Human Intestinal Organoid Kit

1. Preparation of Complete Culture Medium for Human Intestinal

Prepare the complete organoid medium according to the components below, mix thoroughly, and keep on ice. Perform all steps under sterile conditions.

2. Primary Tissue Processing and Isolation of Epithelial Cells/Glandular Structures

a. Place fresh tissue in pre-cooled primary tissue storage solution and process as soon as possible.

b. Under aseptic conditions, wash thoroughly with Human Intestinal Organoid Basal Medium A or PBS to remove blood, fat, connective tissue, and other non-target components.

c. Cut the tissue into small pieces suitable for dissociation. Use the tissue-specific dissociation solution or EDTA treatment as appropriate to loosen epithelial cell clusters, glands, or crypt structures.

d. Release the target epithelial structures by gentle pipetting or mechanical agitation and remove large tissue debris using an appropriate cell strainer.

e. Centrifuge at 250-300 × g for 3-5 min at 4°C, collect the target cell/gland pellet, and wash 1-2 times with Human Intestinal Organoid Basal Medium A.

3. Construction of Organoid

a. Keep the procedure on ice and resuspend the isolated epithelial clusters, glands, or crypts in MCE Basement Membrane Matrix. Use 100% Basement Membrane Matrix when possible; if dilution is required, maintain a Basement Membrane Matrix-to-organoid-medium ratio > 2 : 1.

b. Using a pre-wetted 200 μL pipette tip, dispense approximately 25-35 μL per well into a 24-well plate, avoiding bubbles. Incubate at 37°C with 5% CO₂ for 15-30 min until gelled.

c. After gelation, slowly add 500 μL Human Intestinal Organoid Complete Medium along the well wall without disrupting the gel.

d. Replace 500 μL pre-warmed Human Intestinal Organoid Complete Medium every 3-4 days and monitor organoid growth and differentiation according to tissue source.

4. Organoid Passaging

a. Aspirate the culture medium, add an appropriate amount of Human Intestinal Organoid Basal Medium A, detach the matrix/organoids using a cell scraper or pipette tip, and transfer to a centrifuge tube.

b. Gently pipette to separate organoids from the matrix and centrifuge at 250-300 × g for 3 min at room temperature.

c. Resuspend in Human Intestinal Organoid Basal Medium A and disperse organoids into smaller clusters. If mechanical dissociation is insufficient, briefly use organoid dissociation solution and stop when clusters of approximately 10-50 cells are obtained to avoid over-digestion.

d. Centrifuge and wash 1-2 times with Human Intestinal Organoid Basal Medium A or PBS.

e. Re-embed on ice in Basement Membrane Matrix, dispense 25-35 μL per well, and incubate at 37°C with 5% CO₂ for 15-30 min until gelled.

f. Add 500 μL Human Intestinal Organoid Complete Medium per well and continue culture, replacing the medium every 3-4 days.