An In Vitro Assay for Evaluating the Immunomodulatory Impact of Monocytes on Leukocyte Proliferation

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Density Gradient Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs)

1. Add 25 ml heparinized whole blood into a 50 ml tube by a 25 ml pipette.
   1. Dilute cells with 25 ml of phosphate-buffered saline (PBS).
2. Add 15 ml of Ficoll-Paque density gradient into a new 50 ml tube, and while tilting the tube, very slowly and carefully add in 25 ml of the diluted cell suspension over the density gradient so that there is no mixing of the whole blood with the density gradient, i.e., no disturbance of the blood-density gradient interface.
3. Centrifuge the suspension from step 1.2 at 400 × g for 30 to 40 min in a temperature-controlled swinging-bucket rotor without brake at 20 °C.
   Note: Three distinct layers should be apparent after centrifugation: the upper layer being plasma; the bottom clear layer being the Ficoll-Paque density gradient; and a thin, middle cellular layer being the PBMCs.
4. Aspirate and discard the upper layer by suction with a Pasteur pipette, with care not to disturb the interphase layer of mononuclear cells (i.e., lymphocytes, monocytes, and platelets).
5. Carefully collect this mononuclear cell layer with a 10 ml pipette to a new 50 ml centrifuge tube.
6. Fill the tube with 20 ml of PBS, mix well, and centrifuge at 300 × g for 10 min at 20°C. Remove the supernatant completely after centrifugation.
7. Resuspend the cell pellet in 20 ml of PBS and centrifuge at 200 × g for 10-15 min at 20 °C. Remove the supernatant completely after centrifugation.
   Note: This step removes the platelets-which are unwanted-within the PBMCs; this step can be repeated to ensure complete removal of the platelets.
8. If no selection of specific populations is desired, resuspend cell pellet in leukocyte complete medium (10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin/streptomycin in RPMI-1640 medium) for culturing after performing a cell count to suspend 1 ml of medium per 10⁷ PBMCs before proceeding to the next step.

2. Magnetic Labeling of Leukocyte Populations

1. Centrifuge PBMC suspension at 300 × g for 10 min and aspirate supernatant completely.
2. Resuspend cell pellet in 80 µl of PBS per 10⁷ total cells.
3. Add 20 µl of specified magnetic beads (i.e., CD14 magnetic beads for isolation of monocytes, CD4 magnetic beads for isolation of T lymphocytes) per 10⁷ total cells.
4. Mix well and incubate for 15 min at 2 to 8 °C in the refrigerator.
5. Add 1-2 ml of PBS per 10⁷ cells to wash, and centrifuge at 300 × g for 10 min.
6. Aspirate supernatant completely and resuspend up to 10⁸ cells in 500 µl of PBS.

3. Magnetic Selection of Leukocyte Subpopulations

Note: A variety of magnetic bead separators and columns are available for isolation of specific population of leukocytes by cell surface marker from a number of manufacturers. Isolation can be by positive selection based on one or a few positive markers or by negative selection after depletion of unwanted populations. A general approach of performing positive selection using one marker (i.e., CD4 for T helper lymphocytes, or CD14 for monocytes) is outlined below.

1. Prepare and prime magnetic separator including separation column according to manufacturer's instructions.
2. Apply tube containing the sample of labeled PBMCs. Provide two 15 ml centrifuge tubes for collection of the positively labeled and unlabeled cell fractions, following manufacturer's instructions.
3. Count cell number and resuspend the positively selected leukocytes (i.e., CD4⁺ T lymphocytes or CD14⁺ cells) in leukocyte complete medium, using 1 ml of medium per 10⁷ cells.

4. Carboxyfluorescein succinimidyl ester (CFSE) Staining of Leukocytes for Assessment of Proliferation

Note: CFSE has been widely used in immunological investigations, for both in vivo and in vitro studies. Our protocol has been optimized for in vitro study of MSC/PBMC (or other leukocyte) interactions. While the general steps involved in conducting CFSE in vitro labeling are similar, there may be differences in the specific dose and timing of the protocol. This may be due to a number of factors, i.e., the specific cell type used, cell numbers used-which can likely affect the intensity of the CFSE fluorescent signal.

1. Dilute the CFSE stock solution (10 mM) in PBS to the desired working concentration of 10 µM (CFSE-working solution). Prepare 10⁷ cells (i.e., PBMCs or T cells) for labeling with the CFSE-working solution.
2. Centrifuge at 300 × g for 10 min to obtain a cell pellet and aspirate the supernatant.
3. Resuspend the cells gently in 1 ml of pre-warmed (37 °C) CFSE-working solution and incubate the cells for 10 min at 37 °C.
4. To wash off excess CFSE, dilute the cell suspension with 10x (by volume) of precooled (4 °C) RPMI medium containing 10% FBS. Sediment the cells by centrifugation at 300 x g for 5 min and discard the supernatant. Wash the cell pellet in this manner twice more.
5. Re-pellet the cells by centrifugation and count cell number. Resuspend 10⁷ cells (either PBMCs or T cells) in 1 ml fresh prewarmed leukocyte complete medium.

5. Effector Suppression Assay Magnetic Bead-selected, MSC-induced Immunomodulatory Leukocytes on Activated CFSE-labeled Effector CD4 ⁺ T Cells

1. Seed MSCs at 250,000 cells in 3 ml of MSC complete medium in 6-well plates (MSC density: 25,000 cells/cm²) for attachment O/N in a 37 °C incubator, allowing for the stem cells to reach 80% confluence.  At least 3 6-well plates are necessary to ensure enough MSC-cocultured PBMCs for subpopulation selection. 
2. Aspirate medium and add PBMCs separated as per Step 1 but in 6-well plates at 2.5 x 10⁶ cells/well (cell density: 250,000 leukocytes/cm²) with 3 ml of leukocyte complete medium. Co-culture for 48-72 hr in a 37 °C incubator.
3. Magnetic bead-select specific population of MSC-induced immunomodulatory leukocytes (i.e., CD14⁺ cells) as per Sections 2-3.
   Note: Intracellular cytokine staining for flow cytometric analysis can be performed at this point to assess for changes in leukocyte cytokine expression profile, i.e., expression of interleukin-10-as modulated by MSCs.
4. Add CFSE-labeled allogeneic CD4⁺ T cells generated as per Sections 2-4 in 24-well plates in 1 ml of leukocyte complete medium (T cell density: 250,000 cells/cm²) to bead-selected MSC-induced leukocytes at various ratios, i.e., 1:10, 1:5, 1:2, and 1:1 (cell to cell) ratios.
5. To stimulate CD4⁺ T cells, add α-CD3/28 conjugated microbeads to obtain a bead-to-cell ratio of 1:1.
6. For a negative control, plate 500,000 CD4⁺ T cells/well in a 24-well plate (T cell density: 250,000 cells/cm²) with 1 ml leukocyte complete medium only; for positive control, in addition to plating the same number of CD4⁺ T cells/well, add α-CD3/28 conjugated microbeads to obtain a bead-to-cell ratio of 1:1.
7. On the 3^rd day of co-culture, assess proliferation of CFSE-labeled CD4⁺ T cells (placed in round-bottom tubes) by flow cytometric analysis with 488 nm excitation and emission filters appropriate for fluorescein.