UA Bioscience: Cytokine Optimization Strategy – Key Technology for Enhancing the Reliability of Small Intestinal Organoid Models

Mouse Normal Small Intestinal Organoids, as a revolutionary model for studying intestinal epithelial biology in vitro, rely entirely on a meticulously designed combination of cytokines for their miraculous growth and highly biomimetic structure. These minute signaling proteins act like a precise "conductor," directing intestinal stem cells through self-renewal, differentiation, and tissue construction, perfectly recapitulating the micro-physiological unit of the in vivo intestinal crypt-villus axis.

Part 1: Core Cytokines for Maintaining Stem Cell Stemness

The successful culture of small intestinal organoids is built upon a "core triad" of cytokines that collectively create and maintain a niche allowing intestinal stem cell survival:

• Wnt Signaling: The "Engine" of Crypt Vitality
  
  Wnt proteins (e.g., Wnt3a) are the primary signals maintaining the stemness and proliferative capacity of small intestinal crypt stem cells. They constitutively activate downstream pathways, drive cell division, and provide the initial impetus for organoid cyst and bud formation, mimicking the dynamic stem cell zone at the base of the crypt in vivo.
  
  

• R-spondin 1: The "Super Amplifier" of Wnt Signaling
  
  R-spondin 1, by binding to cell surface receptors, significantly enhances and stabilizes the Wnt signaling pathway. Acting synergistically with Wnt, it forms the most potent combination for maintaining stem cell pool stability and promoting continuous organoid growth, ensuring organoids can be passaged indefinitely without loss of stemness.
  
  

• Noggin: The "Morphological Engineer"
  
  As an inhibitor of Bone Morphogenetic Protein (BMP) signaling, Noggin's role is crucial. In vivo, BMP signaling is highly active at the crypt top and villus, promoting cell differentiation. By inhibiting this signal, Noggin creates a "low-BMP zone" within the organoid analogous to the crypt base, thereby preventing premature stem cell differentiation and allowing them to continuously proliferate and form budding 3D structures.
  
  

Part 2: Auxiliary Factors Regulating Cell Proliferation and Differentiation

A stemness-maintaining environment alone is insufficient for forming functional organoids. Other cytokines play roles in finely regulating differentiation:

• Epidermal Growth Factor (EGF): The "Booster" for Widespread Proliferation
  
  EGF provides a fundamental mitogenic signal for all epithelial cells (including stem and progenitor cells), further driving the overall growth and expansion of the organoid.
  
  

• Notch Signaling: The "Cell Fate Decision-Maker"
  
  Within the organoid, high Notch signaling activity tends to drive cells toward an absorptive lineage (e.g., enterocytes), while low Notch activity drives cells toward secretory lineages (e.g., goblet cells, Paneth cells, enteroendocrine cells). Manipulating Notch signaling can influence the proportion of different cell types within the organoid.
  
  

Part 3: Mouse Small Intestinal Organoid Model Construction

Preparation of Complete Medium for Mouse Small Intestinal Organoids: Combine basal medium, cytokines (from parts 1-2), and various supplements in established proportions to prepare the complete medium.

1.Primary Culture

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

(2) Holding one end of the small intestine with forceps, cut it into small segments (approx. 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 to the tube containing the intestinal tissue for digestion. Digest on a shaking platform at 4°C for 20-30 minutes, frequently checking under a microscope. Crypt detachment indicates the endpoint for digestion; total digestion time should not exceed 40 minutes.

(4) Discard the digestion solution and add pre-cooled PBS, gently swirling to remove EDTA.

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

(6) Retain the supernatant and pass it through a 70 µm cell strainer. Evenly distribute the filtered cell suspension into two 15 ml centrifuge tubes. Centrifuge at 1000 rpm for 5 minutes. Discard the supernatant after centrifugation.

(7) Repeat steps 5-6 twice to increase crypt yield.

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

(9) Mix the crypt suspension with Matrigel at a suitable ratio. Using a 24-well cell culture plate as an example, plate 25-30 µl of the Matrigel-crypt mixture per well (perform at 4°C).

(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 pre-warmed (room temperature) complete medium for mouse small intestinal organoids and begin culture.

2. Organoid Passaging

(1) Aspirate the culture medium using a pipette. Add 1-2 ml of 4°C PBS per well and incubate for 2 minutes.

(2) Gently pipette to disrupt the Matrigel and collect the contents into a 15 ml centrifuge tube. Adjust the volume to 10-14 ml with PBS. Let it stand at 4°C for 20-30 minutes to dissolve the Matrigel (pool 3-5 wells per group). Centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and retain the pellet.

(3) Resuspend the organoids in an appropriate amount of fresh Matrigel. Plate 25-30 µl of the Matrigel-organoid mixture per well in a 24-well plate. Place in the incubator for 20-30 minutes for solidification. Add an appropriate amount of complete medium for mouse small intestinal organoids.

3. Organoid Cryopreservation

(1) Aspirate the culture medium using a pipette. Add 1-2 ml of 4°C PBS per well and incubate for 2 minutes.

(2) Gently pipette to disrupt the Matrigel and collect the contents into a 15 ml centrifuge tube. Adjust the volume to 10-14 ml with PBS. Let it stand at 4°C for 20-30 minutes to dissolve the Matrigel (pool 3-5 wells per group). Centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and retain the pellet.

(3) Add an appropriate amount of organoid freezing medium. Gently pipette to resuspend. Using a 24-well plate as a reference, cryopreserve the contents of 2-3 wells per cryovial (1 ml volume per vial).

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

4. Organoid Thawing

(1) Pre-aliquot 10 ml of DMEM/F12 basal medium into a 15 ml centrifuge tube.

(2) Retrieve the frozen organoid vial from liquid nitrogen and quickly thaw it in a 37°C water bath.

(3) During thawing, gently agitate the cryovial to ensure complete thawing within 1-2 minutes.

(4) Quickly transfer the thawed organoid suspension to the 15 ml tube containing basal medium. Gently pipette 6-8 times. Centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and collect the organoid pellet.

(5) Resuspend the pellet in Matrigel. Plate 25-30 µl of the Matrigel-organoid mixture per well in a 24-well plate. Place in the incubator for 20-30 minutes for solidification. Add an appropriate amount of complete medium for mouse small intestinal organoids.

Small Intestinal Organoid Cytokine Set, Mouse / Mouse Normal Small Intestinal Organoid Cytokine Set_UA090043_UA BIOSCIENCE Official Website

Small Intestinal Organoid Cytokine Set, Mouse/小鼠正常小肠类器官细胞因子套装_UA090043_优爱(UA BIOSCIENCE)官网