Glutamate plus

Placental biopsies were washed in Cytowash (consisting of DME/H-21 (Gibco), 2.5% fetal bovine serum (HyClone), 1% glutamine plus (Atlanta Biologicals, Minneapolis, MN), 1% penicillin/streptomycin (Invitrogen), and 1% gentamicin (Gibco)⁴² (link) and dissected into 1.0 cm² chorionic villi explants. A single villus was placed in one well of a 24-well plate containing 0.5 mL of prewarmed cell free media [DME/H-21, 2% Nutridoma (Roche, Basel, Switzerland), 1% sodium pyruvate (Sigma-Aldrich, St. Louis, MO), 1% HEPES buffer (Invitrogen), 1% glutamate plus (Atlanta Biologicals), and 1% penicillin/streptomycin (Invitrogen)] with 10% fetal bovine serum and incubated at 37°C in 5% CO₂/95% O₂ for 30 min. Vaccine mixtures for explant exposure were then prepared such that final vaccine concentrations were: BNT162b2 and mRNA-1273 vaccine 0.1 μg/mL and 1 μg/mL, as well as control medium. Testing concentrations were based on estimates of localized circulating levels of mRNA vaccines in organs of mice vaccinated for influenza.²⁸ (link) Explants were exposed for 0.5 or 4 h and incubated at 37°C, after which 200 μl of supernatant were collected and stored at -4°C for cytokine analysis. Exposed explants were washed in PBS and then stored at −80°C until RNA extraction, or formalin-fixed for 24 h at room temperature, then washed in 70% ethanol and stored at -4°C until undergoing paraffin embedding.