title: A novel culture system for adult porcine intestinal crypts.
abstract: 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.
authors.affiliation: Department of Surgery, UCLA David Geffen School of Medicine, Los Angeles, CA, USA.
authors.forename: Hassan A
authors.initials: HA
authors.lastname: Khalil
authors.affiliation: Department of Surgery, UCLA David Geffen School of Medicine, Los Angeles, CA, USA.
authors.forename: Nan Ye
authors.initials: NY
authors.lastname: Lei
authors.affiliation: Division of Gastroenterology and Nutrition, Department of Pediatrics, Mattel Children's Hospital and the David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA, USA.
authors.forename: Garrett
authors.initials: G
authors.lastname: Brinkley
authors.affiliation: Department of Surgery, UCLA David Geffen School of Medicine, Los Angeles, CA, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Scott
authors.affiliation: Division of Gastroenterology and Nutrition, Department of Pediatrics, Mattel Children's Hospital and the David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA, USA.
authors.forename: Jiafang
authors.initials: J
authors.lastname: Wang
authors.affiliation: Division of Gastroenterology and Nutrition, Department of Pediatrics, Mattel Children's Hospital and the David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA, USA.
authors.forename: Upendra K
authors.initials: UK
authors.lastname: Kar
authors.affiliation: Department of Surgery, UCLA David Geffen School of Medicine, Los Angeles, CA, USA.
authors.forename: Ziyad B
authors.initials: ZB
authors.lastname: Jabaji
authors.affiliation: Department of Pathology & Laboratory Medicine, VA Greater Los Angeles Health System, Los Angeles, CA, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Lewis
authors.affiliation: Division of Gastroenterology and Nutrition, Department of Pediatrics, Mattel Children's Hospital and the David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA, USA.
authors.forename: Martín G
authors.initials: MG
authors.lastname: Martín
authors.affiliation: Department of Surgery, UCLA David Geffen School of Medicine, Los Angeles, CA, USA.
authors.forename: James C Y
authors.initials: JC
authors.lastname: Dunn
authors.affiliation: Department of Surgery, UCLA David Geffen School of Medicine, Los Angeles, CA, USA. stelzner@ucla.edu.|Department of Surgery, VA Greater Los Angeles Health System, Los Angeles, CA, USA. stelzner@ucla.edu.
authors.forename: Matthias G
authors.initials: MG
authors.lastname: Stelzner
mesh_terms: D000375:Aging / Q000502:physiology*; D000818:Animals; D015925:Cryopreservation; D017077:Culture Media, Conditioned / Q000494:pharmacology; D007150:Immunohistochemistry; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000166:cytology*; D051379:Mice; D058628:Myofibroblasts / Q000166:cytology / Q000187:drug effects; D034421:Sus scrofa; D013696:Temperature; D046509:Tissue Culture Techniques / Q000379:methods*; D014161:Transduction, Genetic
chemical_list: D017077:Culture Media, Conditioned
keywords: Enteroid; Intestinal spheroid; Intestinal subepithelial myofibroblast; Porcine intestinal culture; Transduction of intestinal epithelium