Microfluidic Coculture Device for Monitoring of ... - ACS Publications

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A microfluidic co-culture device for monitoring of inflammation-induced myocardial injury dynamics Xiaoni Ai, Wenbo Lu, Kewu Zeng, Chun Li, Yong Jiang, and Peng-Fei Tu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04833 • Publication Date (Web): 13 Mar 2018 Downloaded from http://pubs.acs.org on March 14, 2018

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Analytical Chemistry

A

microfluidic

co-culture

device

for

monitoring

of

inflammation-induced myocardial injury dynamics

Xiaoni Ai†, Wenbo Lu†, Kewu Zeng†, Chun Li‡, Yong Jiang*†, Pengfei Tu*†

Affiliations †

State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical

Sciences, Peking University, Beijing 100191, China ‡

Modern Research Center for Traditional Chinese Medicine, Beijing University of

Chinese Medicine, Beijing 100029, China.

*Corresponding authors: Phone/Fax: +86-01-82802750; E-mail: [email protected], [email protected].

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Emerging awareness of cardiac macrophages’ role in inflammation after myocardial infarction indicates that overabundant pro-inflammatory macrophages induce accentuated myocardial injury. The investigation of macrophages-cardiomyocytes interaction and inflammation-induced dynamic damage in myocardial infarction, especially in a spatiotemporally controlled manner, remains a huge challenge. Here, we developed an in vitro model using a microfluidic co-culture system to mimic inflammatory cardiac injury. To our knowledge, on-chip pathological models focused on inflammation-induced myocardial injury have not been reported. The device consists of two sets of thin interconnecting grooves that isolate heterogeneous cells spatially but maintain their soluble factors communication. The mass transportation is visually characterized and the complete diffusion reaches equilibrium within 100 seconds. We investigate the dynamic interaction between the macrophages and the cardiomyocytes in the spatiotemporal controlled microenviornment, mimicking a key aspect of the in vivo pathophysiological process. The results show that the activated macrophages induce time-lapsed apoptotic responses of the cardiac cells and damage mitochondria membrane integrity. The anti-inflammatory and cardio-protective effects of quercetin were explored on the chip. The extent of caspase-3 activation is asynchronous in the individual cardiac cells, suggesting the different apoptosis dynamics. We further demonstrate that the mechanism of activated inflammation is associated with the up-regulation of several inflammatory cytokines and NF-κB pathway. Thus, the developed microfluidic co-culture device provides a useful tool on real-time monitoring of inflammatory response for myocardial disease and holds potential for anti-inflammatory drug screening.


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INTRODUCTION Myocardial infarction (MI) is one of the leading causes of morbidity and mortality in the world. Significant loss of functional cardiomyocytes leads to deteriorated heart failure.1,2 Although ischemia/hypoxiainduced pathological injury of myocardial cells has been more concerned, the failure therapies indicate that other pathological mechanisms may be critically involved in the MI process.3,4 In the past decades, evidence suggests that MI triggers an intense inflammatory reaction.5,6 Early activation of inflammatory cytokine pathways might be important for the clearance of dead cells and the stimulation of downstream reparative cascades. However, prolonged and excessive induction of inflammatory signaling is associated with accentuated myocardial injury.7 In this complex inflammatory process, macrophages secrete abundant inflammatory mediators that play important roles in the modulation of inflammatory response, recruitment of pro-inflammatory cells and eventually promoting cardiac apoptosis.8,9 Macrophages are abundant in healthy myocardium and after the process of MI. Though rapid immunological progress in various organ systems, little is known about macrophages in the heart.8 Recently, the anti-inflammatory therapeutic approaches in the management of heart failure have been more concerned. To understand the mechanism of inflammation-induced heart failure, examining the interaction between macrophages and cardiomyocytes during the MI process is considered as a matter of urgency. Conventional in vivo (animal-based) and in vitro (petri dish-based cell culture) models have their own shortcomings. The in vivo model is under uncertain cues and difficult to monitor inflammatory kinetics in real time.10-12 Recently, we established an inflammatory injury model in vitro by lipopolysaccharide



(LPS)-stimulated macrophage-conditional medium.13 The conditional medium containing 1 µg/mL LPS were collected from the macrophage cell supernatants after 24 h incubation, then stimulated the cardiac cells for another 24 h to induce inflammatory injury. However, the reported in vitro inflammatory injury model faces the disadvantages of ignoring the reliable spatiotemporal construction of cell-cell cross talk dynamics, endpoint detection, laborious and time-consuming. It is thus necessary to develop a system that enables a spatiotemporally controlled investigation on the dynamic communication between macrophages and cardiomyocytes during the myocardial inflammatory injury process. Microfluidics is a powerful methodology for spatial and temporal controlling cellular microenvironment and investigating cell-cell signaling process.14-16 Various co-culture techniques have been developed for spatiotemporal manipulation of multiple types of cells, including surface pattern,17,18 cell printing,19 geometrical confinement,20-22 microvalves,23 multiple-layered sandwich,24,25 hydrogel confinement,26-28 et al. Moreover, a few attempts have been made on construction of cardiac microtissues and controlling their microenviornment by microfluidic co-culture systems. The myocardium hosts multiple types of cells, such as fibroblasts, myocytes, endothelial cells, macrophages, and nerve cells. The cellular dynamic communication takes place through chemical, mechanical and electrical signals, and precisely regulates heart function and development.29 Several groups have developed microfluidic systems to mimic vascularization in myocardium by endothelial cells and cardiomyocytes co-culture.30-32 The ischemic stress induced cardiomyocytes to secrete chemokines that subsequently trigger fibroblasts migrating toward the ischemic region.33 Biomaterials create three-dimensional (3D) physiological microenvironment for cardiac tissue modeling.34,35


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MI models were also established through microfluidics, however, most models were based on hypoxia-induced, oxidative stress-induced or reperfusion-induced myocardial injury.36,37 To the best of our knowledge, none of these on-chip pathological models have focused on dynamic inflammation-induced myocardial injury in the MI process. Here, we have developed a co-cultured microfluidic platform for investigating inflammation-induced myocardial injury dynamics. We designed and fabricated two sets of thin interconnecting grooves that decouple a central cell culture chamber from two side microchannels. Furthermore, the activated macrophages and the cardiac cells were separately introduced to their individual channels for mimicking continuous inflammatory process. Next, the inflammation-induced myocardial injury dynamics, namely, cell viability, nuclei morphology, caspase-3 activation, and membrane potential of mitochondria, were recorded and quantified on the chip. We further investigated the protective effect of quercetin on the cardiac cells. Finally, the inflammatory injury mechanism was elaborated by the release of multiple inflammatory mediators and NF-κB nuclear translocation. EXPERIMENTAL SECTION Materials and reagents. Negative photoresist (SU-8 2050 and 2007) and developer were purchased from MicroChem (Newton, MA, USA). Polydimethylsiloxane (PDMS) and curing agent were purchased from Dow Corning (Midland, MI, USA). Fetal bovine serum (FBS), Dulbecco’s Modified Eagle medium (DMEM), antibiotics, and trypsin were from Hyclone (MA, USA). Lipopolysaccharide (LPS; from Escherichia coli, serotype 055:B5), Hoechst 33342 and poly-L-lysine coated glass slide were purchased from Sigma-Aldrich (St Louis, MO, USA). Alexa Fluor 488 donkey anti-mouse IgG



(H+L), DyLight 594 Goat anti-rabbit IgG (H+L) and LIVE/DEAD Viability/Cytotoxicity Kit were purchased from Invitrogen (Carlsbad, CA, USA). The NucView 488 caspase-3 assay kit was purchased from Biotium, Inc. (Hayward, CA). All solutions were prepared using ultrapurified water supplied by a Milli-Q system (Millipore). Fabrication of microfluidic device. The PDMS microfluidic chip was designed in AutoCAD software and fabricated using two-layer soft lithography fabrication technology.38 The first layer with the interconnecting grooves was generated by spinning a 7 µm-thick negative photoresist (SU-8 2007) onto a cleaned silicon wafer. Then, the three main channels with 120 µm thick were patterned by spin coating the SU-8 2050 onto the mentioned above thinner layer. After the exposure through a high-resolution transparency mask (25 000 dpi) and development, a master of desired microstructure was obtained. Next, 10:1 mixture of PDMS prepolymer was poured onto the master and then cured at 70 ºC for 2 h to achieve a fully cross-linked PDMS replica-molded. The piece of PDMS was plasma bonded to a poly-L-lysine coated glass slide irreversibly after punching holes for inlets and outlets (Harrick Plasma, PDC-32G). Cell culture. Murine macrophage cell line (RAW264.7) was purchased from Peking Union Medical College, Cell Bank (Beijing, China) and Rat myocardial H9c2 cells was obtained from Institute of Biochemistry and Cell Biology (Shanghai, China). Cells were cultured in DMEM medium supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37 °C under a humidified atmosphere of 5% CO2 and 95% air. Characterization of mass transportation with fluoresceins. To evaluate the time-lapsed mass diffusion among the microchannles, 1 mM fluorescein sodium salt


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solution or 50 µg/ml FITC labeled dextran (Mw=40 kDa, Sigma Chemical Co., St. Louis, MO) was introduced into the two lateral channels as a model cue, while kept the central channel filled with DI water at the same time. Sequences of optical images of replacing the DI water with the fluorescent dyes were captured over time. The images acquired at a speed of 1 Hz using a fluorescence microscope with a 10× objective to achieve fluorescence intensity profile. Co-culture of macrophages and cardiomyocytes on the microfluidic device. The microfluidic device was sterilized with UV light for 30 min before rinsing twice with PBS. The supplemented DMEM medium was firstly loaded into the two adjacent channels to provide a gravitational difference between the central and adjacent channels, meanwhile, supplied the culture nutrients for H9c2 cells. Then a 3 µL H9c2 cell suspension at a density of 2 × 106 cells/mL was added into the central channel inlet, and a negative pressure was generated at the outlet by pipet suction until the cells were filled the channel. The device was then placed overnight at 37 ºC under a humidified atmosphere of 5% CO2 and 95% air. The next day, the 6 h LPS (1 µg/mL) stimulated or normal RAW264.7 cells with a density of 1 × 107 cells/mL were digested with 0.25% trypsin, washed by PBS, and then were seeded onto the two lateral channels by pipet suction. Following, fresh FBS-free DMEM medium with or without 20 µM quercetin was changed every 8 h and experiments were carried out for co-culture of different periods (24 h, 48 h, 72 h). The cells on the chip could be retrieved by trypsin digestion for culture and future analyses. On-chip cell staining. The H9c2 cell viability assessment was performed using a Live/Dead assay kit after 24 h co-culture. Briefly, the Live/Dead assay kit containing 2



µM calcein AM and 4 µM ethidium homodimer-1 (EthD-1) was introduced into the cell culture chamber and incubated at 37 °C for 40 min. Then, fresh DMEM medium was introduced for final rinse. For a clear visualization of nuclear morphological changes, the H9c2 cells were stained with 4 µg/mL Hoechst 33342 staining at 37 °C for 20 min. After washing twice with fresh DMEM medium, apoptosis rate was calculated as the number of Hoechst 33342-positive cells divided by the total number of cells. At least 300 cells were counted per group, and each assay was carried out in triplicate. The caspase-3 activity of the H9c2 cells was analyzed to evaluate the inflammation-induced injury dynamics of the cardiac cells. 1 µM NucView 488 caspase-3 substrate was introduced into the cell chamber for monitoring the caspase-3 activity in real time. The mitochondrial membrane potential (MMP) was assessed using a JC-1 detection kit (Beyotime Biotechnology, Jiangsu, China) according to the manufacturer’s instructions. The solution of JC-1 was loaded into the cell culture microchannels and incubated for 20 min at 37 °C before rinsing. Off-chip measurement of cell viability. Cell Counting Kit-8 was used to evaluate the cytotoxic effect. Approximately 1 × 104 of RAW 264.7 cells and 5 × 103 of H9c2 cells were seeded into 96-well plates. The RAW 264.7 cells were treated with or without LPS (1 µg/mL) for 6 h, washed by PBS, after which the medium containing the LPS was replaced by fresh DMEM medium without FBS. After 24 h of incubation, the fresh DMEM medium without FBS was used as conditional medium. The H9c2 cells were then stimulated by the conditional medium with or without 20 µM quercetin for 48 h at 37 °C


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in a 5% CO2 incubator. Cells were not treated with LPS or quercetin served as a control well in the experiment. Afterwards, the CCK-8 working solution was added to the each well for 2 h at 37 °C, and then absorbance was detected at 450 nm by a microplate reader. The data were expressed as the mean percentage of the absorbance in treated vs. control cells. The value of the control was set at 100%. All experiments were performed in triplicate. Nitric oxide (NO) assay, ELISA assay and immunofluorescence assay. NO production was determined from RAW264.7 cell supernatants by a NO assay kit. The RAW264.7 cells were seeded in 48-well culture plates with a density of 5, 10, 20, 40 × 104 cells per well, respectively. The cells were treated with or without LPS (1 µg/mL) for 6 h, washed by PBS, then replaced by fresh DMEM medium without FBS (conditional medium) for 24 h treatment. The NO released from the cells was quantitated from the conditional medium on Griess method. The optical density was measured in 96-well plates at 540 nm, and sodium nitrite was used as a standard curve. ELISA assay was used for the measurement of other inflammatory cytokines released from the activated RAW264.7 cells. The 24 h conditional medium were collected and centrifuged at 4 °C, 16000 rpm for 10 min. The supernatants were then used for detecting tumour necrosis factor alpha (TNF-α), interleukin-6 (IL-6), PGE2 and MCP-1 by ELISA kits. For intracellular staining of NF-κB p65, the LPS stimulated or normal RAW264.7 cells were seeded onto the microchannels. The cells were fixed with 4% paraformaldehyde for 20 min, followed by permeabilization (0.5% Triton X100 in PBS) and blocking (5% BSA in PBS) for 30 min at room temperature. The cells were incubated



overnight with a primary antibody against NF-κB (1:250) at 4 °C and then with a secondary antibody (Alexa Fluor 594, 1:500) for 1 h at room temperature. The cells were further stained with DAPI (5 µg/ml in PBS) for 20 min at 37 °C. Imaging and data analysis. Phase contrast and immunofluorescence staining images were captured using an inverted fluorescence microscope (OLYMPUS IX73, Tokyo, Japan) equipped with a DP80 digital camera and mercury lamp (Olympus, U-RFLT50). Image-Pro Plus 6.0 software (Media Cybernetics, Inc.) and GraphPad Prism 6 (GraphPad, Inc.) were used for image and statistical analysis, respectively. All data were presented as mean ± S.D. of at least three independent experiments. One-way analysis of variance (ANOVA) was used to determine the statistical significance of the different treatment groups. A value of p

Microfluidic Coculture Device for Monitoring of ... - ACS Publications

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