Innervation of Human Intestinal Organoids

Our research focuses on the generation of tissue engineered intestines as a solution for short bowel syndrome. Our lab is focused on optimizing the integration of an enteric nervous system and optimizing spatial organization, cellular diversity, and function. We hope that one day we can create a life-changing solution for children suffering from intestinal failure by creating bioengineered intestine.

Innervating human intestinal organoids requires mimicking human intestinal development, and this can be challenging. The development of the enteric nervous system requires both biochemical signaling as well as biomechanical forces. Our lab's future studies are focused on optimizing enteric nervous system development within human intestinal organoids.

We're studying spatial organization as well as cellular diversity, and we've recently been using a novel 3D imaging technique developed by Dr.Herath which can robustly characterize and quantify the ENS within our intestinal organoids. This will help us refine our model and recapitulate human intestinal development and function. First, take the plate of day 15 to 21 cultured enteric neural crest cells or ENCC spheroids and carefully remove the media from the well.

Add one milliliter of a warm enzymatic cell detachment reagent to the well and incubate at 37 degrees Celsius for 30 minutes. Then tap the side of the plate to facilitate spheroid dissociation. Using a P1000 micropipette with a wide-mouth tip, titrate the mixture by pipetting gently up and down until the cells are homogenous.

Transfer the ENCC suspension to a 15-milliliter conical tube. Wash the well with one milliliter of warm neural crest cell media to collect any remaining cells, then add this to the conical tube. Now centrifuge the cell suspension at 300 G for one minute.

Then aspirate the supernatant using a sterile pipette tip. Afterward, using a wide-mouth pipette tip, resuspend the cell pellet in one milliliter of warm neural crest cell media. Collect the cultured mid-hindgut spheroids from all wells of a human intestinal organoid plate using a wide-mouth pipette tip and transfer them to a 15-milliliter conical tube.

Add the calculated volume of ENCC suspension into the conical tube containing the mid-hindgut spheroids. Using a wide-mouth pipette tip, gently tritate the mixture to uniformly mix the spheroids and ENCCs. Centrifuge the combined cell suspension at 300 G for one minute.

Carefully aspirate the supernatant using a sterile pipette tip. Then resuspend the resulting cell pellet in a cold phenol-free growth factor reduced basement membrane matrix. Next, pipette the 30 microliter droplet of the resuspended mid-hindgut spheroids and ENCCs in the clear basement membrane matrix to the center of each dry well.

Once all droplets are plated, cover the plate, invert it, and place it in an incubator at 37 degrees Celsius with 5%carbon dioxide for 30 minutes. On day zero, once the clear basement membrane matrix has polymerized, cover each droplet with 500 microliters of warm day zero to three HIO media. On day three, carefully aspirate the day zero to three HIO media from the side of each well without disturbing the clear basement membrane matrix droplet.

Replace it with 500 microliters of warm day three to 14 HIO media and return the plate to the incubator. On day 14, examine each well under a microscope to assess the number of developing HIO+ENS structures in each droplet of clear 3D basement membrane matrix. Add 30 microliters of clear basement membrane matrix to two-milliliter microcentrifuge tubes and keep them on ice.

Under a laminar flow hood, use fine forceps to carefully remove the clear 3D basement membrane matrix droplet containing the suspended HIO+ENS. Gently tease apart individual HIO+ENS structures using the forceps. Using either fine forceps or a wide-mouth pipette tip, place each isolated HIO+ENS structure into the microcentrifuge tubes containing 30 microliters of clear basement membrane matrix.

Then pipette a 30 microliter droplet of the resuspended HIO+ENS in a clear basement membrane matrix into the center of each dry well. Once the droplets have polymerized, add 500 microliters of warm fresh day three to 14 HIO+ENS media to each well and incubate the plate overnight. On day 15, carefully aspirate the day three to 14 media from the side of each well without disturbing the polymerized droplet.

Replace the media with 500 microliters of warm day 15 to 28 HIO+ENS media. The HIO-ENS coculture showed significant growth with the development of mesenchymal structures, forming thin, hazy projections, creating a halo-like border around epithelial involutions by day 30. ENCCs migrated towards the HIOs, integrated within the mesenchyme, and survived in vitro, as shown by GFP fluorescence within HIOs, confirming adequate survival and differentiation signals from the mesenchyme.

Transplanted HIO+ENS cocultures expressed neuronal markers, including HuCD, and glial markers such as S100 beta after in vivo growth, confirming differentiation into both neurons and glia.