This protocol displays the use of a novel microfluidic co-culture model that enables independent culture of eukaryotic cells into bacteria to test the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal e coli biofilm among hela cells, followed by introduction of entero hemorrhagic e coli or EHEC into the commensal island. In a sequence that mimics the sequence of events in GI tract infection.
The resulting extent of epithelial cell colonization by EHEC is visualized using fluorescence microscopy. Hi, I'm from the laboratory of molecular system biotechnology in the Department of Chemical Engineering at Texas and M University. Today we will show you a procedure for cultivating bacteria and eCard cell together in a micro device in our manner that mimics the organization in the human gastrointestinal tract.
We use this procedure in our laboratory to study how pathogenic bacteria colonization of the epithelial cell is affected by signal that they encounter in the tri tract prior to colonization. So let's get started. Prior to experimentation, prepare a silicon master designed and fabricated according to intended experimental use.
For example, the Stanford Microfluidics Foundry will fabricate designed masters from which subsequent experimental replica molds can be made Molds for two membranes for the eukaryotic cell culture layer and the pneumatic layer and one bacterial channel are required for the tri layer device described in this protocol, the channel heights for the bacterial, pneumatic and eukaryotic cell culture layers are 5, 200 and 100 micrometers respectively. Before preparing a replica mold, expose the SU eight master to fluorocycline vapor in a desiccate attached to a vacuum source. To facilitate easy release of PDMS mold from the master, add a drop of fluoro lane to a paper towel in the vacuum chamber and apply vacuum for one minute.
Remove the vacuum and allow 30 minutes for deposition of fluoro. Keep the SU eight master in a Petri dish for future use to create replica molds. Prepare PDMS pre polymer and crosslinker at a 10 to one weight ratio and mix thoroughly degas the mixture in a vacuum chamber for one hour.
To remove air bubbles, place the SU eight master in a Petri dish and carefully pour PDMS on the master using five grams of PDMS for the and 30 grams of PDMS for the bacterial layer. Degas the PDMS again for 10 minutes to remove air bubbles. Next, spin the mold for one minute to spread the PDMS into an even membrane.
In this protocol, the eukaryotic cell culture membrane is spun at 1200 RPM for a 150 micrometer thickness, and the pneumatic membrane is spun at 800 RPM for a 250 micrometer thickness. After spinning, keep the Petri dish containing the spun PDMS and SU eight master mold for 15 minutes at room temperature prior to heating on a hot plate at 80 degrees Celsius for one hour. To cure the PDMS after curing wet the PDMS with methanol and carefully peel from the SU eight master molds using clean forceps.
Place the bacterial layer on a stereoscope and using a 20 gauge blunt end needle. Drill three holes, two bacteria inlets, and one outlet. The tri layer device is assembled in the following order, bonding of the pneumatic layer to the bacteria layer bonding of the eukaryotic cell culture layer to the bacterial plus pneumatic layer.
Place the pneumatic membrane and bacterial layer on a glass slide and expose to plasma in an oxygen plasma chamber for 40 seconds at an oxygen pressure of 10 standard cubic centimeters per minute, or SCCM and radio frequency or RF power of 100 watts. Adding methanol in the membranes facilitates free movement of the layers and makes alignment easier. Within 10 minutes of oxygen plasma exposure, place the bacterial layer in a Petri dish using forceps and a stereoscope.
Align the middle pneumatic membrane to the bacterial layer based on the inlet and outlet holes for the bacteria. After aligning, set the Petri dish with the aligned PDMS structure on a hot plate at 80 degrees Celsius for eight hours to bond. When this is complete, drill a hole in the pneumatic layer for controlling the pneumatic channel.
Next, repeat steps from assembly to alignment to bond the eukaryotic cell culture channel to the assembled composite PDMS device. Drill holes in the inlet and outlet ports used for seeding eukaryotic cells. Connect tigon tubing to the ports and operate the pneumatic layer by pulling with the 20 milliliter syringe.
Place the composite PDMS structure and a pre-cleaned glass slide into an oxygen plasma chamber. Turn on plasma for 20 seconds without applying the vacuum. Remove the composite PDMS structure from the oxygen plasma chamber and connect a 20 milliliter syringe to it.
Pull immediately creating a vacuum to prevent contact between the valves and the glass. Slide within one minute. After plasma exposure, gently press the composite PDMS structure onto the glass.
Cure the PDMS device on a hot plate at 80 degrees Celsius for 10 minutes. After this, connect tigon tubing to each port and sterilize the device under UV for 15 minutes. Next, fill 500 microliters of sterile PBS in a one milliliter syringe and introduce into the channel to remove any debris.
If an air bubble is observed. Clamp the outlet tubing with an einor micro centrifuge tube and apply slight pressure to force the bubble out through the gas permeable PDMS layers. Fill sterile water into the pneumatic channel to prevent formation of air bubbles in the channel.
A homemade air to liquid pressure converter is used to prevent air bubble formation in the pneumatic channel. To prepare for eukaryotic cell seeding on the glass, introduce 500 microliters of fibronectin into the cell culture region to the eukaryotic cell culture inlet. Using a syringe, keeping the valve closed and incubate for 40 minutes at 37 degrees Celsius.
Finally wash the excess fibronectin with one milliliter of growth media using the syringe pump. The device is now ready to use for the experiment to form a commensal biofilm dilute bacteria in M nine minimal media to OD 600 of about one. In this example, we use a lab strain of e coli, but any non-pathogenic bacterial strain can be used.
Close the valve that separates the bacterial and eukaryotic cell culture regions by applying positive liquid pressure. Introduce 0.2 milliliters of e coli at an OD 600 of about 1.0 into the islands using a syringe pump and allow the bacteria to attach for one hour at 32 degrees Celsius. After incubation.
Perfuse a dilute bacterial suspension OD 600 of about 0.05 and fresh M nine, minimal media at 0.25 milliliters per hour for 12 hours. Using the syringe pump, rinse the biofilm with 0.5 milliliters of fresh serum free and antibiotic free DMEM media at 0.5 milliliters per hour using a syringe pump to remove loosely attached bacteria and prepare for eukaryotic cell attachment image the biofilm. Using a Leica T-C-S-S-P five confocal laser scanning microscope and visualize the biofilm architecture using the MS software.
Trypsin eyes, heela cells from a tissue culture flask and resus. Suspend them in DMEA media to a total volume of 0.1 milliliters at a seating density of five times 10 to the fifth cells per milliliter. Introduce heela cells into the eukaryotic region of the device at a flow rate of two milliliters per minute using a pico plus syringe pump.
Next, using a light microscope, ensure that the valve separating the commensal bacterial region from the eukaryotic cell region is closed completely so that the bacterial biofilm is separate from the helo cell region. Using an einor micro centrifuge tube clamp the cell outlet to reduce the movement of the cell suspension in the channel and incubate for six hours at 37 degrees Celsius in a 5%carbon dioxide incubator. Following incubation, perfuse the device using a syringe pump with 50 microliters DMEM growth medium at 0.5 milliliters per hour.
To remove unattached cells, refresh the DMEM media for hela and commensal e coli regions every six hours. Prepare e coli oh 1 5 7 H seven, or EHEC in M nine media to infect hela cells at a multiplicity of infection or MOI of 100 to 200 EHEC cells to one helo cell wash out loosely attached commensal bacteria as described in the previous section. Introduce a 100 microliter dilute EHEC suspension OD 600 of about 0.1 into the islands via a syringe pump at a flow rate of 0.25 milliliters per hour.
Following this, incubate the device at 37 degrees Celsius in a 5%carbon dioxide incubator for six hours without flow to allow EHEC to be exposed to signals present in the commensal biofilm. After the incubation, open the valve separating the commensal bacterial and eukaryotic cell regions by using a vacuum pump to form a vacuum and keep the valve open for six hours. This will expose the HEC in the commensal bacterial biofilm to heela cells following this incubate again for six hours at 37 degrees Celsius in a 5%carbon dioxide incubator.
Introduce live dead stain into the device prepared according to manufacturer's instructions. To stain for helo cell viability inject 0.1 milliliters of standing solution to the eukaryotic area of the device using a one milliliter syringe as a control for estimating helo cell death. In the absence of EHEC, repeat the previous three steps and introduce sterile serum free and antibiotic-free DMEM media into the device.
Instead of EHEC image multiple locations on a Zeiss AIOt 200 M fluorescence microscope, and estimate the number of live epithelial cells in green among the dead epithelial cells in red by counting the number of green cells from images obtained at a minimum of seven locations. Now we will show some representative images of the trial layer microfluidic device. In this image, we use inert food color to represent the different culture regions.
The purple dye represents the eukaryotic cell culture region, while the green dye represents the commensal bacterial biofilm. In this image, we demonstrate the feasibility of keeping the eukaryotic and bacterial regions distinct from one another as the purple dye in the bacterial region is distinct from the surrounding yellow dye. The colonization of the green protein expressing commensal bacterial biofilm by pathogenic red protein expressing bacteria is shown here.
As shown in this image, the EHEC and commensal bacteria are distinct from the hela cells that surround the bacterial biofilm. We have just shown you how to assemble a dry layer micro device for co culture of bacteria and epithelial cell and usually investigating the effect of so signals on pathogen colonization. When doing this procedure, it's important to remember to remove any bubbles in the device and use this pathogen at appropriate multiple cell infection.
So that's it. Thank you for watching and good luck with your experiment.
