol. The lysis was stored at −80 ◦C until genomic DNA degradation with DNase I and reverse transcription-specific target amplification (RT-STA) using the CellsDirect One-Step qRT-PCR Kit. Biomark HD (Fluidigm Corporation, San Francisco, California, USA) was used to perform the real-time qPCR according to the manufacturer's protocol. All samples were run as duplicates in Fluidigm Dynamic array-plates and the 2-ΔΔCT [36] method was used to calculate relative expression. TATA-box binding protein (*TBP*), eukaryotic translation elongation factor 1 alpha 1 (*EEF1A1*), and glyceraldehyde-3-phosphate dehydrogenase (*GAPDH*) were used as endogenous control genes for data normalization. In assessment of the relative expression, day one samples were used as a calibrator for the data. These samples were similar to the controls samples, the cells were plated to glass coverslips, but cells were lysed one day after plating. Cells were collected from four coverslips (*n* = 4). The TaqMan assays used are listed in Table 2.


**Table 2.** TaqMan assays used in the Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR).
