2Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland
3Department of Medical Physiology, Faculty of Health Sciences, Collegium Medicum Jagiellonian University, Kraków, Poland
Investigating intestinal physiology in vitro remains challenging due to the lack of an effective primary enterocyte culture system. Recently developed protocols for growing organoids containing crypts and villus from adult mouse intestinal epithelium in Matrigel present an attractive alternative to the classical techniques. However, these approaches require the use of sophisticated and expensive serum-free medium supplemented with epithelial growth factor (EGF), Wnt agonist (R-spondin 1), and bone morphogenetic protein inhibitor (Noggin) in high concentrations. Here we demonstrate that is possible to use an isolated chicken embryonic intestinal epithelium to create such an organoid culture. Structures formed in Matrigel matrix in the first two days following isolation survive and enlarge during ensuing weeks. They have the appearance of empty spheres and comprise cells expressing cytokeratin (an epithelial cell marker), villin (a marker of enterocytes), and Sox-9 (a transcription factor characteristic of progenitors and stem cells of intestinal crypts). With chicken embryonic tissue as a source of organoids, prostaglandin E2 is as effective as R-spondin 1 and Noggin in promoting sustained growth and survival of epithelial spheroids.
Intestinal epithelium is one of the most rapidly renewing tissues in vertebrate organisms. Intestinal stem cells located at the bottom of the crypts have a profound potential for proliferation. Nevertheless, establishing primary intestinal epithelial cell cultures using standard techniques has met with substantial difficulties, regardless of tissue source (fetal or adult, small or large intestine) and the species of experimental animal used (1, 2). The lack of a proper in vitro model system has hampered both the fundamental analysis of gut biology and applied research of intestinal tract physiology.
During the last few years, however, the accumulation of knowledge on crypt stem cell behavior has led to significant progress in the field of intestinal cell culture. It was reported that murine intestinal crypt stem cells immersed in extracellular matrix formed organoids in the presence of Paneth cells producing WNT3 (3, 4). The maintenance of self-organizing crypt-villus structures in culture requires a medium supplemented with epithelial growth factor (EGF; 50 ng/mL), Wnt agonist (R-spondin 1; at least 500 ng/mL), and bone morphogenetic protein (BMP) inhibitor (Noggin; 100 ng/mL) (3, 4).
Relatively high concentrations of R-spondin 1 were essential for supporting the Wnt signaling pathway in proliferating cells, a crucial event for the growth and survival of organoids. Noggin is the BMP antagonist produced in vivo at the crypt bottom. It inhibits mesenchyme generated BMP signals that limit stem cell proliferation through modulation of Wnt activity during the process of cell differentiation along the crypt-microvillus axis (5).
A similar approach was used by Ootani et al. who successfully established long-term cultures of intestinal spheres within collagen gel at the air-liquid interface (6). The possibility of getting intestinal tissue by direct differentiation of human pluripotent stem cells using R-spondin 1 containing medium and Matrigel matrix was also recently reported (7).
It is now clear that the proliferation of mammalian intestinal epithelial cells is only possible in a three-dimensional (3-D) environment, mimicking the situation encountered by stem cells and progenitors in the intestinal crypt niche. As mentioned above, the second condition required to culture this tissue in vitro is the presence of WNT ligand and R-spondin 1. Using nearly micromolar concentration of R-spondin 1 substantially increases the costs and limits the potential applications for long-term organoid cultures. Here, we report the growth of organoids derived from embryonic chicken intestine in Matrigel. We also demonstrate that prostaglandin E2 (PGE2) provides a cost-effective alternative to R-spondin 1 and Noggin treatment, as the medium containing PGE2 supports the growth of embryonic chicken epithelial cell organoids in Matrigel as efficiently as protein Wnt pathway agonists.Materials and methods Tissue isolation
Epithelial tissue was obtained from embryonic chicken intestine using the method developed by Barker et al. This method was originally used to isolate crypts and villi fractions of adult mouse intestine, and we adapted it for chicken tissue (8). Embryonated eggs from Ross 308 hens were obtained from a local professional hatchery (Krak-Drób, Ściejowice near Kraków, Poland). For each isolation, material was pooled from four chicken embryos. Small intestines were isolated from 18-day-old embryos immediately following decapitation and washed in a Petri dish filled with ice-cold phosphate buffered saline containing Mg2+ and Ca2+ [PBS (Ca2+/Mg2+)]. Then the intestine was separated from loose mesenchymal tissue and visible blood vessels, transferred to a new dish with cold PBS (Ca2+/Mg2+), cut into approximately 2-cm-long fragments, and dissected longitudinally with surgical forceps. Open pieces of the intestine were moved to a precooled 15-mL tube filled with cold PBS lacking Mg2+ and Ca2+, but containing 2.5 mM EGTA and 0.5% glucose (PBS-EGTA-Glu), and incubated with shaking at 4°C for 15 min. The tube was then shaken vigorously 10 times, allowed to stand for 2 min on ice, and the supernatant decanted and discarded. The fresh portion of PBS-EGTA-Glu (10 mL) was added, and the tissue was again incubated with shaking for 45 min from the initial transferring into PBS-EGTA-Glu. At that moment, the shaking step was repeated, the tissue placed back on the shaker with a third portion of PBS-EGTA-Glu, and incubated for the next 45 min.