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

Organoides esofágicos humanos e protocolos de cultura de interface ar-líquido

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.

Autor em destaque: Investigando a fisiopatologia da esofagite eosinofílica

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

We are investigating the pathophysiology of eosinophilic esophagitis and trying to unravel the inflammatory cascade inducing and promoting the chronic inflammatory process of EOE. The identification of new immune mediators that contribute to the disease pathogenesis centered around the impairment of epithelial barrier and the approval of therapeutic antibodies against said immune mediators. We have identified the IL-20 subfamily cytokines as inflammatory mediators that impair epithelial differentiation and keratinization resulting in epithelial barrier impairment via the MAP kinase pathway.

Cultura de organoides esofágicos derivados de pacientes usando células isoladas de biópsias de esôfago

Para começar, colete amostras de biópsia de esôfago imersas em meio livre de soro de queratinócitos, ou KSFM. Substitua o KSFM por um mililitro de Dispase I e incube as biópsias por 10 minutos em temperatura ambiente. Depois disso, usando uma pipeta de 1.000 microlitros, aspire o Dispase sem tocar nas biópsias ou no pellet de detritos celulares.Em seguida, enxágue as biópsias com DPBS. Depois de centrifugar as biópsias, use uma pipeta de 1.000 microlitros para aspirar o sobrenadante. Em seguida, adicione 500 microlitros de tripsina-EDTA às biópsias e incube a 37 graus Celsius por 10 minutos enquanto agita a 800 rpm.Em seguida, execute pipetagem repetida até obter uma suspensão unicelular. Usando a cabeça do êmbolo de borracha de uma seringa de tuberculina, filtre as células através de um filtro de células de 70 micrômetros. Em seguida, lave o coador com dois a quatro mililitros de inibidor de tripsina de soja.Em seguida, filtre as células através de um filtro de células de 35 micrômetros com uma tampa de encaixe em um tubo de poliestireno de fundo redondo de cinco mililitros. Depois de centrifugar a suspensão celular, use uma pipeta de 1.000 microlitros para aspirar o sobrenadante. Ressuspenda o pellet celular na matriz de hidrogel do extrato da membrana basal e forme gotículas de 40 microlitros em uma placa de cultura de células de suspensão pré-aquecida.Em seguida, incube a placa sem meio por 20 a 30 minutos a 37 graus Celsius para garantir a solidificação das gotículas do extrato da membrana basal. Adicione KSFM pré-aquecido suplementado com 10 micromolares de Y27632. No nono dia, a estrutura multicamadas semelhante a uma cebola em um núcleo queratinizado em crescimento no centro do organoide foi observada.

patient-derived-esophageal-organoids-culture-using-cells-isolated