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

The intestinal epithelium closely interacts with the underlying intestinal mesenchymal stromal cells (iMSCs) to regulate development, homeostasis, and regeneration. However, standardized methods for sequentially isolating both epithelial and stromal fractions from porcine tissue are limited. This protocol describes an optimized stepwise process for isolating crypts and iMSCs from the same jejunal segment of piglets. The method uses an ethylenediaminetetraacetic acid/dithiothreitol (EDTA/DTT)-based dissociation buffer to release crypts, followed by collagenase/dispase digestion of the extracellular matrix to isolate iMSCs. This approach minimizes spatial variations in the isolated cells. The isolated crypts were successfully cultured and formed typical expanding, two-dimensional (2D) enteroid monolayers on collagen hydrogel-coated plates. The cultured iMSCs adhered to culture flasks and expressed stromal markers such as platelet-derived growth factor receptor alpha (PDGFRα) and cluster of differentiation 81 (CD81), as determined by immunostaining. Overall, this optimized isolation protocol yields both epithelial and stromal cells from the same piglet jejunal segment, providing a reproducible foundation for future research involving intestinal organoids and iMSC co-culture systems to elucidate mechanisms controlling epithelial-stromal interactions in pigs, a translationally relevant model for replicating human physiology and advancing animal sciences.