Glass bottom petri dishes

Cultivation and cell patterning was carried out as described by Nehls³⁰ . The pattern was produced with plasma-induced microcontact printing. Microstructured 5 × 5 mm² PDMS stamps, with a base to cure ratio of 10:1 cured for 4 h at 70 ^∘C, were produced using passivated microstructured wafers. These stamps were placed into glass-bottom Petri dishes (Ibidi) cleaned with ultrapure water and ethanol. After a 90 s oxygen plasma exposure, a solution of poly-l-lysine-graft-polyethyleneglycol (0.5 ml, PLL (20)-g[3.5]-PEG (2)/tetramethylrhodamine; SuSoS, Dübendorf, Switzerland) was added to each stamp. After a 1 h incubation at 23 ^∘C, the PDMS stamps were discarded, and the dishes were washed three times with PBS. A Collagen I coating was applied as described above to facilitate cell adhesion. Before seeding with 100,000 MDCK II cells in 2.5 ml M10F with penicillin/streptomycin (0.2 mg ml⁻¹; PAA, Pasching, Germany), 0.25 mg ml⁻¹ amphotericin B (Biochrom), and 40 mM HEPES (Biochrom)), the dish was washed three times with PBS and twice with MEM. After 60 min of incubation at 37 ^∘C, the samples were washed to remove nonadherent cells, and the AFM measurements were carried out.

To demonstrate the effects of EVs on cell morphology, cells were imaged using 3D holotomography (NanoLive 3D Cell Explorer Microscopy, Tolochenaz, Switzerland). MG63 cells were seeded in glass bottom Petri dishes (ibidi GmbH, Munich, Germany) and incubated for 24 h. After 24 h, cell culture medium was replaced with new CO₂ Independent Medium (Gibco, Grand Island, NE, USA) containing EVs at different concentrations (100 EVs per cell, 1000 EVs per cell, and 10,000 EVs per cell). After 1 h incubation with EVs containing medium, images were obtained with a 60× magnification objective lens and processed using STEVE software (v. 1.6.3496, 2020, NanoLive, Tolochenaz, Switzerland).

We conducted 2D under agarose migration assays as described elsewhere [27 (link)]. Briefly, 35 mm glass bottom Petri dishes (ibidi, Martinsried, Germany) were coated with 50 µg/mL of fibronectin (BD) for 1 h at 37 °C, washed twice with sterile water, and dried prior to casting the agarose gel. Next, 2% (w/v) agarose was boiled in plain RPMI1640 medium (Biochrom, Berlin, Germany) without any supplements and mixed with an equal amount of preheated (50 °C) complete RPMI1640 medium to yield a final agarose concentration of 1% (w/v). Finally, 2 mL of the warm and still fluid mixture were quickly added to the culture dish, covered with the dish lid, and allowed to solidify at room temperature.
To assess migration, two wells were punched 1.5 mm apart using a template and a 3 mm sterile skin punch. The prepared culture dishes were then equilibrated for 1 h at 37 °C and 5% CO₂ prior to seeding of the cells. Freshly isolated BMCs (~2000 cells) were seeded into the first well and allowed to settle for 20 min. Subsequently, 10 µg/mL of the chemoattractant LTB₄ (Cayman Chemical, Ann Arbor, MI, USA) was added to the second well, and the dish was carefully placed onto a heated microscope stage (35 °C) with additional CO₂ gassing (5%). Movement of cells was recorded at 1 frame per minute with a Zeiss AxioCAM MRm for a total period of 90 min.