Mouse Colonic Organoid Kit

1. Preparation of Complete Mouse Colonic Organoid Medium

Combine Mouse Colonic Organoid Basal Medium A with Organoid Culture Supplement B (50×), Organoid Culture Supplement C (250×), and Organoid Culture Supplement D (250×) at their corresponding working concentrations, mix thoroughly, and keep on ice.

2. Isolation of Colon Crypts

a. Collect fresh colon tissue and rinse repeatedly with pre-cooled PBS to remove luminal contents and residual blood.

b. Open the tissue longitudinally, cut into small pieces, and wash repeatedly with PBS until the supernatant is largely clear.

c. Prepare an EDTA working solution from the EDTA stock supplied with the kit and incubate the tissue to loosen epithelial crypts from the surrounding tissue.

d. Remove the EDTA solution, add pre-cooled PBS, and release crypts by vigorous shaking or gentle pipetting.

e. Remove large tissue fragments using an appropriate cell strainer and centrifuge at 250-300 × g for 3-5 min to collect the crypt pellet.

f. Wash the crypts 1-2 times with Mouse Colonic Organoid Basal Medium A and evaluate crypt integrity and quantity microscopically.

3. Construction of Organoid

a. Keep the procedure on ice and resuspend the crypts in MCE Basement Membrane Matrix, distributing them evenly.

b. Using a pre-wetted 200 μL pipette tip, dispense approximately 25-35 μL per well into a 24-well plate and avoid bubbles.

c. Incubate at 37°C with 5% CO₂ for 15-30 min until gelled, then slowly add 500 μL Mouse Colonic Organoid Complete Medium per well.

d. Replace with fresh complete medium every 2-3 days and monitor crypt budding and organoid expansion.

4. Organoid Passaging

a. When organoids enlarge, show extensive budding, or become overly dense, aspirate the medium, add an appropriate amount of Mouse Colonic Organoid Basal Medium A, and detach the matrix/organoids from the well.

b. Mechanically pipette organoids into smaller clusters. If needed, briefly use organoid dissociation solution and avoid excessive digestion to single cells.

c. Centrifuge at 250-300 × g for 3-5 min, discard the supernatant, and wash with Mouse Colonic Organoid Basal Medium A.

d. Re-embed on ice in Basement Membrane Matrix, dispense 25-35 μL per well, gel at 37°C, add 500 μL Mouse Colonic Organoid Complete Medium, and continue culture.