Immunoprecipitation

Samples containing 5 g organoid protein extracts were incubated for 3 h at 4°C on the shaking platform with 1 L rabbit polyclonal anti-SOX-9 (PAB12736; Abnova, Taipei City, Taiwan). Afterwards, 4 L Protein A/G Plus Agarose (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were added to each sample, and they were incubated overnight at 4°C, with gentle agitation. Complexes were washed three times with radioimmunoprecipitation buffer as described elsewhere (9). Subsequently, 10 L Western blot analysis sample buffer were added to each pellet, the samples were denatured for 5 min at 98°C, separated by 10% SDS-PAGE, transferred to PVDF membrane, and subjected to the regular Western blot analysis procedure [blocking in 5% fat-free milk in PBS-T for 2 h, incubating in the first antibody against SOX-9 diluted 1:3000 and with goat polyclonal anti rabbit IgG HRP-conjugated antibody diluted 1:20,000 (Dako Cytomation) for 2 h at room temperature]. To visualize bands, a West Pico chemiluminescent kit (Thermo Scientific) was used.

RNA isolation and RT-PCR

Organoids released from Matrigel were lysed with TRI-reagent (Sigma-Aldrich), and total RNA was isolated according to the manufacturer's instruction. One microgram RNA was transcribed into cDNA using a Transcriptor Reverse Transcriptase kit (Roche, Indianapolis, IN, USA) with oligo(dT) primers. PCR was performed with specific primers for chicken cdxA (forward 5'-TAGGTTGCCCAG-AGGGGCCG-3', reverse 5'-CTCCTG TGTCCCAGCACGCC-3') and cdxB (forward 5'-AACAAGTTCCC-TGTTCCCACCAC-3', reverse 5'-GCAGCAGCACG-AACTCCCTGA-3') genes.

Enzymatic assay

Sucrase-isomaltase is a brush border enzyme typical of differentiation state. Its sucrose activity was measured in 1-week-old organoids according to Messer and Dahlqvist (10). The organoids released from Matrigel with Matrisperse were centrifuged (600× g for 5 min) and resuspended in 200 L modified Tyrode solution containing 5 mM sucrose instead of glucose and sonicated for 30 s on ice. After incubation for 3 h in 37°C, the samples were centrifuged (2000× g for 5 min), and the amount of glucose produced by cells was measured in the supernatants by using glucose oxidase (GOX from Aspergillus niger, G6125; Sigma-Aldrich), peroxidase, and orto-dianisidine (11). The enzyme activity is expressed as milliunits per milligram (mU/mg) of protein. One unit is defined as the activity that hydrolyzes 1 mol substrate/min at 37°C.

Results and discussion

Until now, a method for culturing intestinal epithelial cells as organoids has not been used for nonmammalian cells. In avian embryo and adult birds, proliferation of intestinal epithelial cells is not restricted to the crypt bottom (12). Therefore, we tested the methodology of organoid culture applied previously to adult mouse intestine for embryonic chicken tissue expecting to obtain a substantial amount of differentiated cells. We observed the growth of large organoids during the first week of culture in medium containing EGF, R-spondin 1, and Noggin (Figure 1).

Figure 1. Formation of intestinal organoids in Matrigel matrix. (Click to enlarge)

Time-lapse video recording enabled us to track the behavior of embryo-derived epithelium in culture. From this observation, we conclude that the chicken organoid growth pattern is different from mouse. During the first 6 h, the planar epithelial fragments closed into 3-D structures and formed crypt-like protrusions. Next, they evolved into regular spheres with expanding lumen, enclosed by a thin layer of cells. The organoid lumen appears to be more alkaline than the surrounding medium as determined by the reddish color of the media. The difference in pH between the inside and outside of the spheres indicates that they are covered by a nonleaking layer of tightly bound differentiated cells. Some of the spheres enlarge and fuse with neighboring organoids or change shape to move inside the Matrigel matrix. Organoids remained viable for several weeks, but formation of crypt-like structures was seldom observed at later stages of culture. They were also hardly visible on the histological section (Figure 1, Figure 2, C and D, and Supplementary Video S1).

Figure 2. Organoid appearance on day 4 of culture in Matrigel. (Click to enlarge)

Various culture conditions showed that some small spheres were also formed in serum-free medium supplemented only with EGF, but they were obviously less numerous and did not reach the same size as in the Wnt-agonist– and BMP-inhibitor–supplemented medium. The presence of fetal calf serum (FCS), even at 1% concentration, substantially slowed down the process of organoid formation, regardless of R-spondin 1 and Noggin presence (Figure 3). In the medium supplemented only with EGF, spheroids deteriorated when kept in Matrigel longer than 1 week (data not shown).