This protocol is significant as it allows studying a bacterial species effect on cells from the enteric nervous system for the first time. Since the enteric nervous system is not easily accessible, this model allows for studying the effect of the gut microbiome on cells from the enteric nervous system. In the future, this model can be used to see how the gut microbiome of diseased patients impact cells from the enteric nervous system, for example, in Parkinson's Disease.
A possible future aim would be to connect this model to other organ-on-chip models to build a model representative of the gut-brain axis. The visual demonstration of this technique is critical as the protocol includes many steps and each step is crucial for this successful realization of this model. Demonstrating this procedure will be Catherine Sedrani, a PhD candidate from the System Ecology Laboratory.
To begin, tighten the screws, securing the top and bottom parts of the clamp, and transfer the coated and dried bottom polycarbonate lid from the square Petri dish to the top of the clamp base by holding the four screws at the corner of the lid. Use sterile tweezers to position the epithelial chamber gasket with the membrane facing up on the polycarbonate lid. Align the gasket and lid with screws, ensuring the corner inlet and outlet ports align with the gasket openings.
Place the sandwich gasket on top of the collagen gasket with the top side facing up. And then place the top polycarbonate lid on top of the sandwich gasket. Ensure that the barbs on the clamp lid align with the membrane assemblies inlet and outlet opening, and that the bottom lid screws fit on the opening of the top lid.
To close the device, place the clamp lid on top of the polycarbonate lid and close the latches. For the priming of the tubing lines, insert the aeration needles with filters in the septum of each inflow and outflow bottle. Using clean sterile tweezers, insert the 120 millimeter needles in the 250-millimeter serum bottles and the 80-millimeter needles in the 15-millimeter serum bottles.
Insert the 40-millimeter needles at the end of each tubing line into an outflow serum bottle. The 40-millimeter needles of the epithelial and neuronal tubing lines are connected to the same outflow bottle for medium discarding. The bacterial tube line goes to the second outflow bottle.
Insert the pump tubing lines into the pump cassettes. Set the peristaltic pump to direct media from the inflow to the outflow bottle at a speed of five RPM and press the start button to start the pump. Ensure that the three-way stopcocks of the tubing lines are all open.
Once the media is dropping into the outflow bottles, make sure no leaks or air bubbles are present in the tubing lines and connection points. When all the tube lines drop into the outflow bottles, set the flow rate to two RPM to connect the initial device. To connect a line, close the three-way stopcock valves, starting with the outflow side.
Disconnect the short tubing from the female lure connector and attach it to the device's outlet port by pushing the tubing over the barb. For a secure leak-free connection, push the tubing all the way down over the connector, ensuring it contacts the lid. Open the stopcock once one line is completely connected to the device.
Set the flow rate of the pump to two RPM and increase the pump speed to a maximum of 2.5 RPM to avoid leakages due to pressure buildup. Allow the pump to prime the chambers. Reduce the pump speed to 0.5 RPM once all the chambers are filled with the cell culture medium and no bubbles remain in the device.
For the epithelial cells, detach the Caco-2 cells from the flask using trypsin EDTA. Re-suspend them in RPMI 1640 plus 10%FBS, and count them in a Neubauer cell counter using the trypan blue exclusion assay. Centrifuge the Caco-2 cells suspension for three minutes at 300G and discard the supernatant to remove the remaining trips in EDTA.
Resuspend the Caco-2 cells in RPMI 1640 plus 10%FBS to obtain a suspension of 0.35 million cells per milliliter. Transfer 1.5 milliliters of the Caco-2 cell suspension to a sterile two milliliter syringe, and remove any remaining air bubbles in the syringe. Close the three-way stopcock valves on the tubing of both the bacterial and neuronal chambers and disconnect the tubing from the pump by removing the cassettes with the respective tubing from the rotor.
Open the cap of the stopcock valve of the inflow tubing leading to the epithelial chamber and turn the valve to redirect the medium flow from the device to the open connector. Allow the medium to flow until a drop of the medium appears at the open end of the stop valve. Insert the syringe with epithelial cells into the open connector using the drop-drop connection method to prevent air bubble introduction during the insertion.
Turn the valve of the stopcock to stop the flow of the medium from the inflow bottle, and to allow flow from the connected syringe to the initial device. Disconnect the epithelial chamber from the pump and slowly press the syringe to inoculate the epithelial chamber with 1.5 milliliters of cell suspension. Close the valve of the outflow stopcock and disconnect the syringe.
Close the open end of the stopcock with the cap and keep the chamber closed for at least two hours. In the meantime, inoculate the neuronal cells. After removing the device from the incubator and removing the tubing lines, slowly open the clamp and remove the lid.
Remove the top polycarbonate lid with care. Collect the medium in a 1.5 milliliter micro centrifuge tube and place it on ice. Gently remove the sandwich gasket, while collecting the media from the epithelial chamber without touching the cell layer.
Place the sandwich gasket in a square Petri dish and add sterile 0.9%sodium chloride and water solution to the bacterial chamber until the chamber is fully covered. Slowly remove the collagen gasket while collecting the media from the neuronal chamber. After transferring the media to a micro centrifuge tube, place the tubes on ice.
Place the collagen gasket into a square dish and gently add a few milliliters of PBS to the Caco-2 layer until the cell layer is fully covered. Place the bottom polycarbonate lid in a square Petri dish and gently add approximately two milliliters of PBS on top of the neuronal cells so that they do not dry out during the sampling process. Centrifuge the media tube for five minutes at four degrees Celsius.
After transferring the supernatants of each tube to a new micro centrifuge tube, place it on dry ice immediately. The CFU count was assessed for two different initial device setups. L.reuteri co-cultured with Caco-2 and L.reuteri in the human microbial crosstalk device.
In both setups, the CFU counts were significantly different from the HuMix inoculum and the harvested cells indicating the growth of the bacterial cells inside the initial device. To evaluate if culturing the enteric neurons in the device alters the cell phenotype, their gross morphology was observed using an inverted phase contrast microscope. The establishment of a confluent neuronal network indicated that the cells had attached well to the coated devices polycarbonate lid.
The edge between the confluent neuronal network and the gasket delineated spiral was clearly apparent. The potential of neuro humics lies in answering questions that were previously challenging to address, concerning the interactions between the human-gut microbiome and the enteric nervous system due to the lack of a representative model.
