Adapting Gastrointestinal Organoids for Pathogen Infection and Single Cell Sequencing under Biosafety Level 3 (BSL-3) Conditions

This protocol allows us to understand how individual cell types present in the human intestinal epithelium response to viral infection. We here present three independent ways to seed human intestinal organoids to address how the route of infection can impact how the cell respond. We highlight how to prepare samples for single cell RNA sequencing considering the biosafety regulations that need to be respected when working with Category 3 pathogens.

This method can easily be adapted to other pathogens and organoid cultures. The most important consideration is to ensure that your organoid models are fully differentiated with the chosen seeding method and can support viral infection. Before harvesting the infected organoids, remove the single cell gel beads from minus 80 degrees Celsius and warm them to room temperature.

Additionally, equilibrate the RT reagent, reducing agent B, and RT enzyme C to room temperature and resuspend the template switch oligo as described in the manufacturer's instructions. Next, to collect 3D organoids infected with the BSL-3 pathogen, remove the cell culture plate from the incubator and place it in the cell culture hood. Then using a P1000 pipette, remove the differentiation media from each well of the 24-well plate, add 500 microliters of ice cold PBS to each well and incubate at room temperature for three minutes.

Next, to fully disrupt the extracellular matrix solution, pipette up and down 10 times to resuspend the PBS, extracellular matrix, and organoids, then transfer the resuspended organoids into a 15 milliliter conical tube and place the tubes on ice. Collect each infection condition in separate 15 milliliter conical tubes. After changing gloves, clean the outside of the tube and remove the tube from the cell culture hood.

Spin the samples for five minutes. Move the tube back into the cell culture hood and remove the PBS, avoiding resuspension of the organoid's pellet from the bottom of the tube. Next, resuspend the pellet in one milliliter of dissociation enzyme.

then change gloves, clean the tube, and incubate the samples at 37 degrees Celsius for 30 minutes. During the incubation, move the tube back into the cell culture hood every 10 minutes and resuspend the organoids by pipetting up and down. To determine if the organoids are dissociated into single cells, place 10 microliters of the organoid suspension in a disposable plastic cell counter slide using a P10 pipette, then seal the sample input port with clear tape.

After changing gloves, clean the outside of the cell counter and using a Brightfield microscope, confirm that a single cell suspension is created. Following confirmation of single cell suspension, stop the digestion by adding one milliliter of DMEM F12 media containing 10%FBS and pipette up and down 10 times. Then change the gloves, clean the tube and spin the sample.

After centrifugation, carefully remove the media containing the dissociation enzyme, taking care not to disturb the cell pellet at the bottom. Then resuspend the single cells in a minimal volume of PBS containing 0.1%BSA. After passing the cell suspension into a FACS tube through a filter to remove large clumps, place the tube on ice.

Next, to determine the number of cells per microliter, add 10 microliters of the cell suspension to a disposable plastic cell counting chamber. Then seal the sample input port with clear tape before removing the sample from the cell culture hood. Change gloves, clean the cell counting chamber, then count the number of cells using a Brightfield microscope.

Next, inside the cell culture hood, prepare a master mix of RT reagent, template switch oligo, reducing agent B, and RT enzyme C in a 1.5 milliliter tube as per the manufacturer's instructions depending on the number of samples in the experiment. For each sample, aliquot 33.4 microliters of master mix into a PCR tube, then add the cells and water to the master mix according to the target cell number. After changing gloves, move the single cell controller into the cell culture hood and prepare the chip.

Next, add the single cell chip into the chip holder and fill the unused lanes with 50%glycerol. Add the master mix, beads, and partitioning oil to the lanes containing samples per the manufacturer's instructions and cover the chip with a gasket. After loading the chip into the controller, start the program.

Upon completion of the program, remove the chip and the gasket, Then using a multi-channel pipette, transfer 100 microliters of the emulsions to clean PCR tubes. Ensure that each emulsion has a uniform white color indicating that a complete emulsion has occurred. After changing gloves and cleaning the tubes, transfer the PCR tubes to a PCR machine and start the program.

At the end of the PCR run, most enveloped viruses will be inactivated. The representative Brightfield images at days one, three, five and seven post-splitting organoids are shown here. On average, the organoids are split once a week when the centers become dark.

By changing the media conditions, removing Wnt-3a and reducing R-spondin and noggin, the cellular complexity found within the human intestine can be mimicked. Normally, cellular differentiation toward Paneth cells, goblet cells, and enterocytes requires four days of differentiation media. Analysis of single cell sequencing data from SARS-CoV-2 infected human colon and ileum-derived organoids showed that only a subpopulation of human intestinal epithelial cells supported the infection of SARS-CoV-2 after 12 and 24 hours.

Analysis of innate immune responses in SARS-CoV-2 infected colon-derived organoids showed that SARS-CoV-2 induced a pro-inflammatory signal cascade in infected cells while noninfected bystander cells showed an interferon mediated immune response. Additionally, single cell RNA sequencing showed that infected cells were unable to sense interferons due to virus-mediated blockage of the pathway. The organoids must be well-differentiated and healthy.

This can be controlled by determining the level of cell differentiation markers by qPCR prior to infection. If the cells are not well-differentiated, they will not support viral infection and lead to uninformative results. This technique allows us to infect mucosa from both the luminal and the tissue side.

This is very key as it allows us to unravel mechanisms used by mucosal surfaces to tolerate a dirty environment on one side and a sterile one on the other.