Kds 100

The PDMS-based substrate was immersed in the chosen NP dispersion for 10 min (Figure 1a) and then rapidly dried by blowing off any remaining dispersion with compressed air. In another test, the microfluidic device and a syringe filled with the NP dispersion were connected with polytetrafluoroethylene tubes (inner diameter = 0.5 mm, outer diameter = 1.59 mm), and the NP dispersion was introduced into the device using a syringe pump (KDS100, KD Scientific, Holliston, MA, USA) at a constant flow rate (0.7 mL/h for polystyrene NPs; 0.7, 1.4, and 3.5 mL/h for exosomes) for 10 min (Figure 1b). In the experiments that used polystyrene NPs, the flow rate was fixed at 0.7 mL/h to compare the adsorptivity in static and dynamic environments, while in the experiments that used exosomes, the different flow rates were used to investigate the adsorptivity in dynamic environments with different flow rates. The flow rate range and temperature (20 °C) were determined on the basis of previous studies concerning the handling of exosomes using microchannels [5 (link),21 (link)]. Because the ambient temperature in most laboratories is ~20 °C, this temperature was chosen for NP analysis. Subsequently, the PDMS-based microchannels were quickly peeled off, and their surfaces were promptly dried by blowing off any remaining dispersion with compressed air.

Carbonyl cyanide 4‐(trifluoromethoxy)phenylhydrazone (FCCP, C2920, Sigma‐Aldrich, UK) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 100 mM. It was then diluted in stem cell culture medium to obtain a final concentration of 1.5 µM. Oligomycin (75351, Sigma‐Aldrich, UK) was dissolved in 70% v/v ethanol at a concentration of 20 µg mL⁻¹. It was subsequently diluted in stem cell culture medium to a final concentration of 0.2 µg mL⁻¹.
Mouse embryonic stem cells were seeded in the microfluidic culture device at 5 × 10⁵ cells cm⁻² and left to attach in the chamber in static culture conditions (no flow) overnight. The culture was perfused at 300 µL h⁻¹ with a syringe drive (KDS100, KD Scientific, USA) with stem cell medium for the first 2.5 h, then stem cell medium supplemented with Oligomycin for 3.5 h, and finally stem cell medium supplemented with FCCP until the end of the assay.

MnCl₂ solution was injected into all mouse cohorts 23 h before the MEMRI scan (Figure 1). A solution of 100 mM MnCl₂ (MnCl₂⋅4H₂O; Sigma-Aldrich, St. Louis, MO, United States) was made with distilled water and diluted to 50 mM with saline (osmolarity, 275 mOsm/kg). The MnCl₂ solution was infused slowly through the tail vein using a syringe pump (KDS-100; KD Scientific, Holliston, MA, United States) for 60 min to a final dose of 75 mg/kg (final volume, 11.92 ml/kg) under continuous anesthesia (0.5–1.5% isoflurane; Mylan Inc., Tokyo, Japan) with monitoring of SpO₂ with a SomnoSuite (Towa Science, Tokyo, Japan); the mice were placed on a thermoregulated plate to maintain body temperature (Hattori et al., 2013 (link)). After MnCl₂ administration and confirmation of animal recovery from anesthesia, the mice were returned to their home cage and allowed to move freely with free access to food and water until the MRI acquisition. The mice were taken out of the home cage for <1 min for (1) intraplantar injection of formalin or saline (for all three cohorts) and (2) repeated (twice in total) intraperitoneal injections of CNO (for the third cohort). Details of these drug administrations are provided below.