GoKawiilAnimals and cell lines
Animal work
Mouse (Mus musculus) strains C57BL/6J (strain no. 000664) and Cdx2:CreERT2;APCfl/fl;KrasWT (strain no. 035169)50 were obtained from The Jackson Laboratory. Mice were housed at room temperature and ambient humidity in individually ventilated cages at a maximum density of five mice per cage with ad libitum access to food and water in a specific-pathogen-free facility accredited by the Association and Accreditation of Laboratory Animal Committee. Cages contained Anderson’s Bed-o’Cob bedding (The Anderson Inc.), one nestlet (Ancare, 2 × 2-inch2 compressed cotton squares) and a red mouse hut (Bioserv). The colony room was kept on a 12 h–12 h light–dark cycle. All animal handling and experiments were conducted in accordance with procedures approved by the Institutional Animal Care and Use Committee at Harvard University (protocol no. 19-10-362). For tumour formation experiments, euthanasia criteria were weight loss of more than 20%, persistent grossly bloody stool for greater than or equal to 3 days and/or excessively lethargic or moribund state, as determined by veterinary care. These criteria were not exceeded in any experiments.
Cell culture
Human embryonic kidney 293T (HEK293T) cells (American Type Culture Collection (ATCC), CRL-3216; authenticated by short tandem repeat profiling and tested for mycoplasma by ATCC) were grown in DMEM (Thermo, 11965-092) with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. Cells were incubated at 37 °C in 5% CO 2 and maintained in exponential phase.
Mouse organoid derivation and culture
Colitis organoids were derived from whole colonic tissue 11 days following cessation of the third cycle of DSS. Animals were anaesthetized with 2,2,2-tribromoethanol (Sigma, T48402-25G) and cardiac perfusion was performed with PBS to remove peripheral immune cells. Epithelium was removed by incubating colonic tissue in EDTA solution (section ‘Colon tissue processing and cell sorting’ below) supplemented with 100 μg ml−1 primocin (Invitrogen, ant-pm-05) for 20–30 min and scraping the luminal surface with a glass slide. Epithelial fragments were washed once with Advanced DMEM/F12 (ADMEM) and resuspended in Crypt Basal (ADMEM, 10 mM HEPES, 1× GlutaMax (Thermo, 35050061), 1× Pen-Strep (Thermo, 15140122), 1× N2 Supplement (Thermo, 17502048), 1× B27 Supplement (Thermo, 17504044), 1 mM N-acetylcysteine (Sigma, A9165-5G)) before mixing with an equal volume of Matrigel (Corning, 47743-722). Crypts were plated as roughly 30-l domes in a six-well plate and allowed 10–15 min to polymerize. Colon organoids were grown in WENR media: 50% ENR (Crypt Basal with 50 ng ml−1 epidermal growth factor (EGF) (Thermo, PMG8041), 100 ng ml−1 Noggin (Peprotech, 250-38), 1:100 Rspondin conditioned media) and 50% Wnt conditioned media. Conditioned media was generated in-house from L WNT3A cells (ATCC, CRL 2647) or HA-R-Spondin1-Fc 293T Cells (R&D, 3710-001-01). Organoids were passaged every 7–10 days by mild dissociation in TrypLE for 8–10 min, triturating every 4–5 min and quenching with 10% FBS in ADMEM. When collected for SHARE-seq, organoids were dissociated to near single cell for 15 min in TrypLE and quenched before treating with 1:100 recombinant DNase (Roche, 04716728001) in ADMEM at room temperature for 5 min to reduce dead cell DNA contamination. Cells were then washed and frozen in CryoStor at −80 °C before SHARE-seq.
Human organoid derivation and culture
Human organoid lines were derived from de-identified biopsies from grossly unaffected tissue in patients undergoing endoscopy at Boston Children’s Hospital. Informed consent and developmentally appropriate assent were obtained at Boston Children’s Hospital from the donors’ guardian and the donor, respectively. All methods were approved and carried out in accordance with the Institutional Review Board of Boston Children’s Hospital (Protocol number IRB-P00000529).
Organoids were derived from biopsies as previously described in ref. 58. Briefly, intestinal crypts were isolated from frozen tissue and then resuspended and plated in 40-μl Matrigel domes. Once established, human rectal organoids were sustained in specialized growth media that has been previously described58. Media changes occurred every 2 days during expansion, with organoids being passaged once every 6–8 days as necessary. To induce differentiation, organoids were grown in growth media for 2 days postpassage to allow for stem cell expansion; after which, the organoids were transitioned to differentiation media. Media was changed every 2 days for the length of the experiment, with organoids being collected for analysis after a total of 10 days.
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