Recapitulation of Particulate matter Effected Human Alveolus on Chip Inflammatory Responses CURRENT STATUS: POSTED

Background Particulate matter (PM10) pollution around the globe is a major risk factor for inducing respiratory diseases, How-ever such environmental conditions are a challenge to study in trans-well culture system. Micro-physiological system offers potential for mimicking these phenomena for better understanding of possible hazards to human respiratory health. In this study we are introducing an efficient alveolus on a chip for reconstituting this environmental condition in a microfluidic device by in house built TEER sensor, system embedded pH sensor and portable microscope for continuous monitoring of the alveolus on chip. Results Three environmental conditions with respect to particulate matter in air Moderate 7.5 μg/ml , Poor 37.5 μg/ml and very poor 151 μg/ml of particulate matter exposure was executed in our microfluidic system. Our study provides physiological and toxicological data of the stimulated environmental condition by inflammatory markers of respiratory disease which leads to the identification of asthma and COPD, our claims were validated by confocal microscopy and ELISA. Significant increase in IL-13, IL-6 and Mucin advocated the incidence of asthma and COPD like conditions in our organ on chip. Conclusion This study will lead to identification of potential therapeutics to study physiological and pathological conditions and will help in preventing chronic life-threatening toxicities. This device has provided the preliminary information for making this breakthrough in organ-on-chip technology. In future his device would help to make complex tissue mimetics of human for toxicity testing and drug discovery with system integrated pathological sensors for replacing time consuming molecular analysis of pathological conditions. directly in contact with human Lung pulmonary alveolar epithelial type-I and type-II cells for continuously monitoring trans epithelial electrical resistance after every half an hour till end of the experiment. Impedance values provide complete information of monolayer membrane integrity in control chip with flow rate of 80μl/min and shear stress of 12dyne/cm 3 , four chips were introduced to our organ on chip platform for physiological and mechanically controlled growing conditions. Particulate matter exposure was introduced after four days of stable culture on chip for eight hours daily for consecutive four days with three different concentrations to mimicking routine exposure to humans in daily life. Cell monolayer consisting of Human pulmonary alveolar type-I and type-II primary cells exhibited functions of properly differentiated epithelial cells. AT-I mucus secreting lung goblet cell and AT-II ciliated cells were confirmed by confocal microcopy. Particulate matter exposure induced inflammatory response in Human pulmonary alveolar type-I and type-II cells right after 12 hours showed sudden drop in impedance values correlates with cytokine secretion analysis and confocal microscopy ROS level also complements the dose dependent oxidative damage to the monolayer permeability.


Abstract
Background Particulate matter (PM10) pollution around the globe is a major risk factor for inducing respiratory diseases, How-ever such environmental conditions are a challenge to study in trans-well culture system. Micro-physiological system offers potential for mimicking these phenomena for better understanding of possible hazards to human respiratory health. In this study we are introducing an efficient alveolus on a chip for reconstituting this environmental condition in a microfluidic device by in house built TEER sensor, system embedded pH sensor and portable microscope for continuous monitoring of the alveolus on chip.
Results Three environmental conditions with respect to particulate matter in air Moderate 7.5 μg/ml , Poor 37.5 μg/ml and very poor 151 μg/ml of particulate matter exposure was executed in our microfluidic system. Our study provides physiological and toxicological data of the stimulated environmental condition by inflammatory markers of respiratory disease which leads to the identification of asthma and COPD, our claims were validated by confocal microscopy and ELISA. Significant increase in IL-13, IL-6 and Mucin advocated the incidence of asthma and COPD like conditions in our organ on chip.
Conclusion This study will lead to identification of potential therapeutics to study physiological and pathological conditions and will help in preventing chronic lifethreatening toxicities. This device has provided the preliminary information for making this breakthrough in organ-on-chip technology. In future his device would help to make complex tissue mimetics of human for toxicity testing and drug discovery with system integrated pathological sensors for replacing time consuming molecular analysis of pathological conditions. Background 3 Particulate matter have become a major concern owing to their increased level in air pollution correlating with higher respiratory diseases in metropolitan cities of the world, Human health is a challenge due to rapid advancement in cities and urbanization due to emission of hazardous gases from engines of heavy vehicles and burning of fossil fuels and mining (1). Coal burning power plants emit organic pollutants including polycyclic aromatic hydrocarbons, benzene, toluene and multitude of different gases. Weathering of soil turns clay particles into mineral dust composed of huge number of oxides and carbonates of metals. Dust storms in spring season carry the dust particles Flinches in Mongolia and Siberia moves dust particles to low-pressure areas such as eastern China, Korea, and Japan by south eastern to northwestern dust storm. Particulate matter stimulates clinical manifestations according to epidemiology and toxicology studies and contribute in developing lethal diseases such as respiratory disease cardiovascular disease, and mortality at an early age (3). Human lungs are the first organ to face airborne environmental stresses such as pollutants, toxicants and pathogens as they are involved in gaseous exchange (4). Environmental stress stimulants (particulate matter, cigarettes smoke and aerosols) are believed to be involved in the development of asthma, chronic obstructive pulmonary disease (COPD) and interstitial lung diseases (5).
Particulate matter induces release of inflammatory and allergic responsive cytokines in human airways effecting bronchiolar and alveolar passages, experimentally controlled human exposure studies suggest that interleukin-6 and tumor necrosis factor alpha (TNFα) are key mediators of airway inflammation (6)(7)(8). Allergic airways release proinflammatory cytokines and cause Airway hyperresponsiveness (AHR). The immune response to allergen exposure in asthma is associated with TH2 inflammation and IL-13 is a key cytokine involved in directing allergen-induced airway inflammation and remodeling (9). Regrettably animal models of asthma and allergy have failed to offer therapeutics with high efficacy, so human lung physiological mimetics are required for thorough understanding of toxicological disease analysis because animal models of inhaled toxicants and pollutants express different molecular markers from that are being produced in human subsequently resulting data usually lead to erroneous therapeutic development (10). Animal models need to be replaced by physiologically relevant human mimetics therefore considerable advancements have been made in development of the substitutes of organs and tissues to study such stimulation based studies (11) Organ on chip technology helps in revealing physiologically relevant organs by providing technologically controlled biochemical, mechanical and dynamic fluid flow conditions for recapitulating cellular microenvironment to access diseased and stressed conditions (12) .
Many in vitro lung-on-a-chip models have been introduced in the past to study impacts of toxins under dynamic flow conditions almost all the previous models are PDMS based.
PDMS absorbs hydrophobic biomolecules so PDMS based models fail to provide complete information of molecular markers and cytokine analysis in the culture medium, absence of real time monitoring or probe based off line monitored sample analysis provide vague information of stress response. To study acute or chronic stress response of the cells, real time microscopy is required to analyze the morphological changes in normal cells which is lacked by previous microfluidic models. Trans-epithelial electrical resistance (TEER) measurement is a rapid and conservative technique for the determination of integrity and differentiation of epithelial monolayers trans-well cultures because the electrical impedance across an epithelium is interrelated to the vigorous construction of tight junctions with neighboring cells (13). Cell monolayer integrity measurement by TEER has become a standard technique in trans-well culture system, technically TEER measurement of organ-on-chips is challenging because there is no valid and practical approach 5 available. Micrometer sized closed microfluidic channels create hindrance in TEER measurements in organ-on-chips to contact epithelium. Therefore, measurement of permeability changes incessantly using TEER in microfluidic is almost impossible (14) .
Printed TEER electrodes integrated into PDMS based microfluidic devices have been developed but only a few have been used to monitor in situ within organ chips epithelial barrier function (15). Conventional metal patterning techniques have been used in organ on chips microfluidic culture devices for construction of cell culture chambers around electrodes or integrating glass or polymeric substances (16)(17)(18). Previous studies with cells cultured in organ chips provide large variability in measurement, low sensitivity and they are affected by non-uniform cell cultures. TEER electrode location also significantly change in TEER values in these cultures, mathematical models can help to reduce these variations (14,18,19) Therefore successful fabrication of a robust on-chip TEER sensing capability enabled us to measure barrier function electrochemically., this method measures electrical impedance it can also help to study other behaviors of the cells on a microfluidic chip such as cell proliferation, migration, ion channel activity, tissue conductivity and dissolved gases. pH sensors have been recently reported for real time monitoring by fabricating PDMS based organ chips with fully integrated electrodes.
Embedded sensor in organ on chip platform are stringently required for future research applications for example many sensors such as strain sensor, humidity sensor for health monitoring have been developed for implantable applications (20,21).
In an effort to address these problems, this study was designed to introduce human lung-

Materials:
Particulate matter particles PM10 reference material organic components ERM CZ-100 and inorganic ERM CZ-120 were purchased from European Joint research commission JRC were characterized for their size with scanning electron microscopy (MIRA-3 TESCAN).Three 7 concentrations of both materials were prepared according to guidelines provided by daily update of climate control department South Korea, According to these guidelines moderate, poor and very poor conditions are available Following these concentrations particulate matter doses for exposure were determined for analysis. Stock solutions of particulate matter material were prepared in Class II biological safety cabinet to ensure the endotoxin free and sterile environment, 151μg/ml of particulate matter material was suspended in alveolar epithelial complete growth medium and was sonicated in probe sonicator for 60 minutes for disagglomeration, Further diluted for two less concentrated particulate matter solutions by adding more media with resulting concentrations of 37.5μg/ml and 7.5μg/ml respectively. Particulate matter was kept at 4°C either diluted or in original form.

Chip fabrication and sensor's development
Chip is made up of combination of two soda lime glasses each 1.1mm thick, 41 mm wide and 56mm long. Channel for cell culturing was printed on the bottom glass of the chip 15mm channel area was allocated for cell culture compartment. To launch a TEER sensor, indium tin oxide (ITO) coating was printed to make a square electrode of 4 mm length with a thickness of 500 nm. Screen printing technique was used for printing ITO 112 on the glass. 3D inkjet printer was used to print the pattern of silicon elastomer (Musil 113 medical grade silicon MED-6033) (900 nm width) to create channel and cell culture chamber on the chip. Top and bottom chip glasses were fixed in a chip holder at a place where ITO electrodes are exactly crossing each other to complete circuit for measuring electrical resistance. One ITO based TEER electrode is 4mm 2. The impedance was 8 measured in Ohms (Ωmm 2 ). LabVIEW based software was used to monitor the TEER data from the chip and the connectors were coupled to the ITO electrodes. A120 portable microscope was developed for real time monitoring of cell growth on transparent ITO based TEER sensor printed chip, A commercially available Plan Achromatic Objective 121 (AmScope TM) with 10X magnification power, a white LED for light source, and a SCMOS series 122 USB2.0 eyepiece camera (ToupTek TM) were assembled in a 3D printed assembly. A blue wavelength 469±17.5nm filter was used. A camera control software was used for high speed visualization of the images and video processing ToupView (ToupTek TM).
pH sensor was developed for real-time pH monitoring of media. A white LED was inserted in a 3D printed assembly with a photodiode and an optical filter. The pH measurement was calculated on the principle of change in light intensity. A media carrying biocompatible microfluidic, extremely transparent, tube was passed through the sensor. Sensor was programmed to measure the minor discrepancies in color of media with changes in phenol red color due to the acidification of media with time. An Arduino microcontroller was used to quantify an optical signal. To calibrate and characterize the optical pH sensor standard pH media samples ranging from 6.0 to 8.0 were used , calibrations of real time microscope, pH and TEER sensors have mentioned in our previous paper (22). A peristaltic pump was connected to the chip for constant media circulation to mimic dynamic conditions. The shear stress on the cell monolayer induced by media was calibrated to mimic the alveolar environment. The fluid shear stress in in-vivo human lung physiology has been characterized as 8 dyne/cm2 (2). The media flow rate was regulated at 80μl/ml to maintain the 8 dyne/cm2 shear stress upon the monolayer of Alveolar epithelial cells.
The media shear stress induced in the microchannel of the designed chip was calculated by the use of following equation (23); τ= 6μQ/ (wh^2) In this equation, μ represents the viscosity of the media, Q represents the media flow rate, w represents the width and h represents the height of the microfluidic channel.
Optical pH sensor was attached to measure real-time pH before the media inlet.
Connectors were immobilized upon glass chip with epoxy raisin through which tubing was connected to chip for the media circulation controlled by peristaltic pump.

Microfluidic cell culture maintenance
Human pulmonary alveolar type-I and type-II cells (Cat#3200) were purchased from science cell and revived according to manufacturer's protocol, Poly-L lysine (Sigma Aldrich) was coated on T-25 flask (Corning) at concentration of 2μg/cm 3 incubated at 37°C overnight and flask was rinsed before adding culture medium containing Alveolar epithelial cells medium (AEpiCM, Cat. #3201) minimal eagle medium, Epithelial growth supplement(5ml), 10% FBS ,1%pencilin streptomycin solution, with 5% carbon dioxide at 37°C. Before seeding cells on chip bottom glass was sterilized for 1 hour in UV and ECM Collagen solution type-I from Rat tail (Sigma Cat # C3867) was coated at concentration of 10μg by using 0.01% solution in DPBS overnight. At reaching confluency <90 % at passage 2 Human pulmonary alveolar type-I and type-II cells at density of 3.27×10 5 were seeded on to the chip initially cells were allowed to adhere to the ECM coated channel of the chip for 4 hours then chip was introduced to the microfluidic platform for continuous circulation of media with help of peristaltic pump at 120μl/hour, TEER, pH sensors and microscope for real time monitoring were attached to the chip after assembling the chip components after 12 hours media flow was increased to 80μl/min, after 48 hours fresh media was added to media reservoir.

Exposure scheme of particulate matter:
After 4 days of stable culture of Human pulmonary alveolar type-I and type-II cells on chip evaluated by impedance values of the TEER sensor, pH sensor and Real time microscope 3 chips representing moderate (7.5μg/ml), poor(37.5μg/ml) and very poor(151μg/ml) conditions of particulate matter were allowed to exposed to particulate matter containing culture medium, for consecutive 8 hours daily for 5 days, to mimic daily exposure of particulate matter to human lung, was then exchanged with normal medium after exposure. All the chips were kept in same culturing conditions except different concentrations of particulate matter excluding control chip Fig. 2a.

Determination of Membrane barrier integrity:
Epithelial cell tight junctions' integrity was characterized by impedance values provided by in-house developed TEER sensor for Real time monitoring. Alveolar type I-II cells were

DCFDA assay for ROS analysis
As a consequence of cell damage and functional impairment activated defense mechanism release production of intracellular Reactive Oxygen Species (ROS) and anti-oxidant species, pro-and anti-inflammatory cytokines, and induce genotoxicity (25)(26)(27). ROS are responsible for mitochondrial damage and for making cellular environment more acidic environment which leads to the incidence of chronic inflammation and cancer long term exposure to the xenobiotic and air pollutants are responsible of many respiratory diseases such as COPD and asthma. For ROS evaluation on exposure to particulate matter we have analyzed three concentrations of particulate matter after four days of exposure along with normal healthy control and All the chips were washed with PBS and the cells were

Analysis of cytokines release and ROS representing diseased conditions
Our particulate matter model on chip mimicking in-vivo diseased conditions was characterized by complete information of cytokine signaling. Neutrophil uptake increases on the onset of inflammation in airway cells so here is the list of cytokines produced as consequence of allergy and asthma TNF-α, IL-6, IL-13, Mucin and Reactive oxygen species(ROS) detection is the indicator of incidence of asthma due to prolonged exposure of particulate matter to airway cells, Presence of IL-13 provide data about the incidence of COPD, TNF alpha , for oxidative stress and ROS production leading to apoptosis which ultimately cause cancer in the respective tissues. Mucus metaplasia and mucus hypersecretion in human lung airway are pathological changes which occurs on the onset of a severe respiratory disease asthma. These are associated with the CD4 + Th2 type of immune response activation in the lung. This immune response is evaluated by by IL-13 secretion, with minimal production of the Th1 type of cytokines (e.g. IFNγ) (28). The destiny of effector CD4 + T cells is decided by the cytokine environment, which is one of the most important factors, so that the local immune response is affected by cytokines production by lung physical foundations. Although, lung epithelial cells are not part of the 13 immune system but also contribute to the type of immune response by secreting explicit cytokines. One of the cytokines that is produced by lung epithelial cells is IL-6 (29, 30) IL -6 is the cytokine that is produced by lung epithelial cells and asthmatic patients data provide relatively increased secretion of IL-6 as compared to healthy controls (31), (32).In response to PM exposure IL-6 and tumor-necrosis-factor alpha (TNF-α) key intermediaries of the inflammatory response of lung epithelial cells in many in-vivo, in-vitro and human sample based studies(6-8).
In current study TNF-α, IL-13, IL-6 and Mucin were selected as biomarkers for asthma and COPD all the cytokines were detected in media samples collected after every 24 hours from all control and experimental media reservoirs, TNF-α, IL-13, IL-6 ELISA was performed by ELISA kits by standard protocol of manufacturer and ELISA for mucin was performed by sandwich method (33).

Characterization of Particulate matter
Particulate matter (PM-10) include organic and inorganic components named ERM-CZ 100 and ERM-CZ120, purchased from European joint research commission Mainly contain PAH's, Dioxins, PCB's and Zinc, cadmium, mercury, cobalt, SiO2 respectively, particle size of Particulate matter is less than 10μm and characterized by Scanning electron microscope. Particle size detected in under scanning electron microscopy was less than 10μm Shown in fig. 3a.

Impedance data demonstrating barrier integrity:
According to impedance data of our TEER sensor, values in positive control chip started to increase gradually with increasing barrier junctions and increasing confluency of the cells on chip recorded after half an hour interval throughout the experiment. Impedance data demonstrated that the epithelial cells junctions increase with increasing confluency and stable culture for four days at fully confluent monolayer value of impedance was recorded

Monolayer Membrane integrity and differentiated epithelium
Immunofluorescence analysis by confocal microscopy revealed that this platform has

Confocal imaging of Goblet cell hyperplasia and ciliated cells diminishing
Polymeric mucin MUC5AC is a low charge glycoform of MUC2 is major secretary protein in patients with chronic respiratory disease asthma, Mucins hypersecretion due to chronic inflammation results in airway diseases such as chronic obstructive pulmonary disease, and cystic fibrosis (CF). (34,35) Goblet cell hyperplasia occurs when ciliated cells become unable to maintain airway homeostasis by shutting down mucociliary clearance and macrophage activation and this loss leads to chronic pulmonary disease Fig. 6

ROS estimation
Cellular injury occurs in lung cells when exposed to particulate matter (PM) (36). Cr, Co, Ni, Mn, Zn, Cu, and, Fe are the most commonly found elements in airborne PM (37). IL-6 is a cytokine has been considered as biomarker of inflammation rather than a regulatory cytokine with ability to modulate immune responses (45). In response to allergen stimulation to lung airway, IL-6 is released from lung epithelial cells as immune response. (30) Recently IL-6 is involved in adaptive immune response in the differentiation of effector CD4+T cells, Particularly it has function in suppressing Th1 and thus induce TH-2 differentiation of CD4+T cells via independent cytokine regulatory pathways (46). Many Release of mucin is linked to IL-13, Increase in concentration of mucin release from alveolar cells is proof of defective function of the ciliated cells which are involved in the mucociliary clearance Mucin ELISA graph is augmenting the confocal micrographs and validate our platform for recapitulating human physiology at alveolar level by showing increasing concentration of Mucin at high concentrations of particulate matter and for long time exposure, such biomarker could provide incidence of many diseases in our automated and sophisticated organ on chip platform. IL-6 is a proinflammatory cytokine produced in asthma and COPD conditions and is related to acute impaired lung function (50). In our data IL-6 concentration showed gradual increase in the production of this cytokines and this is associated with evidence of asthma patients sputum samples as published by a study(51) TNF-α is expressed by increasing ROS production in airway epithelium, Oxidative stress and free radical production occurs as inflammatory response of associated to PM exposure reported previously in many studies (52-54) Several evidences have provided information of validity of our platform for successful toxicological analysis and reconstituting organ pathophysiology, future target of our group is to study the complex tissue physiology with integration of embedded sensors. Graph representing mucin levels in Fig. 8c showing increase in mucin release in supernatant is also comparable with confocal micrographs.

Statistical analysis
The data are expressed as mean ± standard error of the mean (SEM). For statistical analysis, the experimental data was compared to their controls. Using full-factorial ANOVA with Tukey's multiple comparison. All statistical analysis was performed by Microsoft Excel. A p value of < 0.05 was considered to be significant (*) and < 0.01 as highly significant (**). All the experiments were performed in triplicates.

Conclusion
Technological advancements in organ on chip technology is offering robust and efficient solutions for drug discovery and therapeutic evaluation. These solutions are in demand now a days. Our microfluidic alveoli on chip device have potential to mimic normal human 22 physiology as well as environmental and pathological conditions. According to the results obtained from this study, have validated that this well-equipped system with reconfigurable microchip, portable microscope for Real time monitoring of cells, system integrated sensors pH and TEER sensors would help in the development of better human physiological and pathological mimetics. High metabolic activity of the alveolar epithelial cells was analyzed in this dynamic fluid flow environment as compared to the static culture conditions. High levels of cytokines were observed in this device due to optimized recirculation media conditions. The primary challenge of building an organ on chip is to duplicate in vivo environment for housing an organ with ease of handling and robustness.
Second challenge of organ on chip technology is manufacturing of cost effective, toxicity evaluation with help of system embedded sensors. This device has provided the preliminary data for making this breakthrough in OOC technology. Future perspective of our group is to make complex tissue mimetics of human for toxicity testing and drug

Competing interests
Authors declare that they have no competing interests

Authors' contributions
Faiza Jabbar and Kim Youngsu designed and performed the experiments, did the analysis 25 of all obtained data and drafted the manuscript. Arun helped in characterizations, Afaque managed the implementation of sensors. Choi Kyung Hyun, Lee Sang Ho, Cho Young Jae supervised the study and revised the manuscript. All authors read and approved the final manuscript.