A heart-on-a-chip platform for online monitoring of contractile behavior via digital image processing and piezoelectric sensing technique

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A heart-on-a-chip platform for online monitoring of contractile behavior via digital image processing and piezoelectric sensing technique Mutsuhito Sakamiya a,b,c, Yongcong Fang a,b,c, Xingwu Mo a,b,c, Junying Shen a,b,c, Ting Zhang a,b,c,∗ a

Biomanufacturing Center, Department of Mechanical Engineering, Tsinghua University, Beijing 100084, PR China Biomanufacturing and Rapid Forming Technology Key Laboratory of Beijing, Beijing 100084, PR China c “Biomanufacturing and Engineering Living Systems” Innovation International Talents Base (111 Base), Beijing 100084, PR China b

a r t i c l e

i n f o

Article history: Received 22 May 2019 Revised 7 September 2019 Accepted 3 October 2019 Available online xxx Keywords: Heart-on-a-chip Biofabrication Contractile behavior Image processing Piezoelectric sensing

a b s t r a c t Heart-on-a-chip devices have recently emerged as a viable and promising model for drug screening applications, owing to its capability of capturing important biological and physiological parameters of cardiac tissue. However, most heart-on-a-chips are not developed for online and continuous monitoring of contractile behavior, which are the main functional characteristics of cardiac tissue. In this study, we designed and investigated on a heart-on-a-chip platform that provides online monitoring of contractile behavior of a 3D cardiac tissue construct. The contractile behavior include contraction force, frequency, and synchronization. They can be evaluated by an image processing system and a piezoelectric sensing system simultaneously. Based on the deformation of a micro-pillar array embedded within the 3D cardiac tissue upon subjected to cardiac contraction, the image processing system provides in situ multi-site detection of the contractile behavior. At the same time, the piezoelectric sensing system measures the contractile behavior of the entire cardiac tissue construct. A 3D cardiac tissue construct was successfully fabricated. Then the heart-on-a-chip platform was validated by applying various motion patterns on the micro-pillars, which mimicked the contraction patterns of the 3D cardiac tissue. The drug reactivity of the 3D cardiac tissue construct after a treatment of isoproterenol and doxorubicin was evaluated by measuring the contractile behavior via the image processing and the piezoelectric sensing systems. The results from the drug reactivity provided by both these measurement systems were consistent with previous reports, demonstrating the reliability of the heart-on-a-chip platform and its potential for use in cardio-related drug screening applications. © 2019 IPEM. Published by Elsevier Ltd. All rights reserved.

1. Introduction Cardiovascular disease is the leading cause of death worldwide [1]. Recent studies have shown that it has accounted for one-third of deaths worldwide over the past 25 years. This proportion is still increasing, and is further exacerbated by global ageing [2,3]. Drug development is a time-consuming and expensive process. On average, developing a new drug takes more than 10 years and costs approximately 1.5 billion USD [4]. However, the failure rate of the drug candidates remains high (approximately 60%), owing to the lack of efficiency and safety during the drug screening process [5]. In addition, drug induced cardio-toxicity is a reality that impacts the quality of life. Statistics show that cardio-toxic drugs

∗

Corresponding author. E-mail address: [email protected] (T. Zhang).

account for 45% of the total post-marketing drug withdrawal from the market [6]. Hence, improving the efficacy of cardiovascular drugs and ensuring the safety of new drugs are key areas of focus during the drug screening process. Conventional methods used for drug development, including two-dimensional static cell culturing and animal models, are either mono-layer cultured or unsuited to imitate the human physiology [7,8]. Therefore, there is a pressing demand for developing advanced methods that can precisely mimic the human physiology and pharmacology. With the emergence of biofabrication, microfluidic and biosensing techniques, research in the area of ‘organ-on-a-chip’ is attracting great attention. Heart-on-a-chip, as one of the most advanced models for cardio-related drug screening in vitro, has recently emerged as a promising approach to drug screening, because of its potential to capture cardiac functions [9–11]. For example, biofabricated 3D cardiac tissue constructs

https://doi.org/10.1016/j.medengphy.2019.10.001 1350-4533/© 2019 IPEM. Published by Elsevier Ltd. All rights reserved.

Please cite this article as: M. Sakamiya, Y. Fang and X. Mo et al., A heart-on-a-chip platform for online monitoring of contractile behavior via digital image processing and piezoelectric sensing technique, Medical Engineering and Physics, https://doi.org/10.1016/j.medengphy. 2019.10.001

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with human induced pluripotent stem cell-derived cardiomyocytes is a viable model for personalized drug screening. Vascularized cardiac tissue can enable the delivery of nutrients and removal of metabolic products, which is vital to increase the viability of cardiomyocytes [12–16]. Heart-on-a-chip integrated with microfluidic techniques can provide flow distribution that simulates both real biology and physiology in vivo [17–19]. Moreover, techniques based on a combination of 3D printing and use of bioinks to biofabricate a biomimetic cardiac tissue construct directly are also promising approaches to achieve the construction of tissue models in vitro [20,21]. For cardiac tissues, contractile behavior are some of the main characteristics of the cardiac functions, which can help evaluate the drug efficacy and safety during the drug screening process [22]. Contractile behavior representative of cardiac systolic functions include contraction frequency, force, and synchronization over time. Currently, a variety of studies to assess the contractile behavior with heart-on-a-chip have been published [24–28]. Detecting the deformation of flexible materials upon cardiac contractions is considered mainstream approach for evaluating the cardiac contractile behavior. For example, cardiomyocytes are cultured on the top part of a thin film, composed of filaments to guide the cardiomyocytes to form anisotropic laminar tissues. The contractile behavior is measured by an integrated strain sensor through determining the wire stretching [24]. Pillar based single cell contraction force measurement is a similar approach that uses flexible materials. Isolated single cardiomyocytes are seeded on top of a replicated polydimethylsiloxane (PDMS) pillar array(diameter = 3 μm), and the contraction force can be derived from measuring the displacement of the pillar [25]. Another promising approach to measure the contractile behavior is using the micro-cantilever of an atomic force microscope (AFM) as a probe to detect the deformation of the contracting cardiac tissue [26]. Furthermore, image processing has emerged as an efficient method for its non-invasive detection and potential to realize global analysis of contractile behavior [27]. However, most heart-on-a-chip devices are not developed for online and continuous monitoring of the contractile behavior for 3D tissue constructs [29]. For increasing the throughput of the heart-on-a-chip, it is of great significance to develop the same with online monitoring function for the 3D tissue. Here, we designed and investigated the performance of a heart-on-a-chip platform that provides online monitoring of the contractile behavior of 3D cardiac tissue constructs. Firstly, a 3D cardiac tissue construct was fabricated and cultured in the culture chamber. A micro-pillar array was embedded within the 3D cardiac tissue so that in situ multi-site contractile behavior of the cardiac tissue could be evaluated by capturing the deformation of the micro-pillar array by a digital microscope, followed by digital image processing. A piezoelectric sensing system with a Polyvinylidene Fluoride(PVDF) film combined with the micro-pillar array was designed to simultaneously evaluate the contractile behavior of the entire cardiac tissue based on the deformation of the micropillars. To validate the feasibility of the heart-on-a-chip platform, various motion patterns provided by controlled thin titanium rods were applied to the micro-pillar array to mimic the contraction patterns of the 3D cardiac tissue construct. The contractile behavior of the 3D cardiac tissue construct following a treatment with isoproterenol and doxorubicin were measured and evaluated by the digital image processing and the piezoelectric sensing systems. 2. Materials and method 2.1. Design of the heart-on-a-chip platform This heart-on-a-chip platform mainly consists of a culture system, sensor system, and data acquisition system, as illustrated

in Figure 1a. The 3D cardiac tissue construct was fabricated with a polydimethylsiloxane (PDMS) micro-pillar array embedded within the culture chamber. The culture chamber was perfused with culture medium through a peristaltic pump. The data acquisition system comprises an image processing system with a digital microscope and a piezoelectric sensing system with a charge amplifier, a low pass filter, and a PVDF film. The digital image processing system and the piezoelectric sensing system evaluate the contractile behavior of cardiac tissue (Fig. 1b). Upon cardiac tissue contraction, the deformation of the micro-pillar array is captured by the digital microscope. The micro-pillars with the PDMS membrane are distorted, and the resultant stresses are exerted on the PVDF film, which is connected to the piezoelectric sensing system. The signals and data are then transmitted to a computer and analyzed further with our custom-designed code R R written in MATLAB software (The MathWorks , Natick, MA). In the image processing system, the displacement and bending frequency of the micro-pillar array, which represent the contractile behavior of the 3D cardiac tissue construct, are calculated by the MATLAB code. In brief, the RGB images (1280 × 960 pixels) of the captured video are converted to binary images using an RGB color separation method. The centroid of each pillar is determined by the captured connected region and reconstruction in each image frame. For each pillar, the displacement is estimated from the spatial shift of centroids between each frame and the first frame (reference frame). The displacement curve with time is plotted, and from these data, the bending frequency of the pillars, as well as the equivalent force, is calculated. Based on force-deflection mechanics and pure bending theory, the equivalent force (F) on the micro-pillar can be calculated as follows [30]:

F=



h3 3EI

+

 

d2 (1+γ )h 4EI

+

h2 2EI

(H − h )

Where  is the displacement of the centroid of pillar; h is the height at which equivalent force of the cardiac contraction is applied; H is the height of the pillar; d is the diameter of the pillar; and E and γ are the Young’s modulus and Poisson’s ratio of PDMS, respectively. In the piezoelectric sensing system, the voltage output of the piezoelectric sensor is recorded and processed to assess the contractile behavior of the 3D cardiac tissue construct. Based on the piezoelectric effect, the output voltage (Vo ) of the sensor is described as follows:

Vo = A · k · h · F /



π · d4



Where k is the piezoelectric voltage constant of the PVDF film and A is a constant. 2.2. Heart-on-a-chip fabrication The micro-pillar array made of PDMS was fabricated by a molding technique (Fig. 1c–d). Firstly, a Poly(methyl methacrylate) (PMMA) mold (1.14 mm thickness) was fabricated with a laser cutting system (VLS2.30, UNIVERSAL, USA) to obtain a micro-hole array with a diameter of 0.4 mm. PDMS (Sylgard 184, Dow Corning, Midland, MI) was prepared with PDMS pre-polymer curing agent at a weight ratio of 1:10 and poured into the PMMA mold. PDMS was degassed in vacuum and then treated with spin coating technique to generate a thin PDMS membrane (30 μm) on top of the micro-pillars. The samples were later placed in an oven at 80 °C for 4 h. After peeling off each sample carefully from the mold, the micro-pillar array with the PDMS membrane was physically attached onto a PVDF film to fabricate a piezoelectric sensor, and covered with a culture chamber using an epoxy adhesive (Devcon 14,250). It should be noted that these two parts must be attached

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Figure 1. Heart-on-a-chip platform design and fabrication flow chart. (a) Schematic design of heart-on-a-chip platform consists of a culture system, sensor system, and signal acquisition system; (b) Detection of deformation of micro-pillar array via digital image processing and piezoelectric sensing techniques; (c) Schematic of the replica molding process of the heart-on-a-chip; (d) Photograph of the heart-on-a-chip platform and magnified image of the micro-pillar array structure.

together tightly with no air between them to ensure accurate detection by the piezo sensor. In other words, the piezoelectric sensor consists of the tightly bonded micro-pillar array with the PDMS membrane and the PVDF film. For the purpose of image processing, the top side of each pillar was marked in green color with an oil-free green fluorescent pen (Juice Paint-light green, Pilot, Japan) as shown in Figure 1d. The whole heart-on-a-chip device was autoclaved and sterilized before use.

2.3. Simulation of tissue contraction To validate the feasibility of the heart-on-a-chip platform, the micro-pillar array was pushed and bent by thin titanium rods (diameter at 0.8 mm) to mimic the contraction pattern of the 3D cardiac tissue. The motion of the titanium rods was controlled by a motion platform (KSA200-11-X, China) to achieve various patterns of motion of the micro pillars. For the image

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processing system, a predefined pattern of cyclic reciprocating motion with varied amplitude (0.5, and 0.7 mm) and frequency (0.35, and 0.5 Hz) was applied on the micro-pillar array to mimic the synchronous contraction of the biofabricated cardiac tissue. Random motion was also applied to mimic the chaotic beating of the cardiac tissue. Furthermore, two adjacent pillars were applied with the same motion to demonstrate the stability of the platform. For the piezoelectric sensing system, due to the accuracy of acquisition, cyclic reciprocating motion with higher input amplitude (0.7, and 2 mm) and frequency (0.5, and 0.7 Hz) was applied. 2.4. Fabrication and culture of 3D cardiac tissue construct Cardiomyocytes were harvested from neonatal rat hearts (1 to 2 days old) using a commonly used protocol as previously reported [31]. Briefly, the hearts were harvested, and ventricular tissues were isolated, minced, and digested overnight in a trypsin solution without EDTA (0.05% wt; Sigma, Germany) at 4 °C overnight. After trypsin digestion, the cells were serially digested using Type II collagenase solution (1 mg/mL; Worthington Biochemical Corp., USA) until they dissociated into a single cell-suspension. The dissociated cells were then collected, filtered with a strainer (70 μm), and centrifuged at 1200 rpm for 5 min to yield isolated cell pellets. These cells were plated and incubated for 60 min in tissue culture flasks to enrich the cardiomyocytes. The cells that remained unattached to the flask were used. A 3D cardiac tissue construct was prepared by mixing freshly isolated cardiomyocytes from neonatal rats with R collagen Type I from rat tail (0.6 mg/ml, Cellmatrix ; Japan) and Matrigel (20% v/v, BD Bioscience; Germany) at 5.0 × 107 cells/ml. The pH was neutralized by titration with NaOH (0.02 N). The reconstitution mixture was pipetted into the culture chamber and incubated for 30–45 min at 37 °C and 5% CO2 . Later, 500 μl of serum containing culture medium (H-DMEM, 10% FBS, 100 U/mL penicillin, and 100 g/mL streptomycin) was added to each chamber. The culture medium was replaced every 12 h. 2.5. Immunohistochemical staining After 4 days of culture, the 3D cardiac tissue construct was fixed in situ with 4% paraformaldehyde, rinsed with PBS, permeabilized with 0.1% Triton X-100, and blocked with 2% BSA. Cells were then stained with primary antibodies — sarcomeric alpha actinin (mouse monoclonal antibody) and connexin-43 (rabbit monoclonal antibody) at 4 °C overnight and secondary antibodies — goat R anti-mouse IgG H&L (Alexa Fluor 488) and goat anti-rabbit IgG R H&L (Alexa Fluor 594) at ambient temperature for 1 h. Samples were then stained with DAPI (4‘, 6-diamidino-2-phenylindole) for 15 min before imaging. Confocal microscopy images were acquired using a laser microscope (LSM880, Zeiss), and image reconstruction of z-stacks was performed using the z-project function with the maximum intensity setting. All the antibodies were purchased from Abcam, USA and used as supplied, unless otherwise specified. 2.6. Calcium transient measurement After 4 days of culture, calcium transient was assessed by monitoring calcium dye fluorescence (Fluo 4-AM, Invitrogen) to evaluate the electrophysiological function of the fabricated cardiac tissue construct as previously reported [32]. Briefly, the samples were first washed with HBSS (Hank’s balanced salt solution, R Thermal Fisher ) to remove the residue serum from decomposing Fluo 4-AM. Then, 10 mM Fluo 4-AM in 0.1% Pluronic F-127 was added and incubated at 37 °C for 30 min. The sample was washed with HBSS again and further incubated for 30 min at 37 °C.

Time-lapse fluorescence images were recorded at 10 frames/s on a fluorescence microscope (Nikon Ti-E, Nikon). 2.7. Drug testing To evaluate the drug reactivity, and to demonstrate the stability and effectiveness of the platform, two drugs (isoproterenol and doxorubicin) were selected in view of their positive inotropic action and cardio-toxicity, respectively [23,33]. When the 3D cardiac tissue construct was cultured for 4 days and was beating synchronously, the two drugs were added separately. The isoproterenol (Abcam, USA) was added at a dosage of 5 μM and the sample was further incubated for 15 min to test positive for inotropic action on the 3D cardiac tissue. Doxorubicin (Beyotime) was added at a dosage of 100 μM and the sample was further incubated for 24 h to test for the cardio-toxicity of the 3D cardiac tissue. The contractile behavior of the 3D cardiac tissue following the treatment of the aforementioned two drugs were measured with image processing system and the piezoelectric sensing system. 2.8. Statistical analysis Statistical analysis was performed by Prism software (version 6.0) by GraphPad, with statistical significance set at p < 0.05 (∗∗∗ = p < 0.001). All experimental statistics were expressed as ‘mean ± standard deviation’. Two-tailed student t-test was used as specified in the figure captions. 3. Results and discussion 3.1. Heart-on-a-chip platform construction The heart-on-a-chip platform was successfully fabricated as shown in Figure 1d. The platform was composed of culture system, sensor system and data acquisition system. The culture chamber of the platform was made of PDMS with a micro-pillar array on the bottom. The culture chamber was perfused with culture medium through silicone tubes, which were connected to peristaltic pump. The piezoelectric sensing system was successfully developed in the platform for detection of stress exerted on the PVDF film due to the deformation of pillars. The digital microscope on top of the culture chamber captured the displacement of the micro-pillars upon cardiac tissue contraction. The images and voltage outputs of the piezoelectric sensing system were transmitted to a computer and further analyzed with our custom-designed MATLAB code. As mentioned earlier, the micro-pillar array, as the basic sensing unit, was fabricated through molding technique. The diameter, height, and center-to-center distance were the main parameters of the micro-pillar array, which would affect the efficiency and accuracy of the sensing system. A series of micro-pillar arrays were tested in this study. With due consideration to the thickness of the cardiac tissue and the pillar’s minimum bending force, a diameter of 0.4 mm and a height of three times the diameter were selected for the pillar dimensions. The culture chamber in this platform also provided a mold for the fabrication 3D cardiac tissue construct, ensuring the in situ monitoring of the contractile behavior of the cardiac tissue without transfer. Upon the formation of cell hydrogel, the micro-pillar array was integrated within the 3D cardiac tissue construct and worked as a sensing unit. The deformation of the pillars was captured by the digital microscope and the piezoelectric sensor simultaneously to evaluate the contractile behavior, namely contraction force, frequency, and synchronization. Thus, the heart-on-a-chip platform provided for an in situ continuous online monitoring of the contractile behavior of the fabricated 3D cardiac tissue.

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Figure 2. Feasibility validation of the image processing and piezoelectric sensing technique. (a) Various motion patterns provided by controlled thin titanium rods were applied to the micro-pillar array to mimic the contraction patterns of the 3D cardiac tissue; (b–d) The equivalent force over time measured through image processing at (b) Pillar 1, (c) Pillar 4, (d) Pillar 7; (e–f) Schematic of single input and dual input applied with titanium rods; (g) Voltage output recorded by piezoelectric sensing technique upon various inputs: (i) Single input, amplitude: 2 mm, frequency: 0.7 Hz; (ii) Single input, amplitude: 1.2 mm, frequency: 0.7 Hz; (iii) Single input, amplitude: 1.2 mm, frequency: 0.56 Hz; (iv) Dual input, amplitude: 2 mm, frequency: 0.7 Hz.

3.2. Feasibility validation To demonstrate the feasibility of the heart-on-a-chip platform, the image processing system and the piezoelectric sensing system were separately tested by applying various motions on the micro-pillar array, as shown in Figure 2. The micro-pillar array was pushed and bent by controlled thin titanium rods to mimic various contraction patterns of the 3D cardiac tissue. To test the efficiency and reliability of the image processing system, a predefined pattern of cyclic reciprocating motion with varied amplitude (0.5 and 0.7 mm), and frequency (0.35 and 0.5 Hz) was applied on the pillar arrays to mimic the synchronous contraction of the fabricated cardiac tissue. As shown in Figure 2b, the

equivalent force was regular and periodic in response to the cyclic reciprocating motion. Moreover, the amplitude of the equivalent force increased as input amplitude increased from 0.5 to 0.7 mm. Similarly, the frequency of the equivalent force increased as input frequency increased from 0.5 to 0.7 Hz. When applied with the same motion, pillar 1 and pillar 4 exhibited an identical pattern (Fig. 2c) of equivalent force variation over time, which demonstrated that the image processing system had good stability. A random motion was also applied on the pillar to mimic the chaotic beating of the cardiac tissue. As shown in Figure 2d, the pattern of equivalent force variation over time was irregular. Furthermore, by analyzing the time delay between the peaks of different pillars, the direction of signal transmission could be determined, which is

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Figure 3. Fabrication of 3D cardiac tissue construct. (a) Photograph of a 3D cardiac tissue construct integrated with micro-pillar array; (b–c) Immunostaining results of sarcomeric alpha-actinin (green), connexin-43 (red) and DAPI (blue) indicating that the cardiomyocytes remained the predominant cell type present within the 3D cardiac tissue construct and were maturated on day 4; (d) Calcium transient at two specified points was assessed by Fluo 4-AM, indicating spontaneous and synchronous calcium transient. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

useful for mapping the electrophysiology of the 3D cardiac tissue construct. For the piezoelectric sensing system, cyclic reciprocating motion with higher input amplitude (0.7 and 2 mm) and frequency (0.5 and 0.7 Hz) was applied on single (Fig. 2e) and dual pillars (Fig. 2f), respectively. As shown in Figure 2g, the voltage output was regular and periodic upon the input of cyclic reciprocating motion. For a single input, the amplitude of the voltage output increased as input amplitude increased from 1.2 mm (Fig. 2g (ii)) to 2 mm (Fig. 2g (i)). Similarly, the frequency of voltage output increased as input frequency increased from 0.56 Hz (Fig. 2g (iii)) to 0.7 Hz (Fig. 2g (ii)). For the dual input (Fig. 2g (iv)) there was a large amount of noise found in the pattern of voltage variation over time due to the interference of the signals caused by the asynchronous stimulation of the bending pillars. Thus, the image processing system and the piezoelectric sensing system were demonstrated to be capable of detecting deformation of the micro-pillar array, which was vital to evaluate the contractile behavior of the cardiac tissue. In this study, the equivalent force applied on the micro-pillars, frequency, and synchronization of the equivalent force were measured by the image processing system, while the amplitude and frequency of output voltage were recorded by the piezoelectric sensing system. Thus, the image processing system and the piezoelectric sensing system together provided comprehensive information and insight into the evaluation of the contractile behavior of the 3D cardiac tissue construct in terms of the contraction force, frequency, and synchronization.

3.3. Characteristics of the 3D cardiac tissue A 3D cardiac tissue construct was successfully fabricated in this study as shown in Figure 3a. The cardiomyocytes from neonatal rat were mixed with collagen/Matrigel and the mixture was molded into the culture chamber. The hydrogel gelated after incubating at 37 °C and 5% CO2 for 30 min. Upon the formation of the cardiac tissues, the micro-pillar array was successfully integrated within the cardiac tissue as shown in Figure 3a. The contraction of the

cardiac tissue was initially observed on Day 3 and the maximum contraction was reached on Days 5–7 (see Movie M1). Immunostaining results on Day 4 were positive for both sarcomeric alpha-actinin and connexin-43, indicating that cardiomyocytes remained the predominant cell type present after 4 days of culture as shown in Figure 3b. Cell elongation and the organization of the internal cell structures, including sarcomere and gap junction (Fig. 3c), were observed within the cardiac tissue, demonstrating the maturation of the cardiomyocytes. To characterize the electrophysiological performance of the 3D cardiac tissue, calcium transient was assessed at two specified points by recording the calcium fluorescence intensity (Fig. 3d) (see Movie M2). The 3D cardiac tissue displayed apparent spontaneous electrical activity at each site and calcium transience with synchronism, indicating the functionality of the cardiac tissue. These results demonstrate that the 3D cardiac tissue exhibited good performance of contraction and could be further used as a drug-screening model. 3.4. Evaluation of contractile behavior via image processing To demonstrate the drug reactivity, and to demonstrate the stability and efficiency of the image processing system, isoproterenol and doxorubicin were added to the 3D cardiac tissue construct. In a previous research, isoproterenol was reported to increase the heart rate and contractile force upon binding to the β -receptors of cardiomyocytes [23]. In contrast, doxorubicin, a common chemotherapeutic drug, was known to be cardio-toxic [33,34]. The micro-pillar array provided the measurement at nine positions of the cardiac tissue, enabling in situ multi-site detection of the contractile behavior simultaneously. The variation patterns of the equivalent force applied on the pillars over time signified the contraction of the corresponding areas of the cardiac tissue. A delay in the contraction between pillars was observed as shown in Figure 4a, suggesting asynchronous beating of the fabricated cardiac tissue construct after the addition of isoproterenol. The frequency pattern of each pillar in Figure 4b shows the frequency

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Figure 4. Contractile behavior evaluated by the image processing system. (a) Comparison of equivalent force on pillars over time upon treatment of isoproterenol showing apparent time delay between pillars; (b) Contraction frequency of each pillar and corresponding area of the 3D cardiac tissue construct upon isoproterenol treatment; (c) Equivalent force of pillars following the treatment with isoproterenol and doxorubicin. (d) Beat-to-beat variation upon drug treatment.

of different cardiac tissue areas, which further confirms the asynchronous beating of the entire cardiac tissue. Moreover, for comparison purpose, we also evaluated the contractile behavior of the cardiac tissue following the treatment of doxorubicin. The changes of equivalent force of each pillar in Figure 4c showed the beating disorder after the addition of both isoproterenol and doxorubicin. The beating frequency of cardiac tissue increased after the addition of isoproterenol, whereas it decreased after the addition of doxorubicin (Fig. 4d), which was consistent with previous reports [23,35]. The contraction force, frequency, and synchronization were the main characteristics of contractile behavior of cardiac tissue. The contraction force and frequency of cardiac tissue have been extensively studied in literature [36,37,38]. However, the research on contraction synchronization has not been actively reported. In this study, the contraction synchronization could be determined by analyzing the time delay between the peaks corresponding to different pillars. This capability is very useful for mapping the electrophysiology of the 3D cardiac tissue.

3.5. Evaluation of contractile behavior via piezoelectric sensing technique Drug reactivity on the 3D cardiac tissue construct was performed via the piezoelectric sensing system at the same time as the image processing. The piezoelectric sensing system offered the detection of general contractile behavior of the entire cardiac tissue construct, in contrast to the image processing system that can provide multi-site detection of the cardiac tissue. Figure 5a shows the variation of the voltage output with time after the addition of isoproterenol and doxorubicin. As can be seen from Figure 5b, the amplitude of the voltage output decreased after the addition of isoproterenol and doxorubicin (). On the other hand, the contraction frequency increased after the addition of isoproterenol while it decreased after the addition of doxorubicin, as shown in Figure 5c. These were matching the results provided by the image processing system. As mentioned earlier, the two measurement methods were employed to evaluate the contractile behavior of

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Figure 5. Contractile behavior evaluated by the piezoelectric sensing system (a) Output voltages over time; (b) Output voltages of the cardiac tissue with and without drug treatment; (c) Contraction frequency of the 3D cardiac tissue constructs (∗ ∗ ∗ p < 0.001).

the cardiac tissue in the heart-on-a-chip platform. The image processing system captured the displacement of the pillars and thus measured the equivalent force applied on the pillars, which reflected the contractile behavior of the corresponding areas of cardiac tissue. On the other hand, the piezoelectric sensor detected the stresses exerted on the PVDF film due to the deformation of the pillars, which represented the contractile behavior of the entire cardiac tissue. The two different measurements together provided comprehensive and mutually coherent information on the contractile behavior of the cardiac tissue construct and demonstrated the platform’s potential for drug screening applications. 3.6. Future prospects This study reported a novel heart-on-a-chip platform equipped with image processing and piezoelectric sensing systems for online monitoring of contractile behavior of a 3D cardiac tissue construct. Contractile behavior are some of the main characteristics of cardiac functioning, which are vital in the evaluation of drug efficacy and safety during drug screening processes. The contraction force and frequency of cardiac tissue have been extensively studied and are available in the literature. However, contraction synchronization has not been actively reported. In this study, the image processing system offered in situ multi-site detection of the contractile behavior and the piezoelectric sensing system provided the contractile behavior of the entire cardiac tissue construct. These two different measurements together provided comprehensive information on the evaluation of the contractile behavior of the cardiac tissue. As a proof of concept, the feasibility of the heart-on-a-chip platform was demonstrated by analyzing the reactivity of isoproterenol and doxorubicin, and detecting the tissue contraction equivalent force, frequency, and synchronization. The heart-on-a-chip platform may have the potential for cardio-related drug screening applications owing to its multi-site online monitoring of the contractile behavior of the cardiac tissue. However, the reliability and accuracy of the platform may need to be improved by optimizing the design of the micro-pillar array in future for drug screening applications. For further research on the controllable alignment of cardiomy-

ocytes, several trials can be implemented in the system. These may include stretching the micro-pillar array to apply mechanical stimulation and coating conductive materials on the micro-pillar array for electrical stimulation.

4. Conclusions In this study, we designed and developed a heart-on-a-chip platform that could provide online monitoring of the contractile behavior of 3D cardiac tissue constructs. The contractile behavior in terms of the contraction force, frequency, and synchronization could be evaluated by an image processing system and a piezoelectric sensing system simultaneously. A micro-pillar array, as the main sensing unit, along with a PVDF film, was embedded within a 3D cardiac tissue construct in the culture chamber. The image processing system measured the deformation of the micro-pillar array during contraction and offered in situ multi-site detection of the contractile behavior. The piezoelectric sensing system, on the other hand, provided the contractile behavior of the entire cardiac tissue constructs by measuring the stresses exerted on the PVDF film, resulting from the deformation of the pillars. The heart-on-a-chip platform was initially validated by applying various motion patterns through controlled thin titanium rods on the micro-pillars, thus mimicking the contraction patterns of the 3D cardiac tissue. The contractile behavior of the 3D cardiac tissue construct upon treatment with isoproterenol and doxorubicin were measured and evaluated by the image processing system and the piezoelectric sensing system. In summary, the drug reactivity of the 3D cardiac tissue measured by the heart-on-a-chip platform was consistent with previous reports, demonstrating the reliability of the platform and its potential for use in cardio-related drug screening applications.

Declaration of Competing Interest None.

Please cite this article as: M. Sakamiya, Y. Fang and X. Mo et al., A heart-on-a-chip platform for online monitoring of contractile behavior via digital image processing and piezoelectric sensing technique, Medical Engineering and Physics, https://doi.org/10.1016/j.medengphy. 2019.10.001

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Please cite this article as: M. Sakamiya, Y. Fang and X. Mo et al., A heart-on-a-chip platform for online monitoring of contractile behavior via digital image processing and piezoelectric sensing technique, Medical Engineering and Physics, https://doi.org/10.1016/j.medengphy. 2019.10.001