Intestinal Stem Cell Organoid Culture

Isolated crypts or single cells were cultured as previously described^{2 (link)} with minimal modification. Briefly, crypts or single cells were entrapped in Matrigel and plated at the center of wells in a 24-well plate. Following polymerization of Matrigel (growth factor reduced; BD Bioscience), 500 μl of culture medium (Advanced DMEM/F12 (Life Technologies)) was added, containing growth factors including EGF (50 ng/ml, Life Technologies), Noggin (100 ng/ml, PeproTech) and R-spondin 1 (500 ng/ml, R&D) and small molecules including CHIR99021 (3 μM, Stemgent) and valproic acid (1 mM, Sigma-Aldrich). (See Supplementary Table 1 for details.) For comparison of different culture conditions, small molecules or growth factors were added to freshly isolated crypts immediately after plating in Matrigel to test the ability to minimize potential differentiation of the ISCs within the crypts and thus sustain crypt cultures. Cell culture medium was changed every other day. For single-cell culture, cells were embedded in Matrigel containing Jagged-1 peptide (1 μM; AnaSpec), and Y-27632 (10 μM; Tocris) was added for the first 2 d. Cells were passaged either as cell colonies as previously described^{2 (link)} or as single cells. For single-cell passage, cell culture medium was removed and Accutase (Life Technologies) was added. After incubation at 37 °C for 10–20 min, cell colonies were dissociated into single cells by pipetting. Cells were then washed, embedded in fresh Matrigel and plated into 24-well plates. Cells cultured in the CV condition were passaged every 6 d at a 1:20 split ratio.