Low glucose dmem

Male mice offspring were euthanized to collect liver tissue samples as described previously. Fragments of hepatic tissue were incubated at 37 °C for 2–4 hours in 24-well culture plates containing DMEM low glucose (Invitrogen, USA). Next, the medium was removed, and the tissue washed with 1X PBS. Tissue fragments were incubated at 37 °C for 10 minutes in 24-well culture plates containing 100 nM insulin in DMEM low glucose (Invitrogen, USA). Tissues were then collected, froze in liquid nitrogen and stored at −80 °C until processing for Western blot analysis.

Adipose tissue was collected sterilely from donor Corriedale male sheep weighing ~30 kg as described in the animal model section. Muse cells were isolated under a stress procedure as described previously with minor modifications [16 (link), 17 (link)]. Adipose Tissue was cut into small pieces, rinsed twice with PBS and then incubated with (0.1% (wt/vol) collagenase (Merck, Kenilworth, NJ, USA, cat. #17100017) in DMEM low glucose (Gibco, Waltham, MA, USA, cat. #31600034) for 60 minutes at 37°C with constant agitation. Successively samples were incubated overnight at 4°C and 50 rpm. The next day, the samples were centrifuged at 1500 rpm and the supernatant was discarded, and the pellet was incubated with red blood lysis buffer and washed twice with PBS. Isolated cells were cultured in low glucose DMEM (1g/L) (Gibco, Waltham, MA, USA), 20% SFB (Natocor, Cordoba, Argentina), 1% ATB (Gibco, Waltham, MA, USA) and incubated at 37% with 5% CO₂ in p60 non-adherent plates (Greiner Bio-One, Frickenhausen, Germany), each one with 3,5x10⁶ cells. The culture medium was changed after 48 hours; in addition, 5 days after isolation the cells were cultured in adherence bottles and sub-cultured until passage 4 and 80% confluence for the subsequent experiments. Adipose-derived mesenchymal stromal cells (ASCs) were also extracted from the same donors as control as we reported previously [18 (link)].

To assess the effect of microRNAs within the EV-MSCs on fibroblast differentiation, an in vitro model of the TGFb-induced differentiation of fibroblasts into myofibroblasts was used. For this, primary human dermal fibroblasts (8–12 passages) were seeded in culture plates in complete growth medium at a rate of 15.000/cm². After 24 h, the plates were washed with DMEM low-glucose (Gibco) and left in the second batch of DMEM low-glucose for overnight deprivation. After deprivation, appropriate solutions were added to the cells in each group to induce differentiation: negative control “DMEM” group—DMEM LG; positive control “+C” group—DMEM LG + 5 ng/mL TGFb; “EV” group—DMEM LG + 5 ng/mL TGFb + EV of native ASC52telo; “EV C1” and “EV C4” groups—DMEM LG + 5 ng/mL TGFb + ASC52telo EV treated with scramble gRNA (clones C1 and C4, respectively); “EV 21.6” and “EV 21.7” groups—DMEM LG + 5 ng/mL TGFb + EV ASC52telo with the impaired hsa-mir-21 gene (clones 21.6 and 21.7, respectively); “EV 29c.16” and “EV 29c.19” groups—DMEM LG + 5 ng/mL TGFb + EV ASC52telo with the impaired hsa-mir-29c gene (clones 29c.16 and 29c.19, respectively). After 4 days of incubation, the cells were used for further analysis.