Gaspak ez pouch system

The bacterial strains used in this study are listed in Table 1. E. coli strain EC100Dpir⁺ was used for cloning, and E. coli strain SM10λpir was used to conjugate plasmids into V. cholerae. V. cholerae O1 El Tor strain N16961 ΔlacZ was used as the WT control in all experiments. Bacteria were routinely grown in LB broth or on LB agar at 37°C. Modified T medium was prepared as previously described (21 (link)). Anaerobic conditions were achieved with the BD GasPak EZ Pouch System. Antibiotics were used at the following concentrations: streptomycin, 100 µg/ml; carbenicillin, 100 µg/ml; kanamycin, 50 µg/ml.

100,000 maturated hiPSC‐derived cardiomyocytes/well (40‐45 days post differentiation) were seeded in a 96 well plate. Cells were incubated with 150 μL of fresh maturation medium and incubated for 4 days. Subsequently, sEV subpopulation samples next to a PBS control were added to the cells and incubated for 48 h in 1% hypoxia conditions using a GasPak™ EZ Pouch System (Becton, Dickinson and Company). A plate incubated in normoxia conditions was also prepared with only the PBS control and incubated for the same period. For analysis, after incubation cells were fixated and cell death was determined using staining with Hoechst 33342 (1:10,000) (ThermoFisher Scientific) and ethidium homodimer‐1 (1:500) following the instructions of the Invitrogen™ LIVE/DEAD™ Viability/Cytotoxicity Kit for mammalian cells (ThermoFisher Scientific). After 30 min, staining solution was replaced by maturation medium and cells were scanned using a CellInsight™ CX High Content Screening HCS Platform (ThermoFisher Scientific) and analyzed using the Cell Health Profiling bioapplication to determine the percentage of dead cells. Relative cell death of was calculated relative to the negative controls for independent biological replicates.

Bifidobacterium bifidum (ATCC® 29521™) and Bacteroides fragilis (ATCC® 25285™), purchased from the American Type Culture Collection (Manassas, VA), were cultured in modified reinforced clostridial medium (ATCC® Medium #2107) and modified chopped meat medium (ATCC® Medium #1490), respectively. These bacteria were incubated in a static air-tight chamber, at 37°C, that utilizes a GasPak™ EZ pouch system (Becton, Dickinson & Co., Sparks, MD) to generate anaerobic conditions. Lactobacillus casei was obtained from the culture collection of the Department of Food Science, the University of Tennessee Institute of Agriculture (Knoxville, TN), and grown in tryptic soy broth (TSB) medium at 37°C under aerobic conditions. The TSB medium was prepared using casein peptone (17 g/L), sodium chloride (5 g/L), soy peptone (3 g/L), dextrose (2.5 g/L), and dipotassium phosphate (2.5 g/L) purchased from Alfa-Aesar (Haverhill, MA), where the pH was adjusted to 7.1. Isolated colonies were obtained and maintained on respective media.

DPC were treated 1 day after seeding for 24 h with the inflammatory markers IL-1β at 10 ng/ml (PeproTech Austria, Vienna, Vienna, Austria), TNFα at 10 ng/ml (PeproTech Austria) or TGFβ at 10 ng/ml (isoform TGFβ1; PeproTech Austria) in serum-free αMEM medium (Gibco) with penicillin and streptomycin (Gibco). The time frame of cell treatment was chosen to correspond to a biologically and clinically relevant situation where hypoxic conditions and inflammation occur due to e.g., trauma and normally should undergo regenerative therapy. Hypoxic conditions were mimicked with the hypoxia mimetic agent L-MIM at 1 mM or hypoxia at < 1% O₂ was induced using the BD GasPak EZ Pouch system (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) as it matches the biologically relevant range (Agata et al., 2008 (link), 2013 (link)). The procedure was previously described in detail (Janjić et al., 2018a (link)). DPC were additionally treated in combination with inflammatory markers and L-MIM or hypoxia, respectively. Untreated DPC under normoxia were used as control. All treatment solutions were prepared in serum-free αMEM medium with antibiotics. The choice of IL-1β (Weng et al., 2012 (link)), TNFα (Paula-Silva et al., 2009 (link)), TGFβ (Loots et al., 2012 (link)) and L-MIM (Janjić et al., 2018a (link)) concentrations was based on previous publications.