Eagle s modified essential medium

Grivet (Chlorocebus aethiops) kidney epithelial Vero (American Type Culture Collection [ATCC]; CCL-81) and Vero E6 (BEI Resources; NR596), Vero E6/TMPRSS2 (JCRB Cell Bank, Japan; JCRB1819), human colorectal adenocarcinoma Caco-2 (ATCC; HTB-37), HEK293T (ATCC; CRL-3216), human hepatocarcinoma HepG2 (ATCC; #HB-8065), human hepatocarcinoma Huh-7 (a gift from NIH/National Institute of Allergy and Infectious Diseases [NIAID]/Rocky Mountain Laboratories, Hamilton, Montana, USA), human lung adenocarcinoma Calu-3 (ATCC; HTB-55), and human lung carcinoma A549 cells (a gift from University of Rochester Medical Center) were maintained at 37°C and 5% CO₂ in DMEM (Life Technologies) containing 10% heat-inactivated FBS. Primary human hepatocytes from an anonymous 41-year-old woman with colorectal cancer metastasized to the liver were obtained from Liver Center Resources, Pittsburgh Liver Research Center, Pittsburgh, Pennsylvania, USA, and maintained at 37°C and 5% CO₂ in Eagle’s modified essential medium (Gibco).

Tumour resection specimens were processed into single-cell suspension by mechanical and enzymatic digestion, mixed with Matrigel^® (Corning, USA) and incubated in 50µl droplets on 24-well plates, as previously described by Sato et al. (38 (link)). Organoid culture media (OCM) Human Intesticult™ (Stemcell Technologies Inc, Canada) containing 0.2% Primocin™ (In vivogen, USA) and passaged once confluent. Isogenic KRAS HCT116 (Horizon Discovery, Cambridge, UK) and C80 (#12022904-1VL, Sigma Aldrich, USA) CRC cell lines were cultured in McCoy’s 5a Medium (Gibco, USA) with 10% fetal bovine serum (FBS, Gibco, USA), Iscove’s modified Dulbecco’s media (Gibco. USA) containing 10% FBS, 2mM L-Glutamine (Gibco, USA) and Eagles modified essential medium (Gibco, USA) with 2mM L-Glutamine containing 1% non-essential amino acids (Sigma-Aldrich, USA), respectively, and one hundred units/ml penicillin and 100 μg/ml streptomycin (Gibco, USA). Cells were passaged when sub-confluent at a split ratio of 1 in 3. All cultures were tested for mycoplasma using EZ-PCR Mycoplasma Test Kit (Biological Industries, Israel).