Advanced three‐dimensional culture of equine intestinal epithelial stem cells

• Published in: Equine veterinary journal 2018-03, Vol.50 (2), p.241-248
• Main Authors: Stewart, A. Stieler, Freund, J. M., Gonzalez, L. M.
• Format: Article
• Language: English
• Subjects: 3D culture; Animals; Budding; Cell culture; Cell Culture Techniques - veterinary; Cell Differentiation; Cryopreservation; Drug efficacy; enteroid; Epithelium; Female; Freeze-thawing; Growth factors; horse; Horses; Immunofluorescence; Intestinal Mucosa - cytology; Intestine; Male; Models, Biological; organoid; Polymerase chain reaction; stem cell; Stem cells; Stem Cells - physiology; Tissues; Viability
• ISSN: 0425-1644; 2042-3306
• DOI: 10.1111/evj.12734

Summary Background Intestinal epithelial stem cells are critical to epithelial repair following gastrointestinal injury. 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. 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. Objectives To describe the isolation and culture of small intestinal stem cells into three‐dimensional (3D) enteroids in horses without clinical gastrointestinal abnormalities. Study design Descriptive study. Methods Intestinal samples were collected by sharp dissection immediately after euthanasia. Intestinal crypts containing intestinal stem cells were dissociated from the underlying tissue layers, plated in a 3D matrix and supplemented with growth factors. 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 were cryopreserved immediately following collection and viability assessed. Results Intestinal crypts were successfully cultured and matured into 3D enteroids containing a lumen and budding structures. 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. Previously frozen crypts were successfully cultured following a freeze‐thaw cycle. Main limitations Tissues were all derived from normal horses. Application of this technique for the study of specific disease was not performed at this time. Conclusions The successful culture of equine intestinal crypts into 3D “mini‐guts” allows for in vitro studies of the equine intestine. Additionally, these results have relevance to future development of novel therapies that harness the regenerative potential of equine intestine in horses with gastrointestinal disease (colic).