Mouse Colon Organoids and Cytokines: The Molecular Blueprint for Building an In Vitro Micro-Colon

The successful establishment and long-term maintenance of mouse colon organoids are entirely dependent on precise signal regulation provided by specific cytokine combinations. These factors collectively mimic the molecular characteristics of the in vivo colonic stem cell niche, enabling the organoids to recapitulate the structure and function of the colonic epithelium.

Part 1: Fundamental Supporting Role of Core Growth Factors

Epidermal Growth Factor (EGF)

EGF is the most fundamental mitogen in colon organoid culture. By activating the EGFR signaling pathway, it provides continuous proliferation signals for colonic epithelial cells, ensuring stable expansion of the organoids. Studies have shown that withdrawal of EGF leads to organoid growth arrest, confirming its indispensable role in maintaining basic cell division.

Fibroblast Growth Factor (FGF)

Specific members of the FGF family (such as FGF2 and FGF10) play important regulatory roles in mouse colon organoid culture. They not only enhance the self-renewal capacity of stem cells but are also involved in regulating the establishment of polarity in colonic epithelial cells and the morphogenesis of crypt-like structures, which is crucial for maintaining the stability of long-term organoid culture.

Part 2: Precise Regulatory Network of Key Signaling Pathways

Wnt/β-catenin Signaling Pathway

Wnt signaling is the core factor maintaining the stemness of colonic stem cells. In organoid culture, the combination of Wnt3a and R-spondin potently activates β-catenin signaling, promoting stem cell proliferation and inhibiting their premature differentiation. It is noteworthy that colon organoids exhibit an extremely high dependence on Wnt signaling, which is highly correlated with the pathological feature of aberrant Wnt pathway activation in colon cancer.

BMP Signaling Pathway

Similar to small intestinal organoids, culturing colon organoids requires the inhibition of BMP signaling. By adding BMP inhibitors such as Noggin, the low BMP microenvironment found at the base of the colonic crypt in vivo can be recapitulated in vitro. This is essential for maintaining stem cell function and promoting the budding growth of organoids.

Notch Signaling Pathway

The Notch signaling pathway plays a key role in determining the fate of colonic epithelial cells. High Notch activity promotes the differentiation of absorptive cells, whereas inhibition of Notch signaling leads to an increased proportion of secretory cells (such as goblet cells). This finely balanced regulation allows researchers to manipulate the cellular composition of organoids by modulating Notch signaling.

Part 3: Detailed Experimental Protocols

Preparation of Complete Medium (for Expansion): Mix the basal medium, cytokines (1-4), and various additives in established proportions to prepare the complete medium.

1.Primary Culture

(1) In a biosafety cabinet, completely remove the mouse colon tissue and place it in pre-cooled PBS (supplemented with penicillin, streptomycin, and primary antibiotics). Using sterilized scissors, cut the intestinal segment open longitudinally, then spread it out with the lumen facing up. Use a glass slide to scrape off intestinal debris and residues, scraping back and forth 2-3 times. Rinse the colon with pre-cooled PBS.

(2) Hold one end of the colon with forceps and cut it into small segments, approximately 3-5 mm. Collect and transfer them to a 50 ml sterile centrifuge tube. Add pre-cooled PBS and wash 2-3 times.

(3) Add 30 ml of 5 mM EDTA/PBS solution (30 ml PBS + 300 µl 0.5 M EDTA) to the centrifuge tube. Place the colon tissue in the tube for digestion. Digest on a shaking platform at 4°C for about 30 minutes, frequently checking the progress under a microscope. The detachment of crypts signals the endpoint of digestion.

(4) Discard the digestion solution. Add pre-cooled PBS and gently swirl to remove EDTA.

(5) Add 30 ml of pre-cooled PBS containing 0.1% BSA. Vortex to detach the crypts from the colon tissue.

(6) Keep the supernatant. Pass it through a 100 µm cell strainer. Evenly distribute the filtered cell suspension into two 15 ml centrifuge tubes. Centrifuge at 1000 rpm for 5 minutes. After centrifugation, discard the supernatant.

(7) Repeat steps 5-6 twice to increase the yield of crypts.

(8) Resuspend the pellet in an appropriate amount of basal medium or PBS.

(9) Mix the Matrigel and crypts at a suitable ratio. For a 24-well cell culture plate, plate 25-30 µl of the Matrigel mixture per well.

(10) Place the plated culture plate in a 37°C incubator for 20-30 minutes to allow the Matrigel to solidify. Add an appropriate amount of Mouse Colon Organoid Complete Medium (brought to room temperature) to initiate culture.

2. Organoid Passaging

(1) Aspirate the medium using a pipette. Add 1-2 ml of 4°C PBS to each well and let it sit for 2 minutes.

(2) Gently pipette to dissociate the Matrigel. Collect the contents in a 15 ml centrifuge tube. Adjust the volume to 10-14 ml with PBS. Let it sit at 4°C for 20-30 minutes to dissolve the Matrigel (pool 3-5 wells into one group). Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and keep the pellet.

(3) Add 1 ml of organoid digestion solution to the collected pellet. Pipette to mix. Digest at 37°C for 2-5 minutes. Add DMEM/F12 basal medium to stop the digestion. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and keep the pellet.

(4) Resuspend the organoids in an appropriate amount of Matrigel. Plate 25-30 µl of the Matrigel suspension per well in a 24-well cell culture plate. Place the plate in the incubator for 20-30 minutes for the Matrigel to solidify. Add an appropriate amount of Mouse Colon Organoid Complete Medium.

3. Organoid Cryopreservation

(1) Aspirate the medium using a pipette. Add 1-2 ml of 4°C PBS to each well and let it sit for 2 minutes.

(2) Gently pipette to dissociate the Matrigel. Collect the contents in a 15 ml centrifuge tube. Adjust the volume to 10-14 ml with PBS. Let it sit at 4°C for 20-30 minutes to dissolve the Matrigel (pool 3-5 wells into one group). Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and keep the pellet.

(3) Add an appropriate amount of organoid freezing medium. Gently pipette to resuspend. For a 24-well plate, freeze the organoids from 2-3 wells per cryovial, with a volume of 1 ml per vial.

(4) Label the vials appropriately. After programmed freezing, transfer them to liquid nitrogen for long-term storage.

4. Organoid Thawing

(1) Add 10 ml of DMEM/F12 basal medium to a 15 ml centrifuge tube.

(2) Retrieve the frozen organoids from the liquid nitrogen tank and quickly thaw them in a 37°C water bath.

(3) During thawing, gently shake the cryovial to ensure the freezing medium completely thaws within 1-2 minutes.

(4) Quickly transfer the thawed organoids to the 15 ml centrifuge tube. Gently pipette 6-8 times. Centrifuge at 1000 rpm for 5 minutes, then remove the supernatant and collect the organoid pellet.

(5) Resuspend in Matrigel. Plate 25-30 µl of the Matrigel suspension per well in a 24-well cell culture plate. Place the plate in the incubator for 20-30 minutes for the Matrigel to solidify. Add an appropriate amount of Mouse Colon Organoid Complete Medium.

Colon organoid Cytokine Set, Mouse/小鼠结肠类器官细胞因子套装_UA090047_优爱(UA BIOSCIENCE)官网

Colon organoid Cytokine Set, Mouse / Mouse Colon Organoid Cytokine Set_UA090047_UA BIOSCIENCE Official Website