Microfluidic Model of Necrotizing Enterocolitis Incorporating Human Neonatal Intestinal Enteroids and a Dysbiotic Microbiome

Research in this field is challenging because the pathophysiology of necrotizing enterocolitis is complex, and this complexity is not reflected in many commonly used in vitro models. Studies using human samples are often limited by small sample volumes and the rarity of samples obtained at the time of intestinal surgery. Our goal is to determine the mechanisms that lead to the development of NEC in preterm infants and to identify new treatment strategies.

The NEC-on-a-Chip system improves upon previous models by more closely reflecting the physiology of the neonatal intestine. In this model, we combined patient-derived intestinal organoids, endothelial cells, and the microbiome from a neonate who had severe NEC onto the microfluidic chip. To begin, place the chip and chip carrier into the chip cradle located in a sterile tissue culture dish, and keep it aside in the tissue culture hood while preparing the chip activation reagent.

Add one milliliter of the CR2 solution to the vial containing CR1 powder. Transfer the solution from the vial to a 15 milliliter tube wrapped in aluminum foil. Repeat this process until four rinses and four milliliters of CR2 have been flushed through the CR1 vial.

For the last rinse, cap the vial and invert it to remove residual powder. Next, add six milliliters of CR2 to the 15 milliliter tube containing CR1. Mix the final 10 milliliter solution with a pipette without introducing bubbles.

Using a 200 microliter pipette tip, add 20 microliters of CR1 solution to the bottom channel inlet of the chip until the solution begins to exit the bottom channel outlet. Add 50 microliters of CR1 solution through the top channel inlet until the solution begins to exit the top channel outlet. Then place the chips under ultraviolet light for 15 minutes.

After exposure, remove any CR1 solution from the top and bottom channels. Wash both channels with 100 microliters of CR2 solution. Then wash twice with 100 microliters of DPBS.

Prepare the extracellular matrix solutions for the top and bottom channels of the chip. Add 50 microliters of extracellular matrix solution to the bottom channel inlet until a drop appears on the outlet. Then add 50 microliters to the top channel inlet until the drop appears on the inlet.

Add DPBS to the chip cradle reservoir. Place the lid on the plate and incubate the chips overnight at 37 degrees Celsius and 5%carbon dioxide incubator. The next day, flush the epithelial or top channel of the chip with 100 microliters of prewarm chip expansion media.

Then flush the endothelial or bottom channel with 100 microliters of prewarm HIMEC media. After counting, add 10 microliters of HIMECs to the endothelial channel. If required, add organoid growth media or chip expansion media to the epithelial channel and check the seeding density of the HIMECs.

Next, invert the chip into the chip cradle placed in the cell culture dish. Add DPBS to the reservoir in the chip cradle. And place the chip at 37 degrees Celsius for two to three hours to allow HIMECs to attach to the chip membrane.

After incubation, return the chip cradle to an upright position. Gently wash the endothelial channel with 100 microliters of warm HIMEC media and the epithelial channel with organoid growth media or chip expansion media, leaving the media in the channel. For harvesting enteroids, gently aspirate the media and wash the wells containing cell culture matrix hydrogel with 500 microliters of warm DPBS.

Add 250 microliters of cold cell recovery solution to each well, and scratch the bottom of each well with a fresh pipette tip to disrupt cell culture matrix hydrogel. Add the disrupted cell suspension to a cold 15 milliliter tube on ice and incubate for 45 minutes. After incubation, centrifuge the suspension and remove the supernatant.

Then add two milliliters of warm enteroid dissociation media to the pellet and place the tube in a 37 degrees Celsius water bath for 60 to 120 seconds. Next, add 10 milliliters of cold tissue washing media to the tube. Then centrifuge and remove the supernatant before resuspending the pellet in 200 microliters of chip expansion media.

Using a P200 pipette, dissociate the enteroids and add the suspension into a 1.5 milliliter microcentrifuge tube. Count the cells and adjust the volume of chip expansion media to achieve a concentration of six times 10 to the six cells per milliliter. Then add 30 microliters of resuspended cell suspension to the top channel of the chip.

After returning the chips to the chip cradle, add DPBS to the reservoir and incubate the chips overnight at 37 degrees Celsius and 5%carbon dioxide. The next day, flush the epithelial channel twice with 100 microliters of chip expansion media and the endothelial channel with 100 microliters of HIMEC media. Then add two milliliters of the chip expansion media to the top channel inlet reservoir and 300 microliters to the top channel outlet reservoir.

Add two milliliters of the HIMEC media to the bottom channel inlet reservoir and 300 microliters to the bottom channel outlet reservoir. Prime the pods and add the chips to the pods. Then add the pods to the culture module, set a flow rate of 30 microliters per hour, and initiate the cycle.

The intestinal epithelial cells grown using the neonatal intestine on a chip formed a confluence cell monolayer, and subsequently developed into mature, three-dimensional villus-like structure. The addition of a dysbiotic microbiome from a neonate with severe necrotizing enterocolitis to the neonatal intestine on a chip model showed increased expression of TNF alpha compared to the control.