Materials and reagents

Biological materials

1. Mouse intestinal organoids [13], grown in 50 μL Matrigel drops in a 24-well plate; see General note 1

Reagents

1. DPBS (Thermo Fisher scientific, catalog number: 14190136)

2. Gibco DMEM F12-Glutamax (Thermo Fisher scientific, catalog number: 11514436)

3. Gibco 100× antibiotic-antimycotic (Thermo Fisher scientific, catalog number: 15240096)

4. B27 supplement 50×, serum-free (Thermo Fisher scientific, catalog number: 17504001)

5. N2 supplement 100× (Thermo Fisher scientific, catalog number: 15410294)

6. Mouse EGF (Peprotech, catalog number: 17872513)

7. Mouse FGF (Peprotech, catalog number: 17844573)

8. Murine Noggin (Peprotech, catalog number: 250-38)

9. R-spondin (R&D Systems, catalog number: 3474-RS-050)

10. Nicotinamide (Sigma-Aldrich, CAS: 98-92-0)

11. Chir-99021 (Euromedex, catalog number: AB-M1692-100MG; CAS: 252917-06-9)

12. Rock inhibitor Y27632 (ATCC, catalog number: ATCC-ACS-3030)

13. Accumax (Sigma-Aldrich, catalog number: A7089)

14. Trypan blue stain (Invitrogen, catalog number: T10282)

15. Collagen I, rat tail, 100 mg (Corning, catalog number: 354236)

Solutions

1. Stock solutions

2. Cell dissociation solution (see Recipes)

3. Stop solution (see Recipes)

4. ENR medium (see Recipes)

5. Plating medium (see Recipes)

6. ENR-CNY medium (see Recipes)

Recipes

1. Stock solutions

Aliquot and store at -20 °C.

2. Cell dissociation solution

For 12 Matrigel drops, 1 mL of dissociation solution is needed. Keep dissociation solution on ice. Make fresh each time.

3. Stop solution

For every 1 volume of dissociation solution, 2 volumes of stop solution are needed. For 1 mL of dissociation solution, 2 mL of stop solution is needed. Pre-heat the stop solution to 37 °C. Make fresh each time.

4. ENR medium

ENR medium can be kept at 4 °C for 2–3 weeks and will be used as the base medium for the monolayers throughout the experiment. Use 700 μL for each fluorodish.

5. Plating medium

ENR medium can be kept at 4 °C for 2–3 weeks. Each gel should be plated with 45 μL of plating medium + cells. Add Chir and Y27 to the ENR medium at the needed volume right before plating.

6. ENR-CNY medium

Use this medium for cell spreading in the first 48–72 h. Use 700 μL for each fluorodish. Pre-heat to 37 °C. ENR medium can be kept at 4 °C for 2–3 weeks. Add Chir, Y27, and Nicotinamide to the ENR medium at the needed volume right before adding medium to the plated cells.

Laboratory supplies

1. 35 mm glass‐bottom dish (WPI FluoroDish FD35‐100) with 18 mm polyacrylamide gels (PAA), coated with collagen type-1 and washed with PBS, or any alternative substrate for cell attachment. See Baghdadi et al. [14], supplementary table. See General notes 2 and 3.

2. 20 G needle (TERUMO, catalog number: AN*2038R1)

3. 23 G needle (BD Microlance, catalog number: 300800)

4. 24 G needle (TERUMO, catalog number: NN-2425R)

5. 10 mL syringe (TERUMO, catalog number: MDSS10SE)

6. 2.5 mL syringe (TERUMO, catalog number: SS*02SE1)

7. Cell strainer 40 μm (FisherBrand, catalog number: 22363547)

8. 15 mL conical tubes (Sarstedt, catalog number: 62.554.002)

9. 50 mL conical tubes (Sarstedt, catalog number: 62.548.101)

10. 10 μL pipette tips (STARLAB, catalog number: S1121-2710)

11. 200 μL pipette tips (STARLAB, catalog number: S1120-8710)

12. 1,000 μL pipette tips (STARLAB, catalog number: S1122-1730)

Equipment

1. Laboratory centrifuge with rotors for 15 mL conical tubes, temperature-controlled, chilled to 4 °C (Eppendorf Centrifuge, model: 5804 R)

2. Cell incubator

3. Cell counter (DeNovix CellDrop FL counter)

4. Microscope for evaluating cell dissociation

Procedure