Boyden Chamber Chemotaxis Assays

The chemotactic migration of cells was measured using a variety of Boyden chamber migration assays, depending on the experiment performed. First, the migration toward an MCP-1 concentration by the monocytes from the PBMC populations was evaluated. PBMCs were quickly thawed from cryopreservation and resuspended in complete cell media at a concentration of 1x10⁶ cells ml⁻¹. PBMCs (100,000 cells) were placed into the top chamber of a Boyden chamber in a 24 well plate using a PET filter with 5 μM pores (Corning). The bottom chamber was then filled with complete cell media supplemented with 0, 5 10, 25, 50, 100, 250, or 500 ng ml⁻¹ of MCP-1 (n=3 wells per concentration). Following 4 hours of incubation at 37 °C and 5% CO₂, the filters were removed and fixed in 70% ethanol for 10 minutes. The insides of the filter was then wiped with a cotton swab to remove cells that did not migrate and the filter was stained with hematoxylin for 10 minutes to visualize migrated cells. The filter was then washed in water, cut from the frame, and mounted onto a glass slide. The number of migrated cells were counted with a high power field (20x, Zeiss microscope) from 5 regions of each filter and the average migrated cells per area was calculated. Three filters were tested for each MCP-1 concentration and the entire experiment was performed with each of the three PBMC lines to confirm similar results between individuals. In all cell migration experiments the number of migrated cells was counted from bottom of the filter. This was to provide consistency between experiments, since future experiments contain tissue samples and upon observation there were very few cells suspended in the media.

Next, the migration of PBMCs toward MCP-1-loaded or non-loaded tissue was measured. MCP-1 loading into decellularized leaflet and conduit sections was performed as described earlier (n=5 per tissue type). Control, non-loaded leaflet and conduit sections were also included and were incubated for 24 hours in a solution of PBS with 0.1% BSA prior to testing (n=5 per tissue type). The MCP-1-loaded or non-loaded tissue sections were then used in a Boyden chamber assay using 5 μm pore filters as described previously. The tissue samples were placed into the bottom chamber along with complete cell media. A control group with only complete cell media in the bottom chamber was also included (n=4). PBMCs were prepared as previously described and placed in the top chamber (100,000 total cells). The plate was then incubated for 4 hours at 37 °C and 5% CO₂. Following incubation, the insides of the filters were similarly wiped with a cotton swab and the whole filter was then fixed, stained, washed, cut from the frame, and mounted onto glass slides. Again, the number of migrated cells were counted with a high power field (20x) from 5 regions of each filter and the average migrated cells per area was calculated.

Finally, the successive migration of MSCs following PBMC migration was evaluated. Conduit sections of decellularized pulmonary valve were loaded with MCP-1 in a 100 ng/mL loading solution as described earlier. Only conduit sections were used due to the small amount of leaflet tissue available from each valve. PBMCs were thawed, resuspended at a concentration of 1x10⁶ cells ml⁻¹, and labeled with DiO cell labeling solution (5 μL/mL of media; ThermoFisher Scientific) for 20 minutes. MSCs were cultured until sufficient numbers were available and were labeled with DiI cell labeling solution (5 μL/mL; ThermoFisher Scientific) for 20 minutes. The MSCs were then washed and resuspended in complete cell media at a concentration of 1x10⁵ cells ml⁻¹. The MSC migration study included six groups, the test group and five controls (Figure 1). In the test group, MCP-1-loaded tissue sections were placed in the bottom chamber of a Boyden chamber assay using a 5 μm filter and the top chamber was filled with labeled PBMCs (100,000 cells). After four hours incubation, the PBMC filter was removed and an 8 μM pore filter was placed in the well with labeled MSCs (10,000 cells) in the top chamber. This group tested the subsequent migration of MSCs following PBMC migration (n=5). Additional groups (n=5 per group) included only MCP-1-loaded tissue in the bottom chamber, labeled PBMCs (100,000 cells) placed directly in the bottom chamber, and a negative control media only group. A positive control group was also included using the known stem cell chemoattractant stromal cell-derived factor 1 (SDF1) at a concentration of 100 ng/mL. Lastly, since FBS is a known chemoattractant, an additional negative control media only group without FBS was included using insulin-transferrin-sodium selenite supplement (Sigma Aldrich). For all groups, MSC migration occurred for 8 hours in an incubator (37 °C and 5% CO₂) using 8 μM pore filters. After incubation, the insides of the filters were wiped with a cotton swab and the filters were fixed in formalin, washed in PBS, cut from the frame, and mounted onto glass slides with a DAPI fluorescent mounting media. The whole filter was imaged using tiled images at 5x. Since the cotton swab could not wipe the outside edges of the filter, they were excluded from cell counting. The area of the counting region and number of migrated MSCs were calculated using Zeiss software.

Groups included for the MSC migration assay. (+)MCP-1 & Migrated PBMCs used MCP-1-loaded tissue to recruit PBMCs for 4 hours before the filter was switched and the top chamber was filled with MSCs. A (+)MCP-1 tissue control group used MCP-1-loaded tissue directly with MSC migration. A PBMC control group included PMBCs placed directly in the bottom chamber. A media only control used only media in the bottom chamber.