Isolation, culture and identification of adipose-derived mesenchymal stem cells
Abdominal subcutaneous adipose tissues were collected aseptically at Affiliated Animal Hospital, Department of Veterinary Medicine of Foshan University. The tissues were cut into tissue blocks about 1 mm2 in size and were digested with 1 mg/mL collagenase type Ⅰ at 37℃ for 2 ~ 3 h. The digestive juices were filtered with 200-mesh cell strain and centrifuged at 800×g for 5min to collect AD-MSCs. Approximately 5000 isolated suspended cells per cm2 were transferred to cell culture flask (Corning, USA) in Dulbecco's Modified Eagle's Medium supplemented with 10% exosome-free Fetal Bovine Serum (FBS, Biological Industries, Israel), 1% Pen-Strep (Gibco, USA), and 1% L-glutamine (Gibco, USA) and placed into the incubator at 37°C in a humidified incubator containing 5% CO2. After 24 h, the medium was replaced for the first time to remove most of the blood cells and replaced every 3 d thereafter. AD-MSCs were digested with 0.25% trypsin and passaged routinely when 80 ~ 90% confluence was reached.
The AD-MSCs were characterized by multipotential differentiation and flow cytometry analysis. In vitro adipogenic and osteogenic differentiation were examined using MSCs Adipogenic Differentiation Kit (Cyanogen, China) and MSCs Osteogenic Differentiation Kit (Cyanogen, China) following the manufacturer’s protocol for each kit. Cells were stained with Oil Red O solution to assess adipogenic differentiation and alizarin red solution to assess osteogenic differentiation.
Flow cytometry analysis was performed using a CytoFLEX flow cytometry instrument (Beckman, USA). Data acquisition and analysis was performed with CytExpert (Beckman, USA). Briefly, AD-MSCs of passage 2 were stained for 30 min with FITC -conjugated or phycoerythrin (PE)-conjugated monoclonal antibodies at 37˚C. The following monoclonal antibodies were used: anti-CD34-PE (cat.no.ab23830; Abcam), anti-CD44- FITC (cat.no.MA1-10229; Invitrogen), anti-CD45-FITC (cat.no.ab27287; Abcam), anti-CD90-PE (cat.no.11-0900-81; Invitrogen), anti-CD105-FITC(cat.no.ab11415; Abcam) ,and anti-HLA-DR-FITC (cat.no.L243-347400; BD Biosciences). Chilled PBS was used to wash and remove unbound antibodies, and then a total of 2x105 cells from each sample tube were acquired for analysis using Flow Cytometer.
Preparation of cell culture medium samples
50–80% confluent AD-MSCs at passage 2–5 were washed twice in PBS and further cultured in an exosome-free medium as described above. Briefly, cell culture medium was harvested after 48 h of incubation with exosome- free medium and stored at -80 ℃ for subsequent experiments.
Preparation of plasma samples
Samples were mixed from 3 female and 2 male felines presented at Affiliated Animal Hospital, Department of Veterinary Medicine of Foshan University. Blood samples are collected into acollections tubes containing anticoagulant and the cell components were removed by centrifugation (800 ×g, 4 ℃, 15 min). The supernatant was diluted with phosphate buffered saline of the same volume (1:1) and stored at -80 ℃ for subsequent experiments.
Preparation of Urine samples
Urine samples were mixed from 1 female and 2 male felines presented at Affiliated Animal Hospital, Department of Veterinary Medicine of Foshan University. Samples are collected into tubes and stored at -80 ℃ for subsequent experiments.
Isolation of exosomes
Exosomes were isolated by differential centrifugation. Briefly, Cell culture medium (50 mL) were centrifuged at 4℃,300×g for 10 min to remove dead cells, followed by centrifuging at 12,000× g for 30 min at 4°C to remove cell debris. Supernatant was collected and filtered through 0.22 mm filters (Merck Millipore, USA) to remove contaminating microvesicles. Following this, supernatants were transferred to polycarbonate tubes for ultracentrifugation in ultra-speed centrifuge (Beckman Coulter XL-90, SW28Ti rotor; Beckman Coulter; USA) at 100,000 × g for 70 min at 4℃. Diluted plasma samples (5 ml) were centrifuged at 12,000× g for 30 min at 4 ℃ to remove cell debris. Clarified supernatant was then ultracentrifuged at 50,000 × g for 70 min at 4℃ to remove large proteins and microvesicles. Following this, supernatants were transferred to polycarbonate tubes for ultracentrifugation at 100,000 × g for 70 min at 4℃. Urine samples (25 ml) were centrifuged at 4℃༌300×g for 10 min to remove dead cells, followed by centrifuging at 12,000× g for 30 min at 4°C to remove cell debris. Clarified supernatant was then ultracentrifuged at 50,000 × g for 70 min at 4℃ in ultra-speed centrifuge remove large proteins and microvesicles. Following this, supernatants were transferred to polycarbonate tubes for ultracentrifugation at 100,000 × g for 70 min at 4℃. The all final pellets were resuspended in 100 µl PBS and then stored at -80 ℃ until further analysis.
Transmission electron microscopy (TEM)
Exosome samples were diluted in PBS, dropped on a carbon-coated copper grid, and then stained with 1% uranyl acetate for 1 min. Grids were imaged under a Hitachi H-7650 transmission electron microscope.
Flow nano analyzer
Exosome samples were diluted 1:100 and analyzed using the Flow Nano Analyzer (NanoFCM Inc.) according to manufacturer’s protocol. Briefy, the lasers were calibrated using 200 nm control beads (NanoFCM Inc.), which were then analyzed as a reference for particle concentration. Additionally, a mixture of diferent sized beads (NanoFCM Inc.) was analyzed to set reference for size distribution.
Exosome samples were denatured in protein loading buffer (10% sodium dodecyl sulfate (SDS), 250 mM Tris-HCl (pH 6.8), 0.5% Bromophenol blue, 50% glycerin, 5% β-Mercaptoethanol) at 95°C for 10 min. Proteins were separated by 10% sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE), and were then transferred to a transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, USA). The membranes were blocked were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 for 1 hour at room temperature and afterwards incubated at room temperature for 1 hour with antibodies against TSG101 (Santa Cruz, sc-7964, 1:1000), CD81 (Affinity, DF2306, 1:1000), CD63 (Santa Cruz, sc-5275, 1:1000) and CD9 (Affinity, AF5139, 1:1000), followed by incubation with horseradish peroxidase conjugated secondary antibodies at room temperature for 1 hour. Luminescent visualizationwas done using an ECL kit (Tanon, China) to identify immunoreactive protein bands.