RESUMEN
With the rapid increase of the number of patients with gastrointestinal diseases in modern society, the need for the development of physiologically relevant in vitro intestinal models is key to improve the understanding of intestinal dysfunctions. This involves the development of a scaffold material exhibiting physiological stiffness and anatomical mimicry of the intestinal architecture. The current work focuses on evaluating the scaffold micromorphology of gelatin-methacryloyl-aminoethyl-methacrylate-based nonporous and porous intestinal 3D, intestine-like constructs, fabricated via digital light processing, on the cellular response. To this end, Caco-2 intestinal cells were utilized in combination with the constructs. Both porous and nonporous constructs promoted cell growth and differentiation toward enterocyte-like cells (VIL1, ALPI, SI, and OCLD expression showed via qPCR, ZO-1 via immunostaining). The porous constructs outperformed the nonporous ones regarding cell seeding efficiency and growth rate, confirmed by MTS assay, live/dead staining, and TEER measurements, due to the presence of surface roughness.
Asunto(s)
Hidrogeles , Andamios del Tejido , Humanos , Porosidad , Hidrogeles/química , Células CACO-2 , Andamios del Tejido/química , Proliferación Celular , Gelatina/química , Intestinos/citología , Metacrilatos/química , Ingeniería de Tejidos/métodos , Diferenciación CelularRESUMEN
The colonic epithelial barrier is vital to preserve gut and host health by maintaining the immune homeostasis between host and microbes. The mechanisms underlying beneficial or harmful host-microbe interactions are poorly understood and impossible to study in vivo given the limited accessibility and ethical constraints. Moreover, existing in vitro models lack the required cellular complexity for the routine, yet profound, analysis of the intricate interplay between different types of host and microbial cells. We developed and characterized a broadly applicable, easy-to-handle in vitro triple coculture model that combines chemically-induced macrophage-like, goblet and epithelial cells covered by a mucus layer, which can be coincubated with complex human-derived gut microbiota samples for 16 h. Comparison with a standard epithelial monolayer model revealed that triple cocultures produce thicker mucus layers, morphologically organize in a network and upon exposure to human-derived gut microbiota samples, respond via pro-inflammatory cytokine production. Both model systems, however, were not suffering from cytotoxic stress or different microbial loads, indicating that the obtained endpoints were caused by the imposed conditions. Addition of the probiotic Lactobacillus rhamnosus GG to assess its immunomodulating capacity in the triple coculture slightly suppressed pro-inflammatory cytokine responses, based on transcriptomic microarray analyses. TNF conditioning of the models prior to microbial exposure did not cause shifts in cytokines, suggesting a strong epithelial barrier in which TNF did not reach the basolateral side. To conclude, the triple coculture model is tolerable towards manipulations and allows to address mechanistic host-microbe research questions in a stable in vitro environment.
Asunto(s)
Técnicas de Cocultivo/métodos , Colon/inmunología , Células Epiteliales/inmunología , Microbioma Gastrointestinal , Interacciones Microbiota-Huesped , Mucosa Intestinal/inmunología , Macrófagos/inmunología , Colon/citología , Colon/metabolismo , Colon/microbiología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Lacticaseibacillus rhamnosus/fisiología , Macrófagos/citología , Macrófagos/metabolismo , Macrófagos/microbiología , TranscriptomaRESUMEN
Organs-on-a-chip (OoC) are cell culture platforms that replicate key functional units of tissues in vitro. Barrier integrity and permeability evaluation are of utmost importance when studying barrier-forming tissues. Impedance spectroscopy is a powerful tool and is widely used to monitor barrier permeability and integrity in real-time. However, data comparison across devices is misleading due to the generation of a non-homogenous field across the tissue barrier, making impedance data normalization very challenging. In this work, we address this issue by integrating PEDOT:PSS electrodes for barrier function monitoring with impedance spectroscopy. The semitransparent PEDOT:PSS electrodes cover the entire cell culture membrane providing a homogenous electric field across the entire membrane making the cell culture area equally accountable to the measured impedance. To the best of our knowledge, PEDOT:PSS has never been used solely to monitor the impedance of cellular barriers while enabling optical inspection in the OoC. The performance of the device is demonstrated by lining the device with intestinal cells where we monitored barrier formation under flow conditions, as well as barrier disruption and recovery under exposure to a permeability enhancer. The barrier tightness and integrity, and the intercellular cleft have been evaluated by analyzing the full impedance spectrum. Furthermore, the device is autoclavable paving the way toward more sustainable OoC options.
Asunto(s)
Técnicas de Cultivo de Célula , Sistemas Microfisiológicos , Electrodos , Impedancia Eléctrica , Espectroscopía DieléctricaRESUMEN
Correction for 'Organ-on-a-chip with integrated semitransparent organic electrodes for barrier function monitoring' by Denise Marrero et al., Lab Chip, 2023, https://doi.org/10.1039/d2lc01097f.