Organs-on-a-chip: a new tool for drug discovery.

Review

Expert Opin. Drug Discov. Downloaded from informahealthcare.com by University of Washington on 08/22/14 For personal use only.

Organs-on-a-chip: a new tool for drug discovery Alessandro Polini, Ljupcho Prodanov, Nupura S Bhise, Vijayan Manoharan, Mehmet R Dokmeci & Ali Khademhosseini†

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Introduction

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Organs-on-a-chip-systems

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Expert opinion

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Harvard University, Wyss Institute for Biologically Inspired Engineering, Boston, MA, USA

Introduction: The development of emerging in vitro tissue culture platforms can be useful for predicting human response to new compounds, which has been traditionally challenging in the field of drug discovery. Recently, several in vitro tissue-like microsystems, also known as ‘organs-on-a-chip’, have emerged to provide new tools for better evaluating the effects of various chemicals on human tissue. Areas covered: The aim of this article is to provide an overview of the organson-a-chip systems that have been recently developed. First, the authors introduce single-organ platforms, focusing on the most studied organs such as liver, heart, blood vessels and lung. Later, the authors briefly describe tumor-on-a-chip platforms and highlight their application for testing anti-cancer drugs. Finally, the article reports a few examples of other organs integrated in microfluidic chips along with preliminary multiple-organs-ona-chip examples. The article also highlights key fabrication points as well as the main application areas of these devices. Expert opinion: This field is still at an early stage and major challenges need to be addressed prior to the embracement of these technologies by the pharmaceutical industry. To produce predictive drug screening platforms, several organs have to be integrated into a single microfluidic system representative of a humanoid. The routine production of metabolic biomarkers of the organ constructs, as well as their physical environment, have to be monitored prior to and during the delivery of compounds of interest to be able to translate the findings into useful discoveries. Keywords: biomimicry, body-on-a-chip, drug discovery platforms, in vitro models, in vitro reactors, metabolic biomarkers, microenvironment, microfluidics, organs-on-a-chip, sensors, tissue engineering Expert Opin. Drug Discov. (2014) 9(4):335-352

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Introduction

One of the most challenging aspects in modern drug discovery involves identification and optimization of new drug candidates [1,2]. Drug developers constantly strive to discover and reduce the potential side effects of lead compounds and to bring new drugs to the market in an expedited manner. Conventional in vitro platforms are useful to study and identify different signal molecules (enzymes, receptors and ligands) related to a variety of physiological processes. However, such platforms rarely mimic the complicated cell-to-cell interactions in the body and do not mimic the extracellular mechanical environment [3]. Some of the drawbacks of the conventional in vitro cell culture methods are the static conditions with excessive amounts of nutrients, and this cannot generate the time-changing mechanical or chemical stimuli (signaling molecules) that are important for the normal cellular function. On the other hand, microfluidics can generate not only different mechanical stimuli but also concentration gradients of certain signaling molecules (including drugs) that can be applied in an automated and 10.1517/17460441.2014.886562 © 2014 Informa UK, Ltd. ISSN 1746-0441, e-ISSN 1746-045X All rights reserved: reproduction in whole or in part not permitted

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Article highlights. . . . .

Several in vitro platforms have been developed in order to mimic specific organ functions and drug responses. Though a variety of reports are available, the organs-on-a-chip field is still at an early stage. Automated platforms with on-board real-time sensing capabilities are not available. Effective drug toxicity tests will require the implementation of individual organs and the interactions between them.


This box summarizes key points contained in the article.

time-controlled manner. Thus, microfluidics can aid the selection of potential drug candidates and in determination of drug concentrations in a more realistic and time-efficient manner when compared with standard in vitro models. Nevertheless, the use of microfluidics and biomicroelectromechanical systems (BioMEMS) technologies could result in the development of platforms in which the cellular microenvironments can be precisely controlled, and signals commonly found in the body (i.e., mechanical, electrical and chemical) can be applied to the cellular constructs with high precision [4-14]. Since 2D cell culture models are not always reliable alternatives for mimicking the structural complexity around the cells, organs-on-chips can also utilize three dimensional (3D) cell culture models that better mimic tissue function and architecture [15,16]. The 3D cell culture models, unlike the two dimensional (2D) monolayer cells grown in plastic (coated or not), exert force on one another and move as they do in vivo. In addition, cells form more prevalent gap junctions in 3D, which are important in cell communication processes, tissue integrity and function. In terms of drug diffusion, drugs in 2D culture diffuse faster than in 3D culture, where drugs need to diffuse across multilayers of cells to their final target. Moreover, cells grown in 3D also form tight junctions that bind cells tightly and block or slow the diffusion of drugs, which is not the case in 2D culture. Thus, using 3D culture in microfluidics rather than 2D is an important aspect that needs to be implemented in microfluidic drug-related studies. Most of the early drug development studies use animal models to predict human pharmacokinetic responses [17,18]. Although, animal models are still the main source of obtaining in vivo data for predicting pharmacokinetic responses in humans, there are metabolic and physiological differences between humans and animal models that cannot always predict the outcome of new drugs [19]. These cross-species differences could be partly avoided for certain studies by using only human cells, and within a more appropriate physicochemical environment provided by the use of microfluidic chips. With microfluidics, tissues with different origins can be connected, and thus the response of multiple tissues upon drug exposure could be systematically analyzed in a manner similar to that with animal models. With proper 336

scaling to reassemble a functional organ unit on a chip, these miniaturized systems could possibly reduce some of the costs associated with animal testing, and could probably reduce the negative results, to some extent, obtained due to the cross-species differences. Novel models could further combine an artificially engineered, physiologically relevant cell culture microenvironment with a potential for high throughput testing and could revolutionize the developmental process of pharmaceuticals that rely traditionally on animal testing and clinical trials. Moreover, the application of microfluidics in small chips could potentially improve the distribution of nutrients and other soluble molecules throughout 3D tissue constructs. In addition, microfluidic devices could be made in different dimensions to load only one cell or millions of cells, thus extending the level of flexibility beyond those of conventional cell culture plates [20]. In this review, recent microfluidic and BioMEMS systems for the development of organ-on-a-chip platforms are presented. We will discuss the different approaches in which microfluidic systems are used to perform in vitro cell culture experiments. Specific examples reported in this review include the use of microfluidics for tissue engineering and drug testing on organs-on-a-chip platforms. Finally, we formulate a view on the future of the field and also address the challenges and the problems that need to be solved in future studies. 2.

Organs-on-a-chip-systems

An overview of the main reports on organs-on-a-chip platforms is reported in the following section. In particular, the latest research efforts for mimicking liver, heart, blood vessels and lung are presented. Furthermore, preliminary applications of such platforms for anticancer drug screening are reported in the tumor-on-a-chip subsection. Finally, several preliminary results on other tissues (intestine, muscle and kidney) as well as multiple-organs-on-a-chip systems are highlighted. Liver Liver is the major organ of drug metabolism and thereby a primary target of drug-induced toxicity. In vivo toxicity is one of the major reasons for the failure of » 90% of the drugs after costly Phase I clinical trials [21,22]. In vitro models that can better predict in vivo toxicity than the current animal models that are routinely used for preclinical trials can help to reduce the failure rate of drugs in the late stages of the drug discovery process. In addition, these in vitro models can be used to eliminate the problems of species-specific differences between animal models and human clinical trials. To achieve this goal, engineering hepatic culture systems that optimally mimic in vivo conditions to evaluate drug metabolism and toxicity is highly desirable. Static culture systems, such as culture flasks and multi-well plates, are not ideal as they lack continuous perfusion and have unphysiological medium-to-cell ratio with cells grown in 2D monolayers [23,24]. Microfluidics combined 2.1

Expert Opin. Drug Discov. (2014) 9(4)


Organs-on-a-chip: a new tool for drug discovery

with tissue engineering could provide more suitable dynamic conditions for in vitro liver cultures, termed liver-on-a-chip (Figure 1A) [25]. An important parameter to be considered while developing a suitable culture system is the source of hepatic cells [22]. Studies have been performed using immortal cell lines [26], primary human cells [27] and precision-cut tissue slices [28]. In addition to hepatic cells, subcellular fractions such as microsomes have been used to study the activity of cytochrome p450 enzymes during the early phases of drug development [29]. Microsomes have been embedded in hydrogels and incorporated on a microfluidic chip for drug metabolism analysis [30]. The main advantage of this system is the ease of their availability and use as they can be frozen and stored, whereas the drawbacks include the need to add cofactors for enzyme activity, which results in concentrations outside a range observed in physiological conditions. The major disadvantage of immortalized cell lines is that they do not have the same expression levels of enzymes and transporters as liver cells in vivo [22,31]. Primary human hepatocytes expressing physiological levels of the enzymes used in drug metabolism are the most clinically relevant cells for in vivo pharmacokinetic predictions [32]. However, these cells are difficult to culture for long-term use as their functionality declines with increasing time in culture [33]. To address the challenge of maintaining the functionality of hepatocytes, many systems have employed co-culture techniques by growing hepatocytes with other liver cells, such as Kupffer cells, stellate cells, endothelial cells and fibroblasts, with the primary goal of improving their metabolic activity [34-38]. Reports suggest that, when human placenta-derived mesenchymal stem cells (hpMSCs) are co-cultured with hepatoma-derived HepG2/C3A cells, improved proliferation as well as metabolic activity of the C3A cells are achieved. However, the ratio of co-cultured C3A cells to hpMSCs plays a crucial factor (Figure 1B) [26]. When the number of hPMSCs is significantly higher than C3A cells, the proliferation of C3A cells is rapidly upregulated. For a ratio of 2:1 C3A to hPMSC, the metabolic activities of C3A are elevated, but these effects of hPMSC significantly decrease when the ratio of C3A to hPMSC increases [26]. This and other studies indicate that the ratio of non-parenchymal cells to hepatocytes used for co-culture studies is an important parameter in addition to the cell type chosen for nonparenchymal support [26,39]. Instead of direct cell-cell interactions, some microfluidic co-culture systems have been used to study paracrine effects. For example, to investigate the paracrine effect of rat hepatic stellate cells on 3D culture of rat hepatocyte spheroids, these two cell types were cultured in two separate chips connected such that media from the first chip, containing hepatic stellate cells, flowed through the second chip, containing liver spheroids, thus having a paracrine effect. The paracrine effects were proven to positively affect both the structural integrity and also the functionality of spheroids [34].

Using the currently available technologies from the microfabrication and tissue engineering fields, microfluidic devices have been shown to enhance the function of cells [25,40]. For example, primary rat hepatocytes that were in a microfluidic chip regained their membrane polarity and formed bile canaliculi owing to the design of the chip (Figure 1C) [25], as the microfluidic system was instrumental in aligning the hepatocytes into hepatic cord-like structures and provided an endothelial-like barrier. This enabled the hepatocytes to form bile canaliculi in a much more ordered fashion when compared with a cell culture dish [40]. Moreover, micropatterning and bioprinting techniques allow precise control over cell-cell interactions and help create 3D organoid liver models. Toward the ambitious goal of building a human-on-chip, many groups have started integrating multiple organs-on-chip to build a more functionally reliable platform for drug toxicity evaluation (Figure 1D) [41]. In one such work, 3D aggregates made of human hepatocyte cell line and primary human hepatic stellate cells were co-cultured with skin biopsies to study the interaction of the tissues [28]. Each of the tissues used here were scaled to a miniaturized human scale and shown to be viable and functionally active for a period of 28 days. Due to the crosstalk of the liver and skin prototypes cultured here, the level of albumin was shown to be significantly lower in the co-cultured samples when compared to the monocultured controls due to the consumption of albumin by skin biopsy [28]. The same microfluidic system can integrate as many as seven different tissues with built-in peristaltic micro pumps and a media reservoir [28]. In this system, peristaltic micropumps were made from three poly(dimethylsiloxane) (PDMS) membranes that formed a pneumatic actuator. This microfluidic system also provided the appropriate ratio of cells to media to facilitate the crosstalk between the different tissues [28]. Although these studies have helped elucidate several important aspects relevant to drug studies, some challenges still remain to be addressed. From a clinical perspective, primary hepatocytes are the most relevant cells for culturing, but culture conditions that allow them to express and maintain physiologically relevant enzyme and biomarker levels still need to be well characterized. Furthermore, hydrophobic materials like PDMS used to fabricate most microfluidic devices could cause adsorption of compounds and thereby affect the pharmacodynamics of the system [22]. Methods to treat these materials or novel materials to prevent adsorption should be explored [42]. More in-depth characterization of the cultured organoids in terms of metabolomics, proteomics, genomics and epigenomics analysis will help improve the functional outcome of these studies [43]. Studies investigating proper scaling approaches, integrating multiple organs and evaluating drug delivery vectors using these platforms will prove critical for future development. Heart tissue Microfluidic chips for cardiovascular pharmacology studies are also emerging as potentially useful tools for drug discovery. Cardiomyocytes are highly polarized, aligned and 2.2


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Figure 1. Liver-on-a-chip. A. The viability of hepatocytes cultured in a microfluidic sinusoidal system at high loading density (blue line) is better than the viability of hepatocytes grown in an uncoated 384 well plate (black line). B. Albumin secretion, urea production and activities of CYP1A2 and CYP3A4 in C3A cells at different C3A-to-hPMSC co-culture ratios in microspheres. C. A biomimetic microfluidic system to align the hepatocytes into hepatic cord-like structures and provide endothelial-like barrier to form bile canaliculi. D. Cross-sectional schematic of a multiorgan chip with assay samples first undergoing intestinal absorption and hepatic metabolism and then flows through the target breast cancer cell component. A. Reprinted from [25] with permission of John Wiley & Sons. B. Reprinted from [26] with permission of John Wiley & Sons. C. Reprinted from [25] with permission of John Wiley & Sons. D. Reprinted from [41] with permission of the American Chemical Society. hPMSC: Human placenta-derived derived mesenchymal stem cell.

contracting cells whose functionality also depends on the external physical-chemical environment that facilitates the normal contractility and electrophysiology of heart cells [44,45]. Integration of multiple experimental factors such as cell attachment, elastic properties, microscale topography, flow rate, electrical and biochemical regimes are critical to effectively imitate the cardiovascular tissue on a small chip. Many groups have produced macroscale dynamic cell culture systems and tissue bioreactors [46,47] and recent microfluidic chip studies have shown that cardiac tissue structure can also be replicated on a microchip to obtain more relevant morphometric, electrophysiological, and contractility data [48-52]. One of the most important aspects for microfluidic devices is that the traditional materials used in the fabrication of these 338

devices, such as PDMS, do not necessarily have the biomimetic matrix elasticity and stiffness to facilitate cell attachment [53]. To overcome the challenge of poor cell attachment, Annabi et al. have developed a method for coating microfluidic channels inside a closed PDMS device with a cell-compatible hydrogel layer (Figure 2). Comparing different hydrogel materials, primary cardiomyocytes seeded on these tropoelastin coatings showed preferred attachment as well as higher spontaneous beating rates compared to gelatin coatings, although these results might be related to the different stiffness of the materials and not only to the chemistry [54]. In addition, it was noted that the cellular attachment, alignment and beating were stronger on 5% (w/v) than on 10% (w/v) hydrogel-coated channels [54].



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Figure 2. Heart-on-a-chip. A. Schematic of the coating procedure: i) A hydrogel prepolymer is flowed through the device while being exposed to UV light for crosslinking. ii) The uncrosslinked prepolymer is washed with PBS, while the crosslinked hydrogel layer remains inside the channel and coats the PDMS channel walls. iii) The device can subsequently be loaded with cells and perfused without removing the hydrogel layer. The top figures show the cross-section of a single microfluidic channel, perpendicular to the direction of the flow. The bottom figures also show the channel cross-section, along the direction of the flow. B. Confocal microscopy images showing immunostaining of CM markers inside microchannels coated with (i, ii) 5% (w/v) MeTro and (iii, iv) 5% (w/v) GelMA. Hydrogels stained for (i, iii) troponin (red)/nuclei (blue) and (ii, iv) sarcomeric a-actinin (green)/connexin-43 (red)/nuclei (blue) (scale bar = 50 µm). Reprinted from [54] with permission of the Royal Society of Chemistry. PDMS: Poly(dimethylsiloxane).

The cellular uptake and release of calcium ions are some of the main functional features of the heart cells, which can be affected during acute hypoxic processes. Changes in calcium dynamics are related to arrhythmias, ischemia and heart failure [55,56]. To study the calcium dynamics during the early phase of hypoxia, a microfluidic system for on-line investigation of intracellular calcium ions by confocal microscopy was

developed by Martewicz et al. [57]. Results confirmed that when Fluo-4 loaded cardiomyocytes were exposed to hypoxia, differences in intracellular Ca2+ could be detected and that such changes were reversible below 5% of oxygen partial pressure. Moreover, another ‘heart-on-a-chip’ using a muscular thin film layer (biohybrid constructs of an engineered, anisotropic ventricular myocardium on an elastomeric thin film)


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was developed by Grosberg et al. [58] to measure contractility, quantify action potential propagation and examine cytoskeletal architecture in multiple tissues. This technique was shown to be capable of real-time data analysis when epinephrine (drug used to treat cardiac arrest) ranging from 10-12 M to 10-4 M was administrated to the system. A microfluidic-based heart-on-a-chip system was also developed by Agarwal et al. [59] for pharmacological studies. In this system, a semi-automated fabrication technique was used to process sub-millimeter-wide thin film cantilevers of soft elastomers that were seeded with cardiac cells. Anisotropic cardiac microtissues were created on these cantilevers. Deflections of these cantilevers during muscle contraction were used for calculating diastolic and systolic stresses generated by the engineered tissues. Interestingly, the microdevice was used to test the positive inotropic effect of isoproterenol (drug used in bradycardia or heart block) on cardiac contractility at dosages ranging from 1 nM to 100 µM, demonstrating the capability of this approach to test large ranges of drug concentration [59]. Although, a few promising examples have been developed, important aspects such as electrical stimulation, 3D scaffolds and co-culture to maintain the cardiomyocytes functional in long-term studies should be further exploited in future microfluidic chips. Existing microfluidic cardiac organ-on-a-chip platforms are indeed not yet able to control the contraction of cardiomyocytes, though they can be used for measuring the length-tension relationship in active and passive cardiac tissue as a function of electrical and mechanical stimuli or the administered drugs. In addition, to apply such chips in heart drug discovery studies, it is also important to consider the differences between human and animal cells. Rodent cells are widely used in drug-related studies but exhibit several distinctions from human cells: a shorter action potential, different calcium handling, higher percentage of a-myosin heavy-chain isoform, a higher resting heart rate, faster heart rates and an inverse force-frequency relationship [60,61]. In particular, the differences in ion channels and currents impact the transferability of drug screening and toxicity studies from murine to human models. Despite numerous studies in which different bioreactors were used to culture human cardiomyocytes, microfluidic chips with human cells have not been fully explored. To bypass such a problem, cells of human origin should be used more extensively in future microfluidics studies, especially when drugs are used. Finally, although some progress has been made on engineering heart-on-a-chip platforms, the heart-on-a-chip field is still lacking drug-related studies and is far from being used in predicting a potential drug outcome in human clinical trials. To further advance cardiovascular research and its pharmacology and benefit from the field of microfluidics, disease models on a chip, such as ischemic heart-on-a-chip, should be also considered. Blood vessels Blood vessels--related pathologies are present in many medical conditions, and thus, a key point is to integrate 2.3

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vessel-like elements into organs-on-a-chip systems in order to mimic a more pathological/physiological environment. On the one hand, the possibility of fabricating blood vessels in vitro has been extensively investigated, mostly to address clinically relevant problems. On the other hand, several reports have successfully shown the use of such systems as preclinical models for drug testing, drug delivery and stent assessment, as well as intravascular imaging modalities. Tissue-engineered blood vessels (TEBVs) represent an advantageous model as compared to 2D static models as they are usually tested under conditions such as flow and shear stress, known to affect vascular cell responses in several ways [7]. Pioneering reports by L’Heureux et al. [12,13] and Niklason et al. [14] have proposed TEBVs as models for studying cell interactions, the role of mechanical forces, culture media composition on the blood vessel formation and the effects of various vasoactive agents used to improve such in vitro models. Moreover, these constructs include endothelial, fibroblast and smooth muscle cells to resemble the three-layered organization of blood vessels, have mechanical properties similar to human blood vessels and have been successfully tested in vivo for adult arterial revascularization [11]. Microfabrication approaches have been investigated with the aim of reducing the dimensions of conventional TEBV systems and their integration into organs-on-a-chip platforms. For example, an in vitro microvessel network was fabricated by soft lithography using human umbilical vein endothelial cells (HUVECs) grown on collagen type-1 gel (Figure 3A) [62]. This platform was used to study the HUVECperivascular cell interaction upon treatment with growth factors such as VEGF. The results showed that biomimetic angiogenic sprouts were formed, demonstrating the use of the system as a thrombosis model when pericytes and smooth muscle cells were co-cultured. Recently, an in vitro 3D metabolically active stroma (1 mm thick), including a perfused interconnected human capillary network, has been reported by Moya et al. [63]. This micro-physiological system can work within physiological pressure gradients and interstitial flow ranges and can be useful for studying candidate drugs that affect the microcirculation. Blood vessel-on-a-chip systems represent important platforms to study the process of angiogenesis as well as the effects of different drugs on vessel-like structures. Tremblay et al. [64] tested angiostatic drugs on capillary-like structures in an endothelialized tissue construct, showing drug dose-response effects as expected. In another work, Kim et al. [65] reported perfusable networks of intact 3D microvessels as well as tumor vasculatures produced by spatially controlling co-culture of endothelial cells with stromal fibroblasts, pericytes or cancer cells. These microvessel-like structures showed characteristic morphological and biochemical markers of in vivo blood vessels and exhibited strong barrier function and long-term stability, and were proposed as useful human disease models for pharmaceutical screening.



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Microfluidics can also help to test intact tissue segments, as reported by Gunther et al. [66]. In this example, the authors integrated an intact mouse mesenteric artery segment in an automated microfluidic platform and obtained drug doseresponse curves, proposing this system as a potential platform for controlled delivery of drugs in long-term culture experiments. As recently reviewed by Prabhakarpandian et al. [67], microfluidic devices are also employed as microvascular models for the study of cell-cell and particle-cell interactions, assuming large importance for understanding the behavior of micro/nanoparticles-based drug formulations. Although these studies show great capabilities of microfabrication techniques in developing vessels-on-a-chip system, the field is still far from re-creating the complex structure of blood vessels that combines extracellular matrix, smooth

muscle cells and endothelial cells in an organized manner. Moreover, it is still a challenge to simulate the complex fluid patterns that each vessel experiences during physiological or pathological conditions. The introduction of spatiotemporally defined mechanical stresses in these systems can lead to emulating the different shear stresses and radial deformation that vessels experience in vivo. Blood--brain barrier Microfabrication techniques can also be useful tools to fabricate blood--brain barrier (BBB) constructs, an important interface often studied for drug permeability testing. The BBB consists of a complex network of vascular endothelial cells that isolates the CNS from systemic blood circulation except the circumventricular organs and shows physical and 2.4


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biochemical properties quite distinct from other endothelia. A co-culture in vitro model of endothelial cells (b.End3) and astrocytes (C8-D1A) using a membrane filter has been reported in which the dynamic cerebrovascular environment was effectively mimicked with fluid shear stress simulation (Figure 3B) [4]. In a recent work by Griep et al. [6], an immortalized human brain endothelial cell line (hCMEC/D3) has been cultured on a device where the use of a physical barrier is controlled mechanically by applying fluid shear stress, and biochemically, by using TNF-a. The microfluidic device also had Pt electrodes for investigating the tightness of the barrier by performing experiments for transendothelial electrical resistance measurements. Recently, a microfluidics based, synthetic, microvasculature model of the BBB (SyM-BBB), with microchannels partitioned into two side-by-side chambers, has been proposed [68]. The device allowed a culture of rat brain endothelial cells (RBE4) in the apical chamber under fluidic shear conditions and in continuous contact with an astrocyte-conditioned medium in the basolateral chamber. Improved results in terms of formation of a functional BBB were obtained when compared with a conventional BBB system (Transwells), as shown by upregulation of tight junction proteins (ZO-1 and claudin-1) and permeability glycoprotein, an important membrane drug transporter. Although the research on this topic is still in its early stage, these few studies represent an important starting point for pushing forward the development of a BBB-on-a-chip that is useful for basic biology research as well as for drug screening tests. Lung The smallest structural and functional unit of the lungs is the alveolar-capillary membrane, a bilayer interface constituted mainly of alveolar epithelial cells and microvascular endothelial cells. Gas exchange takes place at this interface with carbon dioxide diffusing from the blood to the alveoli and oxygen diffusing from the alveoli to the alveolar blood vessels. Moreover, this membrane serves as a physical barrier between the body and the external environment, preventing pathogens and other toxic particles from entering the pulmonary circulatory system. Specialized epithelial cells at the interface produce mucus-containing antimicrobial reagents and anti-inflammatory signaling molecules capable of triggering the recruitment of immune cells to the infected area [69]. In order to mimic part of this interface, that is, the alveolar epithelium, reports have focused on the use of a single alveolar epithelial cell line as a lung tissue model for drug delivery studies [70,71]. The lung epithelial barrier is an entrance door for drugs, and is in fact, the largest vascularized network directly exposed to the external environment. Few studies have investigated the possibility of applying fluid flow and mechanical stimulations in cell culture experiments, using a single cell line to emulate a more appropriate air-liquid interface microenvironment where different gas concentrations and mechanical stretching are created similar to the effects observed during breathing [72-74]. 2.5

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Inspired by the human breathing process, Huh et al. [9] fabricated a microfluidic chip to create a human alveolar--capillary interface, having both an air-facing epithelial cell monolayer and a liquid-facing endothelial cell monolayer (representing the wall of the blood vessel). The two layers were attached to the sides of a thin (10 µm), porous, flexible membrane made of PDMS, coated with fibronectin or collagen. Controlled mechanical stretching of the endothelial--epithelial bilayer was carried out by applying vacuum in two adjacent microchannels on the sides of the main alveolar--capillary channel, mimicking the cyclic stretching of the interface during breathing. This device was successfully used to mimic a drug toxicity--induced pulmonary edema (Figure 4), a pathological condition typically appearing in cancer patients who were given IL-2, by using similar doses in a same time period [8]. Several key aspects of the mechanism by which IL-2 induces pulmonary edema were found: i) a vascular leakage results from intercellular gap formation in both the epithelium and endothelium; ii) mechanical breathing motions play a major role in IL-2--induced edema; and iii) the circulating cells of the immune system are not necessarily responsible for the onset and progression of pulmonary edema. The effects of angiopoietin-1 as well as an inhibitor of the IL-2 pulmonary edema were also tested, confirming the importance of this platform as lung-on-a-chip model. Recently, Xu et al. [75] proposed a microfluidics-based drug sensitivity test platform employing a 3D co-culture system (cancer cells and stromal cells isolated from lung cancer tissues). The sensitivities of few anti-cancer drugs were screened using single- and combined-drug chemotherapy in a dosedependent manner for eight patients, representing a unique approach to personalized treatment in patients with lung cancer. These few examples show that mimicking part of the alveolar microenvironment and functions is possible, and disease models can be reproduced in microfluidic systems. The next challenge is to mimic other anatomical structures of the lung to produce a real lung-on-a-chip system and use such platforms for testing new compounds and drugs in a more effective way. Tumor-on-a-chip The tumor microenvironment is a heterogenic and dynamically evolving molecular system in which cancer cells interact with each other by physical and chemical (autocrine, paracrine, hormonal) interactions [76]. Such an environment has a complex microperfusion (blood supply) that affects gas and metabolite diffusion rates [76]. Mimicking such an environment is important for studying the survival, proliferation and spread of the malignant cells. Recent advances in microfluidic cell-based biochips have led to the development of a physiologically relevant tumor microenvironment, which is one of the major factors that affects the efficacy of the anticancer drugs [77,78]. There is an accumulated literature already existing on microfluidic systems for tumor microenvironment 2.6



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Figure 4. Lung-on-a-chip system for studying human pulmonary edema. (A) Representation of the disease condition (fluid accumulation and fibrin deposition in the alveolar air space due to IL-2 therapy-induced vascular leakage). (B) Schematics of the microengineered disease model and phase-contrast image of the apical structure (top-down view, scale bar: 200 µm). (C) Schematics and phase-contrast picture of the reproduced edema on the chip: the liquid leakage from the lower microvascular channel into the alveolar chamber is caused by the endothelial exposure to IL-2; dark bands in the phase-contrast image shows the air (A) -- liquid (L) meniscus; scale bars: 200 µm. (D) Schematic and phase-contrast pictures of the chip (scale bar: 50 µm) upon introduction of prothrombin and fluorescently-labeled fibrinogen to better visualize the fibrin deposits (E) by confocal imaging (F, scale bar: 5 µm). Reprinted from [8] with permission of The American Association for the Advancement of Science.

studies and drug testing and an example is shown in Figure 5 [54,79-83]. When compared with the microfluidic chips developed for other organs, the field for microfluidic tumor systems is growing at a remarkable rate. Moreover, numerous chips have been developed aiming to reproduce the complex tumor microenvironment, to generate different gradients of drug concentration and to test study personalized drug treatments [80]. Using concentration gradient generators to screen the appropriate drug concentration is an important method in all drug screening tests. Recently, Kim et al. [84] developed a fully automated and programmable microfluidic system for drug candidate screening applications that integrates on-chip generation of different drug concentrations with parallel culture of cells. The utility of this platform was demonstrated

in loss of viability of PC3 prostate cancer cells by using combinations of either doxorubicin or mitoxantrone (anti-cancer drugs) with TNF-related apoptosis-inducing ligand (TRAIL) either in a sequential or simultaneous format. The results demonstrated that the microfluidic chip can capture the synergy between different sensitizer drugs and TRAIL, and can be potentially used for screening and optimizing combinatorial drug treatments for cancer therapies prior to starting evaluations in humans [84]. Furthermore, personalized treatment is a promising future method that can be used in different drug sensitivity--related tests in patients, as shown by Yang et al. [85]. An integrated microfluidic concentration gradient chip to characterize various cellular responses was developed and used to analyze apoptosis in human uterine cervix cancer cells. This stepwise concentration gradient generator


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i.

Outlet

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Outlet channel 2

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Outlet channel 1 Central channel

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Figure 5. Tumor-on-a-chip model. Scheme of microfluidic device design for 3D tumor model. (i) The system consisted of three channels separated by an array of square posts, 50 µm (width) 30 µm (height), and spaced 4 µm apart. (ii) Panel images of cell compartment taken before the cell loading of B16.F10 cells and (iii) at 12-h post cell loading under a regular light microscope of in the microfluidic device. Reprinted with permission from [82] with permission of John Wiley & Sons.

was used to test two anticancer drugs, 5-fluorouracil and cyclophosphamide. The drugs were first divided into 65 different concentrations and 65 of their combinatorial concentrations, and subsequently, tested to characterize the different cellular responses. Exploiting the unique high throughput properties of microfluidic devices, the authors were able to study multiple drugs and concentrations at the same time, obtaining a more physiological environment over conventional static culture platforms [85]. Additional examples of recent microfluidic chips used for testing drugs for cancer studies were performed for epigenetic therapy of gastric cancer. A ChIP-on-chip analysis to evaluate microRNA levels that are controlled by histone changes in AGS gastric cancer cells was developed [86]. Further, a device that could provide a convenient in situ assay tool to assess the cytotoxicity of anticancer drugs on tumor spheroids was developed [84]. Quantitative data acquisition of drug-induced cell apoptosis is another important aspect of anticancer drug assessment. To address this phenomenon, a microfluidic platform to assess cell cytotoxicity for anticancer drug screening using Annexin V conjugated quantum dots and Calcein AM as dual apoptotic probes were successfully developed by Zhao et al. [87]. The method was tested with three different adherent tumor cell lines and multiple drugs by using U-shaped cell trapping weirs. Results showed that the dots are fluorescently stable and that this approach can also be used as a multiplex assaying strategy to screen cell toxicity and fundamental cell apoptosis processes. As we discussed in the previous sections, several organ-ona-chip platforms have been developed for studying the 344

physiological mechanism of angiogenesis and how to modulate it by using drugs. Similar ideas have been translated to the study of angiogenesis in tumor microenvironments and to understand how tumor cells regulate this process. Using a microfluidic system, Cross et al. investigated HUVEC invasion and vascular network formation in dense, microfabricated collagen gels in the presence/absence of tumor cells (from human oral squamous cell carcinoma) [88]. The interaction between the two cell lines was shown by the invasion of the endothelial cells (cultured on the gel surface) toward the tumor cells (growing on the bulk of the gel) when co-cultures were monitored for > 3 days. Collagen scaffolds were also employed in microfluidic architectures to study capillary growth and migration of human dermal microvascular endothelial cells (HMVECs) when co-cultured with cancer cells or smooth muscle cells [89]. The communication between HMVECs and the other cells affected the vascular growth and remodeling process, showing this system as a promising platform for studying angiogenesis-related phenomena. While much progress has been made over the past decade in understanding invasion and metastasis, it is notable that both cancer research and microfluidics technology have increasingly progressed. Majority of the work was performed using human carcinoma cells, thus not relying on animal carcinoma cell types, which have a different progression and physiological tumor phenotype. The field still continues to face numerous challenges, such as investigating the effects of angiogenesis, stiffness, low oxygen and tumor-stromal interactions on cancer survival, which are very important parameters in recreating a proper tumor environment.




2.7

Other organs

Besides the above examples of organ-on-a-chip systems, other on-chip models of organs have been developed, and these organ constructs can potentially be integrated into a system to create a more realistic model of a human-on-a-chip, introducing a further level of complexity and allowing more interesting pharmacokinetics and pharmacodynamics studies in vitro. In this context, aiming to produce a portion of the ductal system present in the human mammary gland, Grafton et al. [90] designed a network of branched microchannels with a decreased size and cultured polarized human mammary epithelial cells. This device has been proposed as an in vitro breast cancer platform and used to test the effects of superparamagnetic submicron particles for neoplasia detection and treatment purposes. Markov et al. [91] fabricated a microfluidic 3D culture platform for culturing ‘mammospheres’ from MCF-10A, a human mammary epithelial cell line, and their invasive variants. The platform was successfully used for long-term culture (up to 21 days) and tested for studying the effects of model drugs (proteinase inhibitors) on these 3D cell constructs. Recently, several investigators have focused on the gastrointestinal tract reporting successful models of this highly complex system, body entrance to oral drugs and food constituents. Kim et al. [92] fabricated a microdevice that was able to mimic the intestinal peristaltic--like motions and flow by applying cyclic mechanical stretch to a layer of human intestinal epithelial cells (Caco-2). This stretch behavior showed positive effects on cell alignment and proliferation, and a robust morphogenesis of 3D intestinal villi-like structures were observed. Interestingly, four different types of differentiated epithelial cells (absorptive, mucus-secretory, enteroendocrine and Paneth) were found, assuming positions similar to those observed in native human intestine. As a result of the villi formation, the noteworthy increase of intestinal surface led to a higher metabolizing activity of cytochrome P450 (3A4 isoform) compared with the results obtained from conventional Caco-2 cell monolayers cultured on a conventional, static Transwell system [93]. The importance of a villi-like structure for developing a functional gastrointestinal model was confirmed by Sung et al. [94], who proposed the introduction of such geometries prior to employing microfabrication techniques. A model of the gastrointestinal tract has also been applied to study the effects of dairy products on the immune functions [95]. The authors used a permeable membrane, dividing a confluent layer of epithelial cells and a co-culture of immune cells and monitored the expression of pro-inflammatory cytokines, including interleukin 1 and 6, produced by the immune cells, upon i) the administration of pro-inflammatory stimuli such as lipid polysaccharide; and ii) the application of potentially anti-inflammatory dairy foods. Significant efforts have been made toward the development of artificial muscle constructs in vitro mainly for tissue engineering applications, using surfaces with different patterns as well as materials [96-99], while their implementation

on a microfluidic organ-on-a-chip platform is still in its infancy. A preliminary study by Grosberg et al. [100] showed that microcontact printing technique can be useful to create a thin layer of muscle in a microfluidic system. Contractility measurements confirmed the functionality of the striated as well as smooth muscle layers grown in these devices, paving their potential use for further, more complex on-chip analyses. The kidney is a major organ for drug clearance from the body, playing an important role in the biotransformation of xenobiotics, and is one of the main targets of drug toxicity studies [99]. The early detection of kidney damage is often difficult and a properly defined in vitro model may have beneficial effects in studying any nephrotoxic drug. Aiming at producing a simple kidney-on-a-chip platform for studying the transport barrier functions, Jang and Suh [10] fabricated a microfluidic circuit, in which a thin porous membrane was separated by two stacked microchannels. Rat renal inner medullary collecting duct cells were cultured on the top side of the membrane. By using physiological shear stress, their polarization and a fully differentiated kidney epithelium were obtained. Following this approach, Frohlich et al. [5] integrated porous membranes with several types of pores into a microfluidic system. Human kidney proximal tubule epithelial cells and primary renal proximal tubule epithelial cells grew as a confluent layer on the membrane and expressed biomarkers of differentiated epithelia, that is, of a reabsorptive renal epithelial barrier responsive to mechanical stimulation. Cisplatin-induced proximal tubule nephrotoxicity was demonstrated in an analog system by Jang et al. [101], stating the possibility of using such systems for evaluating humanrelevant nephrotoxic agents. Multiple organs-on-a-chip A major step toward the production of human-on-a-chip platform to simulate and predict the whole body response to drugs is the integration of several individual organ units in a single platform. Advances in this direction have already been reported by several research groups that successfully combined two, three or four organ constructs in one single microfluidic system, using a common overlying media and enabling cellto-cell communications by means of soluble signals [102]. Even though the field is far from really mimicking a wholebody system, these examples represent significant steps in this direction. Aiming at simulating the metabolism of hepatotoxic compounds, liver tissues have often been placed with other tissues in multi-organ platforms. For example, the regulation of bile acid homeostasis has been performed in a study by van Midwoud et al. [33] where rat intestinal and liver slices were placed in adjacent chambers of a microfluidic chip. In this model, metabolites from the intestinal construct were further metabolized by the liver, emulating the in vivo first-pass processes. Moreover, the authors demonstrated the interaction between the two organs by introducing chenodeoxycholic 2.8


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acid, the primary bile acid, into the system and evaluating the regulation of cytochrome P450 7A1. The effects of liver metabolites on adipose tissue have been investigated in a bioreactor system composed of several compartments for culturing primary hepatocytes and adipose cells from rats and by focusing on liver metabolism of urea synthesis, albumin, glycerol, free fatty acid and glucose [103]. Moreover, the introduction of endothelial cells to this system showed metabolic interactions between the three components (liver, adipose and vascular tissues) [104]. Improved physiological analogs of pharmacokinetic models to test drugs and chemicals were developed by integrating three or more organ-models into a single platform. To evaluate oral medicine and food constituents, a three-organ platform was designed by integrating an intestine micromodel (Caco-2 cells), a hepatic module (HepG2 cells) and a bioactivity-testing component (breast carcinoma MCF-7 cells) that resembled intestinal adsorption, hepatic metabolism and target tissue, respectively [41]. This model was successfully tested with anticancer agents and estrogen-like substances. By employing the same platform, the authors were able to mimic gastrointestinal degradation through the use of synthetic gastric juices added before the intestinal compartment [105]. Along the same lines, Shuler’s group has proposed a micro cell culture analog including up to four organs to study more complex drug metabolisms [106-108]. Zhang et al. [109] have made a 3D microfluidic cell culture platform on a single chip with four compartmentalized chambers for liver, lung, kidney and adipose tissue culture, showing that the different tissues were indeed able to communicate via soluble signals. The next step for the upcoming human-on-achip platforms will be the fabrication of more complex organ compartments to better mimic the physiological environment allowing researchers to perform fundamental as well as applied research assays. 3.

Conclusion

In this review, microfluidic cell culture platforms for engineered tissue constructs were discussed. These platforms aim to create an artificially engineered, physiologically relevant cell culture microenvironment and possess a huge potential for drug screening studies. Emphasis was placed on the recent developments in liver, heart, lung. From an engineering point of view, substantial progress has been made in the last decade for reproducing complex mechanical environments such as the ones present in the heart and the lung. In addition, chips applying and measuring electrical signals have been developed for monitoring cardiac cells. A notable progress has been made in studies involving tumoron--chip systems, where platforms mimicking the tumor microenvironment as well as the means to deliver different drugs to these microenvironments have been introduced. Most of the studies performed with tumors have used human tumor cells, which was not the case in the heart- and liver-on346

chip studies. An additional challenge to be addressed in future studies is to create a physical microenvironment with complex architecture that resembles the in vivo complexity. Furthermore, most of the current microfluidic studies still use animal cells and single cell types to evaluate the effects of different drugs. Considering the metabolic and physiological differences between the human and animal models, it is important to use human cells in future organ platforms. The data obtained from in vitro microfluidic platforms can also help in minimizing the number of in vivo animal experiments and predicting proper metabolic activity prior to clinical/ patient testing. Overall, microscale engineering technologies can be used to create and combine multiple organ constructs on a chip to create systems physiologically more relevant than the current in vitro models. 4.

Expert opinion

Organ-on-a-chip models have great potential in boosting the field of personalized medicine with regards to drug toxicity screening, disease modeling for drug target discovery and drug development [110]. Combining these platforms with the rapidly emerging field of induced pluripotent stem cell (iPSC) technology would theoretically allow re-creation of a miniaturized multi-organ human-on-chip potentially matching the genetic profile of a patient [111]. These models are of significant relevance to pharmaceutical companies from the perspective of reducing drug development time and costs, predicting drug response in humans better than animal models, developing safer and more effective drugs [110,112]. However, to successfully harness the commercial potential of this platform, it is critical to address the challenges that have been recognized from early proof-of-concept studies. This section will highlight the key challenges in the organ-on-a-chip field and the approaches undertaken to overcome these hurdles. The microfluidic bioreactors used to culture cells are designed to recapitulate the in vivo microenvironment. However, the current in vitro cell culture platforms have yet to achieve the complexity of physiological 3D microarchitecture, and vascularization of the in vitro construct is still a challenge. Culturing primary, immortalized or stem cells outside the body significantly changes the behavior of the cells [102]. Thus, organ-on-a-chip would be used as a complementary platform to animal models for faster and cheaper screening of novel drug designs to identify potential candidates before moving on to exhaustive and time-consuming animal and human models. PDMS and poly(methyl-methacrylate) are the most commonly used materials for fabricating microfluidic devices. The use of these materials for cell culture applications offer several advantages, including ease of fabrication and handling, low cost and optical transparency for realtime monitoring of the culture [113,114]. PDMS platforms, which were the main focus of this review, also offer the added advantage of being gas permeable to allow oxygenation of the circulating media.




However, the hydrophobic nature of polymer-based microstructures poses a challenge for drug screening experiments due to the nonspecific adsorption of hydrophobic drugs, proteins and other analytes. To avoid this common problem, several different ways of passivating the surface, for example, grafting of hydrophilic polymers, have been reported in the literature [113-116]. It is critical to consider these surface modification strategies in order to prevent the biofouling effect that may alter experimental results, especially for long-term cultures. Most of the organ-on-a-chip platforms consist of 2D cultures on treated PDMS substrates that can capture some of the physiological conditions because of the design of the system, but fail to mimic many morphological aspects, including the in vivo tissue architecture. Furthermore, some studies have suggested that in spite of growing organs in 2D, the physiological functions can be mimicked by simply modifying the design of the system [37,40]. Another critical design consideration is in integration of microfluidics with the microbioreactors and controlling the microfluidic flow through the channels in the multiorgan system. Controlling the flow through the different modules becomes even more important when interconnected multiorgan systems are being developed [117]. Controlled pumping technologies, for example, on-chip peristaltic micropumps and passive capillary-based pumping approaches, have been proposed to improve the microfluidic design of the system [28,118]. Moreover, while integrating the microfluidics and the bioreactor system of a multiple organ platform for drug screening, it is imperative to scale the organ constructs appropriately to achieve the proper pharmacokinetic response upon delivery of the drugs to the organ system and properly study the effects of drugs and their by-products on several organs [20,119]. Furthermore, it is also important that biosensors aimed to monitor the metabolic activity of the organ constructs have to be carefully designed to capture the variations in the biomarker levels before and after drug administration [20,117,119,120]. Real-time monitoring of cellular processes for extended (several weeks) culture periods without disrupting the cultures is still a challenge to be met for evaluating responses of drugs to compounds due to the inaccessibility of the organ constructs and small volume involved in the system. Microbead based optical sensors as well as electrochemical sensors are some of the potential on-chip analytical approaches to monitor biomarkers [112,121]. In addition, the proliferation or movement of cells (in case of cardiac cells) can be inspected using microscopy techniques. The selected microscopy technique should allow for high-resolution images while achieving considerable focus depth to image the cells within the bioreactor. If 3D structures like cell spheroids are being used, techniques such as confocal microscopy, optical coherence tomography and multi-photon microscopy may be the methods of choice for 3D cell imaging. Fluorescence microscopy enables cell

lineage tracing and migration studies by immunolabeling with fluorescent dyes or by introducing a constitutive fluorescent label into the cellular genome using genetic engineering techniques [122]. In order to successfully integrate multiple organs-on-chip, it will be imperative to address the challenge of finding a universal media to perfuse through the entire system while supporting the different tissue types [119]. Since different cell lines require specific growth factors to maintain viability and phenotype, a cocktail that acts as a common blood surrogate is needed. It is important to develop a chemically well-defined blood surrogate supplemented with serum components and additional factors to allow standardizing the experimental outcomes. Moreover for long-time culture maintenance, strategies to remove the metabolic waste products and replenish the depleted nutrients from the media need to be integrated in the microcirculatory network [102]. It would be interesting to investigate the effect of biophysical forces, such as shear and mechanical strain under, flow conditions on cell responses in these multi-organ systems [4-14]. Organ-on-a-chip models using human cell sources could potentially eliminate the effects of cross-species differences introduced by using animal models for clinical drug studies. It is noteworthy to mention that the majority of the developed first-generation organ-on-a-chip platforms used cells based on non-human cell sources. The next generation of organ platforms are expected to use human iPSCs, which opens the application space of this technology for personalized disease and drug screening models [123,124]. This direction brings its own set of challenges including standardizing differentiation protocols, cell-banking protocols, matrix coatings and assessment technologies, such as karyotyping and epigenetic analysis of the pluripotent and differentiated cells. From a commercial standpoint, the technology must also be adopted by the pharmaceutical industry to reduce costs and enhance efficacy of the drug discovery process [125].

Acknowledgement A Polini and L Prodanov contributed equally to this work.

Declaration of interest The authors gratefully acknowledge funding by the Defense Threat Reduction Agency (DTRA; X.C.E.L Program N66001-13-C-2027). The content is solely the responsibility of the authors and does not necessarily represent the official views of the awarding agency. The authors acknowledge also funding from the Office of Naval Research Young National Investigator Award, the National Institutes of Health (HL092836, DE019024, EB012597, AR057837, DE021468, HL099073, EB008392), and the Presidential Early Career Award for Scientists and Engineers (PECASE).


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Affiliation Alessandro Polini1,2, Ljupcho Prodanov1,2, Nupura S Bhise1,2, Vijayan Manoharan1,2, Mehmet R Dokmeci1,2 & Ali Khademhosseini†1,2,3,4 † Author for correspondence 1 Brigham and Women’s Hospital, Harvard Medical School, Division of Biomedical Engineering, Department of Medicine, Cambridge, MA 02139, USA 2 Massachusetts Institute of Technology, Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02139, USA 3 Harvard University, Wyss Institute for Biologically Inspired Engineering, Boston, MA 02115, USA E-mail: [email protected] 4 Tohoku University, World Premier International -- Advanced Institute for Materials Research (WPI-AIMR), Sendai 980-8577, Japan

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