TITLE: SARS-CoV-2       Infection and Viral Replication of Human Lung Organoids
AUTHORS: Morris Baumgardt, Maren Hülsemann, Karen Hoffmann, Mirjana Kessler, Stefan Hippenstiel, Andreas C. Hocke, Katja Hönzke

[DESCRIPTION]
This protocol describes the working steps to infect human alveolar-like organoids with SARS-CoV-2 and quantify the viral replication at defined time points post infection via plaque assay and viral E gene quantitative reverse transcription PCR (RT-qPCR).

[BEFORE_START]
Grow the virus stock (SARS-CoV-2 B.1) on Vero E6 cells (RRID:CVCL_0574), please work with maximum passage 3 and sequence the virus stock initially. 

One day before the plaque assay will be performed, Vero E6 cells need to be seeded in order to ensure confluency and readiness for the plaque assay the next day. Therefore, 175,000 cells per 24-well are seeded in 500 µL Vero E6 medium. The next day, verification is made to ensure confluency of the cells.

[GUIDELINES]
This protocol describes the processing of human alveolar-like organoids which have been grown according to Youk et al., 2020. https://doi.org/10.1016/j.stem.2020.10.004

[STEPS]
SECTION: Cultivation of human alveolar-like organoids
1. Grow your 3D model as described
 
  

SECTION: Cell counting of human alveolar-like organoids
2. A specific cell number is needed to calculate the virus MOI (multiplicity of infection).

Carefully remove organoid medium of one well. 
SECTION: Cell counting of human alveolar-like organoids
3. Add 1 mL (4 °C) cold base medium and collect Cultrex with organoids in a 15 mL  tube, flush well with additional 1 mL base medium.
SECTION: Cell counting of human alveolar-like organoids
4. Centrifuge at 300 x g, 5 min, 4 °C and carefully remove supernatant.
SECTION: Cell counting of human alveolar-like organoids
5. Resuspend pellet in 1 mL TrypLE Express for 15 min in a shaking wather bath (37 °C ) and add 1 mL base medium afterwards.

SECTION: Cell counting of human alveolar-like organoids
6. Produce a single cell solution by resuspending three times with a disposable syringe with needle (27G).
SECTION: Cell counting of human alveolar-like organoids
7. Count single cells using a disposable Counting Chamber (Neubauer improved).
SECTION: Infection of human alveolar-like organoids
8. Carefully remove organoid medium.
SECTION: Infection of human alveolar-like organoids
9. Add 1 mL (4 °C) cold base medium and collect the suspension containing Cultrex and organoids in a 15 mL tube, flush well with additional 1 mL cold base medium.

SECTION: Infection of human alveolar-like organoids
10. Centrifuge at 300 x g, 5 min, 4 °C.

SECTION: Infection of human alveolar-like organoids
11. Carefully remove supernatant.
SECTION: Infection of human alveolar-like organoids
12. Resuspend pellet in 1 mL cold base medium.

SECTION: Infection of human alveolar-like organoids
13. Break up the organoids by repeated (2-3x) resuspension using a disposable syringe with needle (27G).

  

SECTION: Infection of human alveolar-like organoids
14. Pipette virus solution (stock) to the organoids (MOI = 1), mix by pipetting (1 mL total volume per 15 mL tube) and incubate 60 min at 37 °C. Shake the tube gently every 15 mins.

MOI calculation:

(Cell number per well x MOI)/virus stock titer = µL virus solution to be added per well.


  

SECTION: Infection of human alveolar-like organoids
15. Centrifuge at 300 x g, 5 min, 4 °C.
SECTION: Infection of human alveolar-like organoids
16. Transfer 50 µL from the supernatant for input titer control (for viral qPCR later) in a new 1.5 mL tube and freeze at -80 °C at BSL3 (biosafety level 3) until further processing.

SECTION: Infection of human alveolar-like organoids
17. Take 100 µL from the supernatant for input titer control (for Plaque Assay later) and mix 100 µL supernatant with 100 µL gelatin medium in a new 1.5 mL tube and freeze at -80 °C at BSL3 (biosafety level 3) until further processing in the plaque assay.

SECTION: Infection of human alveolar-like organoids
18. Carefully remove and discard as much remaining supernatant as possible from the original tube so that only oganoids remain.
SECTION: Infection of human alveolar-like organoids
19. Wash organoids with 500 µL PBS (1x).

SECTION: Infection of human alveolar-like organoids
20. Centrifuge at 300 x g, 5 min, 4 °C.
SECTION: Infection of human alveolar-like organoids
21. Place tube on ice, resuspend pellet in thawed Cultrex (50 µL and 4 °C) and add into one well of one pre-warmed 24-well plate (37 °C).

  
  

SECTION: Infection of human alveolar-like organoids
22. Incubate 30 min at 37 °C.

SECTION: Infection of human alveolar-like organoids
23. Add 500 µL organoid medium and incubate at 37 °C.

  

SECTION: Plaque Assay
24. Samples for plaque titration are taken from the organoid culture supernatant at different time points/hours post infection (hpi) as described here:
SECTION: Plaque Assay
32.  Infect the Vero E6 cells: (add 200 µL/24-well in duplicates using the desired dilutions).

Exemplary dilutions which can be used:

Input (necessary dilution depends on the virus titer): 10-1, 10-2, 10-3
1 hpi: 10-1, 10-2, 10-3, 10-4
24 hpi: 10-2, 10-3, 10-4, 10-5
48 hpi: 10-2, 10-3, 10-4, 10-5
72 hpi: 10-1, 10-2, 10-3, 10-4
96 hpi: 10-1, 10-2, 10-3, 10-4
SECTION: Plaque Assay
33. Incubate the plate at 37 °C for 60 min.

SECTION: Plaque Assay
34. Remove supernatant and wash the wells once with PBS.
SECTION: Plaque Assay
35.  Mix 2.4% Avicel and 2xDMEM in a ratio of 1:1 and add 500 µL Avicel overlay to each well.

SECTION: Plaque Assay
36. Incubate at 37 °C for 4320 min.

SECTION: Plaque Assay
37. Remove Avicel overlay and wash wells once with PBS.
SECTION: Plaque Assay
38. Inactivate the entire 24-well plate by adding 6% PFA for 30 min in a biotainer and then export from the BSL3.

SECTION: Plaque Assay
39. Remove plates from the biotainer and led them air dry.
SECTION: Plaque Assay
40. Add crystal violet working solution and incubate for 15 min.

SECTION: Plaque Assay
41. Remove crystal violet working solution.
SECTION: Plaque Assay
42. Dry plate under the hood and count plaques.

 
  

SECTION: Plaque Assay
43. Document number of plaques in a table and take a picture of the stained plate.
SECTION: Plaque Assay
44. Calculate PFU/mL (plaques from two dilution levels and two replicates are counted).

1. Sum of plaques given by the identified virus dilutions = Sum of plaques from 4 wells (e.g. 10-3 = 2 wells, 10-4 = 2 wells).
2. Titer of SARS‐CoV‐2 (in PFU/mL) = Number of plaques given by the identified virus dilutions ÷ (lowest dilution factor × 0.44 mL). Calculation is done with the to the lowest dilution referred inoculum volume of 0.44 mL * lowest dilution level.

For example:
10-3 dilution: 34 and 37 plaques/well = 71 plaques/two wells
10-4 dilution: 1 and 4 plaques/well = 5 plaques/two wells

Sum of all counted plaques / total volume of virus 

--> 76 PFU/0.44 mL * 103 = 173*103 = 1.73*105 PFU/mL (the factor 103 corresponds to the lowest dilution level that was counted). 

SECTION: Plaque Assay
45.     

SECTION: viral RT-qPCR
46. Samples for the quantification of SARS-CoV-2 RNA in supernatants of infected organoids were taken as described in step 28. The samples were taken at different timepoints after infection and frozen.
SECTION: viral RT-qPCR
48. Pipet 50 µL of virus containing supernatant into 300 µL of RAV1 buffer with Carrier RNA (as instructed by manufacturer) and vortex.

SECTION: viral RT-qPCR
49. Heat-inactivate the tubes at 70 °C for 10 min in a thermomixer (600 rpm) and export from BSL3.

SECTION: viral RT-qPCR
50. Freeze the samples at -80 °C or directly process them for qPCR by first isolating the viral RNA using Nucleospin RNA Virus Kit according to the manufacturer’s protocol.

SECTION: viral RT-qPCR
51. Quantify the viral RNA copies from your samples (viral qPCR Master Mix table: template RNA) using Platinum SuperScript lll One-Step RT-PCR system with Platinum Taq DNA polymerase kit by preparing the master mix shown in the table below.




 

SECTION: viral RT-qPCR
52. Standards for E gene-based quantitative reverse transcription PCR: SARS-CoV-2 E gene (1x105 copies/μL) as used for clinical diagnostics. In vitro-transcribed  control RNA for the E gene assay can be acquired through the European Virus Archive platform (www.european-virus-archive.com).

SECTION: viral RT-qPCR
53. Prepare 104, 103 and 102 copies/μL from the standard stock using nuclease-free water and use four dilutions (105-102 copies/μL) for qPCR analysis. Caution! avoid contamination of test reaction with the highly concentrated standards.
SECTION: viral RT-qPCR
54. Negative control: PCR-grade, nuclease-free  H2O.
SECTION: viral RT-qPCR
55. Evaluation is performed automatically using the Roche LightCycler Software. 


 



 

 

SECTION: viral RT-qPCR
56.   
  

SECTION: Plaque Assay
25. Pipette Cultrex and medium vigorously up and down (using 1000 µL pipette) in the well until the Cultrex is homogeneously dispersed in the medium and transfer Cultrex, organoids and virus containing medium into a 1.5 mL tube. 

SECTION: Plaque Assay
26. For the complete dissolution of the Cultrex and thereby remove all organoids and viral particles from the matrix, cool the tube with organoid mixture for 10 min at 4 °C using a pre-cooled cooling rack.

SECTION: Plaque Assay
27. After 10 min at 4 °C centrifuge at 300 x g, 5 min, 4 °C.

SECTION: Plaque Assay
28. Take 100 µL from the supernatant at the desired time points (e.g. input (0 h), 1 h, 24 h, 48 h, 72 h, 96 h) and mix 100 µL supernatant with 100 µL gelatin medium in a new 1.5 mL tube and freeze at -80 °C at BSL3 (biosafety level 3) until further processing. At the same time, freeze 50 µL of pure supernatant (without gelatin) in another 1.5 mL tube, as these will be used later for viral RT-qPCR (-80 °C).

SECTION: Plaque Assay
29. Make sure, that Vero E6 cells are confluent before infection (see "Before start"). 
SECTION: Plaque Assay
30. Thaw the obtained supernatants of infected organoids from step 28 and prepare the following dilution serie:
SECTION: Plaque Assay
31. Prepare 10-fold serial dilutions:
Mix 50 µL of thawed supernatant and  450 µL OptiPro in a 1.5 mL tube by vortexing (closed cap, moderate power, ~5 s) to receive a dilution of 10-1. Mix again 50 µL of the 10-1 dilution with 450 µL OptiPro in a new tube to receive a 10-2 dilution and continue this procedure until the desired dilution is achieved. (In the here described condition for infection a supernatant dilution of not exceeding 10-5 is adequate for the detection of SARS-CoV-2 replication in alveolar-like organoids).
SECTION: viral RT-qPCR
47. Thaw the frozen 50 µL supernatant samples at RT.