Cell Isolation and Characterization of Mesenchymal Stem Cells
Cells were obtained from human umbilical cord samples with ethical approval. Cells were isolated using the explant method. Characterization of mesenchymal stem cells was analyzed with multipotency assay and flow cytometry.
For multipotency assay, the passage of cells 5 were seeded at 20,000 cells/well in a 24-well plate. After the cells reached 70-80% confluence, the culture medium was changed to chondrogenic medium (StemPro Chondrogenic Differentiation Kit), adipogenic medium (StemPro Adipogenic Differentiation Kit), and osteogenic medium (StemPro Osteogenic Differentiation Kit). The culture was incubated at 37 0C, 5% CO2 for 21 days, and the medium was changed every 2 days. After 21 days of culturing, the remaining medium was discarded and the culture washed with PBS and fixed with 4% formaldehyde. The cells were stained with Alcian Blue, Oil Red O, and Alizarin Red. The culture was observed with an inverted microscope.
The specific surface marker of mesenchymal stem cell was analyzed with flow cytometry assay using Human MSC Analysis Kit (BD Bioscience) according to manufacturerprotocols. The hWJ-MSC used for flow cytometry assay is passage 5. Flow cytometry was performed using BD FacsLyric.
Proliferation of Cell in Serum Free Medium
The hWJ-MSC passage 5 were seeded at 2,000 cells per well in 96-well plate and cultured in DMEM-FBS or DMEM or RPMI 1640. The culture cells were incubated at 37 0C, 5 % CO2. Assessment of cell proliferation was measured in terms of optical absorbance (OD) by using MTT assay in 6 days of incubation. The absorbance was measured every day with Biorad Microplate Reader at the wave length of 595 nm.
Cell Viability in CoCl2 Medium
The hWJ-MSC passage 5 were cultured in growth medium containing CoCl2 with the concentration 25 μM, 50 μM, 100 μM, 200 μM and 400 μM. The culture cells were incubated at 37 0C, 5 % CO2. The MTT assay was used to assess cell viability in terms of optical absorbance (OD) after 24 and 48 hours of incubation. At a wavelength of 595 nm, the absorbance was measured using a Biorad Microplate Reader.
Exosomes Isolation
The hWJ-MSC passage 5 were cultured in a medium consisting of DMEM LG, antibiotic antimycotic, and FBS 10%. At near 70% of confluence, the cells were washed with PBS. The medium was changed to a culture medium without FBS and the cells were incubated in a humified incubator (37 0C, 5% CO2) for 48 hours. Supernatant was collected and exosomes were isolated. Exosomes were isolated using Invitrogen Total Exosomes Isolation according to manufacturer instructions. Briefly, the conditioned medium was collected and centrifuged at 2000 x g for 30 minutes. The supernatant was collected, added with 0.5 volume Invitrogen reagent, and vortexed to mix. Samples were incubated at 40C overnight. The samples were centrifuged 10,000 x g for one hour at 40C. The supernatant was discarded and the pellet was resuspended in 1x PBS.
Characterization of Mesenchymal Stem Cells-derived exosomes
The presence of exosomes and its morphology visualized with Transmission Electron Microscope. Briefly, ten microliters of exosomes suspension in PBS were placed onto a 300 mesh copper grid with carbon-coated film. The grids were blotted with filter paper and washed with double-distilled water. The samples were stained with uranyless and observed with a Hitachi HT7700 Transmission Electron Microscope.
ELISA was performed to analyze exosomes marker (CD 63). Human CD63 ELISA kit (Thermo Scientific) was used according to manufacturer instructions. Briefly, 100μL of each standard and sample were put into the appropriate well and incubated for 2.5 hours at room temperature. The solution was discarded and washed with wash buffer. The biotinylated antibody was added to each well and incubated for 1 hour at room temperature. The solution was discarded and washed again with wash buffer. Streptavidin-HRP was added into each well, incubated for 45 minutes at room temperature, and the solution then discarded and washed with wash buffer. TMB substrate was added into each well and incubated for 30 minutes at room temperature. Stop solution was added and the absorbance was read with Biorad Microplate Reader at 450 nm.
Exosomes in PBS were visualized by Horiba Scientific ViewSizer 3000 / 0053. Collected data were analyzed with Nanoparticle Tracking Analysis (NTA) software and provide particle size disribution profiles and concentration measurements.
Alcian Blue Assay
Alcian Blue assay was performed to evaluate the presence of glycosaminoglycan (GAG) in the cell culture. The medium of cell culture was aspirated and washed with phosphate buffer saline twice. The cells were fixated with acetone-methanol in 4 C for three minutes. 1% Alcian Blue staining (in 3% acetate acid) was added and incubated in room temperature for 30 minutes. The samples were washed with 3% acetate acid and the washed again with deion. The samples were added with 1% SDS and incubated for 30 minutes with shaker 200 rpm. The absorbance of samples were measured with UV-Vis spectrophotometry at a wavelength of 605 nm.
Immunocytochemistry of Collagen type 2
Medium of cell cultures were discarded and the cells were washed with phosphate buffer saline (PBS). All samples were fixed with paraformaldehyde and permeabilized with PBST (0,05% tween 20 in PBS). BSA was used as blocking agent and the samples were added with primary antibody collagen type 2 and incubated overnight. After incubation, the samples were washed with PBS and added secondary antibody Alexa Flour 488 and counterstaining with DAPI for nucleus staining. All samples were observed with Confocal Laser Scanning Microscope Olympus Fv1200.