Zellshield

Dermal fibroblasts from human foreskin were harvested as previously described after informed consent^{32 (link)}. Cells were expanded on usual tissue culture plastic (Greiner Bio-One, Frickenhausen, Germany) and used for analysis until 4^th passage. Fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Biochrom) supplemented with 10 vol% fetal bovine serum (Biochrom) and 1 vol-% Zellshield (antibiotic; Biochrom) at 37 °C in 5% CO₂ in air at 95% humidity. For analysis of cell fates in 3D, equilibrated collagen matrices were place in 24 well plates (Greiner Bio-One) and 10⁴ fibroblasts were seeded on top of the matrices 24 h after reconstitution as described previously^{18 (link)}.

Experiments involving neonatal and adult mice were conducted according to the ethical guidelines for animal care of the Rostock University Medical Centre. Isolation of neonatal CMs was performed as described previously [30 (link)]. Following enzymatic digestions, cells were seeded on 8 well chamber slides and cultured in DMEM supplemented with 10% FBS (Pan Biotech, Aidenbach, Germany) and 1% Zellshield (Biochrom) on 0.1% gelatin (Sigma Aldrich) coated surfaces.
Adult murine cardiomyocytes were isolated as described elsewhere [31 (link)]. Briefly, adult CM were obtained by injection of collagenase 2/4 solution (Sigma Aldrich) into the left ventricle. Digested heart tissue was cut into smaller pieces and gently triturated with a 1 mL pipette. Isolated cells were seeded on collagen-coated cell surfaces and cultured for one day before being subjected to fluorescence labelling.

Cytotoxicity of the most promising dual therapies was evaluated on human lung epithelial A549 cells (ATCC CCL-185). Cells were grown in Dulbecco modified eagle medium (DMEM, Gibco) supplemented with 10% of fetal bovine serum (FBS, Gibco) and 1% antibiotics (ZellShield™, Biochrom) at 37°C, 5% CO₂. Once having achieved a minimum of 80% confluence, cells were detached, using trypsin, and adjusted to a final concentration of 1 × 10⁵ cells/mL. Then, 100 μL of cell suspension was transferred to each well of a 96-well plate, which was incubated for 24 h at 37°C, 5% CO₂. After this period, cell culture supernatant was removed and 100 μL of dual therapies prepared in supplemented DMEM was added. Fresh culture media with no compounds were also added as a positive control. The plate was incubated for an additional 24 h at 37°C, 5% CO₂. Metabolic activity of cells was determined using the MTS [(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)] inner salt (Promega) assay. Briefly, in the dark, 20 μL of MTS was added to each well and the plate was further incubated for 1 h at 37°C, 5% CO₂. The optical density of the resulting solution was measured at 490 nm. Results were presented as percentage of viable cells compared to the positive control. Two independent experiments, using four replicates, were performed.

All studies involving adult and neonatal mice were performed according to the ethical guidelines for animal care of the Rostock University Medical Centre.
Isolation and culture of adult murine CMs was conducted as described elsewhere [19 (link)]. Upon injection of collagenase 2/4 solution (Sigma Aldrich) into the left ventricle, heart tissue was cut into small pieces and single cells were obtained by trituration with a 1 mL pipette. Isolated CMs were seeded on collagen-coated surfaces and subjected to fluorescence labeling.
Isolation of neonatal CMs was performed as described previously [20 (link)]. Briefly, following enzymatic digestion, isolated cells were seeded on gelatin-coated 8 well chamber slides (Ibidi) or glass coverslips and cultured in DMEM supplemented with 10% FBS (Pan Biotech, Aidenbach, Germany) and 1% Zellshield (Biochrom).

All procedures used to obtain tumor specimens were performed by experienced radiologists. Needle biopsies were performed using a freehand technique under real-time ultrasound guidance (Acuson Sequoia, Siemens Healthcare; or Logiq E9, GE Healthcare). Usually, two biopsy samples were obtained from each liver mass using an 18-gauge biopsy device. One biopsy sample was submitted for pathologic examination. A pathologist was not physically present during biopsies. The number of samples obtained from each mass was based on operator preference and the appearance of the biopsy cores. Specimens were transferred to cold phosphate-buffered saline (PBS) containing 1% Zell Shield (Biochrom AG, Germany).