Culturing Human Intestinal Enteroids for COVID-19 Research

Human intestinal enteroid (HIE) J2 and J3 lines, established from jejunal biopsy specimens of secretor-positive adults (5 (link)), were provided from Baylor College of Medicine under a material transfer agreement. The study protocol was approved by the Review Board of the National Institute of Infectious Diseases in Japan. Wnt3a-producing cells were kindly provided by the Baylor College of Medicine. R-spondin- and Noggin-producing cells were kindly provided by Calvin Kuo (Palo Alto, CA) and Gijs van den Brink (University of Amsterdam, Netherlands), respectively. HIEs were grown as multilobular, 3-dimensional (3D) cultures in Matrigel and were maintained in complete medium with growth factors [CMGF(+)] or IntestiCult organoid growth medium (human) (Veritas) as previously described (5 (link), 10 (link), 19 (link)). Monkey kidney cell line MA104 was maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. VeroE6/TMPRSS2 (JCRB1819, VeroE6 cell overexpressing the transmembrane protease, serine 2 [TMPRSS2]) (21 (link)) was purchased from JCRB Cell Bank (Osaka, Japan) and was maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 mg/ml G418 (Nacalai).