Tissues were all derived from normal horses. Previously frozen crypts were successfully cultured following a freeze-thaw cycle. Immunofluorescence and PCR were used to confirm the existence of stem cells and all post mitotic, mature cell types, described to exist in the horse intestinal epithelium. Intestinal crypts were successfully cultured and matured into 3D enteroids containing a lumen and budding structures. Intestinal crypts were cryopreserved immediately following collection and viability assessed. After several days, resultant 3D enteroids were prepared for immunofluorescent imaging and polymerase chain reaction (PCR) analysis to detect and characterise specific cell types present. Intestinal crypts containing intestinal stem cells were dissociated from the underlying tissue layers, plated in a 3D matrix and supplemented with growth factors. Intestinal samples were collected by sharp dissection immediately after euthanasia. To describe the isolation and culture of small intestinal stem cells into three-dimensional (3D) enteroids in horses without clinical gastrointestinal abnormalities. This study aims to describe the methods of equine stem cell culture and highlights the future benefits of these techniques for the advancement of equine medicine. The culture of intestinal stem cells has quickly become a cornerstone of a vast number of new research endeavours that range from determining tissue viability to testing drug efficacy for humans. Intestinal epithelial stem cells are critical to epithelial repair following gastrointestinal injury.

Equine intestines 3d