Robust Three-Dimensional (3D) Expansion of Bovine Intestinal Organoids: An In Vitro Model as a Potential Alternative...

Area of Science:

• Animal biotechnology
• Stem cell biology
• Gastrointestinal research

Background:

• Intestinal organoids are valuable for disease modeling and research but are understudied in livestock.
• Limited functional characterization exists for adult stem cells in 3D culture for bovine species.

Purpose of the Study:

• To establish and functionally characterize three-dimensional (3D) intestinal organoids from adult bovine small intestine.
• To assess the potential of these organoids as alternatives to in vivo models in animal biotechnology.

Main Methods:

• Isolation and 3D culture of intestinal crypts from adult bovine small intestine.
• Functional characterization using immunohistochemistry, epithelial barrier permeability assays, and RNA sequencing (QuantSeq 3' mRNA-Seq).
• Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for genetic property analysis.

Main Results:

• Successfully established long-term cultivable bovine intestinal organoids that recapitulate crypt structure.
• Organoids exhibited specific marker expression for intestinal stem cells, epithelium, and nutrient absorption.
• Demonstrated high permeability for small molecules (FITC-dextran 4 kDa) and genetic similarity to in vivo tissues.

Conclusions:

• Developed a reliable method for isolating and expanding bovine intestinal organoids in 3D culture.
• Validated that these 3D organoids accurately mimic in vivo tissue topology and functionality.
• Bovine intestinal organoids represent a viable in vitro model, potentially replacing animal experiments in biotechnology.