Intestinal Organoid Culture and Proliferation Assay

Timing: 7 days

WNT mimetic molecules should be evaluated in additional functional assays in vitro and in vivo. This section describes one such in vitro functional system, mouse intestinal organoids (O'Rourke et al., 2016), for the evaluation of WNT mimetics.

Thawing organoids:

Warm IntestiCult™ Organoid Growth Medium to 37°C. Pre-warm a 48-well tissue culture plate at 37°C for at least 30 min.

Quickly thaw a tube of mouse intestinal organoids by holding the tube in hand.

Transfer the organoids in freezing medium into a 15 mL conical tube and add 10 mL DMEM. Spin down organoids at 4°C for 3 min at 150 g. Slowly take out supernatant and repeat the wash with IntestiCult™ Organoid Growth Medium.

After spinning down, slowly take out as much supernatant as possible without disrupting the organoid pellet.

Resuspend organoids in 200 μL Matrigel on ice, mix gently and quickly dispense 25 μL of the mixture into the center of each well of the pre-warmed 48-well plate. Return the plate to 37°C without shaking and allow Matrigel to solidify for 5 min. A Matrigel dome should form in the center of the well.

Gently overlay 300 μL of IntestiCult™ Organoid Growth Medium on top.

Incubate organoids at 37°C for 5–7 days before passaging. IntestiCult™ Organoid Growth Medium should be changed every 3–4 days.

Note: After growing for 5–7 days, organoids should reach a good differentiated state with a slight phase-dark lumen and many phase-light finger-like protruding crypts.

Passaging organoids:

Pre-warm a new 48-well tissue culture plate containing Basal Medium and Gentle Cell Dissociation Reagent to 37°C.

Carefully aspirate the IntestiCult™ Organoid Growth Medium from the organoid culture plate without disturbing the Matrigel dome.

Apply 200 μL Gentle Cell Dissociation Reagent to each well and incubate at 37°C for 5 min.

Collect organoids by gently pipetting up and down a few times and transfer and pool the content of each well to a 15 mL conical tube.

Add 10 mL Basal Medium to the tube and spin down organoids at 150 g for 3 min at 4°C.

Slowly take out the supernatant without disturbing the organoid pellet. Add appropriate amount of Matrigel to the organoid pellet on ice. Using a P200, gently disrupt organoids into smaller pieces on ice 20 times.

Note: we typically do 1 to 4–6 splits, i.e. 1 well of organoid split to 4–6 wells. For example, in a 1 to 4 split, if starting from 1 well of organoid, 100 μl (4x25 μl) of Matrigel should be added to the resuspend organoids. The degree of organoid dissociation, incubator condition, and the time of media change may all affect how the organoids grow. The appropriate split ratio should be determined empirically by the operator.

Quickly dispense 25 μL of the organoid/Matrigel suspension to the center of each well of the pre-warmed 48-well plate and let solidify for 5 min at 37°C.

Gently overlay 300 μL of IntestiCult™ Organoid Growth Medium on top.

Organoids need to be passaged again after growing at 37°C for 5–7 days if not used for the assay below. IntestiCult™ Organoid Growth Medium should be changed every 3–4 days.

Plating organoids for mimetic treatment:

Grow and passage organoids as necessary until organoids are in a good differentiated condition and sufficient amount for WNT mimetic treatments. We generally split organoids 1 to 8 to set up for mimetic treatment.

Prepare WNT mimetic dilution series as desired in Basal Medium with 1 μM IWP2. Keep the protein dilution series on ice. Thaw a few aliquots of Matrigel on ice.

Dissociate organoids.

Pre-warm a new 48-well tissue culture plate containing Basal Medium and Gentle Cell Dissociation Reagent to 37°C.

Carefully aspirate the IntestiCult™ Organoid Growth Medium from the organoid culture plate without disturbing the Matrigel dome.

Apply 200 μL Gentle Cell Dissociation Reagent to each well and incubate at 37°C for 5 min.

Collect organoids by gently pipetting up and down a few times and transfer and pool the content of each well to a 15 mL conical tube.

Add 10 mL Basal Medium to the tube and spin down organoids at 150 g for 3 min at 4°C.

Plate organoids.

Slowly take out the supernatant without disturbing the organoid pellet. Depending on the number of organoids, apply Matrigel to the organoid pellet. Using a P200, gently disrupt organoids into smaller pieces on ice 20 times.

Note: Density and starting size of organoids will affect sensitivity of the assay, as intestinal organoids secrete growth factors and help each other grow. At the time of imaging, ∼50 organoids per well is ideal. (Splitting organoid at a ratio of 1 to 8 has been a good starting point for us.) For example, in a 1 to 8 split, if starting from 1 well of organoid, 200 μl (8x25 μl) of Matrigel should be added to the resuspend organoids.

Quickly dispense 25 μL of the organoid/Matrigel suspension to the center of each well of the pre-warmed 48-well plate and let solidify for 5 min at 37°C.

Gently apply 300 μL of Basal Medium with 1 μM IWP2 or the protein dilution serious to the well. Prepare a minimum of 4 wells for each treatment condition for technical repeats.

Incubate the plate at 37°C for 3 days and change medium with fresh protein dilutions on day 4. On day 7, image organoids under a transmitted light microscope equipped with phase contrast or DIC. Example images of organoids under various treatments are shown in Figure 4.

Treating Mouse Intestinal Organoids with WNT Mimetics

(A–C) Images showing mouse intestinal organoids in Basal Medium (A), Basal Medium+IWP2 (B), and Basal Medium+IWP2+mimetic L1-F1-Fc (C) (Chen et al., 2020). Scale bars: 200 μm.