RESUMO
The measurement of transepithelial electrical resistance across confluent cell monolayer systems is the most commonly used technique to study intestinal barrier development and integrity. Electric cell substrate impedance sensing (ECIS) is a real-time, label-free, impedance-based method used to study various cell behaviors such as cell growth, viability, migration, and barrier function in vitro. So far, the ECIS technology has exclusively been performed on cell lines. Organoids, however, are cultured from tissue-specific stem cells, which better recapitulate cell functions and the heterogeneity of the parent tissue than cell lines and are therefore more physiologically relevant for research and modeling of human diseases. In this protocol paper, we demonstrate that ECIS technology can be successfully applied on 2D monolayers generated from patient-derived intestinal organoids. Key features ⢠We present a protocol that allows the assessment of various cell functions, such as proliferation and barrier formation, with ECIS on organoid-derived monolayers. ⢠The protocol facilitates intestinal barrier research on patient tissue-derived organoids, providing a valuable tool for disease modeling.
RESUMO
Small intestine (SI) maturation during early life is pivotal in preventing the onset of gut diseases. In this study we interrogated the milestones of SI development by gene expression profiling and ingenuity pathway analyses. We identified a set of cytokines as main regulators of changes observed across different developmental stages. Upon cytokines stimulation, with IFNγ as the most contributing factor, human fetal organoids (HFOs) increase brush border gene expression and enzyme activity as well as trans-epithelial electrical resistance. Electron microscopy revealed developed brush border and loss of fetal cell characteristics in HFOs upon cytokine stimulation. We identified T cells as major source of IFNγ production in the fetal SI lamina propria. Co-culture of HFOs with T cells recapitulated the major effects of cytokine stimulation. Our findings underline pro-inflammatory cytokines derived from T cells as pivotal factors inducing functional SI maturation in vivo and capable of modulating the barrier maturation of HFOs in vitro.