Introduction

Intestinal organoids provide researchers with a physiologically relevant cell model and constitute a valuable experimental tool for probing intestinal epithelial cell biology and modeling disease. However, intestinal organoid cultures have a closed luminal compartment—a challenging physical characteristic for experiments requiring access to the apical surface.

To overcome this limitation, researchers can generate monolayers from intestinal organoids to enable easy access to the apical surface, facilitating studies such as those involving apical cell surface receptors, interaction with commensal or pathogenic microorganisms, or modeling the effects of potentially beneficial or harmful compounds in intestinal contents.

In this protocol, we describe a method to generate monolayers from hPSC-derived intestinal organoids using IntestiCult™ Organoid Differentiation Medium (ODM) (Human) (Catalog #100-0214). Using this procedure, hPSC-derived intestinal organoids maintained and expanded using the STEMdiff™ Intestinal Organoid Medium (Catalog # 05140) are dissociated and seeded onto a diluted culture matrix, where they form a confluent monolayer and differentiate to model the intestinal epithelium within 7 days.

Intestinal epithelial monolayer cultures can be established on glass coverslips that enable high-quality imaging or on Transwell® membrane inserts that enable the measurement of active and passive transport of substances across the epithelial layer and electrical barrier function assays. Monolayers can be generated using a range of cultureware, giving flexibility in experimental throughput.

Browse this resource to find technical tips for the optimization of monolayers generated from hPSC-derived intestinal organoids. Researchers working with specific applications may need to further optimize the protocol.

Materials

• IntestiCult™ Organoid Differentiation Medium (Human) (Catalog #100-0214)
• Costar® 6.5 mm Transwell® Inserts (Catalog #38024) or Costar® 12 mm Transwell® Inserts (Catalog #38023)
• Corning® Matrigel® Matrix (e.g. Corning Catalog #354277)
• D-PBS (Without Ca++ and Mg++) (e.g. Catalog #37350)
• 15 mL conical tubes (e.g. Falcon® Conical Tubes, Catalog #38009)
• Y-27632 (Catalog #72302)
• Antibiotics (e.g. gentamicin or penicillin/streptomycin)
• 24-well plate (e.g. Costar® 24-Well Flat-Bottom Plate, Tissue Culture-Treated, Catalog #38017 or Falcon® 24-Well Flat-Bottom Plate, Tissue Culture-Treated, Catalog #38021)
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• Gentle Cell Dissociation Reagent (Catalog #07174)
• DMEM/F-12 with 15 mM HEPES (e.g. Catalog #36254)
• Trypsin-EDTA (0.05%) (e.g. Catalog #07910)
• Pipettor (e.g. Corning® Lambda™ Plus Pipettor, Catalog #38060)
• Pipette tips (e.g. Corning® Filtered Pipette Tips, Catalog #38034)
• Serological pipettes (e.g. Falcon® Serological Pipettes, 10 mL, Catalog #38004)
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I. Coating Cultureware with Corning® Matrigel®

For monolayer organoid culture, cultureware must be coated with Corning® Matrigel®, as described below. For optimal results, Costar® 6.5 mm or 12 mm Transwell® Inserts (Catalog #38024/38023) are recommended; however, standard tissue culture-treated plates may be used. If ALI culture is desired to increase differentiation of the epithelial layer, Transwell® inserts must be used for monolayer culture.

1. Thaw one aliquot of Corning® Matrigel® on ice.
2. Dispense an appropriate amount of cold (2 - 8°C) D-PBS into a 15 mL conical tube and place on ice. Refer to Table 1 below for recommended volumes.
3. Add thawed Matrigel® to the cold D-PBS at a ratio of 1 µL Matrigel® to 49 µL D-PBS. Mix thoroughly.
4. Immediately coat cultureware with diluted Matrigel®. Swirl the cultureware to spread the solution evenly across the surface.
5. Incubate at 37°C for at least 1 hour before use. Do not let the Matrigel® solution evaporate.
   Note: If not used immediately, seal the cultureware with Parafilm® to prevent evaporation; store at 2 - 8°C for up to 1 week after coating. Allow stored coated cultureware to warm to room temperature (15 - 25°C) for 30 minutes before proceeding to the next step.
6. Gently tilt the cultureware to one side and allow the excess Matrigel® solution to collect at the edge. Remove the excess Matrigel® solution using a serological pipette or by aspiration. Ensure that the coated surface is not scratched.
   
   

   Table 1. Recommended Volumes of Diluted Matrigel® for Various Cultureware

   Cultureware

   Volume of Diluted Matrigel® per Well

   6.5 mm Transwell® insert

   100 µL (top)

   12 mm Transwell® insert

   250 µL (top)

   6-well plate

   1000 µL

   24-well plate

   250 µL

   96-well plate

   100 µL

II. Media Preparation

Use sterile technique to prepare IntestiCult™ Monolayer Growth Medium (IntestiCult™ ODM Human Basal Medium + Organoid Supplement + Y-27632). The following example is for preparing 100 mL of complete medium. If preparing other volumes, adjust accordingly.

1. Prepare a 10 mM stock solution of Y-27632 in DMSO. Store at -20°C until ready to use.
2. Thaw Basal Medium and Organoid Supplement at room temperature (15 - 25°C) or at 2 - 8°C overnight. Mix thoroughly.
   Note: If not used immediately, aliquot and store at -20°C for up to 3 months. After thawing the aliquots, use immediately. Do not refreeze.
3. Add 50 mL of Organoid Supplement to 50 mL of Basal Medium. Mix thoroughly.
4. Add 100 µL of 10 mM Y-27632 (final concentration 10 µM). Mix thoroughly.
   Note: If not used immediately, store at 2 - 8°C for up to 1 week.
5. Add desired antibiotics immediately before use (e.g. 50 µg/mL gentamicin or 100 units [100 µg/mL] penicillin/streptomycin).

III. Dissociation of PSC-Derived Human Intestinal Organoids

The following protocol is for dissociating 2 - 3 wells of hPSC-derived intestinal organoids grown in STEMdiff™ Intestinal Organoid Growth Medium (Human) in a 24-well plate for 7 - 10 days in 50 µL 100% Matrigel® domes.

Seeding efficiency may vary with organoid passage number and the number of days of growth. This protocol assumes at least 100 - 150 organoids per well. Matrigel® domes may contain fewer but larger organoids, or a larger number of smaller organoids, but will yield similar results. The number of organoids required may vary with cell line and culture quality and may need to be further optimized.

1. Refer to Table 2 below for the recommended number of wells of intestinal organoids to harvest for various cultureware.
   Note: If ALI culture is desired to increase differentiation of the epithelial layer, Transwell® inserts must be used for monolayer culture.
2. Aspirate all medium from the organoid cultures without disturbing the organoids within the Matrigel® domes.
3. Add 1 mL of Gentle Cell Dissociation Reagent to each well of organoids to be harvested.
4. Incubate at room temperature (15 - 25°C) for 1 minute.
5. Using a 1 mL pipettor, vigorously pipette up and down to disrupt the Matrigel® dome and resuspend the organoids.
6. Pool the harvested wells in a 15 mL conical tube. Incubate at room temperature for 10 minutes with gentle agitation or rocking.
7. Centrifuge at 200 x g for 5 minutes at 2 - 8°C.
8. Remove and discard the supernatant. Add 5 mL ice-cold DMEM/F-12 with 15 mM HEPES to resuspend organoids. Centrifuge at 200 x g for 5 minutes at 2 - 8°C.
9. Aspirate supernatant, removing as much as possible, being careful not to disturb the pellet. Add 1 mL of warm (37°C) Trypsin-EDTA (0.05%) to resuspend organoids.
   Note: If pooling larger numbers of organoid wells, increase the volume of Trypsin-EDTA to ensure efficient dissociation of the organoids.
10. Using a 1 mL pipettor, pipette up and down to mix thoroughly. Incubate at 37°C for 5 - 10 minutes.
11. Mix thoroughly by vigorous pipetting or vortexing to disrupt the organoids as much as possible. Use a microscope to check the organoids for sufficient disruption. Organoids should be dissociated into either individual cells or small fragments. If many large fragments or whole organoids remain, repeat pipetting/vortexing until fragments are sufficiently disrupted.
    Note: Perform the remaining steps as quickly as possible, as cells will start to clump together.
12. Add an equal volume of DMEM/F-12 (e.g. 1 mL DMEM/F-12 per 1 mL Trypsin-EDTA) and pipette up and down to mix thoroughly. Centrifuge fragments at 200 x g for 5 minutes at 2 - 8°C.
    Note: DMEM/F-12 supplemented with 5 - 10% FBS can also be used to neutralize Trypsin-EDTA if needed. Carefully remove and discard the supernatant.
    Note: If a buoyant mucus layer is present, steps 11 and 12 may need to be repeated to properly pellet the organoid fragments.

    Table 2. Recommended Number of Wells of Intestinal Organoids to Harvest

    Cultureware

    Number of Wells of Intestinal Organoids to Harvest (per well to be seeded)

    6.5 mm Transwell® insert

    2 - 3 wells

    12 mm Transwell® insert

    3 - 4 wells

    6-well plate

    6 - 8 wells

    24-well plate

    3 - 4 wells

    96-well plate

    1 - 2 wells

IV. Plating Organoid Fragments for Monolayer Culture

1. Coat cultureware with Corning® Matrigel® and prepare IntestiCult™ Monolayer Growth Medium (see Section II).
2. Add IntestiCult™ Monolayer Growth Medium (prepared in Section II) to organoid fragments and mix gently. Refer to Table 3 below for the volume required for various cultureware.
3. Slowly and gently add the fragment suspension to each Matrigel®-coated well. Incubate at 37°C and 5% CO₂. Monitor growth daily. Perform a full-medium change every 2 - 3 days.
   Note: Monolayers will usually reach 100% confluency within 2 - 3 days; however, if attachment is poor, this may take longer. Monitor monolayers till they reach the desired confluency. Cells will remain viable and the monolayer will remain confluent for at least 3 weeks, with continued full-medium changes every 2 - 3 days.

   Table 3. Recommended Resuspension Volume of IntestiCult™ Monolayer Growth Medium for Various Cultureware

   Cultureware

   Volume of IntestiCult™ Monolayer Growth Medium per Well

   6.5 mm Transwell® insert

   100 µL (top), 500 µL (bottom)

   12 mm Transwell® insert

   500 µL (top), 1.5 mL (bottom)

   6-well plate

   1.5 mL

   24-well plate

   500 µL

   96-well plate

   100 µL

Figure 1. Growth of Monolayers from PSC-Derived Intestinal Organoids in Different Culture Formats

Growth of monolayers from WLS-1C-derived (top) and H9-derived (bottom) intestinal organoids in (A & C) standard 24-well tissue culture-treated plates and (B & D) 24-Well Transwell® plates. Scale bars = 100 µm.

Figure 2. Organoid-Derived Monolayer Cultures

Representative immunofluorescence images of monolayers derived from (A) H9-derived intestinal organoids and (B) primary human intestinal organoids. Immunofluorescent staining for villin (green), E-cadherin (red), and DAPI stain for cell nuclei (blue). Villin staining along the apical edge of the cells indicates cell polarization, and E-cadherin staining indicates the presence of adherens junctions. DAPI stain indicates the presence of the nuclei near the basolateral pole of the epithelial cells.

V. Tips for Further Optimization of PSC-Derived Intestinal Monolayers

The following technical tips could be considered for optimization or improvement of monolayer establishment from PSC-derived intestinal organoids.

• Even if 3D organoids are maintained using STEMdiff™ Intestinal Organoid Growth Medium, they will require IntestiCult™ Monolayer Growth Medium to grow as a monolayer.
• The protocol will require the supplementation of Y-27632 into the medium for the duration of the culture.
• Some hPSC lines work better than others; optimization for seeding densities for particular cell lines is recommended.
• Seeding density will vary depending on the organoid line.
• hPSC-derived organoids tend to attach as small organoid-like clumps, but will usually expand as a monolayer from there, so it may take some time until they form a confluent monolayer.
• Sometimes using a higher Matrigel® concentration when coating the wells helps with attachment (5% vs 2% in the protocol).

Learn how to perform a TEER measurement to evaluate epithelial barrier integrity >

See ICC staining protocol for epithelial cells cultured as monolayers >