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Determination of Benzopyrene-Induced Lung Inflammatory and Cytotoxic Injury in a Chemical GradientIntegrated Microfluidic Bronchial Epithelium System Fen Zhang, Chang Tian, Wenming Liu, Kan Wang, Yuanqing Wei, Huaisheng wang, Jinyi Wang, and Songqin Liu ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b01370 • Publication Date (Web): 03 Dec 2018 Downloaded from http://pubs.acs.org on December 7, 2018
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Determination of Benzopyrene-Induced Lung Inflammatory and Cytotoxic Injury in a Chemical Gradient-Integrated Microfluidic Bronchial Epithelium System Fen Zhang,† Chang Tian,‡ Wenming Liu,*,§,# Kan Wang,† Yuanqing Wei,† Huaisheng Wang,& Jinyi Wang,*,‡,# and Songqin Liu*,† †Key
Laboratory of Environmental Medicine Engineering, Ministry of Education, Jiangsu Engineering Laboratory of Smart Carbon-Rich Materials and Device, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China ‡College
of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China
§School
of Basic Medical Science, Central South University, Changsha, Hunan 410013, China
#College
of Chemistry and Pharmacy, Northwest A&F University, Yangling, Shaanxi 712100, China
&Department
of Chemistry, Liaocheng University, Liaocheng, Shandong, 252059, China
ABSTRACT: Environmental pollution is one of the largest sources responsible for human diseases and premature death worldwide. However, the methodological development of a spatiotemporally controllable and high-throughput investigation of the environmental pollution-induced biological injury events is still being explored. In this study, we describe a chemical gradient generator-aided microfluidic cell system for the dynamic study of representative environmental pollutant-induced bronchial epithelium injury in a throughput manner. We demonstrated the stability and reliability of operation-optimized microfluidic system for precise and long-term chemical gradient production. We also performed a microenvironment-controlled microfluidic bronchial epithelium construction with high viability and structure integration. Moreover, on-chip investigation of bronchial epithelium injury by benzopyrene stimulation with various concentrations can be carried out in the single device. The varying bronchial inflammatory and cytotoxic responses were temporally monitored and measured based on the well-established system. The benzopyrene directionally led the bronchial epithelium to present observable cell shrinkage, cytoskeleton disintegration, Caspase-3 activation, overproduction of reactive oxygen species, and various inflammatory cytokine (TNF-, IL-6, and IL-8) secretion, suggesting its significant inflammatory and cytotoxic effects on respiratory system. We believe the microfluidic advancement has potential applications in the fields of environmental monitoring, tissue engineering, and pharmaceutical development.
Keywords: Microfluidic device, Cell sensor, Bronchial epithelium array, Environmental monitoring, Throughput analysis
Environmental pollution from anthropogenic activities causes adverse effects on human health and is now one of the largest origin of human diseases and even premature death worldwide.1 Exposure to pollutants (e.g., heavy metal, benzopyrene, and dioxin) has been demonstrated to be associated with increases in morbidity and mortality due to various respiratory, cardiovascular, and brain diseases.2,3 Statistically, pollution linked to dirty air, water, and soil leads to at least nine million global deaths every year.4 In particular, it is responsible for over 50% of all deaths from chronic pulmonary disease and over 40% of deaths due to lung cancer. The intentional and
meticulous exploration of the pathogenesis (e.g., minor respiratory irritation) and the development (e.g., inflammatory response and apoptosis) of the pollutantmanaged respiratory diseases has been the subject of much interest and extensive research.5 Previously, the research on these diseases based on the classical in vivo (e.g., rat model) and in vitro (e.g., robotic handler and plate-based cell culture) models has extremely promoted the histopathological understanding of respiratory tissue and its cellular/molecular identification.6,7 The in vivo biological model characteristically exhibits high tissue relevance and the in vitro cell culture model provides the
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desired biological relevance and throughput, avoids ethical concerns, and represents a large fraction of human biology research.8 However, these methods with macrooperation are often cumbersome, laborious, and reagentintensive, as well as might rely on expensive automation equipments. Further, either the real-time monitoring of the changeable pathological process in vivo or the reliable control and reconstruction of extracellular microenvironment dynamics in time and space in vitro has been one of the intractable challenges in respiratory science.9 Therefore, the methodological improvement for an effective investigation of specific respiratory pathology events (e.g., inflammation of the bronchi) remains largely out of reach. As one of the representative microfabrication techniques based on the field of micro-electromechanical systems, microfluidics is becoming a promising platform for tissue/cell microengineering because of its excellent performance in spatiotemporal control and monitoring mammalian cells in vitro and in organizing the cells in well-controlled fluidic microenvironments at microscale, as well as in substantially reducing sample and reagent consumption.10–13 The advantageous capabilities greatly extend microfluidic application to respiratory system research, ranging from structural organization to mechanical activity,14–16 physiological functionality of the alveolar-capillary interface,17,18 cell interaction,19,20 and drug toxicity/nanotoxicity.21–23 Small lung-on-a-chip lined with human airway epithelial cells recapitulated diseaseinvolved features like selective cytokine hypersecretion and increased neutrophil recruitment.24 These studies exceedingly improved the microscale rebuilding and assessment of lung tissue/cell properties, particularly the studies on the physiological functions of respiratory epithelial cells. Recently, an integrated microfluidic system was reported to conduct an excellent protein detection for studying signal transductions of human bronchial epithelial cells in response to carcinogenic particle (PM2.5, particles with a diameter of 2.5 μm or less) exposure.25 Nevertheless, reconfiguration of the mechanical and functional characteristics of lung tissue microenvironment for the exploration of environmental pollutant-induced respiratory diseases is still being explored.26 Importantly, the reported microfluidic lung systems for studying toxicity have not well-achieved the parallel and throughput analysis of lung injury, as well as the functional integration of different microfluidic components (i.e., the integration of the channel/chamber arrays and the chemical gradient generator in the device).10,27 The establishment of lung on a chip with different types of component and subsystem integration for synchronous and throughput cell analysis is quite necessary.11,21 To the best of our knowledge, exploring pathological dynamics of respiratory inflammation and injury induced by ubiquitous and specific environmental pollutants like benzopyrene in microfluidics has been less advanced. Here, we presented a real-time and throughput investigation of bronchial epithelium injury dynamics
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induced by the environmental pollutant with various concentrations in a single microfluidic culture system integrated with a chemical gradient generator. The concentration gradient production at different flow conditions and perfusion times was evaluated systematically. Further, the arranged bronchial epithelium layers were constructed in the chamber array based on the well-controlled and shear-neglected microfluidic culture procedure. Afterwards, different response dynamics including cell proliferation, cell viability, cytoskeleton, Caspase-3 activation, and cell shrinkage during both the culture and the benzopyrene pollutant stimulation were visually recorded and quantitatively analyzed. In addition, various types of inflammatory signals (i.e., reactive oxygen species and three types of inflammatory cytokines including TNF-, IL-6, and IL-8) were monitored by fluorescence and immunodetection at different time points.
EXPERIMENTAL SECTION Device Design. The microfluidic device in this study
was composed of two layers: the fluidic layer and a glass slide (Figure 1). In detail, the fluidic layer contained a chemical gradient generator (i.e., a channel network) and a cell chamber array organized in a 5 × 10 geometry for cell staying and culture-based cell assays. For gradient generator, twelve serpentine channels were spatially arranged with an orderly trapezoid outline and connected with the branched straight channels. Five channel network outputs were finally coupled with five chamber columns for the microfluidic operation of the resulting chemical concentration gradient. Two inlets and five outlets were used to perform sample loading, chamber purging, and waste exclusion. The glass slide was employed for the sealing of the channels and chambers in the fluidic layer.
Gradient Generation and Numerical Simulation.
To conveniently evaluate the on-chip chemical gradient between different terminal chambers and the final concentration in each chamber, two types of model gradient tests were first performed. 100 μM fluorescein in NaHCO3 buffer (pH 8.3) and Fresh NaHCO3 buffer (or 100 μM fluorescein and 100 μM rhodamine B solutions) were introduced from the two inlets respectively into the channel network at the same flow rates (0.1 to 10 µL min–1) using a normal syringe pump (Longerpump, LSP04-1A). Meanwhile, the phase-contrast and fluorescence images of the chemical concentration distribution in the device were recorded. Each experiment was repeated at least thrice. Finite element analysis was performed using the ESI-CFD software (V2010.0, ESI CFD Inc., Huntsville, AL) to evaluate the flow and chemical profiles in the device.
Microfluidic Cell Culture and Pollutant Stimulation. The microfluidic device was first sterilized with UV light for 2 h and coated with collagen-I (200 µg mL–1) for another 2 h. After rinsing thrice with fresh DMEM, human bronchial epithelial cells (16HBE) cells were introduced from the inlets into the chambers by flowing the suspension (5.0 × 106 cells mL–1, 1 µL min–1) for
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5 min. The device was then placed in a humidified atmosphere with 5% CO2 at 37 °C for 2 h to enable the cells to attach. The supplemented DMEM was loaded into the device to provide the cell culture nutrients. For the pollutant treatment of the cultured cells, benzopyrene solution (BaP, 20 μM or 100 μM in fresh supplemented DMEM) was pumped from one inlet into the channel at 0.1 μL min–1. Fresh supplemented DMEM medium was simultaneously introduced from another inlet into the channel at the same flow rate. The BaP treating flow was kept for at least 12 h. Each treating experiment was repeated at least thrice.
Reactive Oxygen Species and Inflammatory Cytokine Activity. The intracellular ROS level was
evaluated using a ROS assay kit according to the manufacturer’s instruction. The cells treated with or without BaP were washed with fresh culture medium. Then, the cells were stained by introducing fluorescent probe DCFH-DA (10 µM in the culture medium) into the chambers and incubated for 20 min at 37 °C, followed by a 10 min rinse with PBS using the flow rate of 0.5 µL min–1. To analyze levels of inflammatory cytokines including TNF-, IL-6 and IL-8, the BaP-contained culture medium passing through the chambers and arriving at the specific outlet of the device at various BaP treating times (0 to 24 h) was collected, and centrifuged at 2000 rpm for 5 min. The supernatant was immediately preserved at −80 °C before use. The TNF-, IL-6 and IL-8 levels in the extracellular medium were quantified using human TNF, IL-6 and IL-8 ELISA kits according to the manufacturers’ protocols (KeyGEN, Nanjing, China). Briefly, the supernatant samples, standards, and blanks were added into the specific wells of each cytokine antibody-coated plate and incubated for 90 min. Biotinylated antibodies for each cytokine were incubated for 60 min. Avidin horseradish peroxidase conjugate was incubated for 30 min. Chromogenic substrate was incubated for 15 min in the dark and reaction was stopped with stop solution (KeyGEN). All incubations were at 37 °C. All plates were washed between each step with wash buffer (KeyGEN). Absorbance was measured at 450 nm by a microplate reader (Model 680, Bio-Rad, USA). Each experiment was repeated at least three times.
Microscopy and image analysis. A confocal laser scanning microscope (Olympus, FV1000) and an inverted microscope (Olympus, CKX41) with a charge-coupled device camera (Olympus, DP72) and a mercury lamp (Olympus, U-RFLT50) were used to obtain phase contrast and fluorescent images. Software Image-Pro® Plus 6.0 (Media Cybernetics, Silver Spring, MD) and SPSS 12.0 (SPSS Inc.) were employed to perform image analysis and data statistical analysis, respectively. Values were represented as means±SD, and statistical comparisons of means were made using one-way analysis of variance (ANOVA). For all statistical tests *P
