The preparation of lung organoid-derived monolayers

The preparation of lung organoid-derived monolayers

Reagents Needed: 

*Please see key resource table for reagent information

• Lung Wash Media 
• Complete PnuemaCult -ExPlus (according to manufacturer)
• 1X DPBS (no calcium, no magnesium) 
• 1X DPBS-EDTA (1:1000 0.5M EDTA)
• Matrigel 

Recipes:

Day 0:

1. Prepare Matrigel diluted in 1X DPBS at a 1:40 ratio (~200 µg/mL final protein concentration) on ice. Mix thoroughly but avoid introducing bubbles. 

2. Coat each transwell insert (6.5 mm diameter, 0.4 µm pore size, Corning) with 150 µl of the 1X DPBS-diluted Matrigel. Place the plate in the 37°C incubator (upright) for the duration of the organoid digestion protocol (30-60 min). 

3. Aspirate media from 6-9 day old organoids derived from human deep lung tissue and cultured in expansion media (AKA Lungbrew, HUMANOID^TM) and add 1 mL DPBS-EDTA to each well.   

4. Scrape the Matrigel button/cells with a pipette tip or cell scraper within the DPBS-EDTA mixture and collect the cell suspension in a 15 ml conical tube. 

5. Centrifuge @ 200g-300g for 4 min 

6. Aspirate and gently resuspend in TrypLE select (2 mL per 12-well plate).  

7. Incubate the cell suspension in a 37°C water bath for 5-7 min. Duration of incubation depends on density, size, and overall health of organoids 

8. Mechanically dissociate the organoids/clusters of cells by pipetting the cell mixture up and down 10-15 times before adding enough lung wash media to inactivate the TrypLE select (1:2 to 1:5 TrypLE: wash media ratio) and pipette again 10-15 times. 

9. Centrifuge @ 200g-300g for 4 min 

10. Aspirate off supernatant and resuspend the cell pellet in 1-2mL of Complete PnuemaCult -ExPlus media. 2mL is commonly used when organoids were grown in a full 12-well plate. 

11. Place a 70 µm filter over a 50 ml tube. Wet the filter with Complete PnuemaCult -ExPlus media filter the cells through the filter. Collect the remaining cell suspension from the opposite side of the filter with a clean pipette tip. 

12. Count the cells and determine the cell concentration.

13. According to your cell count, make your cell mix in Complete PneumaCult™-Ex Plus Medium. For 24-well inserts, you need 1.8 E5 viable cells resuspended in 200 µL of media for each well. 

14. Once your cell mix is ready, carefully aspirate PBS from Matrigel in wells without touching the tip to the bottom of the well.

15. Add 200 µL of cell mixture to each apical side of each transwell and add 700 µL of complete PneumaCult™-Ex Plus Medium to the basolateral compartment. 

16. Check your seeding 1 hr later to examine if cells have adhered evenly across Matrigel coating.

Day 1:

17. Perform a complete media change (apical and basolateral) with Complete PneumaCult™-Ex Plus Medium.

Day 2:

18. Typically, any treatments are applied to the monolayers 48 hrs post-seeding.