MSC isolation and characterization.
MSC were isolated from bone marrow aspirate of male 6-week old C57Bl/6 mice as previously described (12). Cells were used between 6th and 12th passages. MSC were routinely characterized for the presence of specific surface markers (by flow cytometry) and for the potential to generate adipocytes, osteoblasts and chondrocytes, when cultured under appropriate conditions (Fig. 1). Flow cytometry analysis demonstrated the absence of the hematopoietic cell marker CD45 and the presence of surface antigens usually associated to MSC (Sca-1, CD105, CD29, and CD90). Adipocytes, osteoblasts and chondrocytes were evidenced by Oil Red O staining of lipid droplets (Fig. 1a), von Kossa staining of calcium deposits (Fig. 1b), and Alcian Blue staining of acid mucopolysaccharides (Fig. 1c), respectively.
Assessment of the immunosuppressive potential of MSC.
The procedure has three major steps, which are to be performed in different days:
Step 1 (Day 0; ~2 hours): Mitotic inactivation of MSC followed by seeding on tissue-culture wells. Around 1.5 × 10^5 cells are enough for the procedure. This step is performed one day before the interaction of MSC with T cells.
Step 2 (Day 1; ~ 6 hours): Isolation of splenic T cells and fluorescent labeling with Carboxy-fluorescein succinimidyl ester (CFSE) before the addition onto MSC culture in the presence of CD3/CD28 activation beads.
Step 3 (Day 4, ~ 4 hours, including flow cytometry analysis): Assessment of the proliferation index of T cells after three days in culture, based on CFSE dye dilution assay.
MSC irradiation.
MSC were detached with 0.25% trypsin and re-suspended at a density of 10^5 cells/ml in MSC complete medium (DMEM low glucose, supplemented with 10% MSC qualified FBS, in the presence of 1% antibiotic-antimycotic solution). Cell irradiation was performed with a RS-2000 X-ray irradiator at 30 Gy dose, in 1.5-ml microcentrifuge tube. We routinely irradiated the tubes in the middle of the third shelf (for an evenly radiation distribution) for 16 minutes and 36 seconds. After irradiation, cells were seeded in 96-well plate wells, using serial dilutions ranging from 10.000 to 625 cells/well. Technological triplicates were used for each MSC dilution. The irradiated cells were placed in the incubator under 37ºC and 5% CO2 atmosphere and left overnight before interaction with T cells.
Isolation of mouse splenocytes.
Mice were sacrificed by cervical dislocation and prepared for dissection by disinfecting the fur and skin with 70% alcohol. The peritoneum was exposed through a midline incision of the skin. The peritoneal wall was cut along the linea alba and then laterally under the ribcage, towards the left side of the mouse. The bowels were gently grabbed and pulled aside to expose the spleen, which was detached (by cutting the connections with other tissues) and introduced into a 15-ml tube containing 2 ml ice-cold PBS/FBS (PBS supplemented with 2% FBS). Under the laminar flow hood, the tube content (spleen and PBS/FBS) was transferred into a 70-µm cell strainer adapted on the top of a 50-ml tube. The spleen was gently triturated (without cutting in pieces), using the thumb head of a 5-ml syringe plunger, while adding 10 ml of PBS/FBS to wash the strainer. The cell suspension was centrifuged at 400Xg, 4⁰C, for 10 minutes, then the cell pellet was resuspended in 5 ml ice-cold red blood cell lysis buffer and incubated at 24°C for 4 minutes. The lysis was stopped by adding 10 ml ice-cold PBS/FBS and centrifugation at 400Xg, 4⁰C, 10 minutes. Cells were resuspended in 2 ml complete RPMI medium (RPMI ATCC-formula, supplemented with 10% FBS and 50 µM β-Mercapto-ethanol), counted and used in T cell enrichment step. An aliquot of around 20 µl (~ 10^6 cells) was saved for flow-cytometry analysis of splenocytes.
T cell enrichment from splenocytes using the nylon wool column.
The protocol described by Hathcock, with a few minor changes, was used with good results (13). The nylon wool column was prepared in advance by inserting 1 g of nylon wool into a 10-ml sterile syringe and autoclaving at 110⁰C for 15 minutes. Briefly, T cell enrichment followed these steps: 1) the column was placed on the stand and a 3-way stopcock with a 19G needle was attached to the column; 2) with the stopcock in open position, the column was washed 3 times with 8 ml warm RPMI complete medium. During the first wash, the wool was firmly pressed with a 10-ml sterile serological pipette to remove the air bubbles; 3) the stopcock was closed, then 2 ml warm complete RPMI medium was added to the column and the column was allowed to equilibrate in the incubator for at least 45 minutes; 4) the column was moved back in the cell culture hood, the stopcock was opened and the column was let to drain completely; 5) the 2-ml splenocyte suspension was carefully layered on the column surface (not on the wall of the syringe) and the suspension was allowed to drain completely; 6) the column was washed with 0.5 ml warm complete RPMI medium to ensure that all cells had penetrated the wool; 7) the stopcock was closed, and 2 ml warm complete RPMI medium was added onto the column before being placed in the incubator for another 45 minutes; 8) the column was removed from the incubator, the 19G needle was carefully replaced with a 23G needle, the stopcock was opened and T cells were eluted by washing 4 times with 10 ml warm complete RPMI medium; 9) the eluate (~ 40 ml) was centrifuged at 400Xg, 24ºC, 10 minutes, and then the cells were resuspended in 1 ml warm medium. An aliquot of 50 µl was saved for counting (with an intermediate dilution of 1/50) and flow-cytometry analysis. The remaining cells were washed with 10 ml PBS/FBS and centrifuged at 400Xg, 24ºC for 10 minutes. At the end, the cells were resuspended in 950 ul PBS + 1% FBS and transferred in a 15-ml conical tube.
T cell labeling with CFSE.
Uniform labeling of T cells is important for accurate determination of cell proliferation. Our protocol followed the next steps: 1) First, a vortex was brought in the hood and 50 µl CFSE 100 µM was freshly prepared in PBS with 1% FBS (from a concentrated stock solution); 2) While keeping the tube with 950 µl cell suspension in a 45⁰ position, the 50-µl CFSE solution was carefully placed as a stable drop on the upper part of the wall inside the 15-ml tube, making sure that the cell suspension and CFSE solution did not make contact; 3) the vortex was turned on, then the lid on the tube was closed and immediately turned upside down and vortex for 1–2 seconds to ensure the rapid and homogenous dispersal of CFSE solution (the final concentration of CFSE was 5 µM); 4) the tube was then incubated at 24 °C in the dark for 10 minutes; 5) the 15-ml tube was filled up with complete RPMI medium and centrifuged at 400Xg, 4 °C, 10 minutes; 6) the supernatant was decanted and the wash procedure was repeated for another 2 times with 10 ml complete RPMI medium; 7) the cell pellet was resuspended in complete RPMI medium at a cell density of 10^6 cells/ml for plating. An aliquot of 200 µl (around 2 × 10^5 cells) was saved for flow-cytometry validation of the homogenous staining.
Co-culture of T cells with the irradiated MSC.
The immunosuppressive effect of MSC was evaluated by culturing T cells with irradiated MSC in activating conditions. Different MSC: T cell ratios, within the range 1:10 to 1:160, were obtained by serial dilutions of MSC suspension, while keeping the number of T cells constant. The greatest number of MSC per well (104 cells/well) was calculated so that to obtain a confluent layer of inactivated cells. Activation of T cells was done by mixing T cells with anti-CD3/CD28 microbeads in a 1:1 ratio (bead: T cell) in RPMI complete medium. Control wells important in this step: irradiated MSC only (to ensure that after 3 days in culture, irradiated MSC are healthy); activated T cells cultured without MSC (to determine the maximum level of T cell proliferation in culture); T cells cultured in the absence of anti-CD3/CD28 beads (to establish the fluorescence median value of CFSE after three days in culture for accurate assessment of the proliferative index).
Flow-cytometry analysis.
After 3-days of co-culture of T cells with MSC, all cells and beads in the wells were collected for flow cytometry analysis. To achieve this, the following steps were performed: 1) the supernatant of each well was collected in separate 1.5-ml microcentrifuge tubes; 2) the wells were washed with 100 µl PBS/well with recovering of the washed volume in the corresponding tube; 3) the cells were trypsinized by incubating each well with 30 µl Trypsin-EDTA solution (0.25% Trypsin mixed with 0.2 g/l EDTA-4Na in Hank's Balanced Salt Solution) for two minutes, followed by trypsin inactivation with 100 µl complete RPMI medium and collection of whole volume in the corresponding tube; 4) each well was washed with an additional 100 µl PBS, and the washed volume was saved in the same corresponding tubes (the final volume in each tube was around 450 µl); 5) the tubes containing beads were placed in the magnet for 2 minutes, and the cell-containing medium was recovered in fresh tubes; 6) the beads were washed with 500 µl PBS/FBS by pipetting and re-placing the tubes into the magnet for another 2 minutes; the supernatant was recovered in the corresponding fresh tube (the total volume of cells was around 1 ml/tube); 7) the cells were centrifuged at 300Xg, 4 °C, 5 minutes and resuspended in 250 ul PBS/FBS; 8) 2 µg/ml propidium iodide was added to each sample and cells were analyzed using a Beckman Coulter CytoFlex flow cytometer.