Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

The overall goal of this procedure is to examine how tissue resident cells influence the ability of vascular endothelial cells to support the recruitment of leukocytes during inflammation. This is accomplished by first establishing co cultures of endothelial cells with mesenchymal stem cells using one of two in vitro vascular models, the micro slide model and the filter model. These cultures are then treated with cytokine prior to the flow assay.

The flow-based aian system is then assembled in a step-by-step manner with modifications made depending on the model system being used. The final step is to perfuse leukocytes over the endothelial cells and observe their recruitment from flow as well as their subsequent behavior. Ultimately, these multicellular flow-based adhesion assays have shown that crosstalk between mesenchymal stem cells and endothelial cells suppresses neutrophil recruitment during inflammation.

This method can help answer key questions in the leukocyte trafficking field, such as how do tissue resident cells, for example, mesenchymal stem cells, modify this process? Does this change in diseases such as chronic inflammation or cancer? Can we use this knowledge to help develop new therapeutic strategies To begin establish co cultures of endothelial cells with mesenchymal stem cells using the micro slide model or the filter model as described in the text protocol to assemble the flow system.

First turn on the heater and set to 37 degrees Celsius. Then attach a 20 milliliter syringe with the plunger removed, as well as a five milliliter syringe to a three-way tap. Next measure and cut a long piece of 2.4 millimeter tubing that is approximately the distance between the valve and the three-way tap.

Cut an eight to 10 millimeter long piece of 1.3 millimeter tubing and insert it into one end of the thick tubing. Attach the thick tubing side onto the three-way tap and connect the thin tubing. End onto a port of the electronic three-way micro valve to form the wash reservoir.

Then attach the tap to the perspex chamber using micropore tape. Next, cast a six to eight millimeter long piece of thick and thin tubing, and insert the thin tubing into one end of the thick tubing. Attach the thick tubing end onto a two milliliter syringe with the plunger removed.

Now connect the thin tubing end of the two milliliter syringe onto a port on the electronic micro valve to make the sample reservoir. Next measure and cut a long piece of thin tubing that is the distance between the micro valve and the center of the microscope stage. For the micro slide model, attach an eight to 10 millimeter piece of thick tubing to the end of the thin tubing.

Then place an L-shaped connector to the end of the thick tubing. This will connect to the micro slide or the filter model. Attach an eight to 10 millimeter piece of portex blue line manometer connecting tubing to the end of the thin tubing.

Next place the thin tubing end onto the micro valve. The tubing becomes the common output for the wash and sample reservoirs. Bill reservoirs with phosphate buffered saline containing bovine serum albumin.

Abbreviated PBSA prime the tubing by flowing PBSA through to remove any air bubbles, then attach manometer tubing to a 50 milliliter glass syringe. While the micro slide model only attach a 10 to 12 millimeter piece of thick tubing to the end of the manometer tubing leading to the glass syringe. Place an L-shaped connector onto the end of the thick tubing.

Prime the syringe by filling with 10 milliliters of PBSA. Invert the syringe so that the end connected to the tubing faces upwards and push out all air bubbles before refilling with five milliliters of PBSA. Finally, place the glass syringe into a syringe pump for infusion and withdrawal.

Set the refill rate and diameter of the glass syringe to set up the micro slides for flow. Place the micro slide onto the stage of an inverted phase contrast microscope. Turn the three-way tap to allow PBSA to run through the inlet tubing.

Connect the L-shaped connector to the inlet port of a micro slide channel. Next place the L-shaped connector from the syringe pump into the outlook port of the channel. Clean any PBSA that has dripped onto the micro slide.

Alternatively, for the filter model first cut a piece of para film to the same size as the glass cover slip using a metal template. Then cut out a 20 by four millimeter slot in the para film using a metal template to create the flow channel. Use the paraform gasket to mark the flow channel on the glass cover slip.

Align the edges of the six. Well filter on the glass cover slip. Make certain that the filter covers the flow channel markings Carefully cut out the filter using a Type 10 a scalpel.

Next carefully cover the filter with a paraform gasket, ensuring that the flow channel slot is in the middle of the filter. Use a piece of clean tissue and push out any bubbles. Now place the cover slip in the recess of the bottom plate of the Perspex flow chamber.

Position the top perspex plate over the top of the gasket and screw the plates together. Turn the three-way tap to allow the PBSA wash buffer to flow through the valve. Then connect the manometer tubing to the inlet port of the top perspex plate.

Run PBSA through the flow channel to allow bubbles to pass through. Then connect the manometer tubing from the syringe pump into the outlet port of the perspex plate. Lean any PBSA that has dripped onto the upper plate of the chamber.

Finally, place the chamber on the stage of an inverted phase contrast microscope and adjust the focus to visualize the endothelial cells above the filters to perfuse leukocytes over endothelial cells. Set the syringe pump to refill and press run once the focus has been adjusted. To visualize the endothelial cell monolayer, put two milliliters of purified neutrophils into the sample reservoir and leave to warm for two minutes.

Then wash the endothelium with PBSA for two minutes. Turn the valve on to perfuse neutrophils over endothelium. Deliver the neutrophil bolus for four minutes.

Turn the valve off and perfuse PBSA from the wash reservoir for the remainder of the experiment. When performing the experiment, ensure the air bubbles do not pass through at any point during the assay, as this will disrupt the endothelial cell mono layer and cause detachment of adherent neutrophils. Record neutrophil recruitment either during neutrophil flow or post perfusion Using digital image capture software.

Identify the center of the channel by moving the objective to the edge of the channel at the inlet port and identifying the middle of the port. Make all digital recordings of at least five to 10 fields in the center of the flow channel or recording During neutrophil flow. Take images of a single field every 10 seconds for one minute, move along the channel and record another field for one minute.

Repeating this process for the duration of the bolus for recording post perfusion. Make a ten second recording of five to 10 fields down the center of the flow channel for assessing leukocyte behavior. Take images every second within the ten second interval.

This allows sufficient time for capture from flow and the behavior of a adherent neutrophils to be analyzed. Record a single field containing at least 10 trans migrated neutrophils for five minutes, taking images every 30 seconds. This can be used to calculate the velocity of migrated cells, record another series of ten second fields.

This allows neutrophils time to migrate through the endothelial monolayer. Finally, stop the syringe pump and remove the outlet tubing, and then the inlet tubing disassemble the flow chamber and rinse the sample reservoir with PBSA repeat for subsequent filters and micro slide channels before proceeding to analysis of leukocyte recruitment and behavior as described in the text protocol. In the micro slide model, cytokine stimulation with tumor necrosis factor alpha significantly increased neutrophil adhesion to the endothelium in a dose dependent manner.

In addition, A TNF alpha dose dependent decrease was observed in the percentage of neutrophils rolling on the endothelial surface. This was reflected in A-T-N-F-L for dose dependent increase and the percentage of neutrophils migrating through the endothelial monolayer and thus undergoing transgender endothelial migration. Similar results were observed with the filter based flow assay.

For example, TNF alpha stimulation increased neutrophil adhesion in a dose-dependent manner. Co-culture of endothelial cells with bone marrow derived mesenchymal stem cells significantly reduced neutrophil adhesion when compared to endothelial cells. Cultured alone as expected for the micro slide model.

Again, similar effects were observed when the filter based model was used with less adhesion seen on the co-culture versus the monoculture. After watching this video, you should have a good understanding of how to examine how stromal cells communicate with endothelial cells to influence the recruitment of leukocytes during inflammation.