A nephron model for study of drug-induced acute kidney injury and assessment of drug-induced nephrotoxicity

In this study, we developed a multilayer microfluidic device to simulate nephron, which was formed by "glomerulus", "Bowman's capsule", "proximal tubular lumen" and "peritubular capillary". In this microdevice, artificial renal blood flow was circulating and glomerular filtrate flow was single passing through, mimicking the behavior of a nephron. In this dynamic artificial nephron, we observed typical renal physiology, including the glomerular size-selective barrier, glomerular basement membrane charge-selective barrier, glucose reabsorption and para-aminohippuric acid secretion. To demonstrate the capability of our microdevice, we used it to investigate the pathophysiology of drug-induced acute kidney injury (AKI) and give assessment of drug-induced nephrotoxicity, with cisplatin and doxorubicin as model drugs. In the experiment, we loaded the doxorubicin or cisplatin in the "renal blood flow", recorded the injury of primary glomerular endothelial cells, podocytes, tubular epithelial cells and peritubular endothelial cells by fluorescence imaging, and identified the time-dependence, dose dependence and the death order of four types of renal cells. Then by measuring multiple biomarkers, including E-cadherin, VEGF, VCAM-1, Nephrin, and ZO-1, we studied the mechanism of cell injuries caused by doxorubicin or cisplatin. Also, we investigated the effect of BSA in the "renal blood flow" on doxorubicin-or-cisplatin-induced nephrotoxicity, and found that BSA enhanced the tight junctions between cells and eased cisplatin-induced nephrotoxicity. In addition, we compared the nephron model and traditional tubule models for assessment of drug-induced nephrotoxicity. And it can be inferred that our biomimetic microdevice simulated the complex, dynamic microenvironment of nephron, yielded abundant information about drug-induced-AKI at the preclinical stage, boosted the drug safety evaluation, and provided a reliable reference for clinical therapy. (C) 2017 Elsevier Ltd. All rights reserved

Carcinoma-associated fibroblast-derived lysyl oxidase-rich extracellular vesicles mediate collagen crosslinking and promote epithelial-mesenchymal transition via p-FAK/p-paxillin/YAP signaling

Abstract Carcinoma-associated fibroblasts (CAFs) are the main cellular components of the tumor microenvironment and promote cancer progression by modifying the extracellular matrix (ECM). The tumor-associated ECM is characterized by collagen crosslinking catalyzed by lysyl oxidase (LOX). Small extracellular vesicles (sEVs) mediate cell-cell communication. However, the interactions between sEVs and the ECM remain unclear. Here, we demonstrated that sEVs released from oral squamous cell carcinoma (OSCC)-derived CAFs induce collagen crosslinking, thereby promoting epithelial-mesenchymal transition (EMT). CAF sEVs preferably bound to the ECM rather than being taken up by fibroblasts and induced collagen crosslinking, and a LOX inhibitor or blocking antibody suppressed this effect. Active LOX (αLOX), but not the LOX precursor, was enriched in CAF sEVs and interacted with periostin, fibronectin, and bone morphogenetic protein-1 on the surface of sEVs. CAF sEV-associated integrin α2β1 mediated the binding of CAF sEVs to collagen I, and blocking integrin α2β1 inhibited collagen crosslinking by interfering with CAF sEV binding to collagen I. CAF sEV-induced collagen crosslinking promoted the EMT of OSCC through FAK/paxillin/YAP pathway. Taken together, these findings reveal a novel role of CAF sEVs in tumor ECM remodeling, suggesting a critical mechanism for CAF-induced EMT of cancer cells

Application of Microfluidic Chips in Separation and Analysis of Extracellular Vesicles in Liquid Biopsy for Cancer

Extracellular vesicles (EVs) are becoming a promising biomarker in liquid biopsy of cancer. Separation EV from cell culture medium or biofluids with high purity and quality remains a technique challenge. EV manipulation techniques based on microfluidics have been developed in the last decade. Microfluidic-based EV separation techniques developed so far can be classified into two categories: surface biomarker-dependent and size-dependent approaches. Microfluidic techniques allow the integration of EV separation and analysis on a single chip. Integrated EV separation and on-chip analysis have shown great potential in cancer diagnosis and monitoring treatment of responses. In this review, we discuss the development of microfluidic chips for EV separation and analysis. We also detail the clinical application of these microfluidic chips in the liquid biopsy of various cancers

Replacement of soybean meal by urea or starea in beef cattle diet: I. igestibility, nitrogen balance, ruminal and blood parameters; II. performance and III. evaluation of digestibility markers.

Com o objetivo de avaliar a substituição de uma fonte de proteína verdadeira (farelo de soja; dieta deficiente em PDR), por uréia ou amiréia (A-150S - fonte de nitrogênio não protéico de suposta liberação gradativa de nitrogênio; dietas adequadas em PDR), foram realizados três experimentos. Experimento I: Seis machos da raça Nelore, não castrados, com peso médio inicial de 420 kg, foram utilizados em quadrado latino 3x3 duplicado, avaliando-se: a digestibilidade, o balanço de nitrogênio, parâmetros ruminais e sanguíneos (capítulo 3); a estimativa da digestibilidade no trato gastrintestinal utilizando indicadores externo e internos comparados com colheita total de fezes (capítulo 6). O volumoso utilizado foi o BIN (20% da MS). A digestibilidade da MS, MO, CNF, EE, PB e o NDT não diferiram (P>0,05) entre os tratamentos. A digestibilidade da FDA e FDN foram superiores (P0,05) nos valores de pH, AGV total, acetato, propionato, butirato e relação acetato:propionato do fluido ruminal. A concentração de nitrogênio amoniacal no fluido ruminal foi superior (P0,05) entre os tratamentos. A estimativa da digestibilidade utilizando o óxido de cromo apresentou resultados similares (P>0,05) à colheita total de fezes. O mesmo foi observado com a lignina no tratamento deficiente em PDR (FS), mas nos de uréia e amiréia, os coeficientes de digestibilidade foram subestimados (P0,05) entre si. Experimento III: realizou-se outro experimento de desempenho similar ao anterior, utilizando-se apenas outro volumoso (45% de BTPV e 5% de BIN) e os animais estavam na fase de terminação (capitulo 5). O tratamento FS apresentou maior (P0,05) entre si. A amiréia promoveu resultados similares a uréia convencional no consumo dos nutrientes, digestibilidade, parâmetros ruminais e sanguíneos e no desempenho de bovinos de corte confinados.Experiment I: Six Nellore bulls, with 420 kg of body weight, were used to evaluate the replacement of a true protein source (soybean meal-SBM), in an inadequate RDP diet, by urea or starea (non protein nitrogen source supposedly of slow N release), being the last two N sources in an adequate RDP diet. Sugar cane bagasse in natura (BIN) was the only source of diet forage (20% of DM). This trial evaluated: digestibility, ruminal parameters, ruminal ammonia, blood parameters and N balance (chapter 3); total tract digestibility estimated by using internal and external markers compared to total feces collection (chapter 6). DM, OM, NFC, EE, CP and TDN digestibilities (%) did not differ (P>0.05) among treatments. ADF and NDF digestibilities (%) were higher (P0.05) on pH, total VFA, acetate, propionate, butirate and acetate:propionate ratio. Ruminal ammonia N concentration was greater (P0.05) urinary N loss. N retention (g/d and % of ingested) and protein biological value (N retention, % of N digestible) were higher (P0.05) among treatment (chapter 3). The digestibility estimated by using cromium oxide was similar (P>0.05) to that of using total feces collection. Lignin used as an internal marker resulted in similar pattern as feces collection when the diet contained soybean meal (RDP deficient diet), however, when the diet N was urea or starea, the digestibility coefficients were underestimated (P0.05). Experiment III: Another performance trial was done (chapter 5), similar to Exp. II, differing only by the forage portion of the diet (45% hidrolized sugar cane bagasse-BTPV and 5% bagasse in natura-BIN) for finishing cattle. DM intake, ADG and feed conversion were 8.99, 7.43 and 7.69 kg/day; 0.983, 0.368 and 0.404 kg/day and 9.56, 20.14 and 19.54 kg DM/kg gain for SBM, urea and starea treatments, respectively. SBM had the higher (P0.05). Starea showed similar results to urea

Biomimetic tumor-induced angiogenesis and anti-angiogenic therapy in a microfluidic model

We developed a biomimetic microfluidic model to reproduce hallmark events of tumor-induced angiogenesis. The angiogenic capabilities of salivary gland adenoid cystic carcinoma (ACC) cells and oral squamous cell carcinoma (SCC) cells were assessed in this model. The traditional nude mouse xenograft model was used to investigate the physiological similarity of the microfluidic model to animal models, and the results showed that the angiogenic potential of ACC and SCC cells assessed by the microfluidic model was in agreement with the results obtained from the nude mouse model. The microfluidic model was subsequently used to evaluate the effect of antiangiogenic drugs on ACC- and SCC-induced angiogenesis. The antiangiogenic effect of anti-VEGF was further compared between the microfluidic and nude mouse models, and showed that it effectively inhibited tumor-induced angiogenesis in both the microfluidic model and the nude mouse model. Thus, tumor-induced angiogenesis reproduced in the microfluidic model may expand the capabilities of cell culture models, providing a low-cost, timesaving, and rapid alternative to animal models

A microfluidic-based multi-shear device for investigating the effects of low fluid-induced stresses on osteoblasts.

Interstitial fluid flow (IFF) within the extracellular matrix (ECM) produces low magnitude shear stresses on cells. Fluid flow-induced stress (FSS) plays an important role during tissue morphogenesis. To investigate the effect of low FSS generated by IFF on cells, we developed a microfluidic-based cell culture device that can generate multiple low shear stresses. By changing the length and width of the flow-in channels, different continuous low level shear stresses could be generated in individual cell culture chambers. Numerical calculations demonstrate uniform shear stress distributions of the major cell culture area of each chamber. This calculation is further confirmed by the wall shear stress curves. The effects of low FSS on MC3T3-E1 proliferation and differentiation were studied using this device. It was found that FSS ranging from 1.5 to 52.6 µPa promoted MC3T3-E1 proliferation and differentiation, but FSS over 412 µPa inhibited the proliferation and differentiation of MC3T3-E1 cells. FSS ranging from 1.5 to 52.6 µPa also increased the expression of Runx2, a key transcription factor regulating osteoblast differentiation. It is suggested that Runx2 might be an important regulator in low FSS-induced MC3T3-E1 differentiation. This device allows for detailed study of the effect of low FSS on the behaviors of cells; thus, it would be a useful tool for analysis of the effects of IFF-induced shear stresses on cells

Paper Microfluidics for Cell Analysis

Paper microfluidics has attracted much attention since its first introduction around one decade ago due to the merits such as low cost, ease of fabrication and operation, portability, and facile integration with other devices. The dominant application for paper microfluidics still lies in point-of-care testing (POCT), which holds great promise to provide diagnostic tools to meet the ASSURED criteria. With micro/nanostructures inside, paper substrates provide a natural 3D scaffold to mimic native cellular microenvironments and create excellent biointerfaces for cell analysis applications, such as long-term 3D cell culture, cell capture/phenotyping, and cell-related biochemical analysis (small molecules, protein DNA, etc.). This review summarizes cell-related applications based on various engineered paper microdevices and provides some perspectives for paper microfluidics-based cell analysis