Imagine growing a fully-structured "mini-intestine" or "miniature brain" in a lab? This isn't science fiction! Organoids—these amazing "mini-organs"—are making this a reality, opening a whole new door for disease research and new drug development.

Organoids are three-dimensional cell aggregates. They are derived from stem cells or organ progenitor cells. These structures can mimic the key functions and complex structures of real organs. Scientists use them for disease research and drug testing. Compared to traditional methods, their results are closer to the real situation in vivo^[1].

The development of organoids is of great significance to the study of human organ and tissue functions. This is because these miniature organs can well demonstrate human biological characteristics^[2].

Mouse small intestinal organoids are considered one of the most classic and mature organoid models, a position rooted in the pioneering work of Professor Hans Clevers' team in 2009. The team was the first to achieve organoid culture of adult small intestinal stem cells in vitro, laying the foundation for the vigorous development of this field. Mouse small intestinal organoids are widely used in basic research, drug screening, host-microbe interactions, disease modeling and other fields. Compared with traditional two-dimensional (2D) culture systems, 3D organoid culture provides a more physiologically relevant model^[3][4].

Organoid technology provides a powerful platform for modeling core diseases and personalized treatment. These models can highly simulate organ development and disease progression under conditions such as birth defects, cancer, infectious diseases, and metabolic diseases. For example, vascular organoids have been used to reveal the molecular pathways related to vascular dysfunction in diabetes. In the field of drug development, organoid and organ-on-a-chip technologies are expected to reduce the reliance on traditional animal models and be widely used in drug screening, toxicology and pharmacology evaluation, ultimately promoting the realization of personalized treatment^[6][7][8].

Lgr5⁺ intestinal stem cells were first embedded in Matrigel. They were then cultured in a medium containing WENR—which includes Wnt3a, EGF, Noggin, and R‑spondin 1. This process successfully generated an intestinal organoid model. This model allows for the unlimited proliferation of stem cells. It also reproduces the cellular diversity found in the intestinal epithelium. As a result, it provides an ideal platform for the in situ, real-time observation of epithelial development and differentiation^[9][10].

(1) Fast mice overnight or for 24 hours, then euthanize 5 to 8-week-old mice using carbon dioxide asphyxiation. Disinfect the mice by wiping their fur with 70% ethanol before separating the small and colonic intestines.

(2) Cut a segment of the proximal small intestine about 18-20 cm long. For the colon, dissect a segment of about 3-6 cm, preferably a few millimeters below the cecum and a few millimeters above the rectum, to avoid introducing too many toxins and metabolic wastes that could contaminate the culture system, while also considering the high stem cell density in the proximal colon. Then, carefully remove any remaining fat, blood vessels, and outer membrane tissue attached to the surface of the intestinal segment using forceps.

(3) Carefully place the separated intestinal segment into a 10 cm cell culture dish. Add 10 mL of pre-chilled PBS to the dish. Use a 10 mL syringe and a 21G needle to gently rinse the intestinal lumen with pre-chilled PBS until all fecal particles are expelled.

(4) Cut the intestinal segment longitudinally with scissors to form an intestinal slice, and gently rinse the slice with pre-chilled PBS.

(5) Cut the intestinal segment into small pieces of about 2-5 mm and transfer them to a 50 mL centrifuge tube containing 25 mL of pre-chilled PBS.

(6) Gently pipette the small pieces of intestinal segment five times. Let the sample stand for about 20 seconds to allow the tissue fragments to settle to the bottom of the tube by gravity.

(7) After the fragments have settled, discard the PBS. Then, add 25 mL of pre-chilled PBS back to the centrifuge tube and repeat steps (6) and (7) until the remaining PBS becomes clear.

(8) Discard the clear PBS and resuspend the tissue fragments in 20 mL of 0.05% trypsin/EDTA solution. Incubate the sample on a rotary shaker at 37 °C and 20 rpm for 20 minutes.

(9) Discard the trypsin/EDTA solution and resuspend the fragments in 10 mL of pre-cooled 0.1% BSA solution. Gently pipette the fragments three times with a pre-wetted serum pipette.

(10) After allowing the tissue fragments to settle naturally, filter part of the supernatant of the sample through a 70 μm cell sieve and collect the first filtrate.

(11) Add 10 mL of 0.1% BSA solution to the remaining tissue pellet and gently pipette the fragments three times with a pre-wetted serum pipette. Repeat step (10) to collect the second filtrate.

(12) Repeat steps (9)(10)(11) until the fifth filtrate is collected.

(13) Centrifuge the fourth and fifth filtrates at 4-6 °C and 300 g for 5 minutes. Discard the supernatant and resuspend the pellet containing intestinal crypts in 5 mL of DMEM/Nutrient Mixture F-12 medium.

(14) Take a portion of the filtrate sample and transfer it to a six-well plate. Observe the quality of the crypts under an inverted optical microscope.

(1) Resuspend the crypts in DMEM/Nutrient Mixture F-12, then use a pre-wetted micropipette tip to aspirate 10 μL of sample and add it to a cell counting plate to count the number of crypts in the isolate.

(2) Centrifuge the crypt isolate at 4-6 ℃ and 300 g for 5 minutes and discard the supernatant.

(3) Resuspend the crypt isolate in 180 μL of a 1:1 mixture of mouse intestinal organoid growth medium and matrix gel, and mix thoroughly using a slit pipette tip.

(4) Using a slit micropipette tip, add the medium-matrix gel mixture (30 μL per drop) to one well of a six-well plate to form a droplet dome and embed the crypt isolate within it.

(5) Gently invert the culture plate and incubate it in a CO₂ incubator at 37 ℃, 5% CO₂, and 80% relative humidity for 30 minutes to allow the matrix gel to polymerize.

(6) Slowly add 2 mL of mouse intestinal organoid growth medium along the well wall. Then place the culture plate in a CO₂ incubator for incubation.

(7) Change the culture medium daily until the organoids are fully developed and mature.

1. If using 0.25% trypsin/EDTA, further dilution is necessary to avoid cell damage in the tissue.

2. The density of intestinal stem cells in the colon is lower than in the small intestine; therefore, it is recommended to use more than two colons to increase cell density.

3. The mouse intestine must be cut into sufficiently small pieces, otherwise it will affect crypt extraction.

4. The serum pipette must be pre-wetted to prevent intestinal fragments or crypts from adhering to the pipette wall. Pre-pipetting with PBS is recommended as a wetting step.

5. The matrix gel must be handled on ice; it easily thickens or solidifies at room temperature. When mixing the matrix gel with crypts, gentle pipetting is essential; air bubbles can easily cause gel dispersion or even dissolution during later culture.

6. When adding culture medium, do not add it directly to the matrix gel droplets to avoid damaging them. The culture medium must also be brought to room temperature before use; adding refrigerated medium directly to the culture wells can easily damage the gel droplets.

7. To accelerate the complete solidification of Matrigel, the culture plates can be preheated in a CO₂ incubator at 37°C.

8. After organoid construction, organoid characterization is crucial for downstream applications. Initially, morphology can be observed using microscopy and H&E staining; then, Western blotting, qRT-PCR, immunofluorescence, and flow cytometry can be used to detect whether the organoid expresses the corresponding biomarker; gene sequencing can identify whether the cultured organoid has lost certain features; and for some organoids, their functional status can also be determined.

Mouse small intestinal organoids are a revolutionary technology that simulates the structure of the real intestine in three-dimensional culture, providing a powerful platform that closely resembles the in vivo environment for disease research, drug screening, and developmental biology. Successfully culturing mouse small intestinal organoids is an art combining precise manipulation, high-quality matrix gel, and a culture medium containing core factors. By mastering the principles and strictly controlling the details, you can recreate a vibrant "mini-gut" in a petri dish.