How to Generate Mouse Intestinal Organoid-Derived Monolayers Using IntestiCult™

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 the limitation of being a closed luminal compartment; a physical characteristic that presents a challenge for experiments requiring access to the apical surface.

In this protocol, we describe a method to generate mouse intestinal organoid-derived monolayer cultures using IntestiCult™ Organoid Growth Medium (OGM) (Human). Using this procedure, cells expanded as mouse intestinal organoid cultures are seeded onto a dilute culture matrix, where they form a confluent monolayer and differentiate to model the intestinal epithelium within 7 days. These monolayers enable easy access to the apical side of the epithelial monolayer, 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. Intestinal epithelial monolayer cultures can be established on glass coverslips that enable high-quality imaging or on Transwell® membrane inserts that enable 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.

Researchers working with specific applications may need to further optimize this protocol. Browse this resource to find technical tips for optimization of mouse intestinal organoid-derived monolayers.

Why Use IntestiCult™ OGM (Human) for Mouse Monolayers?


Materials

I. Coating Cultureware with Corning® Matrigel®

For growing intestinal organoids as a monolayer, coat cultureware with a dilute Matrigel® solution. The following instructions are for coating cultureware with Corning® Matrigel®; however, defined matrices such as Collagen I, Collagen IV, and Vitronectin XF™ (Catalog #07180) can also be used (as per manufacturer’s instructions).

  1. Thaw one aliquot of Corning® Matrigel® on ice.
  2. Dispense an appropriate amount of cold (2 - 8°C) D-PBS into a conical tube and keep on ice.
  3. Add thawed Matrigel® to the cold D-PBS at a ratio of 1 μL Matrigel® to 49 μL D-PBS. Mix well and keep on ice.
  4. Immediately use the diluted Matrigel® solution to coat tissue culture-treated cultureware. See Table 1 for recommended coating volumes.

    Note: Ensure the solution evenly coats the entire bottom surface of the cultureware; insufficient coating results in poor cell attachment.

    Table 1. Recommended Volume of Dilute Matrigel®

    Cultureware
    Volume of dilute Matrigel® per well
    6-Well Plate
    1000 μL
    24-Well Transwell® Plate
    100 μL
    24-Well Plate
    200 μL
    96-Well Plate
    100 μL

II. Media Preparation

(a) Preparation of IntestiCult™ Organoid Growth Medium (Human)

The following example is for preparing 100 mL of IntestiCult™ Organoid Growth Medium (IntestiCult™ OGM Human Basal Medium + Organoid Supplement). If preparing other volumes, adjust accordingly.

  1. Thaw Basal Medium and Organoid Supplement at room temperature (15 - 25°C) or at 2 - 8°C overnight. Mix thoroughly.
    • Once thawed, use immediately or aliquot and store at -20°C for up to 3 months. After thawing the aliquots, use immediately. Do not re-freeze.
  2. Add 50 mL of Organoid Supplement to 50 mL of Basal Medium. Mix thoroughly.

    Note: If not used immediately, store at 2 - 8°C for up to 1 week.

  3. Add desired antibiotics immediately before use (e.g. 50 μg/mL gentamicin).

(b) Preparation of IntestiCult™ Monolayer Growth Medium (Human)

The following example is for preparing 100 mL of IntestiCult™ Monolayer Growth Medium (IntestiCult™ OGM Human Basal Medium + Organoid Supplement + Y-27632). If preparing other volumes, adjust accordingly.

  1. Thaw Basal Medium and Organoid Supplement at room temperature (15 - 25°C) or at 2 - 8°C overnight. Mix thoroughly.
    • Once thawed, use immediately or aliquot and store at -20°C for up to 3 months. After thawing the aliquots, use immediately. Do not re-freeze.
  2. Add 50 mL of Organoid Supplement to 50 mL of Basal Medium. Mix thoroughly.
  3. Add 10 μM Y-27632. Mix thoroughly.
    • If not used immediately, store at 2 - 8°C for up to 1 week.
  4. Add desired antibiotics immediately before use (e.g. 50 μg/mL gentamicin).

III. Transitioning Mouse Intestinal Organoids From IntestiCult™ OGM (Mouse) into IntestiCult™ OGM (Human)

We suggest transitioning the mouse intestinal organoids cultured and maintained in IntestiCult™ OGM (Mouse) into IntestiCult™ OGM (Human) for at least one passage before use in monolayer cultures.

  1. Aspirate IntestiCult™ OGM (Mouse) from the Matrigel® domes on Day 0 of the current passage.
  2. Add 750 µL of IntestiCult™ OGM (Human) (Media Preparation, Section a) to each well/dome by pipetting the medium gently down the wall of the well.
  3. Place the lid on the culture plate, and incubate at 37 °C and 5% CO2.
  4. Every 2 days, perform a full-medium change with IntestiCult™ OGM (Human), until the organoids are fully developed (5 - 7 days).

IV. Plating Intestinal Organoids as a Monolayer

  1. Harvest 3 - 4 Matrigel® domes of intestinal organoids (approximately 100 - 150 organoids) as described below. See Table 2 for the recommended number of domes to harvest for various cultureware.

    Table 2. Recommended Number of Domes to Harvest When Plating Intestinal Organoids as a Monolayer

    Cultureware to be seeded
    Number of domes to harvest per well to be seeded*
    24-Well Plate
    3 - 4 domes
    24-Well Transwell® Plate
    2 - 3 domes
    96-Well Plate
    1 dome
    *Assuming 50 μL domes being harvested
  2. Add 1 mL of Gentle Cell Dissociation Reagent to each well containing a dome to be harvested.
  3. Incubate at room temperature (15 - 25°C) for 1 minute.
  4. Using a 1000 μL pipettor, pipette vigorously to disrupt the Matrigel® dome and resuspend the organoids.
  5. Incubate the suspension at room temperature for 10 minutes with gentle agitation or rocking.
  6. Pool the harvested wells and centrifuge at 200 x g for 5 minutes at 2 - 8°C.
  7. Discard the supernatant. Add 3 mL ice-cold DMEM/F-12. Centrifuge at 200 x g for 5 minutes at 2 - 8°C.
  8. Aspirate to remove as much of the supernatant as possible, being careful not to disturb the pellet. Resuspend the organoids in 1 mL of warm (37°C) Trypsin-EDTA (0.05%).
  9. Using a 1000 μL pipettor, pipette up and down to mix thoroughly. Incubate the organoids at 37°C for 5 - 10 minutes.
  10. Mix organoids thoroughly by vigorous pipetting or vortexing to disrupt the organoids as much as possible. Check the organoids using a microscope to ensure 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 (Figure 1).
    Note: Perform the remaining steps as quickly and efficiently as possible, as cells start to clump together.
  11. Add 1 mL DMEM/F-12 and mix thoroughly by pipetting. Centrifuge fragments at 200 x g for 5 minutes at 2 - 8°C.
  12. Remove the supernatant and resuspend organoid fragments in IntestiCult™ Monolayer Growth Medium (Media Preparation, Section b) as indicated in Table 3.

    Table 3. Volume of IntestiCult™ Monolayer Growth Medium for Resuspending Organoids

    Cultureware to be seeded
    Volume of IntestiCult™ Monolayer Growth Medium per well
    6-Well Plate
    1.5 mL
    24-Well Plate
    500 μL
    24-Well Transwell® Plate
    100 µL (top), 500 µL (bottom)
    96-Well Plate
    100 μL
    *Assuming 50 μL domes being harvested
  13. Slowly and gently add the cell suspension to each well. Incubate at 37°C and 5% CO2. Monitor growth daily. Perform a full-medium change every 2 - 3 days.
    Note: Monolayers generally reach 100% confluency within 2 - 3 days; in the event of poor attachment, slow proliferation may be observed, but confluency is usually reached if given enough time.

Figure 1. Organoids Must be Disrupted to Single Cells or Small Clumps for Seeding into Monolayers

(A) Single cells are ideal for seeding into monolayers.
(B) Small clumps are acceptable for seeding into monolayers.
(C) Large clumps are not acceptable for seeding into monolayers. If large clumps remain after disruption, repeat pipetting and vortexing (Step 10) to achieve small clumps or single cells.


Figure 2. Growth of Intestinal Monolayers in 24-Well Transwell® Plates

Over time, the monolayer reaches 100% confluency. Time to 100% confluence is generally 2 - 3 days, but may vary depending on the seeding efficiency, which is partly dependent on the passage number of the organoids.
(A) Human intestinal monolayers at low confluency (less than 25%).
(B) Human intestinal monolayers 2 days post seeding (50% confluent).
(C) Human intestinal monolayers after 7 days in culture (100% confluent).
Note: Although we expect the mouse monolayers to look similar to the representative images above, depending on the seeding efficiency, quality of the starting sample, and the cultureware used, you can expect to see some differences in the morphology.


Figure 3. Organoid-Derived Monolayer Cultures

Representative brightfield (left) and immunofluorescent images (right) of monolayers derived from (A) human colon or (B) mouse colon. 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. Scale bars = 500 µm.



V. Tips For Optimization of Mouse Intestinal Organoid-Derived Monolayers

  • Mouse monolayers should be grown using IntestiCult™ Organoid Growth Medium (Human) (OGM) + Y-27632 (IntestiCult™ Monolayer Growth Medium [Human]) as shown in this protocol.
  • Mouse organoids used to seed the monolayers should be grown using IntestiCult™ OGM (Human) as shown in this protocol. Medium can be changed from IntestiCult™ OGM (Mouse) at Day 0 of the current passage.
  • We do not have data suggesting that passaging mouse organoids routinely in IntestiCult™ OGM (Human) improves 2D monolayer formation.
  • Mouse monolayers tend to be more fragile compared to human monolayers (due to a higher rate of cell turnover), and may be difficult to grow for more than a few days.
  • Cells will adhere better to Transwell® inserts versus a culture plate, and are more likely to form a confluent monolayer more quickly. Cells also polarize and mature better in Transwell® inserts than in a culture plate.
  • Mouse organoids used for plating monolayers should not be derived from P0 or directly from the gut/primary tissue. We recommend using at least P1+ cultures.
  • We recommend keeping Y-27632 (ROCKi) for the entire monolayer protocol. It can be removed if needed, but it helps to maintain the monolayer when it is there. If it needs to be removed for experimental purposes, you should wait until the monolayer is entirely confluent, around day 7 or so.
  • Ideally, you should wait until the monolayers are almost confluent before doing any downstream experiments.
  • Intestinal monolayers from mouse small intestinal organoids can be very challenging to work with due to high turnover of cells, and may not hold together after a few days. Using colon-derived intestinal organoids is recommended, as we find that these tend to work better over small intestinal samples.
  • Whenever possible, we suggest starting with more seeding material/higher seeding densities for the monolayer protocol.
  • Seeding efficiency may vary depending on the passage number of the organoids.
  • The optimal seeding density can vary significantly based on the passage number and other variables from the source organoids, so there is a great deal of flexibility in the seeding density; it should be optimized for the start sample and growing conditions.
  • Using 5% Matrigel® instead of a 2% Matrigel® solution may help improve attachment efficiency with some samples. Incubation of pre-coated plates at 37°C for between 2 - 4 hours before adding your cells may also help with improved attachment.
  • If needed for specific experiments, IntestiCult™ Monolayer Growth Medium can be replaced with DMEM/F-12 for at least 24 hours without a significant reduction in cell viability or monolayer integrity. It is important to note that mouse monolayers can be difficult to maintain after a few days.
  • Fully confluent intestinal monolayers can be used for downstream assays such as TEER, FITC-dextran permeability, Ussing chamber analysis, and immunocytochemistry (ICC) staining. Any of the established assays used on cell culture can be applied to intestinal monolayers.

  • Document #PR00042
  • Version 1.0.0
  • March 2021


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