Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

Transcript

The use of this endothelial cell culture system enables assimilation of the situation in the blood flow. And with this situation, we can analyze bacterial infection processes also including the identification of bacterial vectors and also of cell vectors which are involved in this process. This technique paves the way to explore the impact of bacterial infections on the vascular integrity, also including the impact on morphogenesis mechanisms, on inflammation responses and immune regulation.

This technique requires some special handling of the endothelial cells and therefore a visual description is better than just a verbal explanation. This method provides insights into several other areas of research correlated with perfused vascular systems such as endothelial cell differentiation, wound healing, tumor genesis and angiogenesis. One of the key steps is standardized cell cultivation as well as accurate handling of primary endothelial cells.

This technique requires some special cell culture handling which couldn't be clear by just verbal explanation. To begin, work in a sterile environment using a clean bench. Use a pipette to inject 100 microliters of a 2%sterile filtered porcine gelatin PBS solution into a single reservoir of a temperature equilibrated channel slide.

Place the gelatin-coated channel slide on a thin polystyrene or styrofoam plate to prevent a drop in the slide temperature with a one milliliter Luer syringe to add 100 microliters of the four times 10 to the fifth per milliliter HUVEC suspension into the slide to seed and cultivate the HUVEC. Incubate the channel slide with the HUVEC for 60 minutes at 37 degrees Celsius and 5%carbon dioxide. After that, fill up each medium reservoir at both ends of the channel slide with 60 microliters of ECGMS medium.

Incubate for another one hour at 37 degrees Celsius and 5%carbon dioxide. Next, to adjust the microfluidic pump, first connect the equilibrated perfusion set to the pump unit, fill with 13.6 milliliters of ECGMS medium and start the pump control software. In the menu of the fluidic unit setup, select the adequate perfusion set and type of chamber slide using the scroll down windows.

Choose 0.007 dyne second per square centimeter in the software for medium viscosity. Outside of the incubator, connect a glass bottle filled with drying silica beads to the air pressure tubing. Select flow parameters in the software menu, set the pressure to 40 millibar and flush the pump tubes with the liquid medium by starting the continuous medium flow.

We ensure a tight cell attachment by using subconfluently grown HUVECs and additionally focus on getting air bubbles out of the system as well as using independent power supply for the pump system. Tap at the tubing connections to remove air bubbles and ensure that no air bubbles are circulating in the pump system. For connecting the channel slide, stop the flow circulation in the pump control software and hold the medium flow and the perfusion tubing by clamping the tubes near the Luer connection.

Connect the channel slide thereby avoiding air bubbles. In the pump control software advanced tab, use the software tool cycle creator to program the desired shear stress cycles for flow cultivation. Program the shear stress levels for flow circulation and start long-term perfusion.

Start with five dyne per square centimeter which corresponds to a flow rate of 5.44 milliliters per minute for 30 minutes followed by continuous flow at shear stress of 10 dyne per square centimeter which corresponds to a flow rate 10.85 milliliters per minute. Control balanced reservoir pumping. Place the fluidic unit with the connected channel slide in an incubator at 37 degrees Celsius and 5%carbon dioxide.

Start the microscope software control and adjust the principle settings for the fluorescence microscopic monitoring by selecting the appropriate filter settings. For microscopic visualization, place the fluidic unit into the 37 degrees Celsius heating chamber. Place the channel slide on the stage of a prewarmed microscope.

Control the cell morphology and the integrity of the HUVEC layer prior to injection of histamine and bacteria to the flow circulation. Maintain the flow setting. Induce the release of VWF from endothelial viable Palade bodies by injecting 136 microliters of a 100 millimolar histamine stock solution through an injection port into the ECGMS medium circulating in the perfusion tubing.

For immunofluorescence detection of multimerized VWF strings, inject 20 micrograms of a VWF specific FITC conjugated antibody in a volume of 200 microliters of PBS into the circulating 13.6 milliliters of ECGMS medium. To quantify Pneumococcal attachment to the VWF strings generated on HUVEC cell surfaces, inject 1.35 times 10 to the eighth CFU per milliliter RFP expressing Pneumococci in a maximum volume of one milliliter into the ECGMS medium using the injection port. Select a 63X oil immersion objective for microscope magnification and adjust the fluorescence filter settings in the microscope software to the RFP channel with a 540 nanometer detection filter for detection of RFP expressing Pneumococci.

For quantification of bacterial attachment to the VWF strings, create snapshots of Z stacks of at least 30 representative field views each containing approximately 10 morphologically intact HUVEC and count the amount of Pneumococci. To evaluate bacterial attachment after fixation prior to immunofluorescent staining, stop the flow, remove 10 milliliters of ECGMS medium from the pump reservoirs, and add 10 milliliters of PBS supplemented with 5%paraformaldehyde and proceed according to the manuscript. In this protocol, the principle experimental steps begin with a pre-cultivation of primary endothelial cells to subconfluency in cell culture flasks.

Cells are then seeded into a gelatin-coated channel slide. Bacteria are grown on agar plates followed by cultivation in a complex liquid medium to mid log phase. For microfluidic cell culture, a channel slide with endothelial cells is connected to the perfusion tubes of a fluidic unit of the pump system and subjected to constant flow for cell differentiation.

The generation of the VWF strings was induced by histamine injection at a shear stress of 10 dyne second per square centimeter and microscopically monitored by immunofluorescence detection using FITC-labeled VWF specific antibodies. After injection of red fluorescence protein expressing bacteria to the circulating medium, Pneumococcus attachment to the VWF strings was microscopically visualized in real time by fluorescence emission at 450 nanometers. After fixation of the cells using PFA, differential immunofluorescence staining provides the visualization of bacterial attachment at specific infection time points.

The most important step of this procedure is to ensure a tight cell attachment on the slide surface by adjusting physiological shear stress levels and avoiding extreme pump pressure fluctuations. In addition to the microscopic visualization, this technique can be applied to study pharmacological effects of new anti-infective and also to analyze long-term consequences of the vascular infection processes. This pump system perfuses bacterial pathogens by air pressure to prevent any transmission via aerosols.

It is recommended to follow the biosafety precautions.

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Journal (Immunology and Infection)

Journal (Immunology and Infection)

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

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