Assessment Of Barrier Integrity Research Articles

#1343 Microfluidic kidney tubuloids-on-a-chip for improved drug translation

Abstract Background and Aims Current 2D in vitro models of renal epithelium lack key features of the in vivo setting, such as tubular structure and perfusion, resulting in low translatability to human situations and failure in clinical translation. Advanced renal in vitro models are of great importance to study kidney function under healthy and diseased conditions. Kidney tubular epithelial organoids, called tubuloids, are three-dimensional multicellular structures that recapitulate tubular function and have been used to model genetic, metabolic, and infectious renal diseases. To increase physiological relevance of the tubuloids, they can be integrated into an organ-on-a-chip system. This microfluidic technique is becoming increasingly recognized as a valuable tool for adding physiologically relevant cues to traditional cell culture including long-term gradient stability, continuous perfusion, and interaction with other cell types such as vasculature. Method Here, we introduce the OrganoPlate microfluidic platform, which can accommodate up to 64 independent microfluidic chips in a microtiter plate format, allowing the growth of 64 independent kidney tubuloid-derived barrier tissues in the form of perfused tubules. To this end, tubuloids derived from adult kidney tissue were plated as single cells in the OrganoPlate against an extracellular matrix (ECM). These renal tubules can be formed in just four days of culture in the device showing rapid and reproducible cell polarization, tight junction formation, proper expression of renal markers and functional transport. Results When integrated into an OrganoPlate system, kidney tubuloids form leak-tight, perfusable tubes with stable Trans Epithelial Electrical Resistance (TEER) and are suitable for high-throughput screening of compound effects through assessment of barrier integrity by use of OrganoTEER and by real-time imaging of transport. OrganoPlate grown kidney tubes treated with Pgp inhibitor Verapamil show significant reduction of Rhodamine123 transport through kidney tubule barrier which confirms show stable activity of Pgp transporter and usability of the model in studying renal drug clearance. Conclusion Our results demonstrate the suitability of our in vitro microfluidic kidney tubuloid-on-a-chip model in mimicking key physiological aspects of the kidney and offer new ways for studying organ physiology and renal disease mechanisms, toxicity screening, and personalized medicine. Additionally, the use of animal models, as well as the costs of drug development, can be reduced.

Intestinal epithelial barrier integrity investigated by label-free techniques in ulcerative colitis patients

The intestinal epithelial barrier, among other compartments such as the mucosal immune system, contributes to the maintenance of intestinal homeostasis. Therefore, any disturbance within the epithelial layer could lead to intestinal permeability and promote mucosal inflammation. Considering that disintegration of the intestinal epithelial barrier is a key element in the etiology of ulcerative colitis, further assessment of barrier integrity could contribute to a better understanding of the role of epithelial barrier defects in ulcerative colitis (UC), one major form of chronic inflammatory bowel disease. Herein, we employ fast, non-destructive, and label-free non-linear methods, namely coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), two-photon excited fluorescence (TPEF), and two-photon fluorescence lifetime imaging (2P-FLIM), to assess the morpho-chemical contributions leading to the dysfunction of the epithelial barrier. For the first time, the formation of epithelial barrier gaps was directly visualized, without sophisticated data analysis procedures, by the 3D analysis of the colonic mucosa from severely inflamed UC patients. The results were compared with histopathological and immunofluorescence images and validated using transmission electron microscopy (TEM) to indicate structural alterations of the apical junction complex as the underlying cause for the formation of the epithelial barrier gaps. Our findings suggest the potential advantage of non-linear multimodal imaging is to give precise, detailed, and direct visualization of the epithelial barrier in the gastrointestinal tract, which can be combined with a fiber probe for future endomicroscopy measurements during real-time in vivo imaging.

Extended exposure duration of cultured intestinal epithelial cell monolayers in characterizing hazardous and non-hazardous proteins

Recent studies suggest that human derived intestinal epithelial cells (IECs) cultured as polarized monolayers on Transwell® filters may respond differently when exposed to hazardous and non-hazardous proteins. This experimental platform was based on apical exposure of IEC monolayers to test proteins for 24 h followed by assessment of barrier integrity and cell viability. In this study, Caco-2 and T84 IEC polarized monolayers were evaluated for barrier integrity and cytotoxicity following exposure to hazardous and non-hazardous proteins for 24, 48 and 72 h. Hazardous proteins included Clostridium difficile toxin A (ToxA), Streptolysin O (SLO), Wheat Germ Agglutinin (WGA), and Phaseolus vulgaris haemagglutinin-E (PHA-E). Non-hazardous proteins included bovine serum albumin (BSA), porcine serum albumin (PSA), and fibronectin (Fbn). In general, evidence of diminished barrier integrity or cell viability observed following exposure to hazardous proteins for 24 h was more pronounced after 48 and 72 h for both IEC monolayers. Non-hazardous proteins exhibiting no impact following 24 h of exposure elicited minimal effects over longer exposure durations. These results support the utility of using cultured human IEC polarized monolayers to differentiate between hazardous and non-hazardous proteins and suggest that longer durations of exposure may further improve the ability to distinguish between them.