Nc 200

To evaluate the effect of extended periods of AdMSC culture on TIPS microcarriers, 0.5 × 10⁶ AdMSC (passages 4 and 5) were seeded onto ≈17 500 microcarriers as described above for each of the three different PLGA TIPS microcarrier compositions. After 18 h, the cellularized TIPS microcarriers were transferred to 24‐well low bind plates and cultured for a further 11 days under static conditions. The confined conditions of the 24‐well plate resulted in the microcarriers forming a multi‐layered 3D structure. The quantity of cells attached the microcarriers at each time point was measured using an automated counter (NC200; Chemometec, Denmark). As a control condition, AdMSC were seeded at 4.5 × 10⁴ cells per well in 2 mL medium on standard PS tissue culture plastic (“TC”) and cultured for further 11 days under static conditions. The use of cell densities greater than this led to rapid confluency and monolayer detachment before the completion of the study. At predetermined timepoints (days 1, 3, 5, 7, 9, and 11) the supernatant from each well was collected for further analysis and replaced by fresh proliferation medium. The quantity of cells was measured in one sample at each time point by DNA quantification using the DNAeasy Blood and Tissue kit (QIAGEN). DNA concentration in the samples was measured at 260 nm wavelength using a Nanodrop 2000c spectrophotometer (Thermo Scientific).

For scRNA-seq, the left heart ventricle was dissected under a stereomicroscope at E16.5, P1, and P5 (n = 3 litters, each counting 4–8 pups), and enzymatically dissociated using the semiautomatic GentleMACS tissue dissociator system (MACS Miltenyi Biotec; Neonatal Heart Dissociation Kit 130–098-373) according to the manufacturer’s recommendations. Following viable cell counting (NC-200, ChemoMetec), dissociated cells were stained with a fixable viability stain (Fixable viability stain 570; BD Biosciences, 564,995) prior to fixing in methanol for 15 min followed by rehydration to reverse the RNA to its original state. During rehydration, the RNase inhibitor, RNasin Plus (Promega; N2615), was added to prevent RNA degradation and included in all subsequent steps. After rehydration, samples were stored at -80 °C until analysis. All reagents were high grade, RNase free and the environment was kept strictly RNase free to avoid degradation of the RNA. For comparison of fresh and fixed scRNA-seq profiles, mouse myoblasts (C2C12; ATCC, CRL-1772) were used and maintained as recommended.