patient-derived esophageal organoids culture using cells isolated from esophagus biopsies

Human Esophageal Organoids and Air-Liquid Interface Culture Protocols

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 TGF&#946; 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&#160;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.

Author Spotlight: Investigating the Pathophysiology of Eosinophilic Esophagitis

Dreidimensionale Zellkulturmodelle zur Untersuchung der Epithelbarriere bei eosinophiler Ösophagitis 

Kultivierung von patienteneigenen Organoiden der Speiseröhre unter Verwendung von Zellen, die aus Speiseröhrenbiopsien isoliert wurden

Entnehmen Sie zunächst Speiseröhrenbiopsieproben, die in keratinozytenserumfreies Medium (KSFM) getaucht sind. Ersetzen Sie das KSFM durch einen Milliliter Dispase I und inkubieren Sie die Biopsien 10 Minuten lang bei Raumtemperatur. Danach wird die Dispase mit einer 1.000-Mikroliter-Pipette aspiriert, ohne die Biopsien oder das Pellet der Zelltrümmer zu berühren.Spülen Sie dann die Biopsien mit DPBS. Nach dem Zentrifugieren der Biopsien wird der Überstand mit einer 1.000-Mikroliter-Pipette abgesaugt. Geben Sie anschließend 500 Mikroliter Trypsin-EDTA zu den Biopsien und inkubieren Sie 10 Minuten lang bei 37 Grad Celsius unter Schütteln bei 800 U/min.Führen Sie dann wiederholtes Pipettieren durch, bis eine Einzelzellsuspension erhalten wird. Mit dem Gummikolbenkopf einer Tuberkulinspritze filtrieren Sie die Zellen durch ein 70-Mikrometer-Zellsieb. Waschen Sie anschließend das Sieb mit zwei bis vier Millilitern Soja-Trypsin-Hemmer.Als nächstes filtrieren Sie die Zellen durch ein 35-Mikrometer-Zellensieb mit einer Schnappkappe auf einem Fünf-Milliliter-Polystyrolrohr mit rundem Boden. Nach dem Zentrifugieren der Zellsuspension wird der Überstand mit einer 1.000-Mikroliter-Pipette abgesaugt. Resuspendieren Sie das Zellpellet in der Hydrogel-Matrix des Basalmembranextrakts und formen Sie 40-Mikroliter-Tröpfchen in einer vorgewärmten Suspensionszellkulturplatte.Inkubieren Sie die Platte dann ohne Medium für 20 bis 30 Minuten bei 37 Grad Celsius, um eine Verfestigung der Basalmembran-Extrakttröpfchen sicherzustellen. Fügen Sie vorgewärmtes KSFM hinzu, das mit 10 Mikromolaren Y27632 ergänzt wird. Am neunten Tag wurde die zwiebelartige mehrschichtige Struktur in einem wachsenden keratinisierten Kern im Zentrum des Organoids beobachtet.

Research

JoVE Journal

Immunology and Infection

Patient-Derived Esophageal Organoids Culture Using Cells Isolated from Esophagus Biopsies

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

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and ...

Forschung

Immunologie und Infektion

Establishment of Patient-Derived Air-Liquid Interface (ALI) Cultures from Esophagus Biopsies

Etablierung von patientenabgeleiteten Luft-Flüssigkeits-Grenzflächenkulturen (ALI) aus Speiseröhrenbiopsien