Cell Culture
Mesenchymal Stem Cells
Ethical clearance was obtained from Institutional Committee for Stem Cell research (ICSCR), AIIMS, New Delhi (Ref No. IC-SCR/120/21(R); dated 14-06-2021). All research was performed in accordance with guidelines and regulations proposed by ICSCR (AIIMS). Mesenchymal Stem Cells (MSCs) were isolated from healthy donors after obtaining written consent. In the current study, MSCs were derived from Bone Marrow (BM), which was collected from the donors (n=3) undergoing routine medical test procedure in Department of Haematology, AIIMS, New Delhi. BM-MSCs isolated and cultured as per previously established protocols 43,44.
Briefly, the BM-MSCs were cultured in 1X LG DMEM media (Invitrogen-Gibco, USA) supplemented with 10% MSC grade FBS (Himedia, India) and 1% Anti-Anti (Thermo Fischer Scientific, USA). The media was changed on alternate days. Upon reaching 80% confluency (3×104 cells/cm2), the cells were collected using 0.05% trypsin-EDTA (Invitrogen-Gibco, USA) and respectively passaged. BM-MSCs within the passage P3-P5 were used in this study. At P3, the MSCs were characterized for their surface marker profile using monoclonal antibodies specific for CD90-PerCp-Cy5.5, CD73-PE, CD29-FITC, CD105-APC, HLA-ABC-APC, HLA-DR-FITC, CD34/45-PE/FITC (BD Pharmingen, France). Acquisition and data analysis were performed using flow cytometer BD-LSR-II (BD Bioscience, USA) and FACS Diva Software Version 6.2.
The BM-MSCs were also characterized for their trilineage differentiation potential into adipocytes, osteocytes and chondrocytes as per previously established protocols43,44.
For all the experiments, BM-MSCs (n=3) were pooled together at P3, and cultured in STEMPRO® MSC SFM CTS (Thermo Fisher Scientific, USA) up to 75% confluency and then utilised for isolation of sEVs.
HeLa Cells
HeLa cells were procured from NCCS (Pune, India). They were cultured in 1X HG DMEM media (Invitrogen-Gibco, USA) supplemented with 10% FBS (Himedia, India) and 1% Anti-Anti (Thermo Fischer Scientific, USA). The media was changed on alternate days. Upon reaching 75% confluency (3×104 cells/cm2), the cells were collected using 0.05% trypsin-EDTA (Invitrogen-Gibco, USA) and respectively passaged.
Isolation of MSCs derived sEVs
BM-MSCs were cultured upto 75% confluency (3×104 cells/cm2) in 1X LG DMEM media, supplemented with 10% MSC grade FBS and 1% Anti-Anti. The media was then changed to serum free media STEMPRO® MSC SFM CTS (Thermo Fisher Scientific, USA) supplemented with 1% Anti-Anti and the cells were cultured for 24 hours, forth which the conditioned media was collected for sEVs isolation. The sEVs were isolated using one step sucrose ultracentrifugation-based method as previous described 23. Briefly, the conditioned media was centrifuged at 300×g for 10 minutes to remove cellular debris. Further, it was centrifuged at 10,000×g for 30 minutes for the removal of larger vesicles. This was followed by the loading of supernatant carefully over a layer of 30% sucrose solution and centrifugation at 1,00,000×g at 4°C for 90 minutes using Optima XPN100 ultracentrifuge in swinging bucket rotor (Beckman Coulter, USA). After this, the sucrose layer was collected and washed using 1X PBS by centrifugation at 1,00,000×g at 4°C for 90 minutes to pellet down the sEVs. The pelleted sEVs were then resuspended in 500 µl 1X PBS and stored at -80˚C for further use.
Isolation of HeLa Cells derived sEVs
HeLa cells were cultured up to 75% confluency (3×104 cells/cm2) in 1X HG DMEM media, supplemented with 10% FBS and 1% Anti-Anti. The media was then changed to 1X HG DMEM without FBS supplemented with 1% Anti-Anti and the cells were cultured for 24 hours, forth which the conditioned media was collected for sEVs isolation. The sEVs were isolated using one step sucrose ultracentrifugation-based method as previous described 23. The pelleted sEVs were then resuspended in 500 µl 1X PBS and stored at -80˚C for further use.
Characterization of sEVs
Both the MSCs & HeLa cells derived sEVs were characterized & assessed as per the following techniques-
a. Nanoparticle Tracking Analysis (NTA): The sEVs were diluted in 1X PBS at a ratio of 1:100 and analysed by NanoSight NS300 (Malvern Panalytical, United Kingdom). The Brownian motion of each particle was tracked between frames and the size was calculated using the Strokes-Einstein equation via the NS300 instrument. The camera level was kept between 14-16, and the detection threshold was maintained between 4-10. 3 sample replicates were analyzed with videos captured for 60 seconds each.
b. Transmission Electron Microscopy (TEM): The sEVs suspension was diluted at concentration of 1:1000 in 1XPBS and was coated onto Formvar-carbon coated copper grids. The sEVs were allowed to adsorb over the grid for about 5 minutes and then the grids were stained with 2% Phosphotungstic acid solution for about a minute. Grids were then air dried and observed under the transmission electron microscope (Tecnai, FEI, USA).
c. Western blotting: sEVs were lysed with RIPA buffer containing protease inhibitors (Sigma, USA). The Protein concentration of lysates was measured using Bicinchoninic Acid protein assay kit (Pierce, USA). Equal volume (30µL) or equal amount (20µg) of the protein lysate was subjected to SDS-PAGE, based on the requirement of the assay, with 12% concentration in non-reducing conditions for CD63 (1:1000; Thermo fischer, USA) and reducing conditions for ALIX (1:500; Genetex, USA), Calnexin (1:3000; Genetex, USA) and GAPDH (1:3000; Genetex, USA). The molecular weight of proteins was identified using Precision Plus Protein Dual Color Standards (Biorad, USA). It was then transferred to a PVDF membrane (MDI) using wet transfer system (Biorad, USA). The blot was blocked with 5% Non-Fat Skimmed milk in 1X TBS-T solution for 1 hour followed by the incubation in primary antibody overnight at 4°C. The blot was then washed, and incubated with respective HRP-conjugated secondary antibody and visualized using ECL imager (Invitrogen, USA).
Transfection Studies
For gene knockdown & sEVs collection, BM-MSCs and HeLa cells were seeded in 6 well plates (Nest, China) at a density of 1×105 cells per well. After 24 hours media was changed to optiMEM (Gibco, USA) and siRNA (Santacruz) were transfected at a concentration of 50nM using Lipofectamine RNAiMAX (Thermo fischer scientific, USA) as per manufacturer’s protocol. The conditioned media was collected 24 hours after transfection for sEVs isolation. A pool of 3 target specific siRNAs for each gene were used at a concentration of 25pmol/well in 6 well plates as per manufacturer’s instructions (Santa Cruz Biotechnology, USA). FITC-conjugated non-targeting siRNA was used as a control (Santa Cruz Biotechnology, USA).
Immunofluorescence Assay
BM-MSCs and HeLa cells were seeded on glass coverslips in 24 well plates (Nest, China) at a density of 1×103 cells per well. Transfection was performed as previously described, with the siRNA concentration of 5pmol/well, and the cells were then processed for the Immunofluorescence assay as per previously established protocols43. Briefly, the cells were processed for fixation, blocking and permeabilization, followed by overnight incubation with the primary anti mouse antibody-CD63 (1:100; Thermo fischer, USA). The cells were then washed thrice with 1X PBS and incubated with AF488 conjugated anti-mouse secondary antibody (1:500; Thermo fischer, USA) for 40 minutes at room temperature in dark. Finally, after washing thrice with 1X PBS, cells were counter stained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize the cell nuclei. Cells were washed thrice with 1X PBS to remove excess DAPI stain, and imaged using Olympus IXplore Pro Microscope (Olympus, Japan).
RNA Isolation and Real time PCR analysis
For the isolation of total RNA, sEVs from BM-MSCs & HeLa each were lysed with Qiazol Reagent (Qiagen, Netherlands) and miRNA was isolated using miRNeasy mini kit (Qiagen, Netherlands) as per manufacturer’s protocol. For experiments with equal protein concentration, 100 µg of sEVs were lysed, while for experiments with equal starting volume, 100 µL of sEVs were lysed. cDNA was prepared using PCR starter kit (Qiagen, Netherlands) and qPCR was conducted with PCR starter kit (Qiagen, Netherlands) in CFX96 thermocycler (Biorad, USA). Primers for hsa-miR125b-5p, hsa-miR145a-5p, hsa-miR34a-5p, hsa-miR21-5p were commercially synthesised and procured from Qiagen, Netherlands.
Statistical Analysis
Statistical analysis was performed using Two-way ANOVA, post-test Tukey in GraphPad Instant software (GraphPad Software, Inc.). All the data were expressed as mean ± s.d. from n=3, unless mentioned differently. For all analyses, differences with a p value <0.05 were considered statistically significant.