Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality

This protocol shows how to use biopsy-derived intestinal organoids and organ-on-a-chip technology to generate a physiologically relevant intestine chip which can then be used to predict pharmacokinetics and drug-drug interaction and to study intestinal epithelial barrier dysfunction. Uniting organoids in organ-on-a-chip technology allow us to reproduce the complexity of the intestinal epithelium. Additionally, it enables greater experimental control of the mechanical cues, flow distribution, and biochemical gradients.

This method emulates the complex physiology of the human intestine, giving a greater understanding of the cellular and molecular basis of normal and pathological gut functions. This helps to better predict pharmacokinetics and pharmacodynamics and potential drug candidates to prevent inflammatory damage. Seeding is a critical step in the protocol.

Excessive fragmentation or inaccurate seeding density of the intestinal organoid fragments may compromise the development and differentiation of the epithelial barrier. To begin, aspirate the medium from the static organoid culture. Recover media from enough wells to achieve a final seeding density of eight million cells per milliliter.

Add 500 microliters of ice cold BMM dissociation solution to each well and attach the solubilized BMM off the well surface. Collect the suspension into a 15 milliliter low protein binding tube and place it on ice. Gently shake the tube every 15 minutes for one hour.

Centrifuge at 300 times G for five minutes at four degrees Celsius to obtain an organoid pellet. If a transparent gel layer is observed above the pellet, aspirate solution from the tube. Resuspend the pellet and add an equal volume of BMM dissociation solution.

Incubate on ice for 10 minutes and centrifuge. Repeat until no solubilized BMM residues are present. Then discard the supernatant and resuspend the pellet in two milliliters of organoid digestion solution.

Incubate the tube at 37 degrees Celsius for one to three minutes and add eight milliliters of advanced DMEM F12 to stop the enzymatic reaction. Centrifuge and resuspend the pellet in complete organoid growth medium to achieve eight million cells per milliliter. Aliquot 360 microliters in sterile 1.5 milliliter low protein binding tubes.

Next, remove the medium from the top channel of the coated chips and add 30 microliters of the cell suspension. Incubate the chips at 37 degrees Celsius overnight to adhere the organoid fragments to the membrane. Add three milliliters of pre-equilibrated complete organoid growth medium to the top inlet reservoir and three milliliters of pre-equilibrated endothelial cell growth medium in the bottom inlet reservoir.

Add 300 microliters of the same media in the respective top and bottom outlet reservoirs. Transfer the tray carrying the portable modules into the culture module. On the culture module, repeatedly run the prime cycle until sufficient droplets form for successfully connecting the chips.

Then slide the chip carrier into the portable module. Then start the two-hour long regulate cycle to pressurize the culture media in the portable module and chip after which the programmed conditions will resume. Next, change the stretch settings and start the culture module.

After 24 hours, repeat the cycle with 10%stretch and the same frequency. To prepare dosing media or vehicle control, dilute the CYP inducer stock solutions or DMSO in complete organoid growth medium and endothelial cell growth medium. Pause the culture module and bring the trays to the biosafety cabinet.

Replace the media in all the inlet and outlet reservoirs with two milliliters of dosing media with inducers or vehicle control. Return the portable modules back to the culture module and restart the flow at 30 microliters per hour. Replace the media with freshly prepared inducing solution every 24 hours and continue the culture for 48 to 72 hours.

Then bring the trays to the biosafety cabinet and aspirate the dosing medium from all the reservoirs. Wash and replace the top inlet reservoir with warm advanced DMEM F12 medium and the bottom reservoir with endothelial cell growth medium. Replace the wash medium with one milliliter of probe substrate solution.

Perfuse the chips at a high flow rate of 1, 000 microliters per hour for five minutes and aspirate both top and bottom outlet reservoirs. Return the chips to the culture module and incubate for one hour under a constant flow of 300 microliters per hour. After one hour of treatment, stop the flow and bring the trays to the biosafety cabinet.

Collect 100 microliters of effluent from the top outlet reservoir and add it to a tube containing 200 microliters of stop solution. Place the tubes immediately on dry ice and store the samples at minus 80 degrees Celsius. Wash both the channels with 200 microliters of sterile DPBS.

Then block the bottom channel outlet with a 200 microliter filter pipette tip. Perfuse 50 microliters of the dissociation solution through the bottom channel and incubate for two minutes at room temperature. Ensure complete detachment of the endothelial cells and remove the dissociation solution from the channel by pipetting.

Repeat the wash. Next, block the top channel outlet and perfuse 75 microliters of protein lysis buffer. Leave the pipette tip inserted and incubate for five minutes at room temperature.

Collect the cell lysates in a 1.5 milliliter tube by pipetting five to 10 times. Repeat perfusion and collection to obtain complete detachment of cells and store the lysates at minus 80 degrees Celsius until analysis. For RNA lysis, follow the same procedure while using 150 microliters of RNA lysis buffer.

First, prepare an interferon gamma dosing solution by diluting the stock solutions in degassed endothelial cell growth medium. In the biosafety cabinet, remove the medium from the bottom channel inlet reservoirs and replace it with three milliliters of the dosing solution daily. Then place the trays into the culture module and perfuse the chips at a high flow rate of 1, 000 microliters per hour for five minutes.

Switch the flow rate to 60 microliters per hour and continue the fluidic culture. Add 100 microliters of DPBS per well into a 96-well black walled plate. Using a multichannel pipette, add 50 microliters of the effluent from all the reservoirs to the respective wells.

To prepare a standard curve, perform a three-fold dilution of the medium containing 100 micrograms per milliliter of three kilodalton dextran cascade blue in DPBS. Then perform serial delusions using a three-fold dilution of endothelial cell growth medium in DPBS. Distinct morphological features of the duodenum and colon chips were highlighted by the presence of the villi-like formations in the duodenum chip representing the architecture of the small intestine.

In the duodenum chip exposed to the cytochrome P450 inducers rifampicin and vitamin D3, there was an elevated mRNA gene expression for the drug metabolizing enzyme cytochrome P450 3A4 and enhanced catalytic activity of the cytochrome P450 enzyme, indicating appropriate induction responses across all three organoid donors. Upon treating the epithelial monolayer of the colon chip with interferon gamma, compromised cell morphology and loss of the columnar epithelium were observed. Also, treatment with interferon gamma led to increased cytoplasmic signal of tight and adherent junction proteins compared to vehicle control, showing the displacement of tight junction markers ZO-1 and claudin 4 and internalization of occludin and E-cadherin.

A significant increase in the epithelial paracellular permeability was observed following 48 hours of interferon gamma stimulation on colon chips. Similarly, interferon gamma induces activation of apoptosis indicated by increase in the intracellular content of the cleaved Caspase 3. Interferon gamma induced a polarized secretion of pro-inflammatory molecules in the colon chip as shown by the basolateral secretion of VCAM-1 and interleukin-6 and the apical secretion of ICAM-1 and serum amyloid protein A.For a successful intestine chip culture, one should utilize undifferentiated organoids and follow the fragmentation and seeding density instructions.

Following successful establishment of the intestine chip, we can study intestinal absorption, transport, and metabolism, nutrient levels, and intestinal hormone secretion, host microbe pathogen interactions, drug safety and efficacy, patient-specific disease mechanisms, and responses to therapies.