Three-Dimensional Cell Culture Models to Investigate the Epithelial Barrier in Eosinophilic Esophagitis

Published: May 10, 2024

doi:

¹Department of Biomedicine,University of Basel, ²Department of Gastroenterology and Hepatology,University Digestive Healthcare Center, Clarunis

Summary

Here, a protocol for the culture of human esophageal organoids and air-liquid interface culture is provided. Esophageal organoids’ air-liquid interface culture can be used to study the impact of cytokines on the esophageal epithelial barrier.

Abstract

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and microbes. Maintaining the integrity of the epithelial barrier is critical for preventing infections and avoiding inflammation caused by harmless food-derived antigens. This article provides simplified protocols for generating human esophageal organoids and air-liquid interface cultures from patient biopsies to study the epithelial compartment of the esophagus in the context of tissue homeostasis and disease. These protocols have been significant scientific milestones in the last decade, describing three-dimensional organ-like structures from patient-derived primary cells, organoids, and air-liquid interface cultures. They offer the possibility to investigate the function of specific cytokines, growth factors, and signaling pathways in the esophageal epithelium within a three-dimensional framework while maintaining the phenotypic and genetic properties of the donor. Organoids provide information on tissue microarchitecture by assessing the transcriptome and proteome after cytokine stimulation. In contrast, air-liquid interface cultures allow the assessment of the epithelial barrier integrity through transepithelial resistance (TEER) or macromolecule flux measurements. Combining these organoids and air-liquid interface cultures is a powerful tool to advance research in impaired esophageal epithelial barrier conditions.

Introduction

Esophageal inflammation compromises the epithelial barrier integrity¹^,²^,³^,⁴^,⁵, as observed in eosinophilic esophagitis (EoE), a Th2-dominated chronic inflammatory disease of the esophagus⁶. EoE was first described in the 1990s⁷^,⁸ and is predominantly induced by food antigens⁹^,¹⁰^,¹¹^,¹²^,¹³. The most frequently occurring symptoms of EoE in the adult population are dysphagia and food impaction¹⁴. In children, EoE typically manifests with failure to thrive, food refusal, vomiting, and abdominal pain¹⁵. Genome-wide association studies (GWAS) have identified EoE risk genes involved in epithelial barrier integrity, moving the epithelium into the focus of EoE research¹⁶^,¹⁷^,¹⁸. EoE transcriptomics further revealed that an impaired differentiation process and a reactive basal zone hyperplasia cause the compromised barrier function of the esophageal epithelium³^,⁵^,¹⁹^,²⁰^,²¹^,²². The early understanding of EoE being a Th2-mediated disease⁶ led to the discovery of IL-13 as a driving mediator by disturbing epithelial integrity³^,⁴^,²¹^,²³. Experimental systems allowing the dissection of cytokine-mediated effects on epithelial integrity from intrinsic barrier impairment through genetic predisposition provide the possibility to study the complex interplay between immune cells and the epithelium in EoE. Human esophageal organoids and air-liquid interface (ALI) cultures have been proposed as valuable tools to analyze the consequence of cytokine stimulation on epithelial integrity⁵.

The first protocol for generating adult tissue-specific stem cell (ASC)-derived esophageal organoids was established five years after the first published reports of intestinal organoids in 2009 using intestinal Lgr5⁺ ASCs recapitulating the epithelial compartment of the small intestine²⁴. DeWard et al. pioneered generating organoids from murine esophageal epithelial cells²⁵. In 2018, Kasagi et al. generated human esophageal organoids from the immortalized human esophageal squamous epithelium cell line EPC2-hTERT and primary patient-derived cells²⁶. In the same year, Zhang et al. successfully generated induced pluripotent stem cell (iPSC)-derived esophageal organoids. They delineated the significance of TGFβ and bone morphogenetic protein (BMP) inhibition for esophageal progenitor cell (EPC) development and the crucial role of Notch signaling in the differentiation of the stratified squamous epithelium²⁶^,²⁷. Trisno and colleagues complemented these findings by identifying Sox2 as a Wnt inhibitor that directs the developmental fate towards esophageal differentiation²⁸. The subsequent refinements of protocols, medium composition, and culture conditions increased the organoid formation rate and made subculturing and recovering organoids after cryopreservation possible²⁶^,²⁹^,³⁰^,³¹^,³². Although these organoids are powerful tools for studying tissue architecture and expression of potential target genes after stimulation with cytokines, esophageal organoids will not offer the possibility to measure transepithelial resistance (TEER) or macromolecule flux as direct measures for barrier integrity. As previously described by Sherrill and colleagues²², ALI cultures modeling epithelial differentiation⁴ allow direct assessments of epithelial integrity. Combining patient-derived organoids and ALI cultures is a powerful tool for investigating tissue architecture and epithelial barrier integrity in EoE.

Here are procedures with instructions for isolating viable cells from esophageal biopsies and establishing esophageal organoid and ALI cultures that can further be used to study the effects of cytokines on barrier integrity.

Protocol

The procedures were approved by the ethics committee of Northwest and Central Switzerland (EKNZ; Project-ID 2019-00273). All patients provided written informed consent for the experimental use of biopsies before the endoscopic examination. The reagents and equipment used in the study are listed in the Table of Materials. 1. Cell isolation for patient-derived esophageal organoids NOTE: A list of the medium constituents for culturing hu…

Representative Results

Esophageal organoids will grow from primary cells extracted from patient biopsies according to the instructions of the provided protocol, as documented with an inverted brightfield microscope (Figure 1). Epithelial ASCs start forming cell clusters in a self-organizing manner within the first two days of culture after seeding the isolated cells in the basement membrane extract, serving as a scaffold. The size and number of cell clusters, noticeable with an inverted brightfield microscope, inc…

Discussion

The provided procedures allow the cultivation of patient-derived organoids and ALI cultures with high prospects of success. The organoid protocol has been adapted from the first published protocol reporting the generation of human esophageal organoids²⁶ and from a recently published protocol³². Sherill and colleagues have described the ALI model²². Organoids and ALI culture models assist each other in studying the impact of cytokines and other mediat…

Divulgations

The authors have nothing to disclose.

Acknowledgements

The SNSF grant 310030_219210 to J.H.N. supported the publication of this manuscript without restrictions. Figure 1 has been created with the help of BioRender.com.

Materials

1250 µL Griptip – Filter	Integra	4445
300 µL Griptip – Filter	Integra	4435
70 µM cell strainer	Sarstedt	83.3945.070
Ascorbic Acid	Sigma-Aldrich (Merck)	A4544
Bovine pituitary extract	Gibco (Thermo Fischer Scientific)	3700015
Calcium chloride	Sigma-Aldrich (Merck)	21115
Cell Culture Multiwell Plates CELLSTAR for suspension cultures	Greiner Bio-One	7.657 185
Cultrex Basement Membrane Extract (BME), Type 2, Pathclear	R&D Systems (Bio-Techne)	3532-010-02
Dimethyl sulfoxide (DMSO), >99,5% BioScience Grade	Carl Roth	A994
Dispase I	Corning	354235
Dispase II	Sigma-Aldrich (Merck)	D4693
Dulbeccos Phosphate Buffered Saline (DPBS)	Sigma-Aldrich (Merck)	D8537
EVE Automated Cell Counter	NanoEntek	EVE-MC
EVE Cell counting slide	NanoEntek	EVS-050
Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap	Falcon	352235
Fluorescin isothiocyanate (FITC)-dextran	Sigma-Aldrich (Merck)	FD4	average mol wt 3000-5000
Heraeus – Megafuge 40R	Thermo Fisher Scientific	75004518
Human recombinant epidermal growth factor	Gibco (Thermo Fischer Scientific)	3700015
Keratinocyte-SFM	Gibco (Thermo Fischer Scientific)	17005042
Penicillin-Streptomycin	Gibco (Thermo Fischer Scientific)	15140122
Recombinant Human KGF/FGF-7 Protein	R&D Systems (Bio-Techne)	251-KG-010/CF
Screw cap tube, 15 mL	Sarstedt	62.554.502
Single Channel EVOLVE 100-1000 µL	Integra	3018
Single Channel EVOLVE 20-200 µL	Integra	3016
Syringe 1 mL		1134950
ThermoMixer C	Eppendorf	5382000015
Trypsin inhibitor from Glycine max (soybean)	Sigma-Aldrich (Merck)	T9128
Trypsin-EDTA	SAFC Biosciences (Merck)	59418C
Y27632 dihydrochloride	Tocris (Bio-Techne)	1254

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