establishment of patient-derived air-liquid interface (ali) cultures 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

Modelos tridimensionais de cultura celular para investigar a barreira epitelial na esofagite eosinofílica 

Estabelecimento de culturas de interface ar-líquido (LPA) derivadas do paciente a partir de biópsias de esôfago

Para começar, pegue células isoladas de biópsias de esôfago e ressuspenda-as em quatro mililitros de KSFM com 10 micromolares Y27632. Transferir as células para um balão de cultura de células T25. Uma vez que os queratinócitos formam uma monocamada com 60% a 80% de confluência, passam as células P1.Em seguida, semeie queratinócitos primários P2 em inserções TRANSWELL para a cultura de interface ar-líquido. Para realizar o transporte aéreo no sétimo dia, substitua o meio na câmara inferior por KSFM com alto teor de cálcio e aspire o meio da câmara superior.

establishment-patient-derived-air-liquid-interface-ali-cultures-from

Research

JoVE Journal

Immunology and Infection

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

83 visualizações.  University of Basel. Para começar, pegue células isoladas de biópsias de esôfago e ressuspenda-as em quatro mililitros de KSFM com 10 micromolares Y27632. Transferir as células para um balão de cultura de células T25. Uma vez que os queratinócitos formam uma monocamada com 60% a 80% de confluência, passam as células P1.Em seguida, semeie queratinócitos primários P2 em inserções TRANSWELL para a cultura de interface ar-líquido. Para realizar o transporte aéreo no sétimo dia, substitua o mei...

Vídeo: Estabelecimento de culturas de interface ar-líquido (LPA) derivadas do paciente a partir de biópsias de esôfago

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 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.

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.