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

Human tissue was received from colon resection or ileum biopsies from the University Hospital Heidelberg for the following protocol. This study was carried out under the recommendations of the University Hospital Heidelberg with informed written consent from all subjects in accordance with the Declaration of Helsinki. All samples were received and maintained in an anonymized manner. The protocol was approved by the Ethics commission of the University Hospital Heidelberg under protocol S-443/2017.

1. Maintenance and passaging of intestinal and colon organoids

CAUTION: Human intestinal organoids are derived from human tissue or from induced pluripotent/embryonic stem cells, and as such, ethical approval is required. Country-specific regulations need to be followed. Human material is generally not tested and is therefore often considered BSL-2 material. Proper containment conditions need to be confirmed in the country in which the experiment takes place.

1. Prepare intestinal and colon organoids from isolated tissues and/or biopsies using previously described methods². Additionally, more technical details on how to prepare organoids from patient-derived material or from iPSCs can be found in Lees et al. and Mahe et al.^24,25.
2. Once organoid cultures are established, follow the below described splitting routine to prepare organoids to perform infections with enteric pathogens.
3. Seed 20-100 organoids in 50 μL of 100% ECM solution in 24-well non-tissue culture treated plates. Add 500 μL of growth media (Table 1) per well.
4. Change the media every 2 days by removing 250 μL of the old media and adding 250 μL of the fresh growth media to each well. Warm the media to room temperature before changing the media.
   CAUTION: Cold media will liquefy the ECM solution containing organoids.
5. Passage the organoids once per week when the interior begins to become dark due to the accumulation of dead cells. An example of this can be found in Figure 2.
6. On the day of splitting, remove the ECM solution from -20 °C and thaw on ice.
7. Place the new empty cell culture plate that will be used to seed the organoids after splitting at 37 °C (this requires a minimum of 1 h to be warm and could be warmed overnight). Warm the culture media to room temperature.
8. Remove the growth media with a P1000 pipette and add 500 μL of cold 1x phosphate-buffered saline (PBS) to each well for 3 min to partially liquefy the ECM solution and dissociate from the plate.
9. To ensure full disruption of the ECM solution, use a P1000 pipette (set-up at 450 μL). Pipette up and down 10 times to resuspend the PBS, ECM solution, and organoids, transfer the resuspended organoids into a 15 mL conical tube and place them on ice.
10. Collect multiple wells of the same organoids into the same conical tube.
11. If multiple different organoids (different donors, different sections, different pre-treatment, etc.) are split at the same time, collect them in different conical tubes. While collecting, maintain the conical tubes on ice.
12. Spin the samples at 500 x g for 5 min at 4 °C. Carefully remove the PBS with a pipette to maintain the organoid pellet at the bottom.
13. Avoid using a vacuum waste system with a fitted pipette as the pellet of organoids is very loose and can easily be aspirated when removing the PBS too quickly.
14. Add 1 mL of 0.05% trypsin to the conical tube and resuspend organoids by pipetting up and down 10 times with a P1000 pipette. Incubate the conical tube containing organoids at 37 °C for 3 min.
15. Add 2 mL of DMEM/F12 media containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin to stop the digestion and resuspend to disrupt the organoids by pipetting up and down 10 times with a P1000 pipette.
16. Spin the samples at 500 x g for 5 min at 4 °C. Remove conical tubes from the centrifuge and store them on ice.
17. Carefully remove media/trypsin with a 5 mL disposable pipette to maintain the organoid pellet at the bottom. Leave around 500 μL of media and remove this with a P1000 pipette.
18. Once media/trypsin has been removed from all conical tubes, remove the 24-well non-cell culture treated plate from the 37 °C incubator and place it under the cell culture hood.
19. Add 100% ECM solution (kept on ice) to the conical tube with the split organoids at 1:3 to 1:5 ratio depending on the growth habits of the individual donor organoids. (For example, if one well containing 20-100 organoids in a 50 μL drop of ECM solution was passaged, resuspend the organoid pellet into 150-250 μL of ice-cold ECM solution.)
20. Seed 50 μL of ECM solution/organoid mix per well of the 24-well non-cell culture treated plate pre-warmed to 37 °C.
    NOTE: ECM solution will polymerize very quickly once it has warmed. Keep the ECM solution cold at all times (stored on ice) during use. When organoids have been resuspended, seed immediately on a new plate. For beginners, it is recommended to store a box of pipette tips at -20 °C to allow extra time for the seeding.
21. Incubate the 24-well plate at 37 °C for 10-15 min to allow the ECM solution to polymerize.
22. Following polymerization, add 500 μL of growth media (Table 1)²⁶ to each well and incubate the organoids at 37 °C. Check the organoids daily by microscopy. Change the media every 2 days as in step 1.4.

2. Preparation of organoids in two-dimensions (2D) for apical infection

NOTE: The following protocol will describe how to seed intestinal organoids as a monolayer of cells in a cell culture plate to infect intestinal epithelium cells from their apical side. Use the 48-well plate for the sequencing experiments and the 8-well glass-bottom chamber slide to control infection using immunofluorescence approaches.

1. Grow and maintain organoids as described in section 1.
2. Prior to seeding organoids in 2D, coat the 48-well plates and 8-well glass-bottom chamber slide with 200 μL of 2.5% human collagen in water per well for 1 h at 37 °C.
   NOTE: Intestinal organoids are best seeded in 48-well plates and in 8-well glass-bottom chamber slide. Experience has shown that they do not infect well in 96-well plates. Transwell inserts can also be used to allow for both apical and basolateral infection in 2D. If transwells are used, monitor the trans-epithelial electrical resistance (TEER) prior to infection to confirm a confluent monolayer. Normally, intestinal organoids have a tight barrier with a TEER of 450-600 Ohm/cm².
3. Seed 100-150 organoids in each well of a 48-well plate or for one well of an 8-well glass-bottom chamber slide.
4. To estimate the number of organoids, count the number of organoids present in the 50 μL of ECM solution drop of the 24-well plate prepared in section 1. On average, 1-2 wells are needed, which will give approximately 15,000-30,000 cells.
5. To disrupt the ECM solution and trypsinize the organoids, follow steps 1.8-1.17.
6. Remove human collagen from the 48-well plates and the 8-well glass-bottom chamber slide.
7. Resuspend the organoid pellet in the conical tube in 250 μL of growth media/well and add the mixture to the collagen-coated wells. Place the plate in a 37 °C incubator.
   NOTE: When performing the experiments, multiple conditions are compared (mock vs. infected +/- treatment of interest). To minimize variability between each well of 2D seeded organoids, collect the total number of necessary organoids in the same conical tube. Collecting organoids from up to 12 wells of a 24-well plate can use 1 mL of trypsin and 2 mL of neutralizing media. When using 12-24 wells, increase this to 2 mL of trypsin and 4 mL of neutralizing media.
8. After 48 h, remove the plate from the incubator and place it in the cell culture hood. Remove the growth media and replace it with 250 μL per well of differentiation media (Table 1). Repeat this media change 48 h later.
9. After 48 h, confirm differentiation (four days post-switch to differentiation media) by extracting RNA, making cDNA with 250 ng of RNA, and performing SYBR green based qPCR using primers for stem cells (OLFM4 and/ or SMOC2), goblet cells (MUC2), enteroendocrine cells (CHGA), and enterocytes (SI and/or CYP3A4) (Figure 3).
   NOTE: Upon switching from growth media to differentiation media, organoids will decrease their expression of stem cell markers (OLFM4 and/or SMOC2) and increase expression of goblet cells (MUC2), enteroendocrine cells (CHGA), and enterocytes (SI and/or CYP3A4) markers by qPCR. Prepare an extra well and maintain with growth media to compare the expression level of each cell-type-specific marker in growth vs. differentiation media.
10. Upon confirmation of differentiation, organoids are ready for apical infection. Move the organoids to the biosafety level containment required for the pathogen of choice.
    CAUTION: Refer to the institute's local regulations and standard operating procedures when handling pathogens under BSL-2 or BSL-3 containment.
11. To infect the intestinal organoids grown in 2D from their apical side, remove media with a P1000 pipette and add pathogen diluted at the multiplicity of infection required for the experiment in differentiation media (minimum volume of pathogen/media mix is 50 μL per well and maximum volume is 250 μL per well).
12. Allow the infection to proceed for 2 h at 37 °C with either a rocker table located in the cell culture incubator or by manually rocking the plate every 15-20 min. Optimize this time based on the pathogen of choice.
13. After 2 h, remove the media with a P1000 pipette and replace it with 250 μL per well of fresh differentiation media. Incubate cells at 37 °C until the time point of single-cell sequencing.
14. To prepare cells for single-cell sequencing, proceed to section 4.

3. Preparation of organoids in three-dimensions (3D) for apical and basolateral infection

1. Grow organoids as described in section 1 in a 24 well, non-cell culture treated plate.
2. Two days post-passaging, remove the plate from the incubator and place in a cell culture hood.
3. Remove the growth media with a P1000 pipette, replace it with 500 μL/well of differentiation media pre-warmed to room temperature and place the plate in the 37 °C incubator.
4. After 48 h, replace with fresh differentiation media (500 μL/well).
   NOTE: Organoids are maintained in differentiation media for a total of 4 days prior to infection.
5. Confirm differentiation as described in step 2.9.
6. Upon confirmation that differentiation has occurred, organoids are ready for infection.
7. Thaw ECM on ice. Warm the differentiation media to room temperature and warm a 24-well plate at 37 °C.
8. To perform infections, remove the organoids from the ECM solution.
   NOTE: Remove as much ECM solution as possible as viruses prefer to stick to the ECM solution rather than cells. If there is too much ECM solution remaining around the organoids, then infectivity will be severely impacted.
9. Disrupt the ECM by adding 500 μL of cold 1x PBS and incubating for 3 min. Use a P1000 to pipette up and down 10x. Combine organoids in a tube and centrifuge at 500 x g for 5 min. Remove PBS with a P1000.
   NOTE: To minimize variability between each infection condition, combine the organoids coming from several wells of a 24-well plate into the same conical tube. For example, if eight conditions for infection are required, eight wells of a 24-well plate (containing ~100 organoids each) will be combined and subsequently divided into eight wells of a new 24-well plate following needle disruption (see step 3.12).
10. Resuspend the organoids in 1 mL of differentiation media. For apical and basolateral infection follow step 3.10.1 and for basolateral infection only follow step 3.10.2.
    1. Attach a 27 G needle to a 1 mL syringe and resuspend the pellet six times to allow disruption of organoids and for infection to occur at both the apical and basolateral sides. Proceed to step 3.11.
       NOTE: The organoids should be the classical cyst-looking and octopus-budded organoids with a dark center when observed prior to needle disruption. Following the disruption, these organoids will appear smaller with less to no dark interior. When performing infection, there will always be a minimum of two conditions (mock and infection), a minimum of two wells of a 24-well plate will be initially combined into a 15 mL conical tube for needle-based disruption (explaining why a minimum of 1 mL is used for resuspension). When performing more than two conditions, combine up to 10 wells of a 24-well plate in a single 15 mL conical tube and resuspend in 1 mL of differentiation media for needle disruption. Following the disruption, add 500 μL of differentiation media per n + 1 (for example, if using 10 wells, then top up with 4.5 mL to have 5.5 mL total). If more than 10 conditions are needed, use several conical tubes. A 1 mL syringe is recommended as organoids are disrupted better with this volume of the syringe. An alternative to a syringe is to employ a well-flattened P1000 tip.
    2. Resuspend the organoids in 500 μL of differentiation media per well. Be careful not to disrupt the organoids and make sure that the apical side is not accessible. Proceed to step 3.11.
11. Transfer 500 μL of organoid suspensions per well to a 24-well cell culture plate using a P1000 pipette and move the plate to the biosafety level containment required for the pathogen of choice.
    CAUTION: Refer to the local regulations and the institute's standard operating procedures when handling pathogens under BSL-2 or BSL-3 containment. Always perform the needle disruption of the organoids outside the BSL-3 to avoid potential accidents with the needle. Local regulations may also prevent the use of needles in the BSL-3.
12. Add the pathogen diluted in differentiation media to reach the multiplicity of infection required for the experiment. Do not exceed a total volume of 500 μL (to have 1 mL total in the 24-well plate).
13. Allow the infection to proceed for 2 h (this time will need to be adapted to the pathogen of choice) at 37 °C with either a rocker table located in the cell culture incubator or by manually rocking the plate every 15-20 min.
14. Following the 2 h incubation, collect the organoids into one 1.5 mL tube per condition and spin at 500 x g for 5 min at 4 °C.
15. Remove the media containing pathogen with a P1000 pipette and store it on ice. Wash the organoids pellet once with PBS.
16. Resuspend the organoids in 50 μL of 100% ECM solution (which was previously thawed on ice), plate into a 24-well non-cell culture treated plate pre-warmed to 37 °C and incubate the 24-well plate at 37 °C for 10-15 min to allow the ECM solution to polymerize.
17. Following polymerization, add 500 μL of differentiation media at room temperature to each well and incubate at 37 °C until harvesting for single-cell sequencing.
    ​NOTE: If harvesting needs to occur more than 48 h post-seeding, change the media every 2 days by removing the old media and replacing it with 500 μL of fresh differentiation media. However, experience has shown that since organoids are grown under differentiation media, they will not survive much longer than 48 h.

4. Preparation of single cell solution and preparation of Gel beads-in-emulsion (GEMs) in biosafety level 3 (BSL-3) conditions

NOTE: Completion of the following steps requires that the single cell sequencing equipment (Table of Materials) and a PCR machine capable of handling 100 μL reactions are present inside a BSL-3 facility. Organoids are grown as described in section 1 and infected as described in sections 2-3 depending on the pathogen and entry route. At a pre-determined time post-infection, organoids are harvested. Below the method used to harvest intestinal organoids is described.

1. Prior to harvesting infected organoids, remove the Single Cell Gel beads (Table of Materials) from -80 °C and warm to room temperature (at least 30 min prior).
2. Additionally, equilibrate the RT reagent, Reducing Agent B and RT enzyme C (all stored at -20 °C) to room temperature. Resuspend the template switch oligo as described in the manufacturer's instructions.
   NOTE: All the following steps need to be performed in respect to the local biosafety regulations following the established standard operating procedure for the considered pathogen. As a rule of thumb for BSL-3 work, it must be ensured that the exterior of all vessels (plates, tubes, etc.) exiting a cell culture hood are properly disinfected. The same applies for the hands/gloves of the person performing the experiments.
3. To perform infections on organoids seeded in 2D (section 2) proceed to step 4.4. To perform infections on 3D organoids (section 3) proceed to sections 4.5.
4. For infections of 2D organoids, bring the cell culture plate into the hood and remove the differentiation media with a P1000 pipette.
   1. Add 250 μL of 1x PBS at room temperature to each well.
   2. Remove the PBS with a P1000 pipette and add 250 μL of room temperature dissociation enzyme (e.g., TrypLE Express) to each well of a 48-well plate. Change gloves, clean the plate, and place the plate with the dissociation enzyme in the 37 °C incubator.
   3. Observe the cell dissociation by microscopy every 5 min.
      NOTE: Approximately, it takes 15 min to dissociate intestinal organoids cultured in 2D into single cells. This time will need to be adjusted as it will depend on how many organoids were initially seeded and infected as well as on the pathogen as some pathogens are more cytopathic and cause organoids to dissociate much faster than others.
   4. Following confirmation of an apparent single cell suspension, bring the plate back into the cell culture hood and stop the digestion by adding of 250 μL of DMEM/F12 media containing 10% FBS per well of a 48-well plate.
   5. Collect the cells into a 15 mL conical tube using a P1000 pipette.
   6. Change gloves, clean the tube, and spin the samples at 500 x g for 5 min at 4 °C.
   7. Carefully remove the media/dissociation enzyme with a P1000 to maintain the cell pellet at the bottom. Resuspend single cells in a minimal volume of PBS containing 0.1% BSA. For intestinal organoids, resuspend in 250 μL for each well of the 48-well plate.
   8. Pass the cell suspensions into a FACS tube with a filter to remove any large clumps and place the FACS tube containing cells on ice.
   9. Proceed to step 4.6 to continue with cell counting and preparation of the master mix.
5. For infections of 3D organoids, remove the plate from the incubator and place it in the cell culture hood.
   1. Remove the differentiation media from each well of the 24-well plate with a P1000 pipette. Add 500 μL of ice-cold 1x PBS to each well and incubate for 3 min on ice.
   2. To ensure full disruption of the ECM solution, use a P1000 pipette (set to 450 μL). Pipette up and down 10 times to resuspend the PBS, ECM solution and organoids; transfer the resuspended organoids into a 15 mL conical tube and place on ice. Collect each infection condition in its own 15 mL conical tube.
      NOTE: Using a 15 mL conical tube gives a better, more distinct cell pellet than a 50 mL conical tube or a 1.5 mL tube.
   3. Change gloves and remove the tube from the cell culture hood. Clean the outside of the tube.
   4. Spin the samples at 500 x g for 5 min at 4 °C.
   5. Move the tube back into the cell culture hood and remove PBS with a P1000 pipette to avoid resuspending the organoids pellet from the bottom of the tube.
   6. Resuspend the pellet in 1 mL of dissociation enzyme (e.g., TrypLE Express). Change gloves, clean the tube, and incubate the samples at 37 °C.
   7. Every 10 min for 30 min, move the tube back in the cell culture hood and resuspend the organoids by pipetting up and down with a P1000 pipette 10 times.
      NOTE: Normally, intestinal organoids require around 30 min to form a single cell suspension when infected in 3D.
   8. To determine when organoids are dissociated to single cells, take 10 μL of the organoid suspension using a p10 pipette.
   9. Place the suspension in a disposable plastic cell counter slide. Seal the sample input port with clear tape.
   10. Change gloves and clean the outside of the cell counter. Use a brightfield microscope to determine whether a single cell suspension is made.
   11. Following confirmation of a single cell suspension, stop the digestion by adding 1 mL of DMEM/F12 media containing 10% FBS and pipette up and down 10 times with a P1000 pipette.
   12. Change gloves, clean the tube, and spin the samples at 500 x g for 5 min at 4 °C.
   13. Carefully remove the media/dissociation enzyme with a P1000 pipette to avoid resuspending the cell pellet from the bottom of the tube.
   14. Resuspend the single cells in a minimal volume of PBS containing 0.1% BSA. For intestinal organoids resuspend in 250 μL.
   15. Pass the cell suspensions into an FACS tube with a filter to remove any large clumps and place the FACS tube containing cells on ice.
   16. Proceed to step 4.6 to continue with cell counting and preparation of the master mix.
6. Determine the number of cells per μL by adding 10 μL of the cell suspension to a disposable plastic cell counting chamber.
7. Seal the sample input port with clear tape before removing the sample from the cell culture hood, as at this stage, the cell suspension is still infectious.
8. Change gloves, clean the cell counting chamber, and count the cell number using a brightfield microscope.
9. 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 mL tube as per the manufacturer's instructions depending on the number of samples in the experiment. For each sample, aliquot 33.4 μL of master mix into a PCR tube and store on ice.
10. Add the cells and water to the master mix according to the target cell number as described in the manufacturer's instructions.
    NOTE: 50%-60% of the cells are normally recovered (i.e., when loading 10,000 cells on the chip, 5,000-6,000 cells are used for analysis). Therefore, always load 10,000 cells. If the cell density does not allow this, centrifuge the cells and resuspend in a lower volume. Take care not to overload the chip as this will result in clogging of the chip. Additionally, if cells are not properly dissociated, there will be an increased risk of getting multiple cells per bead, which will need to be removed in downstream bioinformatics processing.
11. Change the gloves, move the single-cell controller into the cell culture hood, and prepare the chip as the chip is only covered with a gasket that is not sealed.
    NOTE: The machine needs to be inside the hood to prevent exposure to infected cell suspensions and potential aerosols.
12. Add the single-cell chip to the chip holder and fill the unused lanes with 50% glycerol.
13. Add the master mix, beads, and partitioning oil to the lanes used for samples as per the manufacturer's instructions.
14. Cover the chip with the gasket, load the chip into the controller, and start the program.
    NOTE: It is recommended to only load six of the eight lanes. Issues with improper emulsions often occur in lanes one and eight. The company says that all lanes are independent, and this should not occur; however, if possible, avoid these two lanes and run two chips if eight samples are needed.
15. Upon completion of the program, remove the chip and the gasket.
16. Use a multichannel pipette and transfer 100 μL of the emulsions to a clean PCR tube. Ensure that each well has a uniform white color indicating a complete emulsion has occurred.
17. Change gloves, clean the tubes, and transfer the PCR tube to a PCR machine that can support 100 μL reactions. Run the program: 53 °C for 45 min; 85 °C for 5 min.
18. At completion, store the samples at 4 °C. At this point, process the reaction according to the manufacturer's instructions or store at 4 °C for 3 days or -20 °C for 1 week.
19. After 5 min at 85 °C, most enveloped viruses will be inactivated, remove the cDNA from the BSL-3 according to the normal operating procedure and perform the library preparations in a BSL-1 laboratory.
    NOTE: This experiment needs to be performed as three biological replicates (e.g., on three different days) because the extent of the differentiation of each cell type can slightly vary between each experiment. The resulting sequencing libraries could be differently indexed and sequenced together in one sequencing run.