Pt 3001

We seeded human mesenchymal stem cells (hMSCs, Lonza, PT-2501) in a T75 flask at 5000 cells/cm² in growth media (Lonza, PT-3001). These cells were labelled with 8 ml 250 μg/ml silica nanoparticles in fresh cell culture media without any transfection agents. The cells were then incubated under standard conditions for 7.5 hours. We washed the cells with sterile PBS to ensure all the free silica nanoparticles were removed. Cells were then detached by adding 2 ml of TrypLE Express (Life technologies). We scanned these labelled cells with ultrasound both in vitro and in vivo. hMSCs were labelled with silica nanoparticles at different concentrations (0 to 1000 μg/ml) and studied the cell viability by using CellTiter 96® AQueous One Solution cell proliferation assay (MTS, Promega). After incubation with silica nanoparticles for 4 hours, 20 μl of the assay reagent was pipetted into the samples in 100 μl of growth media. This was allowed to incubate under standard conditions. After 4 hours, we transferred 80 μl of the sample solutions to a new plate and read the absorbance at 490 nm. Epifluorescence microscopy used an Evos microscope (Life Technologies) and Hoechst 33342 (NucBlue® Live ReadyProbes® Reagent, Thermo Fisher Scientific) for nuclear staining.

The hMSCs (Lonza, PT-2501) were seeded at 5000 cells/cm² in growth media (Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 1% Glutamax, and 1% penicillin-streptomycin, Lonza, PT-3001) and grown to 80–85% confluency in a T75 flask. These cells were labeled with PB-PLL nanocomplexes in fresh culture media (50 µg/mL) for 6 h. After washing with sterile PBS three times to ensure all free particles were removed, cells were trypsinized using 0.25% trypsin/EDTA. Detached cells were collected by centrifugation at 300g for 5 min, resuspended, and counted by hemocytometer. For all experiments, cells from passages 4–8 were used.

Poietic hMSCs (Lonza PT-2501) were grown in supplemented media (Lonza, PT-3001) and seeded in a T75 flask at a concentration of 5000 cells/cm². These cells were labeled with nanobubbles and incubated under standard conditions for 6 h unless otherwise noted. The hMSCs were washed three times with PBS to remove free nanobubbles and detached using TrypLE Express (Life Technologies). The cell viability was determined using the Cell Viability Kit 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Biotium). A total of 10 μL of the MTT solution was added to 100 μL of the medium in each well, mixed briefly, and incubated at 37 °C for 4 h; 200 μL of DMSO was added to each well and pipetted to dissolve the resulting formazan salt. The absorbance was read at 570 nm on a spectrophotometer. MTT assays were conducted by plating 8000 cells/well in replicate (n = 8) in 96-well plates and treated at varying time points (0–24 h) at a constant concentration (240 μg/mL) as well as at varying concentrations (0–480 μg/mL) at a constant time (8 h unless otherwise noted). Wells were analyzed in replicate (n = 8). For differentiation studies, adipogenic cells were stained with Oil Red O, and osteogenic-differentiated cells were stained with a Von Kossa stain as described previously.^{66 (link)}

Human mesenchymal stem cells (hMSCs) were obtained from Lonza. Cells were maintained on tissue culture polystyrene using Mesenchymal Stem Cell Growth Medium (MSCGM; Lonza pt3001) per manufacturer’s instructions. Early passage hMSCs (<5) wereused for all studies.