A novel culture system for adult porcine intestinal crypts

• Published in: Cell and tissue research Vol. 365; no. 1; pp. 123 - 134
• Main Authors: Khalil, Hassan A., Lei, Nan Ye, Brinkley, Garrett, Scott, Andrew, Wang, Jiafang, Kar, Upendra K., Jabaji, Ziyad B., Lewis, Michael, Martín, Martín G., Dunn, James C. Y., Stelzner, Matthias G.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01-07-2016; Springer; Springer Nature B.V
• Subjects: Aging - physiology; Analysis; Animals; Biomedical and Life Sciences; Biomedicine; Cell differentiation; Cryopreservation; Culture Media, Conditioned - pharmacology; Digestive system; Epidermal growth factors; Genetic engineering; Glycogen; Glycogen synthesis; Health aspects; Hogs; Human Genetics; Immunohistochemistry; Intestinal Mucosa - metabolism; Intestines - cytology; Lentivirus; Mice; Molecular Medicine; Myofibroblasts - cytology; Myofibroblasts - drug effects; Proteomics; Regular Article; Stem cell transplantation; Stem cells; Sus scrofa; Temperature; Tissue Culture Techniques - methods; Transduction, Genetic; Viral genetics; Transduction of intestinal epithelium; Intestinal subepithelial myofibroblast; Porcine intestinal culture; Enteroid; Intestinal spheroid
• ISSN: 0302-766X; 1432-0878
• DOI: 10.1007/s00441-016-2367-0

Porcine models are useful for investigating therapeutic approaches to short bowel syndrome and potentially to intestinal stem cell (ISC) transplantation. Whereas techniques for the culture and genetic manipulation of ISCs from mice and humans are well established, similar methods for porcine stem cells have not been reported. Jejunal crypts were isolated from murine, human, and juvenile and adult porcine small intestine, suspended in Matrigel, and co-cultured with syngeneic intestinal subepithelial myofibroblasts (ISEMFs) or cultured without feeder cells in various culture media. Media containing epidermal growth factor, noggin, and R-spondin 1 (ENR medium) were supplemented with various combinations of Wnt3a- or ISEMF-conditioned medium (CM) and with glycogen synthase kinase 3 inhibitor (GSK3i), and their effects were studied on cultured crypts. Cell lineage differentiation was assessed by immunohistochemistry and quantitative polymerase chain reaction. Cultured porcine cells were serially passaged and transduced with a lentiviral vector. Whereas ENR medium supported murine enteroid growth, it did not sustain porcine crypts beyond 5 days. Supplementation of Wnt3a-CM and GSK3i resulted in the formation of complex porcine enteroids with budding extensions. These enteroids contained a mixture of stem and differentiated cells and were successfully passaged in the presence of GSK3i. Crypts grown in media supplemented with porcine ISEMF-CM formed spheroids that were less well differentiated than enteroids. Enteroids and spheroids were transfected with a lentivirus with high efficiency. Thus, our method maintains juvenile and adult porcine crypt cells long-term in culture. Porcine enteroids and spheroids can be successfully passaged and transduced by using lentiviral vectors.