Journal Article

A novel culture system for adult porcine intestinal crypts

Jabaji, Ziyad B., Khalil, Hassan A., Scott, Andrew, Dunn, James C. Y., Lewis, Michael, Stelzner, Matthias G., Lei, Nan Ye, Brinkley, Garrett, Martín, Martín G., Kar, Upendra K., Wang, Jiafang

2016

Overview

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.

• Publisher: Springer Berlin Heidelberg,Springer,Springer Nature B.V
• Subject: adults / Aging - physiology / Analysis / Animals / Biomedical and Life Sciences / Biomedicine / Cell differentiation / coculture / Cryopreservation / culture media / Culture Media, Conditioned - pharmacology / Digestive system / digestive system diseases / epidermal growth factor / Epidermal growth factors / Genetic engineering / Glycogen / Glycogen synthesis / Health aspects / Hogs / Human Genetics / humans / Immunohistochemistry / intestinal crypts / Intestinal Mucosa - metabolism / Intestines - cytology / jejunum / juveniles / Lentivirus / Mice / Molecular Medicine / Myofibroblasts - cytology / Myofibroblasts - drug effects / Proteomics / quantitative polymerase chain reaction / Regular Article / Stem cell transplantation / Stem cells / Sus scrofa / swine / tau-protein kinase / Temperature / Tissue Culture Techniques - methods / Transduction, Genetic / Viral genetics