Culture of hp-MSCs, HUVEC, and H9c2 cells
hp-MSCs were obtained from as a gift (17). hp-MSCs were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS, Hyclone Laboratories, Logan, UT, USA), 10 ng/ml human recombinant basic fibroblast growth factor (Wako), and 1% penicillin (100 U/ml)/streptomycin (100 U/ml) solution (Life Technologies). The HUVEC cell line was purchased from PromoCell GmbH (Germany) and grown in endothelial cell growth medium 2 (PromoCell) supplemented with 10% fetal bovine serum and 1% penicillin /streptomycin. The H9c2 cell line was purchased from ATCC (CRL-1446) and grown in DMEM supplemented with 10% fetal bovine serum and 1% penicillin /streptomycin. All Cells were cultured in a 5% CO2 incubator at 37 °C.
Radiation exposure
The culture medium from twice-passaged hp-MSCs culture at 80% confluence was aspirated, cells were washed with phosphate buffered saline (PBS) (Wako, Osaka, Japan) to remove the residual FBS. Fresh culture medium supplemented with 10% exosome-depleted FBS (System Biosciences) was added. Then the hp-MSCs were exposed to 0 or 5 Gy γ-rays at a dose rate of 1 Gy/min using a PS-3100SB γ-ray irradiation system with a Cs source (Pony Industry Co., Ltd. Osaka, Japan) (18).
Isolation of exosomes derived from non-irradiated and irradiated hp-MSCs
After 48 hours of incubation, culture medium from hp-MSCs that irradiated or not was collected and underwent different steps of centrifugation as previously described with minor modifications (19). Briefly, culture medium was centrifuged at 300 g for 3 minutes, at 4 °C and 2000 g for 30 minutes to remove cell debris and apoptotic bodies. The supernatant was ultra-centrifuged at 4 °C and 100000 g for 120 minutes to collect exosomes. Then, the pellet was washed with PBS and underwent another step of ultracentrifugation at 4 °C and 100000 g for 120 minutes to concentrate and purify exosomes. At last, the pellet was re-suspended in PBS and went through 0.22 μm filter (Millex) for further experiments or stored at -80 °C.
Exosomes characterization
Image of exosomes were taken by a transmission electron microscope. Briefly, 5 ul exosomes were dropped on the copper net and incubated at room temperature (RT) for 5 minutes. Then, excess liquids were removed by the filter paper. Add 5 ul 1% phosphotungstic acid to the copper mesh and incubated for 1 minute at RT. Excess liquids were also removed by the filter paper. Add deionized water to the copper mesh to remove excess dye solution. Observe the exosomes under microscope after drying at RT.
The protein concentration of exosomes was tested as described in the instructions (Thermo Scientific 23235). Expression of exosome marker CD63 and TSG101 (System Biosciences) were verified western blot analysis. Exosome miRNAs were analyzed using gene chip miRNA 4.0 by the Filgen company.
Uptake of exosomes
Exosomes derived from non-irradiated (Non-irradiated-exo) and irradiated hp-MSCs (Irradiated-exo) were labeled with the PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich) according to the manufacturer’s protocol with minor modifications. Non-irradiated or irradiated-exo diluted in PBS were added to 1 ml Diluent C (Sigma-Aldrich). In parallel, 4 μl PKH26 dye was added to 1 ml Diluent C and incubated with the exosome solution for 4 minutes. To bind excess dye, 2 ml 0.5% BSA/PBS was added. The labeled exosomes were washed at 100,000 g for 1 hour, and the exosome pellet was diluted in 100 μl PBS and used for uptake experiments. PKH26 labeled non-irradiated or irradiated-exo were cultured with HUVEC and H9c2 cell line, respectively. The images of exosomes uptake were taken when co-culture at 3 and 24 hours using confocal microscopy.
Evaluation of cell proliferation and DNA damage
To evaluate the effects of exosomes on cell proliferation and DNA damage, HUVEC and H9c2 cells were seeded on 4-well chamber culture slides. After 72 hours of culture with non-irradiated-exo or irradiated-exo, the cells were washed with PBS and fixed in 4% paraformaldehyde for 10 minutes. After blocking, the cells were incubated with KI67 Monoclonal Antibody (SolA15, Invitrogen), anti-53BP1 antibody (ab36823, Abcam), or anti-gamma H2A.X (ab2893, Abcam), followed by associated Alexa flour 488-conjugated second antibody. Nuclei were stained with DAPI, and the positively stained cells were counted under fluorescence microscopy with 200-fold magnification, and 20 fields per section were randomly selected for quantitative counting.
Annexin-V flow cytometry
To evaluate the effects of exosomes on HUVEC and H9c2 cell apoptosis, HUVEC and H9c2 cells were seeded on 10 cm culture dishes. After 48 hours of culture with non-irradiated-exo or irradiated-exo, the cells were collected and washed with cold D-PBS by centrifugation for 5 min at 500 × g at 4 °C. Cells treated with 3% formaldehyde in buffer for 30 min were included as positive control. The cell pellets were suspended with 100ul cold D-PBS, then added 5 ul of Annexin V-FTIC solution and 2.5 ul dissolved PI as described in the manual (Beckman coulter). The samples were kept on ice and incubated for 10 min in the dark. Finally, 400ul ice-cold 1×binding buffer were added to the samples for analyze by flow cytometry.
Tube formation
Corning® Matrigel® Matrix (356230) was thawed overnight on ice at 4 °C according to the guidelines in the manual. All pipets and procedures were previously kept on ice. Added 289 ul chilled corning Matrigel matrix in to 24-well culture plates avoiding air bubbles. Plates were incubated at 37 °C for 30-60 minutes. The medium remained was removed carefully without disturbing the matrix layer and the plates were ready to use. HUVEC cells were previously co-cultured with non-irradiated-exo or irradiated-exo for 48 hours. 300 ul cell suspensions were collected and added to each well and incubated at 37 °C, 5% CO2 atmosphere. The tube formation was observed and pictured. The photo was further analyzed by Image J.
Intracellular Calcium detection
The intracellular calcium was examined by loading H9c2 cells with Fluo 3 (Dojindo Molecular Technologies, Inc) according to the instructions. Briefly, H9c2 cells were previously co-cultured with non-irradiated-exo or irradiated-exo for 48 hours. Cells were harvested ant then plated on 96-well plates. Culture medium were carefully removed without injuring the cells. Cells were washed with PBS gently and then incubated with loading buffer at 37 °C for 1 hour. Loading buffer was removed carefully and added warm recording medium. The fluorescence was examined by multifunctional microplate detector.
Statistical analysis
All the results are presented as the mean ± SD. The statistical significance was determined by one-way ANOVA and followed by Turkey’s multiple comparisons test (GraphPad Prism). Differences were considered significant when P < 0.05.