A novel culture system for adult porcine intestinal crypts

Abstract

Background Porcine models are useful for investigating therapeutic approaches to short bowel syndrome and potentially to intestinal stem cell (ISC) transplantation. While 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. Methods 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 glycogen synthase kinase 3 inhibitor (GSK3i) and studied on cultured crypts. Cell lineage differentiation was assessed by immunohistochemistry (IHC) and quantitative PCR (qPCR). Cultured porcine cells were serially passaged and transduced with a lentiviral vector. Results 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. Discussion We describe a method to maintain juvenile and adult porcine crypt cells long-term in culture. Porcine enteroids and spheroids can be successfully passaged and transduced using lentiviral vectors.

Wnt/β‐catenin‐mediated heat exposure inhibits intestinal epithelial cell proliferation and stem cell expansion through endoplasmic reticulum stress

“…Of note, a proper increase of temperatures, such as 39°C or 40°C, can improve the viability of porcine intestinal epithelial cells, which might be related to the relatively high resting physiologic body temperature of pigs. Khalil et al (2016) further found that there was no difference in the expression of differentiation markers between enteroids cultured at 39°C and that cultured at 37°C. Accordingly, exposure to 41°C induced early‐ and late‐phase intestinal cell apoptosis.…”

Large Animal Models: The Key to Translational Discovery in Digestive Disease Research

“…27 Optimum culture methods to maintain neonatal and adult porcine enteroids for several weeks have been established, and transplantation of intestinal crypts into the omentum of pigs has resulted in the growth of histologically complete intestine including villi, crypts, lamina propria, nerve elements, and muscularis mucosa. 27,67,68 Methods for cryopreservation and genetic manipulation of porcine enteroids also have been successful, further establishing the porcine enteroid model as a powerful tool for intestinal studies and preclinical assessment. 68 …”

“…27,67,68 Methods for cryopreservation and genetic manipulation of porcine enteroids also have been successful, further establishing the porcine enteroid model as a powerful tool for intestinal studies and preclinical assessment. 68 …”

Application of porcine gastrointestinal organoid units as a potential in vitro tool for drug discovery and development

“…Consequently, pigs represent a candidate species to develop 3D organoids as models for drug screening and other mechanistic translational research (Table ). Gonzalez et al established organoids from whole small intestinal crypts harvested from juvenile pigs (Gonzalez, Williamson, Piedrahita, Blikslager, & Magness, ), and more recently Kahlil et al published a method for the long‐term culture of organoids derived from adult porcine jejunal crypts (Khalil et al, ). To date, the culture of porcine colonic organoid units has not been described in the published literature; therefore, we have successfully developed a method for the long‐term expansion of small intestinal, colonic and rectal organoids derived from adult porcine tissue that can be maintained in a proliferative state for an extended period (Figure c–h).…”

“…Significant advances in the study of intestinal stem cell physiology have been made using murine models; however, anatomical and physiological differences between rodents and humans limit their translational capacity. As porcine weight, diet and intestinal anatomy is comparable to that of humans (Kararli, 1995), pigs have been extensively used for preclinical studies of intestinal pathophysiology (reviewed in Ziegler, Gonzalez, & Blikslager, 2016 (Khalil et al, 2016). To date, the culture of porcine colonic organoid units has not been described in the published literature; therefore, we have successfully developed a method for the long-term expansion of small intestinal, colonic and rectal organoids derived from adult porcine tissue that can be maintained in a proliferative state for an extended period (Figure 1c-h).…”