Human specimens and organoid lines
Two terminal ileum tissues (donors H0514 and H0515-2) were obtained for immunofluorescent staining and flow cytometry analysis from Diakonessen Hospital Utrecht, with approval by the Medical Ethical Committee of the hospital and with informed consent of each patient.
The PBMCs from patients with CeD were collected from participants of the CeDNN study. CeDNN was approved by the Medical Ethical Committee of the University Medical Center Groningen, with METc no. 2013/440. Written consent was signed by all participants, their parents or legal representatives for participants aged under 16 for CeDNN. Normal PBMCs were purchased from Sanquin, with a signed material transfer agreement.
A total of five human intestinal organoid lines were established in our laboratory and recruited to this study. Three ileum tissues (donor 12339, 4280N/HLA-DQ2.5 #1 and 4403N/HLA-DQ2.5 #2) were obtained from the Netherlands Cancer Institute (NKI); one ileum tissue (donor N39) and one colon tissue (donor P11N) were obtained from the Diakonessen Hospital Utrecht, all with approval by the Medical Ethical Committee of the respective organizations and with informed consent of each patient.
This study was approved by the Ethical Committee of Hubrecht Institute and was in accordance with the Declaration of Helsinki and according to the Dutch law. The study complied with all relevant guidelines and regulations regarding research involving human participants.
Mice
Wild-type C57BL/6 mice were used in this study. Both male (n = 2) and female (n = 8) mice (more than 8 weeks of age) were included. No specific sample size was chosen, and neither randomization nor blinding were necessary, as no comparison was made. No specific ethical guidance was required. In this study, we only collected the intestinal Peyer’s patch tissues from euthanized mice for flow cytometry analysis and immunofluorescent staining, which was approved by the Institutional Review Board of Hubrecht Institute.
Organoid culture and M cell differentiation
Human intestinal organoid culture
Three-dimensional cultured human intestinal organoids were mixed with BME (R&D Systems, 3536-005-02), seeded as ~5 µl droplets, cultured in optimized culture of human intestinal organoids (OCHIO) medium and passaged weekly through mechanical dissociation as per the method previously described14.
... continue reading