3D sheep rumen epithelial structures driven from single cells in vitro

Area of Science:

• Animal Science
• Cell Biology
• Digestive Physiology

Background:

• Ruminants are vital livestock providing high-quality protein.
• Existing 3D organoid models exist for abomasum and intestines, but not the rumen.
• The rumen is crucial for lignocellulose digestion via its microbiota.

Purpose of the Study:

• To establish a method for culturing rumen epithelial organoids from single cells.
• To characterize the properties and potential applications of these organoids.

Main Methods:

• Single rumen epithelial cells were cultured to form self-organized 3D structures.
• Specific growth factors (EGF, Noggin, Wnt3a, IGF-1, FGF-10) and small molecules (CHIR-99021, A83-01, SB202190, Y-27632) were used.
• Organoid stability was tested through passage, cryopreservation, and resuscitation.

Main Results:

• Rumen epithelial organoids self-organized into structures resembling native rumen epithelium.
• Key growth factors and small molecules significantly improved organoid seeding efficiency, formation, and maintenance.
• Organoids retained functionality after cryopreservation and passage, expressing specific genes and uptaking fatty acids.

Conclusions:

• A feasible method for culturing rumen epithelial organoids from single cells was established.
• This novel in vitro system offers a promising alternative for studying rumen function and may reduce experimental animal use.