Growth in a dilute Matrigel® suspension allows the scale-up of cultures beyond what may be practical when plating in Matrigel® domes. Organoids grown in a dilute Matrigel® suspension are typically ready for passage earlier, usually within 4 - 7 days, compared to 7 - 10 days for dome cultures, and supports culture in larger, bioreactor formats (e.g. spinner flasks).

The below protocol describes the expansion of hepatic organoids in a dilute Matrigel® suspension culture using HepatiCult™ Organoid Growth Medium (Human).

Materials

• Healthy hepatic organoid cultures expanded in HepatiCult™ Organoid Growth Medium (Human)
• HepatiCult™ Organoid Growth Medium (Human) (Catalog #100-0385)
• DMEM/F-12 + 15 mM HEPES (Catalog #36254)
• 25% Bovine serum albumin (BSA) solution in water
• Corning® Matrigel® (Corning 356231, ≥ 8mg/mL protein)
• Antibiotics (e.g. gentamicin)
• 15 mL conical tubes (Catalog #38009)
• Ultra-Low Adherent (ULA) 6-Well Plate for Suspension Culture (Catalog #100-0083) OR 100 mL sterile spinner flasks
• Sterile 200 µL and 1000 µL tips
• 5 mL and 10 mL serological pipettes (Catalog #38003 and Catalog #38004)
• Styrofoam box with ice
• Incubator with atmospheric O₂ and 5% CO₂ at 37°C
• Orbital shaker or spinner flask-compatible stirring platform that can be placed in the incubator

Preparation

1. Thaw and store Matrigel® on ice.
2. Thaw HepatiCult™ Organoid Growth Supplement overnight at 2 - 8°C. Prepare 100 mL Matrigel®-supplemented HepatiCult™ Organoid Growth Medium (OGM + Matrigel®) on ice using sterile technique:
   • 90 mL HepatiCult™ Organoid Basal Medium
   • 5 mL HepatiCult™ Organoid Growth Supplement
   • 5 mL Matrigel®
   • Add antibiotics (e.g. 50 µg/mL gentamicin)
   • Mix thoroughly. Store on ice.

Note: Once thawed, use HepatiCult™ Organoid Growth Supplement immediately or aliquot and store at -20°C. After thawing aliquots, use immediately. Do not re-freeze.

Note: Prepare OGM + Matrigel® fresh for every use. Do not store any remaining volumes long-term.

3. Prepare 50 mL of serum-supplemented DMEM/F-12 + 15 mM HEPES (DMEM + BSA):
   • 2 mL 25% BSA solution in water (1% final concentration)
   • 47.9 mL DMEM/F-12 + 15 mM HEPES
   • Mix thoroughly. Store on ice.
4. If using ULA 6-well plates, place the plates at 2 - 8°C for at least 10 minutes. If using spinner flasks, place flasks on ice for at least 10 minutes before use.

Protocol

1. Process organoids based on the culture format in use as follows:
   If passaging from a 6-well suspension culture:

   • Using a serological pipette, transfer the contents of all wells to be passaged to a 15 mL conical tube. Leave tubes at room temperature for 5 - 10 minutes to allow organoids to settle.
   • Remove and discard as much of the spent medium as possible without aspirating any organoids, leaving up to 0.5 mL of medium in the tube.
   • Add 1 mL cold DMEM + BSA to the tube and proceed to step 2.
   If passaging from dome cultures:

   • 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 the next step. If the dome is loose, add cold DMEM + BSA to top up the total volume in the well to 1 mL and let sit for 1 minute, then skip the next step and proceed without aspirating the medium in these wells.
   • Without touching the dome, aspirate and discard the medium in each well to be passaged.
   • Using a 1 mL pipettor, forcefully add 1 mL cold DMEM + BSA to the center of each dome, then let sit for 1 minute. Proceed to step 2.
2. Using a 1 mL pipette tip on the pipettor, vigorously pipette the total volume up and down 45 times, taking care not to generate bubbles.

   Note: This results in mechanical breakdown of organoids and Matrigel® into fragments between 30 - 100 μm in size. If many larger fragments remain, continue pipetting the suspension until the majority of fragments are < 100 μm in size.

   Note: Cell strainers can be used to collect a suspension of more uniformly sized fragments if desired.

3. Determine the volume required to seed the desired number of fragments in the subsequent passage. Transfer this volume to a 15 mL conical tube containing 1 mL of DMEM + BSA.

   Note: Seeding density should be optimized for each donor. ~1000 - 3000 fragments per mL of OGM + Matrigel® is recommended.

4. Centrifuge tubes from step 3 at 290 x g for 5 minutes.
5. Aspirate as much of the supernatant as possible without disturbing the pellets (the pellet is often not visible).
6. Resuspend pellet as described below and store tube on ice.
   • 0.5 mL OGM + Matrigel® per well to be seeded if using a 6-well plate.
   • 3 mL OGM + Matrigel® if seeding spinner flask.
7. Place the pre-cooled 6-well plate or spinner flask on ice in the biosafety cabinet. Add 1.5 mL cold OGM + Matrigel® per well of the 6-well plate or 47 mL cold OGM + Matrigel® to the spinner flask.
8. Add the OGM + Matrigel® + fragment volume from step 6 to the culture vessel, swirling the vessel to mix.
9. Carefully transfer the plate or flask to an orbital shaker set to 80 rpm or stirring platform set to 33 rpm respectively, in an incubator at 37°C and 5% CO₂.
10. Perform a full-medium change every 2 - 3 days as follows:
    • Tilt the culture vessel at a ~45 degree angle and allow organoids to settle for 3 - 5 minutes. If using a spinner flask, be careful not to disturb the central arm, as this will stir the organoids back into suspension.
    • Remove and discard ~75% of the spent medium, taking care not to aspirate any organoids.
    • Add an equivalent volume of fresh, room temperature complete HepatiCult™ OGM.

    Note: OGM does not need to be supplemented with Matrigel® for medium changes. To avoid weekend medium changes, perform medium changes on Mondays, Wednesdays, and Fridays. Hepatic organoids should be passaged before the lumen turns dark and collapses, approximately every 4 - 7 days.