An Ex Vivo Gut Organ Culture System to Study Host-Microbiota Interactions

To study interactions between the host and the gut microbiota ex vivo, begin with a murine colon tissue free from specific gut-colonizing bacteria. Flush the tissue with a medium to remove the feces and transfer to a multi-well plate containing a suitable medium.

Concurrently, assemble the multi-well, ex vivo gut organ culture device. 

Each well of the device comprises two paired input and output ports — the first connecting to the colon lumen and the second providing a continuous supply of medium to the wells to maintain tissue viability.

Transfer the tissue to the device. Connect the colon's proximal and distal ends to the first pair of input and output ports. Start the luminal stimulant flow into the colon lumen and medium flow into the well.

The stimulant contains gut-colonizing bacteria that secrete mucus layer-degrading enzymes, allowing them to attach to underlying epithelial cells. This activates a signaling cascade that triggers resident dendritic cells to extend their dendrites between epithelial cells, capturing bacterial antigens and generating specific immune responses.

Consequently, naïve CD4+ T cells differentiate into Th17 cells.

The Th17 cells produce pro-inflammatory cytokines which activate other immune cells to combat the invading pathogens and maintain the integrity of the gut barrier.

To begin, insert the blunt-end needles to the appropriate position within the device mold and cast approximately 20g of polydimethylsiloxane or PDMS mix for one set of the device and lid. Place the molds in a vacuum chamber for 30 minutes to remove air bubbles from the PDMS mix, then, incubate the molds at 55 degrees Celsius overnight to complete PDMS polymerization.

When the PDMS is set, pull out the needles from the mold and carefully release the culture device and lid from the plastic molds. Remove PDMS residues from the well outline using a surgical blade.

Attach PDMS device in the device cover onto a cover glass of 75 by 50-millimeter microslides using non-toxic silicone adhesive, and leave the parts to set overnight. Apply the glue to the smooth side of the device. Insert 12 22-gauge needles for the lumen and 12 18-gauge needles for the well. Fix all the needles in place using silicone and let it set overnight.

Purge the input syringes, and make sure that the well medium flows out from all tubes into a waste glass. Then, purge the input syringes, and make sure that the stimulations flow out of all the tubes into a waste glass, taking care to not contaminate the different stimulations.

After sacrificing the mouse, use sharp scissors and forceps to dissect it, and take out the digestive tract from the stomach to the anus by cutting all the fat and connective tissues. Cut the colon and place it on a new plate. Minimize contact while holding the tissue gently and only at the edges. Perform the colon flush under the dissection microscope.

Gently flush the colon content with sterile IMDM with the prepared 10-milliliter syringe as described in the text. After removing the feces from the intestinal tissue, place the colon in a new 6-well plate filled with 0.5 milliliters of sterile IMDM. Next, take the colon tissue and carefully connect it to the 22-gauge needle, making a tight tie with the two threads.

Maintain the correct orientation of the colon to the lumen flow such that the proximal and distal is equal to input and output respectively. Repeat colon flushing and needle tying for all the tissues. Connect the input and output tubes to the device, then begin the experiment by starting the pumps at the desired rates.