# Modeling Epithelial Homeostasis and Perturbation in Three-Dimensional Human Esophageal Organoids
## 3 Results  
### Exogenous EGF Is Dispensable for Normal Human Esophageal Organoid Growth in ADF-Based Medium  
ADF-based media are commonly utilized to grow 3D organoids representing a variety of epithelial types and conditions [30; 35; 36; 37]; however, previously reported ADF-based formulations are not particularly efficient for growing and maintaining normal human esophageal 3D organoids [22]. Given human serum EGF concentration estimated at 40 pM (i.e., \(\sim\)0.25 ng/mL) [38], most ADF-based media contain supraphysiological levels (50 ng/mL) of recombinant EGF [30; 35; 36; 37]. Excessive mitogenic stimuli may trigger cell cycle arrest or senescence in human esophageal epithelial cells [39]. We first tested how varying concentrations of recombinant EGF added to ADF-based medium, designated as human organoid medium with or without EGF (HOME) (Table 3) may influence organoid formation with extensively characterized immortalized normal human esophageal cell lines EPC1 and EPC2. Additionally, we included ADF++ (hereafter ADF3+) [23; 40] (Table 3) for a comparison, as ADF3+ was utilized to grow organoids representing human normal oral mucosa, sharing the non-cornifying stratified squamous epithelium. Likewise, we have included KSFMC [22] (Table 3) that was previously utilized to generate human normal esophageal organoids from EPC1 and EPC2 as well as endoscopic biopsies. As visualized under bright-field microscopy and expressed as organoid formation rate (OFR), organoid formation appeared to be significantly better in HOME0 and HOME0.1 compared with HOME1 or HOME50 for both cell lines (Figure 1A-C). Likewise, EPC1 and EPC2 formed organoids poorly in ADF-based medium referred to as advanced DMEM+/+/+ (herein ADF3+) containing 50 ng/mL EGF [40] (Figure 1A-C, Table 3) that was utilized to generate 3D organoids from human oral mucosa consisting of non-cornifying stratified squamous epithelium. Moreover, OFR in HOME0 and HOME0.1 was comparable with that in KSFMC (Figure 1A-C, Table 3). Figure 1: Low EGF concentrations (\(\leq\)0.1 ng/mL) may facilitate organoid formation in ADF-base medium. EPC1 and EPC2 cells were seeded at a density of 5000 cells per 50 \(\upmu\)L Matrigel and fed with KSFMC, HOME, or ADF3+, containing human recombinant EGF at indicated final concentrations and allowed to form organoids. (**A**) Resulting structures were captured by Keyence and photomicro-graphed. Scale bar, 200 \(\upmu\)m. (**B,C**) OFR was determined and plotted in graphs. \({}^{\star}\), \(p<0.05\) vs. 0 ng/mL EGF in HOME (i.e., HOME0); NS, not significant vs. HOME0, n = 3 per group. One-way ANOVA was used for the overall comparison. When the resulting organoids were subjected to morphological analysis by hematoxylin-eosin (H&E) staining, organoids grown in KSFMC, HOME0, or HOME0.1 displayed a normal differentiation gradient (Figure 2). By contrast, differentiation was impaired within structures grown in HOME50 or ADF3+, where degenerative changes or atypical basaloid cells were noted (Figure 2), suggesting that 50 ng/mL EGF may be excessive for normal human esophageal organoids to grow and display a proper squamous cell differentiation. Both EPC1 and EPC2 organoids grown in HOME0 or HOME0.1 had limited cornification that is more compatible with non-cornifying squamous epithelium of the esophagus than those grown in KSFMC (Figure 2). Since endogenous EGF or other EGFR ligands may stimulate organoid cell proliferation or survival in the absence of exogenous recombinant human EGF, we tested the effect of a pharmacological EGFR tyrosine kinase inhibitor AG-1478 upon EPC1 and EPC2 organoid formation in HOME0. Interestingly, AG-1478 decreased OFR as well as cell viability in a dose-dependent manner (Figures 3A,B and S1A), suggesting that organoid formation requires the EGFR activity, which may be activated by EGFR ligands expressed by organoid cells in culture or present in the medium components (e.g., CM-NR; Figure S1B).