Mc03f

MSCs were isolated from human placenta as described previously [21 (link)]. Cells were placed in culture flasks with the complete culture medium DMEM-F12 supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 10% fetal bovine serum (all reagents from Gibco) and maintained in a humidified atmosphere under standard conditions (37 °C, 5% CO₂). Cells were passaged at 80% confluence and collected for transplantation after 3–5 passages. The phenotype of cultured cells checked by flow cytometry was CD34−, CD45−, HLA-DR−, CD105+, CD29+, CD73+, CD90+, which is typical for MSCs. Prior to transplantation cells were labeled with superparamagnetic iron oxide (SPIO) microparticles (MC03F Bangs Laboratories, mean diameter 0.50 ± 0.99 μm) carrying the Dragon Green fluorescent dye (λex = 480 nm, λem = 520 nm) and with the red lipophilic membrane fluorescent dye PKH26 (Sigma-Aldrich, Burlington, MA, USA) as described previously [22 (link)]. Double cell labeling had no influence on cell viability and proliferation [7 (link),22 (link)]. Before transplantation, cell viability was checked by the trypan blue test using an automated cell counter (Invitrogen) and was more than 90%. A dose of 5 × 10⁵ cells in 2 mL of saline was prepared for each IA transplantation.

Cells were labeled with superparamagnetic iron oxide (SPIO) microparticles (MC03F Bangs Laboratories, mean diameter 0.50 ± 0.99 μm) carrying Dragon Green fluorescent dye (λex = 480 nm, λem = 520 nm) and with lipophilic membrane red fluorescent dye PKH26 (Sigma-Aldrich) as previously described (Namestnikova et al., 2017b (link)). Double cell labeling had no influence on cell viability (data presented in the Supplementary Material) and cell proliferation, as shown previously (Namestnikova et al., 2017b (link)).
Determination of the average iron content per cell was carried out by inductively coupled plasma (ICP) atomic emission spectrometry (Agilent 4200 MP-AES, United States) using a calibration curve. Standard solutions with Fe (III) concentrations of 500, 1,000, 1,500, and 2,000 ppb were prepared by dilution of iron standard solution in 2% (w/w) HNO₃ for ICP (Merck, United States). 2% aqueous solution of HNO₃ was used as a blank. Samples were prepared by pelleting 2.4 × 10⁶ SPIO-labeled pMSC or drNPC by centrifugation and dissolving the pellet with 500 μl of concentrated nitric acid (stirring at room temperature, 48 h). Three independent measurements were carried out and the average iron content in cells was calculated as the mean of 3 experiments ± standard deviation.