An Optimized Sequential Isolation of Crypts and Mesenchymal Stromal Cells from Porcine Intestinal Tissue

Area of Science:

• Gastroenterology
• Cell Biology
• Animal Science

Background:

• Intestinal epithelium and mesenchymal stromal cells (iMSCs) are crucial for gut development and function.
• Standardized methods for isolating both cell types from porcine tissue are lacking.
• Porcine models offer translational relevance for human physiology.

Purpose of the Study:

• To develop and optimize a stepwise protocol for the sequential isolation of intestinal crypts and iMSCs from the same porcine jejunal segment.
• To provide a reproducible method for obtaining both epithelial and stromal cell populations for co-culture studies.
• To establish a foundation for research into epithelial-stromal interactions in pigs.

Main Methods:

• Utilized an ethylenediaminetetraacetic acid/dithiothreitol (EDTA/DTT) buffer for crypt isolation.
• Employed collagenase/dispase digestion to isolate iMSCs from the remaining tissue.
• Characterized isolated cells using cell culture, immunostaining, and marker analysis (PDGFRα, CD81).

Main Results:

• Successfully isolated viable intestinal crypts and iMSCs from the same jejunal segment.
• Cultured crypts formed two-dimensional (2D) enteroid monolayers.
• Isolated iMSCs adhered to culture flasks and expressed key stromal markers.

Conclusions:

• The optimized protocol efficiently yields both intestinal epithelial and iMSCs from porcine jejunum.
• This method supports future research on epithelial-stromal interactions using organoid and co-culture systems.
• The protocol provides a valuable tool for advancing swine models in translational research and animal sciences.