With the booming development of the medical industry, the popularity of organoid/stem cell culture continues to rise, and gastric organoids are also a type of organoids that receive considerable attention in organoid culture. Gastric organoids cultured in vitro contain various cellular subtypes and are often derived from gastric stem cells or pluripotent induced stem cells. Their culture environment simulates the microenvironment of the body, so gastric organoids are highly similar to gastric epithelial tissues in terms of cellular composition, spatial structure, and biological functions, providing a favorable platform and tool for the study of human gastric physiology and diseases.

The culture of gastric organoids is relatively complex. The gastric epithelial glands can be divided into gastric fundic glands and gastric pyloric glands, both of which can be used to construct gastric epithelial organoids. The principle of gastric epithelial organoid culture is to culture gastric epithelial stem cells under 3D conditions, which requires the addition of small molecule compounds (such as Gastrin, Y-27632) and growth factors to stimulate stem cell proliferation, differentiation, and formation of spherical structures. Gastrin I is a small molecule compound that must be added to the culture medium for gastric organoid culture, commonly used at a working concentration of 1-10 nM. Below is a summary of the approximate process of gastric organoid culture and the various compounds, biochemical reagents, growth factors, consumables, etc., needed for it.

Compounds, biochemical reagents, growth factors, consumables, etc., required for gastric organoid culture:

Recombinant Human FGF-10 Protein, His Tag	C230418
Recombinant Human EGF Protein	C230328
Recombinant Human Wnt -3a Protein	C230259
Recombinant  Human RSPO1 Protein	C230254
Recombinant Human Noggin Protein,His Tag	C230462

 

Cultivation Protocol

1. Resuscitation of Gastric Organoids

1. Place the frozen storage tube containing the organoids in a 37°C water bath for 2-3 minutes to rapidly thaw.

2. Transfer the organoids from the storage tube to a conical tube and add an appropriate amount of gastric organoid culture medium. Pipette up and down 2-3 times with a serological pipette to resuspend the organoids.

3. Centrifuge at 500g for 3 minutes to pellet the gastric organoids and discard the supernatant.

4. Resuspend the gastric organoids in low growth factor extract, pipette up and down 2-3 times with a serological pipette until fully dispersed, then add to a 24-well plate along with an appropriate amount of resuspension medium.

5. Place the 24-well plate in a cell culture incubator and incubate for approximately 30 minutes.

6. Prepare the passage culture medium for gastric organoids.

7. Add an appropriate amount of gastric organoid passage culture medium to each well.

8. Place the culture plate containing gastric organoids into the cell culture incubator for further cultivation.

2. Cultivation of Gastric Organoids

Refresh the culture medium every 2-3 days by discarding the spent medium and replacing it with fresh culture medium. Continuously culture for 1-2 weeks before passaging.

3. Passaging of Gastric Organoids

1. Observe the growth status of gastric organoids under a microscope. Each well of the 24-well plate can contain 500-1000 organoids.

2. Transfer the 24-well plate containing gastric organoids to a conical tube.

3. Centrifuge at 500g for 3 minutes to pellet the gastric organoids. Aspirate the medium from the bottom of the tube for digestion and extraction.

4. Gently pipette up and down 2-3 times with a serological pipette, then place the conical tube in a 37°C water bath and incubate for 10-15 minutes for digestion and extraction.

5. Add an appropriate amount of FBS to the conical tube to terminate the digestion.

6. Gently pipette up and down 2-3 times with a serological pipette, then filter and separate the organoids.

7. Centrifuge at 500g for 3 minutes to pellet the gastric organoids and discard the supernatant.

8. Resuspend the organoids in culture medium, add to a 24-well plate, and place the plate in a cell culture incubator for 25-30 minutes.

9. Add an appropriate amount of passage culture medium to the 24-well plate and continue cultivation in the cell culture incubator. 

 

Figure: The embryonic components separated from pluripotent stem cells can be used in tissue engineering to generate complex human gastric organoids.

[1] Eicher A K ,  Kechele D O ,  Sundaram N , et al. Functional human gastrointestinal organoids can be engineered from three primary germ layers derived separately from pluripotent stem cells - ScienceDirect.  2021.