Mouse ileal organoid lines

This protocol allows for the establishment of small intestinal organoids from mice and the preparation of cultures for live imaging experiments.

Reagents:
1. EDTA
2. PBS
3. Cell Recovery Solution (Corning, #354253)
4. Advanced Dulbecco’s modified Eagle’s medium/Ham’s F12 (Gibco, #12634010)
5. N2 Supplement (100x) (Gibco; #17502048)
6. B27 Supplement (50x) (Gibco; #17504044)
7. N-acetyl-L-cysteine (Sigma; #A9165)
8. Recombinant epidermal growth factor (Gibco; #PMG8045)
9. R-spondin 1 (Peprotech; #315-32)
10. Recombinant Noggin (Sigma; #SRP3227)
11. Growth factor reduced Matrigel (Corning; #354230)
12. L-Glutamax (Gibco; #35050061)
13. HEPES (Gibco; #15630056)
14. Penicillin/Streptomycin (Gibco; #15140122)
15. 70 μm Cell strainer (BD Falcon; #352350)
16. 24-well cell culture plates (Corning; #3524)
17. 35 mm cell culture dishes (Corning; #430165)
18. 35 mm μ-Dish, high glass bottom (No. 1.5H glass bottom; ibidi; #81158)

Media Formulations:
• Basal Medium
    o Advanced DMEM/F12 supplemented with:
         2 mM L-Glutamax
         10 mM HEPES
         100/100 U Penicillin/Streptomycin
     o Medium is aliquoted and stored at -20 °C
• Organoid Medium
    o Basal medium supplemented with
         1 mM N-acetyl cysteine
         1x N2 supplement
         1x B27 supplement
         50 ng/ml EGF
         100 ng/ml Noggin
         500 ng/ml R-Spondin 1

Isolation of small intestinal crypts
1. A 6-week-old mouse was anesthetized with isofluorane and sacrificed by cervical dislocation.
2. Following sacrifice, the small intestine (last 3 cm) was dissected and flushed with ice-cold PBS using a blunt ended needle and syringe.
3. The small intestine was inverted, and one end tied. The inverted intestine was then filled with ice-cold 5 mM EDTA in PBS and the other end tied off.
4. The inverted small intestine was incubated at 4 °C in 5 mM EDTA/PBS for 1 hour with agitation. The buffer was exchanged every 20 minutes.

5. Following incubation, the tissue was transferred to 10 ml ice-cold PBS and crypts were released by vigorous shaking. The presence of crypts in the supernatant was checked under the microscope. If crypts were not present the tissue was moved to fresh PBS and the process was repeated. Crypts were usually present in fractions 3 & 4.
6. Fractions containing crypts were pooled and kept on ice.

Culture of small intestinal crypts/organoid formation
1. Pooled crypt containing fractions were passed through a 70 μm cell strainer and then collected by centrifugation at 300 x g for 5 minutes at 4 °C.
2. Pellet was resuspended in 10 ml basal medium, and crypts were collected by centrifugation at 150 x g for 90 seconds. This is to remove single cell contamination from the preparation.
3. Repeat step 2.
4. The pellet was resuspended in 140 μl Matrigel and the suspended crypts were kept on ice.
5. 25 μl droplets of suspended crypts were plated in a prewarmed 24-well plate, the plate was carefully inverted and placed in the incubator for 10-15 minutes to allow the Matrigel drops to polymerize.
6. 500 μl of organoid medium was gently added to each well and the cultures were incubated at 37 °C , 5% CO₂.
7. Medium was replaced every 2-3 days.

Passaging of organoid cultures
Organoids were generally passaged once a week by mechanical fragmentation of crypt domains and seeding in Matrigel.
1. Medium was removed and the Matrigel drop containing organoids was scraped into 500 μl of either Cell Recovery Solution (Corning) or 5mM EDTA/PBS.
2. Organoids were fragmented by repeated pipetting through a 200 μl tip, approximately 20 times. 5 ml of basal medium was then added to the fragmented organoids. Take care not to over fragment the organoids.
3. Fragmented organoids were collected by centrifugation at 300 x g for 5 minutes at 4 °C.
4. Pellet was resuspended in 10 ml basal medium, and crypts were collected by centrifugation at 150 x g for 90 seconds.
5. The pellet was resuspended in Matrigel, and the suspended crypts were kept on ice. Cultures were split in the ratio of 1:4 to 1:6.
6. 25 μl droplets of suspended crypts were plated in a prewarmed 24-well plate, the plate was gently inverted and placed in the incubator for 10-15 minutes to allow the Matrigel drops to polymerize.
7. 500 μl of organoid medium was gently added to each well and the cultures were incubated at 37 °C, 5% CO₂.
8. Medium was replaced every 2-3 days.

Seeding of organoids for live imaging experiments
Organoids were passaged as described in above protocol “Passaging of organoid cultures” with one modification of the protocol.
Organoids were seeded in 10 μl drops of Matrigel containing approximately 20 crypts. These droplets were placed in the center of 35 mm diameter cell culture dishes with a glass bottom if using an inverted microscope or standard 35 mm cell culture dishes if using an upright microscope.
Cultures were grown for 5 days before use in experiments to allow for organoid differentiation.