Penicillin streptomicin

hGCs were purified by centrifugation through a discontinuos Percoll (Amersham, Sweden) gradient as indicated in [22 (link)], and cultured individually in 24 wells plate (50 x 10³ cells/well) in McCoy 5A medium (Carlo Erba, Italy) supplemented with 5 % foetal bovine serum (FBS) South America (EU Approved, Carlo Erba, Italy), 2mM L-Glutamine, 1 % Penicillin/Streptomicin and 1 % Amphotericin B (Sigma Aldrich, St. Louis, MO, USA). Primary hGCs culture were mantained at 37 °C under a controlled atmosphere of 5 % CO₂ for 6 days to avoid side effect due to IVF hormones treatment, and subjected to medium changes with fresh culture medium daily.

The A549 human lung
carcinoma cell line
was obtained from the American Type Culture Collection (ATCC), cultured
in DMEM supplemented with 10% fetal bovine serum (FBS, Gibco), 1%
penicillin/streptomicin (Sigma), and 0.1% amphotericin B (Gibco) and
maintained in 20% O₂ and 5% CO₂ at 37 °C
under normoxic conditions or in 1.5% O₂ and 5% CO₂ at 37 °C under hypoxic conditions. Cells were routinely tested
for mycoplasma using a universal mycoplasma detection kit (ATCC).

Human coronary smooth muscle cells from a young healthy donor were purchased from Promocell (C-12511) and cultured in growth medium (Promocell, C-22062), supplemented with 10% FBS and 1% penicillin-streptomicin (Sigma Aldrich). To achieve RS, cells were serially passaged (Bielak-Zmijewska et al., 2014 (link)). Cells were cultured until 70–80% confluence and re-seeded in T75 Flasks (Thermo Fisher Scientific, Nunc EasYFlask, #156499) at a density of 3.5 × 10³ cells/cm² until proliferative arrest (passage 6–8). Cumulative population doublings were counted using the formula cPD = X + 3.322^∗(log Y−log I), X representing the cumulative population doubling of the subculture used to initiate the culture, Y being the viable cell number on harvest, and I the cell number on inoculation. Low passage VSMCs (passage 2–3) were treated with bleomycin to induce DS, as previously reported (Gardner et al., 2015 (link)).

Mice were killed with CO₂, and E13.5 embryos were isolated and kept in cold PBS. Embryos were decapitated and DNA was injected subretinally using an elongated borosilicate glass capillary (Harvard Apparatus). All constructs were co-electroporated with GFP subcloned in the same vector. The success of DNA injection was assessed using 0.03% Fast Green added to the DNA solution. The paddles of the electrode (CUY650P5, Sonidel) were placed at the bottom and at the top of the head, respectively64 . Two poring pulses (square wave, 175 V, 5 ms duration, with 50 ms interval) followed by four transfer pulses (40 V, 50 ms and 950 ms interpulse) were applied. The protocol was repeated with inverted polarities. After electroporation, the retinas were isolated using a 21G needle and kept 24 h in culture medium (DMEM-F12 supplemented with 1mM glutamine (Sigma Aldrich), 1% penicillin/streptomicin (Sigma Aldrich), 0.01% BSA (Sigma Aldrich), 0.07% glucose), in a humidified incubator at 37 °C and 5% CO₂. Non-electroporated retinas were isolated at E14.5. Retinas were cut into 200 μm squares and explants were plated on glass coverslips coated with 100 μg ml⁻¹ poly-L-lysine (Sigma Aldrich) overnight at 37 °C and 20 μg ml⁻¹ Laminin (Sigma Aldrich) for 3 h at 37 °C. Cells were cultured for 24 h in culture medium supplemented with 0.4% methyl cellulose and B27.

Adipose tissue samples (n = 78: 26 SAT, 26 VAT and 26 BAT) were processed as previously described with some modifications to obtain tissue secretomes [9 (link),66 (link)]. In brief, whole adipose tissue explants were processed to remove any contaminants by doing an intensive wash in PBS that was repeated several times. The tissue pieces were transferred to a tube containing 25 mL of PBS and centrifuged for 5 min, at 1800 rpm, at room temperature to eliminate red blood cells and debris. 1 g pieces of each tissue type (VAT, SAT, and BAT) from independent animals were incubated at 37 °C and 5% CO₂ in 5 mL/tissue piece/well with serum-free DMEM medium without phenol red (Sigma-Aldrich, Burlington, MA, USA) supplemented with 1% (v/v) penicillin-streptomicin. The medium was changed after 2 and 24 h. After the last wash (time point 24 h), all dishes received fresh DMEM medium (3 mL/1 g tissue) and were left in culture for an additional 48 h to allow the secretion of vesicles. Then, the media were collected, centrifuged during 5 min at 1800 rpms, and stored at −80 °C for further analysis.

The FaDu cell line and its variants were maintained in Dulbecco's Modified Eagle Medium (DMEM) with high glucose (4.5 g/L, Sigma), supplemented with 8.85% (v/v) of fetal bovine serum (Sigma), 1.77 mM L-glutamine (PAA), 0.885% (v/v) MEM non-essential amino acid solution (PAA), 0.885% (v/v) penicillin-streptomicin (PAA), and 8.85 mM HEPES (Sigma). The 293T cell line used for the production of lentiviral vectors was cultured in DMEM with high glucose (4.5 g/L) medium (Sigma) supplemented with 8.85% (v/v) fetal bovine serum (Sigma) and 20 mg gentamicin (Kirka). Cell lines were cultivated in a humidified 5% CO₂ atmosphere at 37°C. The mycoplasma detection tests (Minerva) were performed routinely during cell line culturing. All cells used in the experiments were the same ones used in the 15^th splitting.