Three-Dimensional Morphogenesis in Canine Gut-on-a-Chip Using Intestinal Organoids Derived from Inflammatory Bowel Disease Patients

Our research goal is to create a method for growing 3D structures using intestinal cells from animals similar to humans, specifically dogs. Intestinal cellular morphogenesis has been largely studied through laboratory animal models, which are costly, time consuming, and do not accurately represent human developmental processes. Furthermore, conventional static 2D cell culture models lack the ability to mimic the complex spatial organization of a 3D abSerial architecture.

Achieving the attachment of individual cells derived from intestinal organoids onto the ECM-coated PDMS surface has posed a significant challenge. A pivotal step in addressing this challenge in both ensuring a uniform ECM coating through surface activation and allowing it to dry overnight. The integration of patient-derived canine organoid technology with in-vitro 3D Morphogenesis offers a promising avenue for conducting translational research into chronic multifactorial diseases, aligning with the One Health Initiative.

The protocol for canine IBD, Gut-on-a-Chip, offers a replicable model for comparative medicine, enabling studies on intestinal morphogenesis, host-microbiome interactions, infections, drug and probiotic screenings, and has cross-species applicability. To begin, place the Gut-on-a-Chip device in a dry oven set at 60 degrees Celsius for 30 minutes to eliminate moisture. Then using a UV ozone generator, expose the device to UV and ozone treatment for 60 minutes.

Secure the inlet, bypass, and outlet tubing of the upper microchannel using binder clips. Clamp the inlet tubing for the lower microchannel, then disconnect the outlet tubing for the lower microchannel. Ensure that the bypass tubing of the lower microchannel remains open.

Using a P100 micropipette, add 100 microliters of a 1%polyethyleneimine solution through the outlet hole of the lower microchannel. Reconnect the outlet tubing for the lower microchannel. Secure the inlet bypass and outlet tubing of the lower microchannel, followed by the inlet tubing for the upper microchannel using binder clips.

Now disconnect the outlet tubing for the upper microchannel, ensuring the bypass tubing remains open. Next, add 100 microliters of a 1%polyethyleneimine solution through the outlet hole of the upper microchannel. Reattach the outlet tubing of the upper microchannel and allow the device to incubate it room temperature for 10 minutes.

Repeat the procedure with 0.1%glutaraldehyde and then with deionized water to remove any excess surface activation solution, dry the chip overnight at 60 degrees Celsius in an oven. The next day, remove the chip from the oven and allow it to cool in a biosafety cabinet for 10 minutes. Secure the inlet, bypass, and outlet tubing of the upper microchannel, including the inlet tubing of the lower microchannel using binder clips.

Disconnect the outlet tubing for the lower microchannel. Add 20 microliters of extracellular matrix, or ECM mixture, through the outlet hole of the lower microchannel. Then reattach the outlet tubing for the lower microchannel.

Secure the inlet, bypass, and outlet tubing of the lower microchannel, including the inlet tubing of the upper microchannel, with binder clips. After disconnecting the outlet tubing for the upper microchannel, add 20 microliters of VCM mixture through the outlet hole of the upper microchannel. Reattach the outlet tubing of the upper microchannel and then remove the binder clip from the outlet tubing of the lower channel ensuring both of the outlet tubes are open.

Place the chip in a 37 degree Celsius 5%carbon dioxide incubator for one hour. To begin, prepare an organoid culture medium containing 10 micromolar Y-27632, and 2.5 micromolar CHIR99021. After coating the ECM onto the Gut-on-a-Chip, disconnect the outlet tubing from the upper microchannel while keeping the bypass tubing of the upper microchannel open.

Using a binder clip, secure both the inlet and outlet of the lower microchannel. Using a P100 micropipette, add 20 microliters of the cell suspension into the outlet hole of the upper microchannel. Secure the bypass and inlet tubing of the upper microchannel using binder clips.

Reattach the outlet tubing to the outlet hole of the upper microchannel, ensuring the tubing remains open throughout the process. Once done, gradually secure the outlet tubing of the upper microchannel using a binder clip Next, under a microscope, verify that the cells are evenly distributed throughout the upper microchannel. Place the Gut-on-a-Chip device in a humidified carbon dioxide incubator at 37 degrees Celsius.

Connect the syringe attached to the upper microchannel of the chip to a syringe pump placed within an incubator. Set the flow rate to 30 microliters per hour and start the continuous flow of the seating medium for the upper microchannel. During this period, leave the lower microchannel clamped.

The day after seeding, replace the medium with an organoid culture medium containing only A8301. Once the monolayer is established for the upper micro channel, initiate the continuous seating medium flow to the lower micro channel. Next, introduce organoid culture medium into both the upper and lower micro channels to initiate the development of three dimensional morphogenesis in the chip.

Increase the flow rate to 50 microliters per hour to achieve sheer stress of 0.02 dine per square centimeter in the Gut-on-a-Chip design. Using a computer regulated bioreactor, apply 10%cyclic strain and 0.15 hertz frequency to the cells cultured on a Gut-on-a-Chip device for two to three days to establish three dimensional morphogenesis. After six to nine days of medium flow, occasional clustering of three dimensional morphogenesis of canine intestinal epithelial cells was observed throughout the microchannel.

Immunofluorescent staining evaluated the three dimensional structure of organoid derived monolayers and villus-like structures in microfluidic chips. Intestinal barrier function measured by teer showed stable teer values on day five of culture.