Culturing and Differentiating Human Intestinal Epithelial Cells

The African green monkey kidney cell line, Vero cells (ATCC®CCL-81™), were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque, Kyoto, Japan) containing 5% fetal bovine serum (FBS; Thermo Fisher Scientific, MA, USA). Vero cells expressing TMPRSS2, VeroE6/TMPRSS2 cells (VeroE6/TMPRSS2, JCRB number; JCRB1819), were maintained in DMEM containing 5% FBS and 1 mg/mL G418 disulfate aqueous solution (Nacalai Tesque) at 37 ℃. IEC#17 cells were differentiated from a human iPSC line, TkDN4-M, as described previously^{33 (link)}. IEC#20, IEC#25, and IEC#29 were also differentiated from the human iPSC lines, 1231A3, 1383D6^{36 (link)}, and TkPP7^{37 (link)}, respectively. To prepare monolayers, IECs were cultured in a multiwell plate at a density of 6.3 × 10⁵/cm² in a culture medium for 2 days. For differentiation, cells were cultured in a differentiation medium for 4 days (for details, refer to Ref.^{38 (link)}). After 6 days, an average of 1 × 10⁵ differentiated IECs were seeded in 96-well plates.