Rat collagen type 1

Immunofluorescence was performed for native and transfected cell lines to further confirm CLDN expression. The cells were cultivated on rat collagen type I (Trevigen, Gaithersburg, MD, USA) coated glass coverslips. Thereafter, cells were washed with PBS, fixed wit (1:1) Acetone/Methanol for 5 min at −20 °C and blocked for 30 min with 1% BSA (bovine serum albumin, Sigma-Aldrich, Taufkirchen, Germany) in PBS at 37 °C. CLDNs were stained with primary antibodies (Table 4) diluted in PBS containing 1% BSA, overnight at 4 °C. Cells were washed with PBS. iFlour™ 488 antimouse (AAT Bioquest, Sunnyvale, CA, USA) and iFlour™ 555 antirabbit (AAT Bioquest) were diluted 1:500 in PBS containing 1% BSA and added to the respective cells as secondary antibodies for 1 h at 37 °C. For nuclei staining, DAPI (2 µM) (Sigma-Aldrich) was used. Cells were stored in PBS at 4 °C for further analysis. As a control for unspecific binding sites, cells were also incubated with only the secondary antibodies. Fluorescent images of cells were taken with a Nikon Eclipse TE2000-E confocal laser scanning microscope (400 nm for DAPI, 555 nm for CLDN-3 and -7 proteins, and 488 nm for CLDN-4), with a 60× water immersion objective and software EZ-C1 3.80 (Nikon, Düsseldorf, Germany).

For single-cell cardiomyocyte-based BCTs (SC-BCTs), the CBs were dissociated after selection, and 1 million CMs were used for tissue preparation as described [22 (link)]. CMs were mixed with the respective number of other cell types (Figure 3B and Figure 4A) in 100 μL of BCT medium [22 (link)] per tissue. An extracellular matrix mixture (150 μL/BCT) composed of 0.9 mg/mL rat collagen type I (Trevigen, Gaithersburg, MD, USA), 10% Matrigel, and 2.5% 0.4 M NaOH was added. The cell-matrix mixture was poured into a custom-made silicon mold containing two titanium rods (distance 6 mm; initial slack length) and solidified at 37 °C for 30 min. Then, the construct was covered by 5 mL of BCT medium with 60 μM of L-ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA) and cultured under standard cell culture conditions with medium change every 1 or 2 days. When BCTs were stimulated with TGFβ1 (5 ng/mL; Peprotech, Rocky Hill, NJ, USA) between D7 and D14, medium was refreshed every day. For all tissues, a growing static stretch (G-stretch) was applied through stepwise elongation by 400 µm on days 7, 11, 15, and 19.
Primer sequences, primary-, and secondary antibodies used to characterize BCTs are listed in Tables S4–S6, respectively.

Human t-PA (rt-PA; Actilyse^®) was purchased from Boehringer Ingelheim GmbH (Rhein, Germany) and dialysed against 0.4M HEPES pH 7.4 to remove the original vehicle components [5 (link)]. Human Glu-plasminogen was from Enzyme research laboratories (South Bend, IN, USA). Fluorescein isothiocyanate (FITC)-conjugated BSA, bovine aprotinin and endothelial cells growth supplement (ECGS) were obtained from Sigma Aldrich (St Louis, MO, USA). HA1077 (fasudil; hydrochloride) was purchased from Cayman Chemicals (Ann Arbor, MI, USA) while KD025 (SLx-2119) from MedChem Express (Princeton, NJ, USA). Matrigel was obtained from BD Biosciences (Australia) and rat collagen type-I from Trevigen (Gaithersburg, MD, USA). All TaqMan^® gene expression assays and reagents were purchased from Applied Biosystems (Thermo Fisher Scientific, Australia).