# Modeling Epithelial Homeostasis and Perturbation in Three-Dimensional Human Esophageal Organoids
## 2 Materials and Methods  
### Cell Culture and 3D Organoid Culture  
All the cell culture reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA) unless otherwise noted. The live cells were determined by Coutness(tm) Automated Cell Counter following exclusion of Trypan Blue dye-stained dead cells. Immortalized normal human esophageal cell lines EPC1 and EPC2 were grown in a monolayer culture in Keratinocyte SFM (KSFM) and utilized to generate 3D organoids as described previously [22; 31; 32]. For both EPC1 and EPC2, we have validated the mycoplasma negative status by the Universal Mycoplasma Detection kit (ATCC, Manassas, VA, USA), and the cell authentication by short tandem repeat profiling (ATCC). Three independent patient-derived organoid (PDO) lines EN1-3 were established from esophageal normal mucosa biopsies that were obtained via upper endoscopy from an adult patient (EN1) at Columbia University Irving Medical Center (CUIMC) and pediatric \begin{table}
\begin{tabular}{l l} \hline \hline
**Abbreviation** & **Name** \\ \hline ADF & advanced Dulbecco’s Modified Eagle Medium/Ham’s F-12 \\ ADF3+ & advanced DMEM +/+/+ \\ CM & conditioned medium \\ CM-NR & CM containing Noggin and R\(\cdot\)Spondin1 \\ EGF & epidermal growth factor \\ FGF2 & fibroblast growth factor 2 \\ FGF10 & fibroblast growth factor 10 \\ HB-EGF & heparin-binding EGF-like growth factor \\ HOME & human organoid medium containing EGF at indicated concentrations \\ HOME0 & human organoid medium containing 0 ng/mL EGF \\ HOME0.1 & human organoid medium containing 0.1 ng/mL EGF \\ HOME1 & human organoid medium containing 1 ng/mL EGF \\ HOME10 & human organoid medium containing 10 ng/mL EGF \\ HOME0 \({}^{\text{AA}}\) & HOME0 devoid of A83-01 \\ KSFM & keratinocyte serum-free medium \\ KSFMC & KSFM medium containing 0.6 mM Ca2+ \\ MOM & murine organoid medium \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Abbreviations of cell culture media and related growth factors. patients (EN2 and EN3) at the Children's Hospital of Philadelphia (CHOP). All the clinical materials were procured from informed-consented patients according to the Institutional Review Board standard and the guidelines of each respective institution. The biopsies at CUIMC were immediately subjected to tissue dissociation to initiate organoid formation on the day of the tissue procurement. The biopsies from patients EN2 and EN3 were frozen in a cryogenic vial containing 90% fetal calf serum (FBS) and 10% Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) and shipped on dry ice to the Organoid and Cell Culture Core (OCCC) facility at the CUIMC and stored in liquid nitrogen until use. To generate the 3D organoids, an esophageal biopsy was dissociated into a single-cell suspension as described [32]. The tissue-derived cells or cultured cells (EPC1 and EPC2) were seeded into 24-well plates in 50 \(\upmu\)L Matrigel at 5000 cells per well, and grown for 11 days with 500 \(\upmu\)L (per well) of advanced DMEM/F12 (ADF)-based medium containing 1X GlutaMAX, 1X HEPES, 1X N2 Supplement, 1X B27 Supplement, 1 mM \(N\)-acetyl-L-cysteine (Sigma-Aldrich, St. Louis, MO, USA), 2.0% Noggin/R-spondin-conditioned media, 10 \(\upmu\)M Y-27632 (Selleck Chemicals, Houston, TX, USA), 5 \(\upmu\)M A83-01 without EGF (HOME0) or with 0.1-50 ng/mL human recombinant EGF (Perrotech, Cranbury, NJ, USA) (HOME0.1, HOME1, or HOME50) (Table 3) unless otherwise noted. To monitor organoid growth in each well, phase-contrast images were captured by EVOS FL Cell Imaging System (Thermo Fisher scientific) and bright-field images were captured by KEYENCE Fluorescence Microscope BZ-X800 (Keyence, Osaka, Japan) equipped with the Keyence software (version 1.3.0); the latter was utilized to determine the number and size of the individual organoids (defined as \(\geq\)5000 \(\upmu\)m\({}^{2}\) spherical structures). Organoid formation rate (OFR) is calculated as the number of organoids formed per number of cells seeded and was expressed as a percentage (%) as described [32].