Re-engineering the "Miniature Stomach": A Full-Process Protocol for the In Vitro Construction of Mouse Gastric Organoids

In the fields of life sciences and medical research, the development of in vitro models capable of accurately simulating the complex structure and function of internal organs has long been a goal pursued by scientists. The emergence of normal mouse gastric organoids represents a major breakthrough in this direction, providing an unprecedented powerful tool for understanding gastric development, physiology, and disease mechanisms.

Gastric organoidsare three-dimensional (3D) miniaturized organs derived from stem cells in laboratory culture dishes. The construction of normal mouse gastric organoids typically begins with the isolation of adult stem cells or embryonic stem cells from mouse gastric tissue. These cells are cultured within a Matrigel matrix containing specific growth factors (such as EGF, Wnt, Noggin, etc.), which precisely simulate the stem cell niche microenvironment of the gastric mucosa in vivo.

Part 1: Core Cytokines for Constructing Gastric Organoids

Within the organoid culture system, several core cytokines form an indispensable "basic formula," each playing a distinct role:

• Wnt Signaling Agonists: The "Guardians" of Stemness
  Factors such as Wnt3a are central to initiating and maintaining the stemness of gastric epithelial cells. Their continuously activated signaling pathways ensure the stability and self-renewal of the stem cell pool, providing the primary driving force for the sustained growth of organoids.
  

• EGF: The "Engine" of Proliferation
  As a potent mitogen, Epidermal Growth Factor directly drives the rapid proliferation of stem cells and progenitor cells. It is the power source enabling the continuous expansion and scaling up of organoids.
  
  

• Noggin: The "Border Guard" Inhibiting Differentiation
  As a highly effective inhibitor of the BMP signaling pathway, Noggin's role is crucial. By inhibiting the differentiation-promoting BMP signal, it creates a "safe zone" for stem cells, shielding them from external differentiation cues, thereby maintaining the undifferentiated state and long-term growth capacity of the organoids.
  
  

Part 2: Shaping Structure and Function in Mouse Gastric Organoids

Beyond the core factors mentioned above, other signaling molecules such as R-spondin 1 (which consolidates the stemness environment by enhancing Wnt signaling) and various Fibroblast Growth Factors (FGFs) participate in more refined regulation. They influence cell polarization and lineage differentiation, guiding some stem cells to develop into functionally specific cells, such as acid-secreting parietal cells or pepsinogen-secreting chief cells. This allows the organoids to evolve from simple cell clusters into miniaturized stomachs possessing functional structures.

Part 3: Protocol for Constructing Mouse Gastric Organoid Models

Preparation of Complete Medium for Mouse Gastric Organoids: Combine basal medium, cytokines (1-6), and various additives according to established proportions to prepare the complete medium.

1. Primary Culture

(1) Quickly dissect the mouse abdominal cavity under a biosafety cabinet, remove the gastric tissue, and place it in pre-cooled PBS (supplemented with penicillin, streptomycin, primary antibiotics). Open the stomach, rinse out the contents, and remove the outer fat and blood vessels.

(2) Collect the cleaned tissue fragments into a 1.5 ml microcentrifuge tube. Mince the tissue pieces as finely as possible (to a homogenate-like consistency) using sterile sharp-pointed ophthalmic surgical scissors. The degree of tissue mincing correlates with the digestion time; higher degree of mincing shortens the digestion time. Transfer the minced tissue to a 15 ml sterile centrifuge tube.

(3) Add 10 times the volume of tissue digestion solution and digest at 37°C with shaking for 10-30 minutes. Monitor the process closely. Digestion can be terminated when a large number of cells are observed leaking out under the microscope, and most cells appear as cell clusters. (Note: Avoid digesting to a single-cell state).

(4) To the confirmed digested tissue suspension, add Fetal Bovine Serum (FBS) to a final concentration of 2-5% or Bovine Serum Albumin (BSA) to a final concentration of 0.1%, then pipette to mix evenly.
(5) Filter the tissue and cell suspension through a 70 µm cell strainer. Centrifuge the filtered cell suspension at 1000 rpm for 5 minutes. After centrifugation, remove the supernatant.

(6) If obvious red pellet (red blood cells) is observed in the precipitate after centrifugation, add 2 ml of red blood cell lysis buffer, pipette to mix evenly, and let it stand for 3 minutes. Add 10 ml of PBS to resuspend and stop the lysis. Centrifuge at 1000 rpm for 5 minutes, then discard the supernatant.

(7) Resuspend the precipitate in an appropriate amount of basal medium or PBS.

(8) Mix Matrigel and crypts/villi fragments in an appropriate ratio. Using a 24-well cell culture plate as an example, plate 25-30 µL of the Matrigel mixture per well (operate at 4°C).

(9) Place the plated culture plate in a 37°C incubator for 20-30 minutes for gelation. Add an appropriate amount of complete medium for mouse gastric organoids (brought to room temperature) to begin culture.

2. Organoid Passaging

(1) Aspirate the culture 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 disrupt the Matrigel, and collect the contents into a 15 ml centrifuge tube. Adjust the volume to 10-14 ml with PBS and let it stand at 4°C for 20-30 minutes. Group every 3-5 wells together. Centrifuge at 1000 rpm for 5 minutes, discard the liquid, and keep the pellet.

(3) Add an appropriate amount of Matrigel to resuspend the organoids. Plate 25-30 µL of Matrigel per well in a 24-well cell culture plate. Place the plate in the incubator for 20-30 minutes for gelation. Add an appropriate amount of complete medium for mouse gastric organoids.

3. Organoid Cryopreservation

(1) Aspirate the culture 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 disrupt the Matrigel, and collect the contents into a 15 ml centrifuge tube. Adjust the volume to 10-14 ml with PBS and let it stand at 4°C for 20-30 minutes. Group every 3-5 wells together. Centrifuge at 1000 rpm for 5 minutes, discard the liquid, and keep the pellet.

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

(4) Label the vials with information. Perform programmed freezing, then transfer to liquid nitrogen for long-term storage.

4. Organoid Thawing

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

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

(3) During water bath 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 Matrigel per well in a 24-well cell culture plate. Place the plate in the incubator for 20-30 minutes for gelation. Add an appropriate amount of complete medium for mouse gastric organoids.

Gastric Organoid Cytokine Set, Mouse/小鼠正常胃类器官细胞因子套装_UA090042_优爱(UA BIOSCIENCE)官网