Huvecs

Human umbilical vein endothelial cells (HUVECs; PromoCell GmbH, Heidelberg, Germany) were used to test the impact on cell-barrier properties. HUVECs were cultured in low-serum endothelial cell growth medium (PromoCell) at 37°C with 5% CO₂ in a humidifying incubator and used at passage p4–p6. Cells were cultured on transparent polyethylene terephthalate Transwell supports (0.4-μm pore size; Greiner Bio-One, Kremsmünster, Austria), coated with Entactin-Collagen IV-Laminin (E-C-L) Cell Attachment Matrix (Merck Millipore, Burlington, MA) diluted in Dulbecco's modified Eagle's medium (10 μg/cm²) for 1 h before seeding, at a density of 40,000 cells/cm², and grown for 3 days.

Human umbilical vein endothelial cells (HUVECs) (PromoCell GmbH, Heidelberg, Germany) were used to test the effect of the bacteria on vascular properties. After thawing, the frozen HUVECs were expanded in low-serum endothelial cell growth medium (PromoCell) at 37°C with 5% CO₂ in a humidifying incubator and used at passages p3 to p6 (30 (link), 44 (link)). Cells were grown to 80 to 90% confluence before being transferred to transparent polyethylene terephthalate (PET) Transwell supports (0.4 μm pore size, Greiner Bio-One, Austria), to a plastic well plate (Corning), to a glass-bottom well plate (Cellvis, Mountain View, CA), and to the insert chip (30 (link)). Before seeding, the uncoated substrates were treated with entactin-collagen IV-laminin (ECL) cell attachment matrix (Merck) diluted in Dulbecco’s modified Eagle’s medium (DMEM) (10 μg/cm²) for 1 h in the incubator. Then, the HUVECs, harvested with trypsin/EDTA solution (Biological Industries), were seeded inside the culture platforms at a density of 250,000 cells/cm² and grown for 48 h. Then, bacteria were added and their effect on cell behavior was tested after 1 h, 4 h, and 24 h.

The HT29 human colorectal adenocarcinoma cell line and the HCT116 human colorectal carcinoma cell lines were obtained from the European Collection of Cell Cultures (Sigma Aldrich, Dorset, UK). Human umbilical vein endothelial cells (HUVECs) were purchased from Promocell (Heidelberg, Germany) and Human dermal fibroblasts (HDFs) from Invitrogen (Paisley, UK). HT29 and HCT116 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 1 g/L glucose, 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin (all from Invitrogen, Paisley, UK). HUVECs were cultured in complete endothelial growth medium (EGM) (Promocell, Heidelberg, Germany) supplemented with 10% FBS and 100 units/ml penicillin and 100 μg/ml streptomycin. HDFs were cultured in high glucose (5 g/L) DMEM supplemented with 10% FBS and 100 units/ml penicillin and 100 μg/ml streptomycin. All cell types were routinely maintained as 2D monolayers at 37 °C in standard cell culture conditions (5% CO₂/air and 95% humidity).

In the current study, we used human umbilical vein endothelial cells (HUVECs) purchased from Promocell (Heidelberg, Germany). The endothelial cell medium (C-22010, Promocell, Heidelberg, Germany) was added to the endothelial cell growth factors (C-39215, Promocell, Heidelberg, Germany) and used to maintain HUVECs. The cells were kept at 37 degrees Celsius (°C) in a 95% humidified atmosphere containing 5% CO₂. After thawing, the cells were seeded in a T75 cell culture flask. The cells were passaged when they reached 90% confluency. For passaging, the cells were trypsinized at 37 °C for 4 min. A total of 5000 cells/cm² were seeded in 10 cm culture plates for protein analysis and 6-well plates for mRNA analysis. For all experiments, endothelial cells at passage 7 were treated with endothelial cell medium containing either 400 millimolar (mM) ethanol (EtOH), 50 nanomolar (nM) Colchicine, or 400 mM ethanol combined with 50 nM Colchicine. The endothelial cells were treated with a higher concentration of ethanol (400 mM) to induce senescence and SASP over a short period. Controls were treated with endothelial cell medium only. For all experiments, the endothelial cells were treated with different conditions for 24 h, with the exception of immunofluorescence staining, for which the duration of treatment was 2 h. Colchicine was purchased from Sigma-Aldrich, St. Louis, MO, USA (C3915).

Human colon adenocarcinoma (HT-29), fibrosarcoma cells (HT-1080), human and mouse melanoma cells (SK-MEL-28 and B16-F1, respectively) and human umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection (Rockville, MD). HT-29 cells were cultured in McCoy’s 5A supplemented with 5% Fetal Bovine Serum (FBS) and HT-1080, SK-MEL-28 and B16-F1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco-BRL) supplemented with 10% FBS.
HUVECs were grown in endothelial cell growth medium (ECGM, PromoCell, France) supplemented with 2% FBS and growth factors according to the manufacturer’s instructions in Nunclon® flasks (Dutscher Brumath, France) at +37°C in a humid atmosphere (5% CO₂, 95% air). For all experiments, a PromoCell DetachKit was used for the detachment of HUVECs according to the manufacturer’s instructions.

HUVEC׳s were purchased from PromoCell (Germany) and were used between passages 3 and 5. Cells were cultured in Complete Endothelial Cell Growth Media (EGM) (PromoCell, Germany), supplemented with 10% FCS (FirstLink,UK) and 1% Penicillin/Streptomycin (Gibco,UK). HBMSCs were obtained from patients at the RNOH undergoing total hip replacements (with informed consent and ethical approval) and were isolated based on the method by Igarashi et al. [20] (link). Cells were cultured in low glucose Dulbecco׳s Modified Eagles Medium (DMEM, Sigma USA) supplemented with 20% FCS and 1% Penicillin/Streptomycin. Cells were detached from tissue culture flasks, following washes with Phosphate Buffered Saline (PBS), and incubation with 0.5% trypsin at 37 °C for 5 min.
HUVEC׳s were pre-tested by PromoCell for cell proliferation and morphology. These were also positive for EC specific markers such as CD31 and von Willebrand Factor. HBMSCs were CD31 negative cells, with mesenchymal cell characteristics and differentiation potential into osteoblasts, chondrocytes and adipocytes.