T75 culture flask

Tissue from biopsy human brains were collected as previously described^{20 (link),71 (link)}. Briefly, the tissue was mechanically dissected and dissociated before enzymatic digestion in Hank’s balanced salt solution (HBSS), containing 2.5 U/mL papain (Worthington) and 100 U/mL DNase 1 (Invitrogen) for 30 min at 37 °C with gentle rotation. This step included a gentle titration at 15 min. Following this, complete media, DMEM:F12 (Invitrogen) was used to stop the enzymatic digestion. To collect the cells, they were centrifuged (170×g, 10 min) and resuspended in complete media. Cells were plated onto uncoated T75 culture flasks (Nunc). For all the experiments presented here, pericytes were passage 5–9 to ensure no contamination of astrocytes or microglia in our pericyte pure cultures. Late passages (> 4) displayed immunocytochemical staining for pericyte markers such as platelet-derived growth factor receptor beta (PDGFRβ), alpha smooth muscle actin (αSMA) and neural/glial antigen 2 (NG2)^{21 (link)}. The cultures also displayed immunocytochemical staining for fibroblast markers prolyl-4-hydroxylase (P4H) and fibronectin. Cells were subsequently incubated at 37 °C with 5% carbon dioxide until seeded for experiments, grown in DMEM:F12 (Invitrogen) containing 10% fetal bovine serum (Gibco) and 1% Penicillin/Streptomycin (Gibco).

The BMSCs were harvested from bone marrow and subsequently isolated following well-established protocols [13 (link)]. In short, bone marrow was obtained during surgery using a heparinized syringe. The heparinized bone marrow components were subsequently layered onto Ficoll-Paque Plus (GE Healthcare Europe, Freiburg, Germany) for phase separation. After centrifugation and repeated washing, the mononuclear cells were transferred to T75 culture flasks (Nunc, Roskilde, Denmark), which were coated with 0.1% gelatin (Sigma-Aldrich). Cultivation occurred under standard cell culture conditions (37 °C and 5% CO₂ in a humidified atmosphere) in expansion medium (EM) that consisted of high-glucose DMEM supplemented with 12.5% fetal calf serum (FCS), 2 mM L-glutamine, 1% non-essential amino acids (NEAA), 50 μM β-mercaptoethanol (all Thermo Fisher), 100 μg/mL penicillin/streptomycin (PenStrep; Biochrom, Berlin, Germany), and 4 ng/mL fibroblast growth factor 2 (Abcam, Berlin, Germany). The first exchange of medium was performed after 24 h of cultivation to remove the non-adherent cells. The EM was changed every third day until 80% confluency was observed, and the cells were thereupon passaged. Experiments were conducted with cells in passages 4 and 5.

RA and OA synovial fragments were washed with sterile phosphate-buffered saline (PBS). The tissues were minced and treated overnight with 1.5 mg/ml of collagenase A (Roche^®) in Dulbecco’s modified Eagle medium (DMEM) (Gibco^®) at 37°C. Dissociated cells were then centrifuged at 3500 rpm for 3 min, washed and resuspended in DMEM supplemented with 20% fetal bovine serum (FBS) and 1% penicillin- streptomycin (Gibco^®). The cell pellet was seeded in T-75 culture flasks (Nunc^®) and cultured at 37°C with 5% CO2. After 72h, non-adherent cells were removed, and adherent cells were kept at 37°C in 5% CO2. The medium was replaced every 3 days. After 10-14 days of culture, adherent cells were trypsinized using trypsin-EDTA (Gibco^®) and transferred to new flasks with a density of 5.10⁵ cells/flask. Passage 3 cells were morphologically homogenous and were used for experiments.

We employed three colon cancer cell lines, SW480, SW620, and DLD-1 in our study. Both SW480 and SW620 were MSS, while the DLD-1 was MSI-H cell line [21 (link)]. All cell lines were purchased from the American Type Culture Collection. The cells were planted in T75 culture flasks (Thermo Fisher Scientific, 156499, Waltham, MA, USA) and maintained in RPMI-1640 medium (Gibco, 31800-022, Waltham, MA, USA) supplemented with 10% FBS (Gibco, 10437-028) and 1% amphotericin B, penicillin, and streptomycin (antibiotic–antimycotic; Gibco, 15240-062) in an atmosphere of 5% CO₂ at 37 °C.

Passage 3 hADSCs harvested from T75 culture flasks (Thermo Fischer Scientific) were seeded on 24-well culture plates (Thermo Fischer Scientific, Waltham, MA, USA), with 2 × 10⁴ cells on each well. Cells were transduced with adenovirus-encoding hBMP-2 (Ad.hBMP-2) or green fluorescent protein (Ad.GFP) (Sirion Biotech, Martinsried, Germany) when reaching 70–80% confluency. hADSCs transduced with Ad.GFP were investigated for transduction efficiency, while Ad.hBMP-2-transduced hADSCs were employed for osteogenic differentiation analysis. Ad.hBMP-2 or Ad.GFP vectors were previously diluted at designed MOI of 0, 10, 20, 50, 100, 200 and 1000 in 1× PBS (Phosphate Buffered Saline) (Thermo Fischer Scientific, Waltham, MA, USA) and then added to the wells. The amount of PBS was enough to cover the whole layer of cells to prevent the cells from drying out. The transduced cells were then incubated in a Binder FED 115 incubator (BINDER) for 1 h and carefully rotated every 20 min. Cells were subsequently washed three times with 1× PBS, and fresh growth medium was added. For the osteogenic differentiation analysis, hADSCs were cultured with osteogenic medium.

HeLa cells were seeded out at a density 150,000 cells/mL in Fluorobrite DMEM media (Gibco) in T75 culture flasks (Thermo Fisher Scientific). One day later the cells were incubated with TNF-α (20 ng/mL, Sigma-Aldrich) at 37°C for 24 h. Cells were washed twice in PBS and incubated in ice-cold Fluorobrite DMEM media with Stx2:Alexa 488 (200 ng/mL, final concentration) for 1 h in 4°C. Cells were washed as above and incubated with A23187 10 μM for 40 min to induce microvesicle release. The cell media was collected and centrifuged at 2,500 × g for 5 min and the supernatant, containing microvesicles, was collected and washed as described above. These microvesicles were used for detection and quantification of microvesicle uptake by DLD-1 cells.