304 Chapter 12 cells for 30 minutes at room temperature. The total recorded events were 1000-25,000. Acquired data were analyzed with FlowJo Version 7.6.2 (Treestar Software, Ashland, OR, USA). RNA Extraction and qPCR. Day 21 cardiac organoids were subjected to RNA extraction by the chloroform method as previously described.33 cDNA synthesis was performed using 500 ng RNA per sample in the qScript cDNA Synthesis Kit (Quantabio). The cDNA produced was then diluted 20 times with nuclease-free water and stored at -20°C for subsequent analysis. qPCR was carried out on an Applied Biosystems CFX384 Fast Real-Time PCR system using the SYBR Green method using 5 μL cDNA per duplicate. Data from the qPCR were normalized to RPL32, which was chosen due to its consistency across the sample groups. Integrated DNA Technologies Primers were designed and are displayed in Supplementary Table 1. Engineered Heart Tissue (EHT) generation. Engineered Heart Tissues were generated with small modifications, as previously described.34 Briefly, hiPSC-CMs were detached and resuspended in RPMI supplemented with 2% B27, 10% KOSR and 10 μM Y-27632. EHTs of 1x106 cardiomyocytes were casted by mixing the reconstitution mix consisting of 79 μl cell suspension, 15.5 µL 2x DMEM (Gibco), and 2,5 µL (0.5 mg) of fibrinogen (Sigma) with 3 μl (100 U/ml) thrombin (Sigma) and pipetting it into the casting molds. Fibrin polymerization (37°C, 7% CO2, 98% RH, 2 h) led to the formation of a muscle strip. The EHTs were transferred to a culture medium (Dulbecco’s modified Eagle’s medium) containing 10% horse serum, 1% penicillin/streptomycin, 10 μg/ml insulin, and 33 μg/ml aprotinin and maintained at 21% oxygen, 7% CO2 and 37°C in a humidified cell culture incubator for 17 days. Contractility measurements were performed in a culture medium, as described before.34 Statistical Analysis. Statistical analysis was carried out on GraphPad Prism 9. Data were subjected to one-way-, two-way- ANOVA (Tukey), and/or unpaired t-tests, as indicated in the different figure legends. Significance was determined as a p-value of < 0.05.
RkJQdWJsaXNoZXIy MTk4NDMw