Establishment of bovine 3D enteroid-derived 2D monolayers

Area of Science:

• Cell Biology
• Gastroenterology
• Animal Science

Background:

• Three-dimensional (3D) intestinal enteroids are valuable in vitro models but their closed structure limits apical surface access.
• This limitation hinders high-throughput studies involving exogenous compounds and pathogens.

Purpose of the Study:

• To develop a method for generating confluent 2D enteroid monolayers from bovine 3D enteroids.
• To establish a defined, cost-effective culture medium for these 2D enteroids.

Main Methods:

• Generated confluent 2D enteroid monolayers from single-cell suspensions of bovine 3D enteroids.
• Developed and utilized a bovine enteroid monolayer (BEM) medium for culture.
• Cultured monolayers on Matrigel-coated plates and transwell inserts.

Main Results:

• Monolayers proliferated extensively and formed confluent cell layers by day 3.
• 2D enteroids maintained epithelial cell lineages present in 3D enteroids and native ileum tissue.
• Formed a functional epithelial barrier with adherens and tight junction proteins (E-cadherin, ZO-1) and measurable electrical resistance for up to 7 days.

Conclusions:

• The described method provides a useful model for studying bovine epithelial cell biology with accessible apical surfaces.
• This 2D enteroid model has potential for investigating host-pathogen interactions and screening bioactive compounds.