Glutamax

IPEC-J2 is a non-transformed cell line, derived from the jejunum epithelium of unsuckled piglets (DSMZ, Braunschweig, Germany). Cells used for the experiment were defrosted from a cryopreserved stock and cell passages of 24–28 were used for the experiments. Cells were routinely grown in a total volume of 100 mL of 1:1 of Dulbecco’s modified Eagle’s medium with stable L-glutamate and Ham’s F-12 mixture (DMEM/F12) (Immunological sciences, Società Italiana Chimici, Rome, Italy), plus 15 mM of HEPES (Sigma-Aldrich, Milano, Italy), 5% heat-inactivated foetal bovine serum (FBS) (Immunological sciences, Società Italiana Chimici, Rome, Italy), 1% penicillin (100 U/mL)/streptomycin (100 mg/mL) (Euroclone, Milano, Italy) and 1% GlutaMAX at 37 °C in a 5% CO₂ atmosphere and subcultivated at 80% confluence. For adhesion assay, IPEC-J2 monolayers were prepared in a 2-well chamber slides system coated with collagen. After collagen coating, the cells were seeded at a concentration of 4 × 10⁵ cells/chamber to reach 80% confluence before lactobacilli addition.

The hUMSCs (StemBioSys, San Antonio, TX, USA) were cultured in α-Minimum Essential Medium Eagle (VWR International srl, Milan, Italy) supplemented with 2.5% heparin-free human pooled platelet lysate, 1% Antibiotic-Antimycotic (VWR International srl), and 1% GlutaMax (Euroclone, Pero, Italy).
Experiments were carried out using the plasma of 10 AMA patients at the time points, T0, T1 and T3, and of 5 patients belonging to the controls at the same time points. For the in vitro experiments, we selected patients/controls characterized by a physiological pregnancy, without the onset of any complications, and for whom samples were available and adequate at the three chosen time points.
Untreated cells were also included in the analysis. In particular, we used specific Transwell inserts in order to analyze cell viability, ROS and NO release in hUMSCs treated with plasma samples, as previously performed. As depicted in Figure 5, 10% plasma samples calculated in relation to total volume of each insert were positioned in the apical surface of the insert itself for 3 h, while hUMSCs were plated in the basal one. After 3 h stimulation with plasma, the inserts were removed and various assays were performed, as described below. Experiments were executed in triplicate and repeated three times on different pools of hUMSCs.