br Isolation and Identification of
Isolation and Identification of BMSCs
Under sterile conditions, 10 mL bone marrow was extracted from the femoral shaft fracture end with a 20-mL syringe (containing 2,000 IU heparin) and mixed with heparin quickly. The bone marrow was centrifuged in a centrifuge tube at 258 g for 10 min in order to re-move the upper adipose tissue, followed by three washes with DMEM, and resuspended using 15 mL medium. Bone marrow was centrifuged in a centrifuge tube containing the same volume of Ficoll-Paque PLUS lymphocyte separation fluid at 716 g for 20 min. Nucleated 4μ8C were noted to be located predominately in the boundary and up-per liquids, while most of the erythrocytes had precipitated to the bot-tom. The nuclear cells were withdrawn from the interface with a straw, centrifuged at 179 g for 8 min, after which the supernatant was discarded. Next, 5 mL cell culture medium was added to make Molecular Therapy: Nucleic Acids
nuclear cells evenly spread. The cell suspension (10 mL) was evenly mixed with 490 mL PBS. After that, 10 mL of mixture was obtained and counted under the microscope. The cells were inoculated in a cul-ture bottle (1 105 cells/bottle) and incubated with 5 mL low-glucose DMEM culture medium at 37 C with 5% CO2 and saturated humid-ity. After 24 h, BMSCs began to adhere to the wall, and half of the me-dium was replaced to remove non-adherent cells. The medium was replaced every 2–3 days, during which a small amount of hematopoi-etic stem cells, as well as the red blood cell suspension that failed to be removed by means of centrifugation, along with the other non-adherent mixed cells, was removed in a progressive manner. Cell adhesion and growth were observed using an inverted phase-contrast microscope. When the monolayer adherent cells grew to 80%–90% confluence at days in vitro (DIV) 10–14, the cells were treated with 0.25% trypsin and sub-cultured at ratio of 1:2–1:3. Flow cytometer was used to detect surface markers CD29, CD34, CD44, CD45, CD71, and HLA-DR of BMSCs. The adipogenic and osteogenic dif-ferentiation of BMSCs was identified according to the ability of inducing differentiation in vitro, and the formation of lipid droplets was observed by oil red O staining under an inverted microscope. The calcium deposits of osteoblast differentiation were observed through the application of alizarin red staining 4 weeks after osteo-blast induction and differentiation.
The exosomes in serum were removed by means of ultracentrifuga-tion of FBS at 100,000 g for 8 h. When BMSCs’ confluence reached around 80%, the supernatant was removed. BMSCS were cultured in 10% exosome-free FBS at 37 C in a CO2 incubator for 48 h. The collected supernatant was centrifuged in a gradual manner at varying speeds according to the following steps: 300 g for 10 min at 4 C with the removal of the precipitation, at 2,000 g for 15 min at 4 C with the precipitation removed, at 5,000 g for 15 min at 4 C with the precipitation removed, and at 12,000 g for 30 min at 4 C following the collection of the precipitation. The supernatant was subsequently centrifuged at 12,000 g for 70 min at 4 C with the precipitation collected. The supernatant following centrifugation was centrifuged at overspeed for 70 min at 100,000 g at 4 C, after which the precipitation was collected, followed by centrifugation for 70 min at 100,000 g at 4 C with the precipitation collected.
Nanoparticles Tracking Analysis
20 mg of exosomes was dissolved in 1 mL PBS and vortexed for 1 min in order to ensure a uniform distribution. NanoSight nanoparticle tracking analyzer (Malvern Panalytical, Worcestershire, UK) was em-ployed in order to directly determine the size distribution.
Transmission Electron Microscopy Observation