Protocol for SARS-CoV-2 infection of kidney organoids derived from human pluripotent stem cells

Summary This protocol presents the use of SARS-CoV-2 isolates to infect human kidney organoids, enabling exploration of the impact of SARS-CoV-2 infection in a human multicellular in vitro system. We detail steps to generate kidney organoids from human pluripotent stem cells (hPSCs) and emulate a diabetic milieu via organoids exposure to diabetogenic-like cell culture conditions. We further describe preparation and titration steps of SARS-CoV-2 virus stocks, their subsequent use to infect the kidney organoids, and assessment of the infection via immunofluorescence. For complete details on the use and execution of this protocol, please refer to Garreta et al. (2022).1


SUMMARY
This protocol presents the use of SARS-CoV-2 isolates to infect human kidney organoids, enabling exploration of the impact of SARS-CoV-2 infection in a human multicellular in vitro system. We detail steps to generate kidney organoids from human pluripotent stem cells (hPSCs) and emulate a diabetic milieu via organoids exposure to diabetogenic-like cell culture conditions. We further describe preparation and titration steps of SARS-CoV-2 virus stocks, their subsequent use to infect the kidney organoids, and assessment of the infection via immunofluorescence. For complete details on the use and execution of this protocol, please refer to Garreta et al. (2022). 1

BEFORE YOU BEGIN
The protocol herein outlines the detailed procedures for using SARS-CoV-2 virus isolates to infect human kidney organoids. Specifically, we describe the steps to prepare SARS-CoV-2 virus stocks and determine virus titers using Vero-E6 Cells. We also detail the methodology to culture and differentiate hPSC into kidney organoids, and how to induce diabetic-like kidney organoids in vitro. Next, we explain how to efficiently infect human kidney organoids with SARS-CoV-2 and assess infection by immunofluorescence and confocal imaging.
CRITICAL: All laboratory procedures related to virus preparation, infection of cell cultures/organoids or collection of infected cells/organoids must be performed in certified Note: Vero-E6 Cells recovery improves by plating cells at high densities post-thaw (3 3 10 4 to 7 3 10 4 cells/cm 2 ). Small 25-cm 2 or 50-cm 2 cell culture flasks can also be used for this purpose. For a 25-cm 2 flask, re-suspend the cells in 5 mL of Complete Growth Medium; for a 50-cm 2 flask, re-suspend the cells in 10 mL of Complete Growth Medium. 5. Incubate flasks at 37 C and 5% CO 2 . 6. Split the cells when 80%-90% confluency is achieved using a split ratio of 1:10 (the dilution ratio Note: After recovery from frozen stock, Vero-E6 Cells need 2-3 passages to reach their regular growth rate. Note: Prepare frozen stocks of Vero-E6 Cells from early passages. A confluent 75-cm 2 flask can be used to make 5 cryovials. To freeze cells, following trypsinization of a 75-cm 2 flask (see steps 6a-e above), add media back to cells to reach a total volume of 10 mL. Pellet cells by centrifugation at 300 3 g for 5 min. Remove the supernatant and add 5 mL of freezing medium consisting of FBS supplemented with 10% Dimethyl Sulfoxide (DMSO) to re-suspend the cell pellet. Transfer 1 mL of the cell suspension using a p1000 micropipette into each labelled cryovial. Immediately place the cryovials with the cells into a Mr Frostyä Freezing Container and store at À80 C for 24 h before to stock them in liquid nitrogen.
Note: 60 mL of VTN-N is diluted in 6 mL of DPBS 13.
c. Place 1 mL of the VTN-N solution into each well of 6-well plate. d. Incubate at 20 C-22 C for 1 h. e. Before plating cells, aspirate the VTN-N solution from each well.
Note: VTN-N coated plates can be stored at 4 C for up to a week. Store the plates with the VTN-N solution. Do not allow drying. Prior to use, pre-warm the culture plate at 20 C-22 C for 1 h. Before plating cells, aspirate the VTN-N solution from each well.
Note: Other plate formats can be used to maintain and expand hPSC although 6 well-pates are recommended.
9. Prepare 0.5 mM EDTA in DPBS 13. a. Dilute 50 mL of 0.5 M EDTA in 50 mL of DPBS 13. b. Filter the obtained solution using a 0.22 mm pore size syringe filter.
Note: The 0.5 mM EDTA solution can be stored at 20 C-22 C for up to 6 months.
10. Thaw a cryovial containing hPSC in a 37 C water bath. Troubleshooting 1. a. After carefully submerging the cryovial in the water bath check the cryovial content every 20 s. As soon as the cryovial content becomes liquid, transfer the cell suspension using a p1000 micropipette to a sterile 15 mL conical tube containing 10 mL of Complete E8 Medium. b. Centrifuge the cell suspension at 300 3 g for 5 min. Remove the supernatant and gently resuspend the cell pellet in 2 mL of Complete E8 Medium to obtain a homogeneous cell suspension. c. Count cells.
i. Take 50 mL of cell suspension in a 1.5 mL Eppendorf tube.
ii. Add 150 mL of Accumax and incubate at 37 C for 5 min to obtain a single cell suspension.
iii. Mix 10 mL of the single cell suspension with 10 mL of 0.4% Trypan Blue Stain and pipette 10 mL of the mixture into a Countess TM cell counting chamber slide. iv. Count viable cells using a Countess Automated Cell Counter. v. Consider that the initial cell suspension is diluted 1:4 with Accumax. Thus, the final number of cells per mL is [Number of viable cells] 3 4.
Alternatives: A Neubauer cell counting chamber can be used to count viable cells under the microscope.
11. Dilute the cells into the volume of Complete E8 Medium required for achieving a cell density between 4.75-5.7 3 10 5 viable cells/mL. Then, plate 2 mL of the cell suspension per well of VTN-N coated 6-well plate (1-1.2 3 10 5 cells/cm 2 ).
CRITICAL: When thawing hPSC, do not add ROCK inhibitor to the medium. hPSC routinely exposed to ROCK inhibitor may lead to inefficient generation of kidney organoids.
CRITICAL: After thawing, hPSC should be passaged at least twice before using the cells for subsequent differentiation methodologies.
13. Routinely, passage hPSC when the colonies cover 80% of the surface area of the culture plate, usually every 4-6 days (see Figure 1). Troubleshooting 2 and 3. a. Aspirate the Complete E8 Medium from each well of 6-well plate. b. Wash with 2 mL of DPBS 13 per well. c. Add 1 mL of 0.5 mM EDTA per well and incubate for 4-6 min at 20 C-22 C. During incubation with EDTA, observe the hPSC colonies under the microscope. When the cells in the colonies start to round up and detach, they are ready to be removed from the well.
CRITICAL: Avoid longer EDTA exposure. Excessive cell dissociation may result in low cell viability.
d. Aspirate the EDTA solution from the wells without disrupting the hPSC colonies. Then, dissociate the colonies by flushing them with 1 mL of Complete E8 Medium (2-3 times) to break up the colonies into small cell clusters. CRITICAL: Do not pipette the cell clusters more than 3 times, because hPSC are sensitive to mechanical stress. Excessive mechanical disruption would result in low cell viability.
e. Re-suspend the cell clusters in Complete E8 Medium and plate 2 mL of the cell suspension per well in a new VTN-N coated 6-well plate. Use a split ratio ranging from 1:3 to 1:4 for the first 2 passages post-thaw and from 1:6 to 1:10 for subsequent passages.
CRITICAL: Do not add ROCK inhibitor to the medium during hPSC passage.
Note: The optimal split ratio and dilution ratio during a split may vary among different hPSC lines and will need to be adjusted to allow growth of the cell colonies for 4-6 days until reaching 80% confluency.
f. Incubate the 6-well plate containing hPSC at 37 C and 5% CO 2 . Refresh with 2 mL of Complete E8 Medium per well the day after cell plating. Change medium every other day. 14. Prepare frozen stocks of hPSC.
a. Prepare Freezing Medium containing 10% DMSO by mixing 9 mL of Complete E8 Medium with 1 mL of DMSO in a sterile 15 mL conical tube. b. When hPSC colonies cover 80% of the surface area of the culture plate, dissociate them from each well of 6-well plate into small cell clusters using 0.5 mM EDTA as detailed in steps 13a-d.
c. Centrifuge the cell suspension at 300 3 g for 5 min. Remove the supernatant. For each well of dissociated hPSC, use 1 mL of ice-cold Freezing Medium to gently re-suspend the cell pellet. d. Transfer 1 mL of the cell suspension using a p1000 micropipette into each labelled cryovial.
Immediately place the cryovials with the cells into a Mr Frostyä Freezing Container and store at À80 C for 24 h. e. The day after, transfer the frozen cryovials from À80 C freezer to a nitrogen tank. Filter the solution using a 0.22 mm pore size syringe filter and store at 4 C, use within 1 week.

Reagent Final concentration Volume
TBS 13 supplemented with 1% Triton X-100 N/A 9.7 mL Donkey Serum 3% 300 mL Filter the solution using a 0.22 mm pore size syringe filter and store at 4 C, use within 1 week.

Total N/A 50 mL
Filter the solution using a 0.22 mm pore size syringe filter and store at 4 C, use within 1 week.

Total N/A 50 mL
Filter the solution using a 0.22 mm pore size syringe filter and store at 4 C, use within 1 week.

Primary antibody dilution ratios for whole mount or paraffin section immunofluorescence
Primary antibody or reagent Dilution ratio Dilute the primary antibodies at the indicated dilution ratios in TBS 13 supplemented with 1% Triton X-100 and 1% BSA for whole mount immunofluorescence or in TBS 13 supplemented with 0.5% triton X-100 and 1% BSA for organoid paraffin sections. Prepare fresh. This step allows the production of virus stock by amplification of a defined SARS-CoV-2 virus strain in Vero-E6 Cells. The infectivity of virus stock is assessed by 2 different means including plaque assay (next section, beginning at step 7) or by tissue culture infectious dose-50 (TCID50) assay (beginning at step 14).
Note: It is desirable to produce large amount of virus at a time to have consistency between experimental replicates. Usually, 10 flasks of Vero-E6 Cells for SARS-CoV-2 infection can be prepared. Vero-E6 Cells need to be 85%-90% confluent for virus inoculation and propagation.
c. Incubate the flasks at 37 C and 5% CO 2 .
CRITICAL: Restart the culture with a new early passage of Vero-E6 Cells if changes in morphology or if virus titers have decreased to below 10 5 plaque-forming units (PFU)/mL. CRITICAL: SARS-CoV-2 should not be passaged more than 3 times in Vero-E6 Cells to avoid viral genome mutations that may decrease or change its infectivity.

Reagent Final concentration Amount
CRITICAL: To produce SARS-CoV-2 virus stocks, it is important to inoculate the virus at low MOI (MOI=0.01 per cell) to ensure that each cell is productively infected while preventing the formation of defective viral particles.

Thaw a vial of SARS-CoV
a. Collect the virus supernatant into sterile 15 mL conical tubes. Discard the mock-infected control. i. Dispose flasks, remaining cells and pipettes as infectious waste. b. Centrifuge the tubes at 450 3 g for 5 min at 4 C to pellet the cell debris. c. Prepare 200-500 mL aliquots of the supernatant in labelled 2 mL screw-tubes. d. Immediately store the aliquots of virus stock at À80 C.
Note: The volume of virus stock aliquots will need to be adjusted to avoid repeated freezing and thawing cycles. This will prevent decrease of virus stock infectivity.

Timing: 4 days
This step is required to determine the virus stock titer by plaque assay, which measures infectious SARS-CoV-2 particles by quantifying the plaques generated in Vero-E6 Cell cultures after infection with serial dilutions of the virus stock using a viscous medium overlay. The viscous overlay is used to restrict the spreading of progeny virions to the initially infected foci of cells. In this manner, each infectious particle will generate a single area of cellular death, so called PFU. After incubation, cell monolayers are fixed and stained to visualize the plaques using crystal violet, which stains cells in purple while plaques remain clear. Plaques are then counted to assess the virus titer in PFU per mL (PFU/mL).   Figure 2B). a. Prepare 10-fold serial dilutions of the virus sample between 10 -1 to 10 -7 . i. For each virus sample to be titrated, take seven 1.5 mL Eppendorf tubes and label them with each dilution (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 ). ii. Add 900 mL of Complete Infection Medium to each tube. iii. Add 100 mL of the virus sample to be titrated to the first tube (10 -1 ) to make the first dilution. iv. Mix by vortexing the tube. This is your 10 -1 virus dilution. v. Continue with serial dilutions by taking 100 mL of the first virus dilution (10 -1 ) and transferring it to the second tube (10 -2 ). vi. Mix by vortexing the tube. This is now your 10 -2 virus dilution. vii. For subsequent virus dilutions repeat steps v-vi by taking 100 mL of the newly created virus dilution and adding it to the next Eppendorf tube until you have reached a 10 -7 dilution.
CRITICAL: Directly discard the tips without pipetting between each dilution.
11. Infect Vero-E6 Cell monolayers prepared in step 7 with the serial virus dilutions. a. Label each well of 6-well plate with the corresponding dilution ranging from 10 -3 to 10 -7 and 1 well for non-infected control. b. Remove the existent Complete Growth Medium from the Vero-E6 Cell monolayers. c. Wash once with 1 mL of PBS 13 per well. d. After vortexing the tube, transfer 500 mL of the 10 -7 virus dilution to one well of the labelled 6-well plate. e. Repeat this step for 10 -6 to 10 -3 dilutions.
Note: It is recommendable to start with the most diluted sample so the same pipette tip can be then used for subsequent pipetting from the most diluted to the less diluted samples.
f. Incubate the plates at 37 C and 5% CO 2 for 1 h to allow virus to attach.
CRITICAL: Redistribute the supernatant by rocking the plates carefully every 15 min to prevent cell monolayers from drying out.
g. During the incubation time, calculate the volume of Plaque Assay Overlay Medium needed, counting 3 mL per well of 6-well plate. Include 3 mL extra as a control of polymerization.
h. After 1 h incubation, remove the viral inoculums from each well and close the plate.
CRITICAL: Change tips between each well.
CRITICAL: The overlay medium has to be prepared just before being distributed in the plate because agarose begins to polymerize rapidly after mixing with the EMEM/14% FBS Medium. k. Incubate the plates at 37 C and 5% CO 2 for 3 days to allow plaque formation.
Note: Crystal Violet stains proteins and DNA within intact cells. In this manner, areas of infected dead cells will appear as clear spots on the purple cell monolayer.
a. Add 1 mL of 25% Formaldehyde solution per well on top of the agarose to allow fixation of the cell monolayers. b. Incubate for 30 min at 20 C-22 C.
Note: The fixation step with 25% formaldehyde solution allows for virus inactivation.
c. Carefully remove the agarose layer using a flat spatula. Dispose as infectious waste.
CRITICAL: Do not touch the cell monolayer with the spatula to avoid scratching the monolayer. CRITICAL: Properly discard formaldehyde and crystal violet solutions as toxic waste, according to institutional rules.
g. Leave the plate inverted to air-dry the stained monolayers.
Pause point: At this point, air-dried stained plates can be stored at 20 C-22 C for further quantification.
a. Count the plaques in each well at each virus dilution.
Note: Discount wells with fewer than 5 or greater than 100 plaques. Take note of plaque size and morphology. The negative control should present a uniform, purple-stained monolayer and is used as a reference control. Note: For instance, if you counted 50 plaques in the well corresponding to 10 -5 dilution in which you have added 0.5 mL of diluted virus inoculum, the titer of the virus stock would be 50/ (10 -5 3 0.5) = 10 7 PFU/mL.

OPEN ACCESS
Note: We recommend to run the titration in duplicate or to repeat the titration step using another tube from the same viral stock.
Titration of SARS-CoV-2 virus stock by TCID50 assay

Timing: 3 days
As an alternative method to the plaque assay, this step details how to determine the virus stock titer by TCID50 assay.  Figure 2D). 15. Infect the Vero-E6 Cells prepared in step 14 with 10-fold serial virus dilutions. a. Remove the 100 mL of media from the first row of wells of the 96-BCB plate containing Vero-E6 Cells. b. Add 110 mL of non-diluted virus stock per well. c. Use a multichannel pipette to remove 10 mL of virus from the first row of wells and place it in the second row of wells. Gently pipette up and down to mix the virus with the media. This is now your 10 -1 virus dilution. d. Repeat step 15c by removing 10 mL of the virus dilution from the second row of wells (10 -1 ) and adding it to the third row of wells. This is now your 10 -2 virus dilution. e. For subsequent virus dilutions repeat step 15c by removing 10 mL of the newly created virus dilution and adding it to the next row of wells until you have reached a 10 -6 dilution. f. Incubate the 96-BCB plate for 24 h at 37 C and 5% CO 2 .
CRITICAL: Change the pipette tips for each dilution and make sure to properly mix the contents of the well before diluting into the next well. Leave the last lane as a mock sample. inactivated. Once completely inactivated, next steps can be performed in the BSL-1/BSL-2 laboratory.
17. Perform immunolabelling of the fixed 96-BCB plate to detect virus infection (see Figure 2D Alternatives: An epifluorescence microscope can also be used instead of the Odysseyâ Imaging System to image the immunolabelled plate. In this case, use PBS 13 supplemented with 5% BSA as blocking buffer and perform the secondary antibody incubation using a secondary antibody conjugated to Alexa Fluor 488 at 1:1000 dilution ratio and the nuclear stain DAPI at 1:2000 dilution ratio in blocking buffer.
18. Determine the number of positive infected wells using the following method: a. Determine the average intensity of mock-infected cells. 19. Use the Spearman-Karber method to calculate the TCID50/mL using the following formula: log 10 50% end point dilution = -(x 0 -d/2 + d P r i /n i ).
x 0 = log 10 of the reciprocal of the highest dilution (lowest concentration) at which all wells are positive. d = log 10 of the dilution factor (dilution factor= 10). Summation is started at dilution x 0 .
20. TCID50/mL can be used to calculate the PFU/mL using the following conversion: PFU/mL = TCID50 3 0.69.

Generation of kidney organoids from human pluripotent stem cells
Timing: 18 days The following steps describe the generation of kidney organoids from hPSC in a 96-well floating culture system. 1,2 This protocol is an adaptation of our previous methodology describing the generation of kidney organoids using the transwell culture system 3 and our previous published works using 96-well floating culture system. 2,4 In the next section, day 16 kidney organoids generated in floating culture conditions are exposed to normoglycemic-like glucose culture regimes or high oscillatory glucose culture conditions to emulate a diabetic-like milieu (beginning at step 26).
21. Seeding of hPSC for differentiation (day -2 to day 0), see Figure 3A. Troubleshooting 8. a. 1 h prior cell dissociation, prepare one 24-well plate with VTN-N coating (as described in before you begin section). Use 0.3 mL of VTN-N solution per well of 24-well plate.
Note: A typical differentiation experiment is started from 1 VTN-N coated 24-well plate.
b. Disaggregate 3 wells of 6-well plate with hPSC at 80% of confluency into small cell clusters, as it is performed for a normal hPSC passage (described in before you begin section).
Note: A typical differentiation experiment can be initiated from 3 wells of 6-well plate with hPSCs at 80% of confluency. The number of cells that can be obtained from 1 well of 6-well plate with hPSC colonies at 80% of confluency normally ranges from 2 to 3 million cells.

OPEN ACCESS
CRITICAL: The starting cell density and colony distribution is crucial for an efficient differentiation and can vary among different hPSC lines. Usually, the optimal cell density ranges between 1-2 3 10 5 cells per well of 24-well plate and should be adjusted for every hPSC line.
i. Calculate the volume of Complete E8 medium needed to achieve a cell density between 2-4 3 10 5 cells/mL. Re-suspend the cells and plate 0.5 mL of the cell suspension per well of VTN-N coated 24-well plate. ii. After 24 h, refresh with Complete E8 Medium. iii. Incubate at 37 C and 5% CO 2 for 24 h. 22. Induction of posterior primitive streak formation (day 0 to day 3), see Figure 3A. Troubleshooting 9. a. Gently aspirate the Complete E8 medium from each well of the 24-well plate prepared in step 21 without disrupting the hPSC colonies (day 0). b. Add 0.5 mL of Complete Advanced RPMI 1640 Basal Medium supplemented with 8 mM CHIR (Posterior Primitive Streak Induction Medium) to each well. Incubate the plate at 37 C and 5% CO 2 . c. Replace the media with fresh Posterior Primitive Streak Induction Medium every 24 h for 3 consecutive days (see Figure 3B). Figure 3A.    26. Maintain organoids in V-bottom MicroWellä plate and expose them to normoglycemic (control) or high oscillatory glucose (diabetic) conditions for 7 days (from day 16 to day 23). a. Use a 12-multichannel pipette to replace the media every day with 100 mL per well of Normoglycemic or Hyperglycemic Medium. Incubate at 37 C and 5% CO 2 . b. Control organoids are cultured in Normoglycemic Medium only. c. Diabetic organoids are exposed to Normoglycemic Medium on even days or Hyperglycemic Medium on odd days.

Induction of intermediate mesoderm formation (day 3 to day 4), see
CRITICAL: When performing medium changes ensure to completely replace the media from each well to guarantee the proper exposure of kidney organoids to the desired glucose concentration. Pay attention not to aspirate organoids by accident.
27. After the 7 day-treatment, use control and diabetic kidney organoids for SARS-CoV-2 infection experiments (beginning at step 28).

Infection of kidney organoids with SARS-CoV-2
Timing: 2 days This step describes the methodology to infect control or diabetic kidney organoids with SARS-CoV-2. Mock and SARS-CoV-2 infected kidney organoids are analysed to detect the viral RNA by qRT-PCR and the virus nuclear protein (NP) by immunofluorescence. For RNA extraction, qRT-PCR analysis and primer sequences, please refer to Garreta et al. (2022). 1 For immunofluorescence protocol see next section (beginning at step 31).
28. Calculate the amount of virus stock needed to infect a given number of kidney organoids with 10 6 SARS-CoV-2 infectious particles per organoid (10 6 PFU per organoid). Keep some of the organoids for the mock control. Prepare 24 organoids for each condition tested, 12 organoids for qRT-PCR and 12 organoids for immunofluorescence. 29. Infect control or diabetic kidney organoids with 10 6 PFU per organoid.
a. Use wide orifice 200 mL pipette tips (wo-tips) to place 1 organoid per well of Low-Attachment Surface 96-well plate with 100 mL per well of the Normoglycemic or Hyperglycemic Medium.
Note: Use always wo-tips to collect or handle organoids.
CRITICAL: Keep mock and SARS-CoV-2 infected kidney organoids in separate plates to avoid cross-contamination of the mock controls with virus from infected organoids. CRITICAL: When performing the washing steps pay attention not to aspirate organoids by accident.
g. Add 100 mL per well of fresh Normoglycemic or Hyperglycemic Medium and incubate the mock or SARS-CoV-2 infected organoids at 37 C and 5% CO 2 for 1 day. 30. Collection of infected kidney organoids for further analysis at day 1 post-infection (1 dpi).
a. For each condition tested, use a wo-tip to collect 24 organoids in two 1.5 mL Eppendorf tubes (12 organoids per tube). b. Wash the organoids 3 times with 500 mL of PBS 13 per tube to remove unbound virus. c. Remove the supernatant and dispose as infectious waste. d. For viral RNA detection by qRT-PCR, add 500 mL of Trizol TM to 12 organoids of each condition tested and store the samples at À80 C for further analysis. e. For NP detection by immunofluorescence use 4% PFA solution to fix 12 organoids of each condition tested. i. In the fume hood, add 500 mL of 4% PFA per Eppendorf tube.
ii. Incubate the samples for 12-16 h at 4 C.
iii. In the fume hood remove the fixative and wash the fixed organoids 3 times with 500 mL of PBS 13. iv. Keep the fixed samples in PBS 13 at 4 C for further analysis.
Pause point: Fixed organoid specimens are stored at 4 C for further analysis in the following weeks. For longer storage of fixed specimens (up to 6-12 months), we recommend storing them in PBS 13 supplemented with 0.02% of Sodium Azide.

Timing: 4-5 days
This section describes the analysis of the extent of SARS-CoV-2 infection in kidney organoids exposed to Control and Diabetic conditions by immunofluorescence. Kidney organoids are analysed at 1 dpi for the detection of NP in combination with Lotus Tetragonolobus Lectin (LTL) to detect proximal tubular-like structures and angiotensin-converting enzyme 2 (ACE2). Immunofluorescence is performed in whole mount (see step 32) or by paraffin-sectioning (see step 33) of the organoids (see Figure 4). 31. Divide the number of fixed organoid specimens (previously fixed with 4% PFA; see step 30) to perform either whole mount immunofluorescence or immunofluorescence in paraffin sections.   CRITICAL: Prolonged incubation with secondary antibody solution for more than 4 h might cause unspecific antibody adhesion to the organoids.

Whole mount immunofluorescence of kidney organoids
k. After secondary antibody incubation, wash the organoids 3 times with 400 mL of TBS 13 per well for 20 min at 20 C-22 C under shaking conditions. l. Counterstain the nuclei with DAPI.

OPEN ACCESS
i. Remove the TBS 13 from each well and cover the organoids with 250 mL of the DAPI working solution (dilute the DAPI stock at 1:5000 ratio in TBS 13). ii. Incubate for 12-16 h at 4 C. iii. Remove the DAPI solution and add 400 mL of PBS 13 to each well. Keep the immunolabelled organoids at 4 C in the darkness until mounting them for imaging.
m. Perform clearing and mounting of the samples for confocal imaging (See Figure 4C). i. Take 1 rectangular cover slip per sample condition and adhere a double-sided sticky iSpacerâ. ii. Using a wo-tip, transfer 1 immunolabelled organoid, trying to collect the minimum amount of PBS 13, onto the cover slip. Use a tip to remove as much as possible any residual PBS 13 solution.
Note: 3 organoids per experimental condition can be cleared and mounted in the same rectangular cover slip.
iii. Add a drop of 100 mL of RapiClearâ 1.47 clearing reagent to completely immerse the organoid in the clearing solution. Avoid introducing bubbles. iv. Incubate for 20 min at 20 C-22 C until the organoid becomes transparent. v. Place another rectangular cover slip on top of the cleared organoid and check that the clearing solution spreads and cover the entire surface. The double-sticky iSpacerâ allows adhesion of both cover slips. vi. Store the mounted organoid samples at 4 C in the darkness until imaging.

Immunofluorescence of kidney organoid paraffin sections
Timing: 4 days 33. For immunofluorescence of kidney organoid paraffin sections proceed as follows (see Figures 4A and 4B). Troubleshooting 16. a. Prepare organoids for paraffin-embedding. i. Heat the 0.8% low melting agarose solution at 60 C in a water bath until having a liquid agarose solution. Maintain the agarose solution warm at 37 C. ii. Place a layer of 100 mL of 0.8% low melting agarose solution into the bottom of a plastic cryomold. Allow to cool down for 5 min. iii. Using a wo-tip, immediately re-suspend the fixed kidney organoids samples (from step 31) in 150 mL of warm 0.8% low melting agarose (37 C) and transfer the organoidagarose suspension to the agarose-coated plastic cryomold.
Note: Place at least 3 fixed kidney organoid specimens per cryomold.
CRITICAL: Ensure that organoids are surrounded by agarose without introducing air bubbles. Place the organoids close to each other.
iv. Place the prepared molds on ice until the agarose is solidified. v. Remove the agarose block containing the organoids from the mold and place it in 1 well of 12-well plate with 1 mL of cold PBS 13.
Pause point: The agarose blocks with organoids can be stored at 4 C, up to 1 week, for further paraffin embedding. We recommend to store agarose blocks in PBS 13 supplemented with 0.02% of Sodium Azide. i. Using a spatula, transfer the agarose blocks prepared in step 33a into embedding cassettes. ii. Proceed to standard dehydration in an automatic tissue processor (Tissue-Tekâ VIPâ 6, Sakura or equivalent) following the standard procedure. iii. Embed in paraffin at 58 C using metallic molds to obtain paraffin blocks in a paraffin station (Tissue-Tek TEC, Sakura or equivalent).
Pause point: The paraffin blocks can be stored at 20 C-22 C.
c. Section the paraffin blocks. i. Cool down the paraffin blocks on a cold plate (Tissue-Tek, Sakura or equivalent).
ii. Prepare organoid sections of 3 mm-thickness using a rotary microtome (Leica RM2255 or equivalent).
CRITICAL: Since organoids are quite small and difficult to distinguish within the paraffin block, do not approach the sample by cutting thicker sections to avoid losing the sample.
iii. Guide the ribbon of paraffin sections into the water bath (Leica HI1210 or equivalent) at 40 C to help unfolding of the sections on the water surface. iv. Collect the paraffin sections using charged glass slides. Prepare 20 slides with 2 sections per slide.
CRITICAL: Properly label each slide.
Note: We recommend performing serial sectioning.
v. Dry the slides in an oven at 60 C for 12-16 h.
Pause point: The slides can be stored at 20 C-22 C in a dry place for 6-12 months.
d. Select the slides for immunofluorescence analysis. i. Perform Hematoxylin and Eosin (H&E) staining in slides number 5, 10, 15 (from a total of 20 prepared slides per paraffin block) following standard procedures. ii. Observe the H&E-stained slides under the microscope to identify the slides that contain organoid sections.
Note: For example, if organoid sections are visible by H&E staining in slides 5 and 10 but not in slide 15, this indicates that organoids sections are majorly present from slide 5 to slide 10.
iii. Select at least 2 slides per sample to perform immunofluorescence analysis of a given combination of primary antibodies. Take an additional slide for performing the secondary antibody control. e. Deparaffinize and re-hydrate the selected slides.
i. Use an automated tissue processor following the standard procedure. f. Perform antigen retrieval.
i. Immerse the slides in citrate buffer (pH 6).
ii. Autoclave the immersed slides at 95 C for 20 min.
iii. Cool down the slides to 20 C-22 C.
Pause point: After antigen retrieval the slides can be stored at 4 C in TBS 13 for 24 h before continuing with the next step. i. Wash the slides with 0.5-1 mL TBS 13 per slide.
ii. Use 0.5-1 mL of TBS 13 containing 1% Triton X-100 and 3% Donkey Serum (3%DS-Blocking Buffer) to cover the organoid sections in each slide. iii. Incubate the samples for 1 h at 20 C-22 C.
Note: Use a box for histology slides to make a humidified chamber and horizontally place the slides inside to perform the blocking and the subsequent immunolabelling steps.
h. Perform an additional blocking step with the Streptavidin/Biotin Blocking Kit as detailed in step 32d. This step is required when biotinylated LTL is used. After that, cover the organoid sections in each slide with 0.5-1 mL of TBS 13 containing 0.5% Triton X-100 and 1% BSA (BSA-0.5%T-Blocking Buffer).
Note: Shaking is not required. iii. Immediately place rectangular coverslip on top of the slide. Carefully remove the mounting media that may come out from the slide using absorbent paper. iv. Seal the borders of the slide with nail polish.
Pause point: The mounted slides can be stored at 4 C in darkness for 1-2 weeks without substantial decrease in fluorescence signal intensity.

EXPECTED OUTCOMES
Following the procedures detailed here we routinely produce SARS-CoV-2 virus stocks and assess virus stock titers in 95% of plates from which virus production are initiated, obtaining virus stock titers ranging from 10 6 and 10 7 PFU/mL.
Additionally we expect the efficient generation of kidney organoids from hPSC in 90% of plates from which differentiations are initiated. Following our methodology kidney organoids develop in free floating conditions using V-bottom 96-well plates in which 1 single organoid forms in each well.
Successful generation of kidney organoids is characterized by the formation of round translucent epithelial structures, so-called RV, visible under the microscope on day 11 of differentiation ( Figure 3H), and then progress into nephron-like structures until day 16 of differentiation ( Figure 3J). Our procedure leads to the generation of kidney organoids containing segmented nephron-like structures as well as stromal-like cells, endothelial-like cells, among others. [1][2][3][4] Recently, we established a high oscillatory glucose regime that leads to early hallmarks of diabetic kidney disease during development and investigated the impact of a diabetic-like milieu on early stages of SARS-CoV-2 infection in hPSC-derived kidney organoids. 1 Our procedure is highlighted in Figures 5A and 5B. Here, we provide representative confocal images of control and diabetic organoids showing that ACE2 expressing cells (ACE2 + ) are mainly detected within LTL positive proximal tubule-like structures (LTL + ) ( Figures 5C and 5D). 1 Here we also detail methods for the detection of SARS-CoV-2 infection in control or diabetic kidney organoids taking advantage of whole mount immunofluorescence and immunofluorescence in paraffin sections. In our recent manuscript both immunolabelling techniques have been optimized and validated for the efficient detection of ACE2 + and NP expressing cells (NP + ) along with different renal markers, such as LTL for proximal tubule structures, WT1 for podocyte-like cells, or CD31 for endothelial-like cells. 1

OPEN ACCESS
Here, we provide the step-by-step procedure for the detection of viral NP together with ACE2 and LTL in mock or SARS-CoV-2 infected kidney organoids under control or diabetic conditions. In Figure 6 we provide representative confocal images of immunolabeled whole mount organoids (Figure 6A) or organoid paraffin sections ( Figure 6B) showing that NP + are mainly detected within LTL + in infected organoids.
Overall, the methodology described herein enables side-by-side comparison of SARS-CoV-2 infectivity between control and diabetic kidney organoids. Our read outs allow for the examination of viral NP expression concomitantly with ACE2 and LTL. Further analysis using markers of major kidney compartments can be found in our recent manuscript. 1

LIMITATIONS
The protocol described herein has been successfully used to study the impact of a diabetogenic-like milieu on early stages of SARS-CoV-2 infection in hPSC-derived kidney organoids. 1 However, we are aware that in vitro models as organoids do not truly recapitulate the full complexity of the human organ. For instance, it should be noted that the procedure described here to generate kidney organoids does not allow for the generation of fully developed vascular networks neither immune cells nor a proper collecting duct system. Nevertheless, our renal differentiation procedure consistently gives rise to kidney organoids containing ACE2 + within tubular-like structures, 1 as well as contain other renal cell populations (i.e., podocyte-like cells, tubule-like cells, endothelial-like cells, among others), 1-4 recapitulating in part the multicellular complexity of the native organ and thus providing a valuable in vitro model to study SARS-CoV-2 infection in the human context.

OPEN ACCESS
In our recent study we have made use of 2 different hPSC lines for the derivation of kidney organoids, including male human embryonic stem cells (ES [4] cell line) and female cord blood iPSCs (CBiPS1sv-4F-40 cell line), confirming infection with SARS-CoV-2 in both genetic backgrounds under non-diabetic and diabetic-like conditions. 1 It is well known that to model infection successfully it is crucial to ensure the accessibility of the different cell types in the organoid model compared to the native organ. 5 In this regard, it is important to highlight that ACE2 + in kidney organoids are spontaneously exposed and accessible to in vitro interventions as SARS-CoV-2 infection. Another important aspect in this regard is that our procedure does not require extracellular matrix (ECM)-related matrices (i.e., Matrigel) for organoid culture and thus viral accessibility is equal between biological replicates. However, as the organoid model system defined here does not recapitulate important features as proper vascularization or immune system, there remains a lack of understanding on complex cell-to-cell interactions and crosstalk upon infection. Further aspects to be studied in the future include exposure time by increasing organoid lifespan and sample analysis upon longer periods post-infection.

Problem 1
Low cell viability after thawing a cryovial of hPSC on VTN-N-coated plates in Complete E8 Medium. 24 h after thawing hPSC have not attached and most of them appear as floating dead cells in the culture plate (before you begin, step 10; see Figure 1B).

Potential solution
Ensure that hPSC colonies are at 80% confluency and have the correct morphology (without spontaneous differentiation) before freezing. When freezing hPSC, ensure to gently dissociate hPSC colonies in cell clusters avoiding single cell dissociation and use E8 medium supplemented with 10% DMSO as freezing media. For successful cryopreservation of cells, use a freezing container (i.e., Nalgeneâ Mr Frosty) to store the cryovials at À80 C. This type of containers ensures a controlled slowdown of the cryovials temperature at 1 C/min cooling rate until reaching À80 C. Keep the cryovials at À80 C for 24 h after which immediately transfer the frozen cryovials to the liquid nitrogen tank. It is also important to thaw quickly the cryovial at 37 C and immediately wash cells to remove the DMSO by adding 10 mL of Complete E8 Medium in a 15 mL falcon tube, centrifuging the cell suspension (5 min, 300 3 g) and gently re-suspending the cell pellet in fresh Complete E8 Medium. Do not use rock inhibitor.

Problem 2
Low cell viability after passaging. High cell mortality and too low cell attachment is observed after performing the passage (before you begin, step 13; see Figure 1C).

Potential solution
Ensure to passage hPSC colonies with the right confluency (80%) and morphology, without spontaneous differentiation. Adjust the timing of exposure to EDTA. Too long EDTA exposure may result in too small cell clusters or single cells, reducing viability. Too short EDTA exposure may result in insufficient cell detachment and exposure of cells to high mechanical stress when trying to detach them, thus reducing viability. After EDTA exposure, use 1 mL of Complete E8 Medium to flush the well and detach de hPSC colonies. They should come out easily. Use another fresh mL of E8 medium to flush again the well and collect the remaining cells. Avoid excessive up and down pipetting of the hPSC clumps suspension. Do not use rock inhibitor to passage the hPSC. Revise that VTN-N coatings have been properly prepared. Avoid using prepared VTN-N coatings that have been stored for more than a week. Remember to routinely check the expiration date and batch of VTN-N and Complete E8 Medium.
Problem 3 hPSC colonies do not have defined edges, or cells appear less compact within the colonies, or large differentiated cells appear in the culture within or surrounding the hPSC colonies (before you begin, step 13; see Figure 1D).

Problem 4
When plates are revealed with crystal violet staining, plaques are found to be grouped around edges of wells or uneven distributed within wells (step 12 of step-by-step method details).

Potential solution
Virus inoculum was not well distributed during incubation of virus dilutions for 1 h. Make sure to distribute the virus inoculum across the wells by gently moving the plates from front to back and side to side every 15 min of incubation to allow homogeneous distribution of virus.

Problem 5
A large area of missing cells or a large plaque-like spot is observed (step 12 of step-by-step method details).

Potential solution
This could be caused by scratching them when removing the agarose with the spoon. To avoid this, be careful not to touch monolayers with the flat spoon to prevent harming the cell monolayers.

Problem 6
The number of counted plaques is lower than 5 in all virus dilutions, or the number of counted plaques is bigger than 100 in all virus dilutions (step 13 of step-by-step method details).

Potential solution
If number of plaques is lower than 5 at all dilutions, this means that virus titer may be less than 1 3 10 2 PFU/mL. Discard the virus stock and consider making new virus stocks to increase the virus titer. If number of plaques is bigger than 100 at all dilutions, this means that virus titer may be higher than 1 3 10 7 PFU/mL. In this case, repeat the plaque assay performing further serial dilutions: 10 -6 , 10 -7 and 10 -8 .

Problem 7
Low virus titers are obtained (step 13 of step-by-step method details).

Potential solution
In our hands, most SARS-CoV-2 virus stock titers are between 10 6 to 10 7 PFU/mL. With this as reference, some methodological points can be considered to achieve optimal virus titers. (a) Routinely check morphology of Vero-E6 Cells and change to low passage Vero-E6 Cell stocks if any change is appreciated or infectivity has decreased. (b) For proper virus production, infect Vero-E6 Cells at low MOI per cell, usually 0.01. (c) To guarantee that virus supernatants are collected at the optimal time (when virus production peaks), prepare additional flasks to collect the virus supernatants at different time points and check the best titer by the plaque assay.
Problem 8 24 h after seeding the hPSC in the 24-well plate for differentiation, hPSC appear all together in the middle of the well or uneven distributed across the well, or heterogeneity on cell density is observed among different wells (step 21 of step-by-step method details).

Potential solution
When plating the hPSC into the 24-well plate, dispense 0.5 mL of the cell suspension per well of the 24-well plate using a p1000-micropipette or a 5 mL-pipette while making sure to homogenize the ll OPEN ACCESS suspension of cell clumps regularly by gently moving the falcon tube. After plating the cells, manually shake the 24-well plate to allow homogeneous distribution of the cell clumps across the wells, repeat after 30 min. Then, keep the plates in a quite incubator, avoiding any vibration or movement (i.e., opening-closing the incubator) for at least 2 h.

Problem 9
Cells massively die after CHIR addition (from day 0 to day 3 of renal differentiation), (step 22 of stepby-step method details).

Potential solution
It is important to take in mind that hPSC seeding density and colony distribution are crucial for achieving efficient renal differentiation and formation of a compact cell monolayer by day 4 of differentiation. If there is excessive cell death upon CHIR stimulation, this might be due to inadequate cell density or inadequate colonies morphology at day 0 of the protocol. For optimal colony density and morphology on day 0 see Figure 3B. If colonies are too small after 24-48 h of plating, replace the media with fresh Complete E8 Medium and wait additional 24 h before starting the differentiation. It is recommendable to optimize the initial cell seeding density for each hPSC line. If above reasons have been excluded, revise that CHIR stocks have been correctly prepared.

Problem 10
On day 4 of differentiation cells have not formed a confluent and compact cell monolayer as expected at this stage. Instead, areas of loose cells or empty areas are observed in the cultures (see Figure 3D, step 23 of step-by-step method details).

Potential solution
This could be due to improper hPSC density and colony distribution when starting the differentiation at day 0 of the kidney organoid generation protocol. If this is the case, repeat the experiment using 2 different initial hPSC seeding densities to ensure that 48 h after hPSC seeding, the required confluency and colony distribution is optimal to start the differentiation process. Also, from day 0 to day 4 of the differentiation protocol, take special care when performing media changes since differentiated cells tend to easily detach from the plate. Thus, dispense media slowly to avoid producing excessive pressure against the cells that would damage the cell monolayer and impair the formation of a compact cell monolayer by day 4 of differentiation.

Problem 11
On day 6 of the kidney organoid generation protocol cell spheroids do not have a compact morphology with defined edges. Instead, cell spheroids appear disaggregated (see Figure 3F, step 24e of step-by-step method details).

Problem 12
On day 11 of the kidney organoid generation protocol RV structures are not visible within organoids under the optical microscope (see Figure 3I, step 24h of step-by-step method details).
Potential solution This is due to failed renal differentiation. Repeat the experiment. Check that the initial hPSC density is the optimal for efficient kidney organoid generation (see Figure 3B) and perform media changes regularly as explained in each step of the protocol. Also, revise that the stocks of factors and reagents for medium preparation and basal medium for kidney organoid generation have not expired. Avoid repeated freezing and thawing cycles of the aliquoted stocks and avoid using reconstituted stocks of CHIR, FGF9, Activin A, and heparin for more than 6 months.

Problem 13
On day 16 of kidney organoid generation nephron-like structures are not observed within organoids under the optical microscope (see Figure 3K, step 25 of step-by-step method details).

Potential solution
This is due to failed or inefficient renal differentiation. It is possible that the yield of kidney differentiation was low, leading to the formation of other cell types that overgrow within the organoid, thus impairing the development of the few renal structures that could have formed. Repeat the experiment to improve the yield of renal differentiation by following specific recommendations detailed in the protocol herein (i.e., proper initial cell density and colony distribution by day 0, formation of a proper homogeneous and compact cell monolayer by day 4, perform media changes regularly as explained in each step of the protocol, among others). Also, revise that the stocks of factors and reagents for medium preparation and basal medium for kidney organoid generation have not expired. Avoid repeated freezing and thawing cycles of the aliquoted stocks and avoid using reconstituted stocks of CHIR, FGF9, Activin A, and heparin for more than 6 months.

Problem 14
After performing whole mount immunofluorescence of kidney organoids, the organoid samples used for secondary antibodies control display a high background signal (step 32 of step-by-step method details).

Potential solution
The secondary antibodies detailed in the protocol herein have been extensively tested and successfully validated for whole mount immunolabelling of kidney organoids with reproducible results. However, make sure to avoid incubating samples with secondary antibodies solution for more than 4 h since this may promote unspecific antibodies binding and cause small antibody precipitates. Also, after secondary antibodies incubation you can perform additional washing steps with increased duration time (i.e., 4 times for 30 min each). Ensure to maintain samples under shaking while preforming the antibodies incubation and washing steps.

Problem 15
After performing whole mount immunofluorescence of kidney organoids weak signal of immunolabelled proteins is observed across the organoid sample (especially in the middle of the organoid) by confocal microscopy (step 32 of step-by-step method details).

Potential solution
This could be caused by inefficient antibodies penetration within the organoid samples. The whole mount immunofluorescence protocol detailed herein has been optimized for the efficient immunolabelling of kidney organoids. Ensure to maintain samples under shaking while preforming the antibodies incubation and washing steps. Primary antibodies incubation step can be extended up to 48 h.

Problem 16
After performing immunofluorescence of kidney organoid paraffin sections, uneven immunolabelling of organoid sections is observed by confocal microscopy (step 33 of step-by-step method details).

Potential solution
This could be caused by heterogeneous exposure of the antibody solution in the sample. Ensure that there has not been any problem during de-paraffinization and re-hydration of organoid paraffin sections. Ensure that organoid sections do not dry during any of the immunolabelling steps, especially during primary antibodies incubation.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Nuria Montserrat (nmontserrat@ibecbarcelona.eu).

Materials availability
This study did not generate new unique reagents.

Data and code availability
The published article includes all datasets generated or analyzed during this study.