I have spent quite some time trying to cultivate colonoids from crypts which I isolated from mice aged 6-10 weeks. However, despite an acceptable number of viable-looking crypts seeded per well only few of them started to form colonospheres which did not even increase in size but remained really small. Also, I had the impression that many of these mini-colonospheres were just sitting loosely together instead of forming a solid organoid body. Unnecessary to mention that hardly any of them survived the first splitting procedure.
I adapted the protocol from several other ones including Sato et al., 2011.
Here‘s the chemicals and protocol that I used for my experiments:
Crypt Chelating Buffer I:
DPPS (no calcium, no magnesium) + D-sorbitol (54.9 mM) + D/L-sucrose (43.4 mM) + DTT (0.5 mM)
Crypt Chelating Buffer II:
Crypt Chelating Buffer I supplemented with EDTA 2 mM
Basic Mini Gut Medium:
DMEM/F12 + Glutamax + Penicillin/Streptomycin + N2 + B27
Mini Gut Growth Medium:
Basic Medium + Noggin (100 ng/ml) + EGF (50 ng/ml) + R-Spondin 1 (1 µg/ml) + Wnt3a (100 ng/ml) (all recombinant)
Mini Gut Differentiation Medium:
Basic Medium + Noggin (100 ng/ml) + EGF (50 ng/ml) + R-Spondin 1 (1 µg/ml) (all recombinant)
Crypt isolation from mouse colon and seeding
Make an incision into the abdominal cavity and extend the incision to the rib cage by cutting abdominal musculature on both sides. Grasp the duodenum and cut the intestine from the stomach at the pyloric sphincter. Pull gently the intestine out of the abdominal cavity. Cut the proximal colon from the cecum and the distal colon at the anal margin.Flush the intestine with ice-cold PBS using a 10 mL syringe mounted with an 18 gauge needle. The needle is placed into the lumen and the flushing proceeds until the removal of chyme is complete.Cut the dissected intestine open lengthwise and cut into 2-3 fragments. Place in a 50 ml conical tube with 25 ml ice-cold PBS. Invert 10–15 times, remove the PBS and replace with 25 ml of ice-cold PBS. Repeat this process until the supernatant no longer contains any visible debris.Mince the fragments thoroughly with 2 scalpels.Place the minced tissue in a 50 ml falcon tube filled with 30 mL of crypt chelating buffer II. Vigorously triturate the fragments by pipetting up and down into a 10 ml pipette 15 times.Bury the tube on ice horizontally and gently shake the tube for 60 minutes on an orbital rocker at 4 °C.Add 10 ml of ice-cold crypt chelating buffer I and then pipet up and down 3 times and shake vigorously for 60 s. Let the fragments settle down under normal gravity for 1 minute and collect this 10 ml supernatant fraction (1) in a separate tube (repeat this procedure to obtain a total of 3 fractions, all tubes need to be coated). Pool all fractions ( ~ 30 ml) and filter through a 100 μm cell strainer (coated) into a coated 50 ml Falcon tube.Filter the solution again, this time through a 70 μm cell strainer (coated) into a coated 50 ml Falcon tube and then centrifuge the filtrate at 1000 rpm for 5 min at 4° C.Resuspend the pellet in 5 ml Basic Mini Gut Medium and centrifuge the resuspension for 5 min at 1000 rpm and 4 °C.Resuspend the pellet in 200 µl Basic Mini Gut Medium and count the number of crypts per 10 μL drops from the crypt suspension. Take the corresponding volume out of the crypt suspension to plate 500 crypts per well, and transfer to a coated 15 mL falcon tube.Using pre-chilled pipette tips, resuspend the crypts pellet in 600 µl Resuspension Buffer + 600 µl Matrigel (500 crypts/50 μL Matrigel) for a 24-well plate.Apply 50 μL of Matrigel suspension per well on the pre-warmed plate. Slowly eject the Matrigel in the center of the well.Place the 24-well plate in a CO2 incubator (37°C, 5% CO2) for 30 minutes to allow complete polymerization of the Matrigel.Overlay the Matrigel with 350 μL of Mini Gut Growth Medium.Culture the plate in the CO2 incubator (37°C, 5% CO2).After 4 days, replace the media with 150 µl fresh Mini Gut Growth Medium.I am very thankful for any ideas or suggestions of what might be the reason for these problems!
