Our research focuses on the development and use of immunocompetent organ-on-chip platforms. By creating these physiologically relevant, humanized in vitro models, we are able to study the complex host-pathogen interaction. The knowledge we create can be used to manipulate and identify molecular targets for the therapy of infectious diseases.
One key challenge is maintaining a living microbiota under homeostatic conditions alongside host tissue in vitro. This includes replicating the organotypic 3D topography of crypt and villus structures and their microbial colonization. Intestine-on-chip platforms address this by using flow to shape the organotypic structures and prevent bacterial overgrowth.
This ensures stable culture conditions. Compared to other available intestine-on-chip platforms, our model enables the detailed study of the human immune response to pathogens due to the inclusion of primary tissue-resident immune cells. Additionally, the model size enables multiple readouts in one single experiment, reducing the overall cost and number of experiments.
Our laboratory is focusing on using induced pluripotent stem cells to develop autologous systems that enable personalized medicine. To begin, prepare 1:100 dilution of a collagen stock solution in DPBS with magnesium and calcium. Add 350 microliters of the diluted stock solution to the respective chamber and incubate for five minutes at room temperature.
Flush all chambers two times using 350 microliters of DPBS with magnesium and calcium to wash out the remaining collagen and acetic acid. Remove the plugs from one site and switch them to the other side. After flushing the chamber again, add 350 microliters of endothelial cell growth medium to it.
Remove the plugs from one site and switch them to the other side. Then, add 350 microliters of endothelial cell growth medium to each chamber. Take HUVECs cultivated at passages 1-3 with 80 to 90%cell confluency in endothelial cell growth medium.
Then, remove the cell culture medium from a T25 cell culture flask and wash the cells gently with three to five milliliters of DPBS without magnesium and calcium. Once the solution is removed, add one milliliter of trypsin dissociation reagent and incubate for five minutes at 37 degrees Celsius until the cells detach from the flask. Transfer the detached cells into a tube using nine milliliters of 5%FBS in PBS without magnesium and calcium Centrifuge at 350 g for five minutes at room temperature.
After moving the supernatant, resuspend the cell pellet in one milliliter of endothelial cell growth medium. Add the respective volume of cells to the chamber and incubate the biochips in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide. Perform a medium exchange of the HUVEC-containing chamber with 350 microliters of endothelial cell growth medium after 24 hours.
If air bubbles appear in the microfluidic biochips, close all ports of the biochip besides the ports of the affected chamber. Tilt the chip to move the air bubble close to one end of the cavity. From the port of the other end, push 700 microliters of cell culture medium through the chamber while holding the opposite plugs.
To begin, thoroughly clean all areas of the incubator and peristaltic pump with disinfectant to ensure a quasi-sterile environment. Then, flush each tubing with 700 microliters of PBS with magnesium and calcium, followed by 500 microliters of C2 or EC-conditioned medium. Use the tubing with the short distance from the luer lock to the peristaltic pump stopper for the left cavity and the tubing with the other symmetry for the right cavity.
After retrieving the biochip with HUVEC and C2BBe1 cells from the incubator, perform a medium exchange with 350 microliters for each chamber. Then, move the plugs from the lower to the upper site. Add 350 microliters of the fresh media to the lower chamber of the biochip.
After that, remove all plugs and fill all ports to the very top. Starting at the left cavity, insert the luer lock adapter into the port of the biochip to connect the first tubing to the right port of the upper chamber. Then, connect the second tubing to the left port of the lower chamber.
Next, take a reservoir and add a small drop of cell culture medium to the bottom of the reservoir. Then, insert the reservoir to the opposite side of the first tubing and repeat for the other chamber. Once all ports are connected to a tubing or reservoir, fill the reservoirs with 3.5 milliliters of cell culture medium.
Then, place the loose side of the tubing, which has the lid attached to it on the top of the reservoir to close the microfluidic system of each chamber. Transport the biochip to the peristaltic pump. Using the peristaltic pump stoppers, connect each tubing to the peristaltic pump so that the medium flows from the reservoir into the cavity and back via the pump.
Then, perfuse each chamber with a flow rate of 50 microliters per minute. Once the pre-perfusion is complete, stop the peristaltic pump. Remove the lid connected to the tube of each reservoir and place it on a sterile tissue next to the pump.
After removing all medium, fill the reservoirs with two milliliters of freshly prepared medium. Reconnect the tubing in the lid and continue the circular perfusion at a flow rate of 50 microliters per minute for an additional 24 hours. Once the peristaltic pump is stopped, open the reservoirs of all cavities, empty the reservoirs and disconnect the tubing and reservoirs from the biochip.
Wash the microfluidic cavities with 500 microliters of cold PBS with magnesium and calcium twice per chamber. Add 500 microliters of ice cold methanol to all cavities. After opening the chip, cut or remove the bonding foil of the biochip.
Cut the tissue containing PET membrane in half to stain in parallel with different immunopanels. Using precision tweezers, transfer each membrane piece to a separate well in a 24-well plate with a blocking and permeabilizing solution. Then, transfer the membrane pieces onto a clean glass slide inside a humid chamber.
Add 50 microliters of primary antibody prepared in staining solution to each membrane piece. Once the samples are transferred to a 24-well plate, wash the membranes twice for five minutes with wash solution, then with PBS containing magnesium and calcium. Using a fluorescence mounting medium and a cover glass, mount the membrane pieces on a clean glass slide and store them at four degrees Celsius until microscopic imaging.
After five days of perfusion, nuclear DNA stained with DAPI showed fully differentiated monocyte-derived macrophages expressing CD68 spreading all over the vascular tissue. HUVECs created a confluent layer showing tissue integrity by the formation of adherens junctions such as VE-Cadherin. Additionally, the von Willebrand factor is highly expressed by HUVECs.
After five days of perfusion, C2BBe1 cells formed polarized epithelial cell layers expressing the junction proteins E-Cadherin and ZO-1. The three-dimensional growth of villus-like and crypt-like structures of the epithelium can be detected in the x and y-sections of the z-stack after six days of perfusion.
