Growing hepatic organoids as organoid-derived monolayers offers further flexibility for experimental design and decreased interference from the extracellular matrix. Organoid-derived monolayers can additionally be grown on Transwell® inserts, enabling different treatments in each of the compartments.
Thaw Matrigel® on ice. Prepare the coating solution by combining thawed Matrigel® with ice-cold PBS at a 1:50 dilution (i.e. 2% solution). Coating volumes required are:
100 µL per well for Transwell® plates
200 µL per well for 24-well plates
Coat culture plate(s) with a dilute Matrigel® solution:
Add an appropriate volume of coating solution to each well to be used for monolayer generation. Ensure the entire well surface is evenly coated.
Incubate at 37°C for at least 1 hour before use. Do not let the Matrigel® solution in the wells evaporate.
Prepare 10 mL of TrypLE™ + BSA:
400 µL 25% BSA solution in water (1% final concentration)
10 mL TrypLE™ Express Enzyme
Mix thoroughly. Store on ice.
Pre-wet all serological pipettes, pipette tips, and conical tubes that will come in contact with the organoid-derived cells, first with Anti-Adherence Rinsing Solution and then with DMEM/F-12 + 15mM HEPES.
Check that the Matrigel® domes to be passaged are intact (i.e. the whole dome remains attached to the plate and no loose Matrigel® pieces or organoids are observed in the well). If the dome is intact, proceed to step 2. If the dome is loose, remove at least 500 µL medium using a 1 mL pipettor, and add 1 mL TrypLE™ + BSA to the well to 1mL; proceed to step 4.
Without touching the dome, aspirate and discard the medium in each well to be passaged.
Using a 1 mL pipettor, forcefully add 1mL of TrypLE™ + BSA to the center of each dome, then let sit for 1 minute.
Using a 1 mL pipettor, vigorously pipette the total volume in the well up and down 45 times, taking care not to generate bubbles.
Note: This results in mechanical breakdown of organoids and Matrigel® into a single-cell suspension. Some smaller fragments may remain that can be further dissociated by incubating the suspensions at 37°C for 5 - 10 minutes with periodic trituration/agitation using a pipette or vortex.
Add 1 mL cold DMEM + BSA to each well. Transfer the entire volume of single-cell suspensions from each well to a 15 mL conical tube on ice.
Note: Pool the contents of up to 4 wells in one 15 mL tube.
Wash the wells that the organoids were harvested from with 1 mL cold DMEM + BSA. Add this volume to the 15 mL tube prepared in step 5.
Add ice-cold DMEM + BSA to each tube so that the total volume of each tube is 12 mL. Centrifuge tube(s) at 290 x g for 5 minutes. Aspirate as much of the supernatant as possible without disturbing the pellet.
Resuspend cells in 2 mL of DMEM + BSA.
Note: If more than 4 wells were dissociated, cells from all processed wells can be pooled into the same 15 mL tube at this stage.
Perform a live cell count and determine the number of wells to be seeded for monolayer generation. The recommended seeding densities are as follows:
1.0 x 105 cells per Transwell®
3.0 x 105 cells per well of a 24-well plate
Note: These densities will typically yield 100% confluent hepatic monolayers within 2 days. Alternative seeding densities can be used if confluent monolayers are desired at a different timepoint.
Centrifuge tube(s) at 290 x g for 5 minutes. Aspirate as much of the supernatant as possible without disturbing the pellet.
Resuspend cells in complete HepatiCult™ OIM to generate a single-cell suspension. Place tube on ice.
Note: If using the recommended seeding densities in step 9, resuspend cells at 2.0 x 106 live cells/mL.
Remove the coated plate from the incubator and place in the biosafety cabinet.
Gently tilt the plate. Aspirate and discard the excess Matrigel® volume that collects at the edge of the well, ensuring that the coated surface is not scratched.
Immediately add complete HepatiCult™ OIM to each well to be seeded, as follows:
100 µL in the upper chamber and 500 µL in the lower chamber per Transwell®
350 µL per well of a 24-well plate
Slowly add the single-cell suspension generated in step 11 to each well, as follows:
50 µL of suspension per Transwell® (i.e. 1.5 x 105 cells/Transwell® if using a 2.0 x 106 live cells/mL suspension)
150 µL of suspension per well of a 24-well plate (i.e. 4.5 x 105 cells/well if using a 2.0 x106 live cells/mL suspension)
Note: Monolayers typically achieve 100% confluence within 2 - 3 days if seeded at the indicated density.
Incubate the plate at 37°C and 5% CO2. Monitor growth daily.
Perform a full-medium change every 2 - 3 days by carefully aspirating the medium and adding 750 µL of fresh complete HepatiCult™ OIM at room temperature (15 - 25°C).
Note: To avoid weekend medium changes, perform medium changes on Mondays, Wednesdays, and Fridays.
Note: Monolayers can be used in assays once they are confluent. Once established, monolayers remain viable for at least 10 days. Multi-layering of cells may be observed if confluent monolayers are maintained for longer periods.
Figure 1. A 3D-Organoid-Derived Hepatic Monolayer on a Transwell® Insert
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Establishment of 3D Organoid-Derived Hepatic Monolayers Using HepatiCult™ Organoid Initiation Medium (Human)
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