- Email: [email protected]
Organs-on-chips and Its Applications
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 44, Issue 4, April 2016 Online English edition of the Chinese language journal
Cite this article as: Chin J Anal Chem, 2016, 44(4), 533–541.
REVIEW
Organs‐on‐chips and Its Applications SUN Wei1, CHEN Yu-Qing1, LUO Guo-An2,3, ZHANG Min3, ZHANG Hong-Yang1, WANG Yue-Rong1, HU Ping1,* 1
Shanghai Key Laboratory of Functional Materials Chemistry, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, China 2 Department of Chemistry, Tsinghua University, Beijing 10084, China 3 Shanghai Key Laboratory of New Drug Design & Modern Engineering Center for TCM, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
Abstract:
Microfluidic chips have been proven to be attractive platforms for cell culture in vitro. Microfluidic chip-based
organs-on-chips technology has received more attention because it can mimic the complex structures and functions of human organs. In this review, the recent advances of organs-on-chips technology in different organs are introduced, including the build of human physiological models, drug discovery, and toxicology research. The development trend of this technology is also proposed. Key Words:
1
Microfluidic chip; Organs-on-chips; Cell; Human organ; Review
Introduction
Since 2011, Barack Obama, the president of the United States of America, announced the official start of the “human-on-chip[1]” special project established by the National Institutes of Health (NIH), Food and Drug Administration (FDA) and United States Department of Defense[2], a worldwide research interest of the “human-on-chip” has been aroused. The so-called “human-on-chip”, as the research level of now or rather, is the organ-on-a-chip systems, also known as organs-on-chips[3], which is a biomimetic system based on the microengineering technology in microfluidic chip to mimic the key functions of human organs[4]. Organs-on-chips not only has the advantages such as miniaturization, integration and low consumption[5], but also it can accurately control the multi-parameters of system such as chemical concentration gradient[6], fluid shear stress[7], cell patterning[8], tissue-tissue interface[9], organ-organ interaction[10] and so on, mimicking the complex structure, microenvironment and physiological function of human organs. The study of human physiology is a significant part of life science research. The conventional two-dimensional cell
culture modes have been extensively used in the human physiological and pathological studies, but these simple modes couldn’t reflect complex physiological functions of human tissues and organs[11]. So the researchers often did animal experiment instead of in vitro culture mode. However, animal experiments also have drawbacks such as long cycle and high cost. What’s more, animal models often fail to predict the human response to various drugs[12]. The organs-on-chips helps solving the problems of animal experiments, establishing more realistic physiological models on chips, and is expected to become a biomimetic, efficient, and energy-saving physiological research and drug development tools. After the rapid development in recent years, researchers have realized the construction of various organs in microfluidic chips, such as liver, lung, intestine, kidney, vessels, heart and multiple organs. The collaboration between well-known research units and pharmaceutical companies has made the organs-on-chips step into the practical stage. According to the reports from websites, the Netherlands biotechnology company Mimetas developed a kidney-on-achip and reached an cooperation agreement with several
________________________ Received 26 October 2015; accepted 23 December 2015 *Corresponding author. Email: [email protected] This work was supported by the National Science and Technology Major Project for Significant New Drugs Development of China (No. 2013ZX09507005). Copyright © 2016, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(16)60920-9
SUN Wei et al. / Chinese Journal of Analytical Chemistry, 2016, 44(4): 533–541
pharmaceutical companies for drug screening[13]. In addition, Johnson & Johnson also planned to use the thrombosis-onchip system, which was developed by Wyss Institute in Harvard University affiliated with Emulate company, for drug experiments and liver-on-a-chip for the test of drug hepatotoxicity[14]. This review summarizes the research progress of organs-onchips in recent years, and the development trend of this technology is also prospected.
2
Design concepts of organs-on-chips
The growth of cells requires the synergetic effect of various complex external and internal environmental factors. Hence, the reality of the external environmental parameters is to be considered in establishment of in vitro physiological models[15]. By combination of microfluidic technology, micromachining and cell biology, environmental parameters can be precisely controlled on organs-on-chips, which may in turn generate fluid shear stress, mechanical stress, biochemical concentration gradient and other physical and chemical stimuli. The cells on the chip can response to these stimuli, develop self-organization and express more realistic physiological functions, exhibiting special advantages of organ-on-chip in the establishment of in vitro physiology model[16]. (1) Generation of fluid shear stress. Fluid shear stress exists in the human body all the time, but is absent in the traditional static culture. Microfluidic technology enables dynamic perfusion in cell culture through micro pumps, which is conducive to give stable nutrients and discharge waste timely. Furthermore, dynamic environment of cells is more similar to that in vivo. In addition, the fluid shear stress in perfusion culture is essential for some physiological functions of the human body such as renal reabsorption[17]. (2) Generation of dynamic mechanical stress. The pressure in human body is related to many life activities, such as blood pressure, pulmonary pressure, skeletal stress, etc. The steady pressure plays an important role in the body's physiological functions including tissue formation, cell differentiation and even tumor formation[18]. Microfluidic technology can produce periodic mechanical stress by utilizing flexible porous membrane on which the cells are cultured. The mechanical stretching of the membrane caused by external force can mimic the partial physiological functions such as lung breathing, intestinal peristalsis and heart contractions, etc. (3) Generation of concentration gradient. The flow of fluid in the microscale is laminar, which is beneficial to producing various types of concentration gradients in the channel[19]. Various concentration gradient-driven biochemical signals play critical roles in many physiological processes such as cell migration, differentiation, immune response and cancer metastasis[20]. Microfluidic technology can be used to achieve a stable three-dimensional biochemical concentration gradient
by transforming the flow rate and channel size and using micro valve, micro pump or unique channel design so as to simulate a variety of complex physiological processes in human body[21]. In addition, the generation of multi-channel concentration gradients makes it possible for high-throughput screening of drugs on chip. (4) Control of cell patterning. Human body is not just cell stacking, but is consisted of a variety of cells constructed elaborately in an orderly arrangement, to form a functional unity through complex interaction. Microfluidic technology shows superior control ability to cell patterning. Template method[22], surface modification[23], electrochemical method[24], laminar flow[25] and micropillar array[26] have contributed to the cell patterning on the chip. This is of great benefit to the construction of an in vitro physiological model with a complex geometric structure. Meanwhile, it provides an ideal platform for studying the cell-cell and cell-matrix interaction.
3 3.1
Research progresses of organs-on-chips Liver-on-a-chip
Liver is the most important organ where drug metabolism occurs. Researches on the hepatotoxicity of drugs is significant important in drug discovery. The hepatocyte cultured in traditional static two-dimensional cell culture model lacks contact with the extracellular matrix and will lose liver-specific function soon[27]. Moreover, due to lacking of continuous perfusion, this model cannot be presented the in vivo microenvironment. Microfluidic and tissue engineering technology can make the hepatocyte self-organize, thereby maintaining the liver function partly. Most liver-on-a-chip researches focused on the establishment of physiological related models of liver on a chip, such as bile duct, hepatic lobule, and hepatic sinusoid model, which are designed for drug screening and toxicology studies. For example, in a early study, Lee et al[28] designed an endothelial-like barrier structure, in which the primary hepatocytes were cultured, and the media was perfused in external channel as shown in Fig. 1. It is the first model of liver sinusoid established on microfluidic chip. The penetrative endothelial-like barrier structure could not only isolate primary hepatocytes and external sinusoid-like region spatially, but also realize mass transport. The cells could survive for seven days without an extracellular matrix coating. Experiment results showed that the drug diclofenac had no hepatotoxicity in 4 h, but the cell would die in a longer period of time (24 h), indicating that hepatocytes in this model had physiological activity. This study laid the foundation for the establishment of liver model on chip. Ho et al[24] used field-induced dielectrophoresis trap to generate radial-pattern electric fields, patterned hepatocytes and epithelial cells in a circular polydimethylsiloxane (PDMS) chip and successfully mimicked the structure of hepatic lobule.
SUN Wei et al. / Chinese Journal of Analytical Chemistry, 2016, 44(4): 533–541
In subsequent studies, this group improved the model and designed the “honeycomb” dielectrophoresis array to mimic hepatic lobule. The results of cell-cell interactions study showed that the structure could effectively enhance the activity of CYP450-1A1 enzyme. Hegde et al[29] developed an on-chip method to culture hepatocytes in a sandwich configuration. They designed a dual-layered chip using the porous PET film to separate the channels. Continuous perfusion flow was in the upper channel to culture rat primary hepatocytes sandwiched by collagen and fibronectin in the lower channel. After more than two weeks of culture, the hepatocytes could form a bile canaliculi structure and some liver function was achieved. Compared with the existing in vitro liver model, the main advantage of liver-on-a-chip is achieving part of the liver function of liver cell clusters in micron scale, thereby establishing a more realistic liver model and maintaining liver specific functions for a long time[30]. Because microfluidic technology has great potential in high content screening, how to realize the multi-parameter, high content and rapid analysis on chip will be the focus in future studies. 3.2
Kidney-on-a-chip
Kidney is an important excretory organ in human body and plays an important role in maintaining osmotic pressure and self-homeostasis in vivo. The basic functional unit of kidney is nephron which can remove metabolites and waste in vivo through production of urine, and retain moisture and other nutrients by reabsorption function[31]. Fluid shear stress is one of the significant influencing factors of nephron function. It can change the function and morphology of nephrocytes, and is closely related with some diseases such as polycystic kidney disease (PKD). Jang et al[32] designed a double-layered chip to simulate the structure of kidney proximal tubule, in which 1 dyn/cm2 of fluid shear stress was introduced. The chip was divided into two layers by an elastic porous membrane, and the primary human renal proximal tubular epithelial cells were cultured in the upper channel. Upon exposure to the fluid shear stress, albumin transport and glucose reabsorption
capacity were 2-fold and 3.5-fold respectively as those of transwell experiment, which fully proved the importance of fluid shear stress on establishment of renal physiology model. These researchers further tested the cytotoxicity of cisplatin and compared with the results from Transwell culture on the renal chip[33] by adding quantitative concentration (100 μmol) of cisplatin solution in the lower channel and perfusing 24 h to cause cell death. Cell viability was detected by lactate dehydrogenase activity and V-FITC/PI staining method. The results showed that under the same conditions, cell viability on chip was higher than that in Transwell cultures in same circumstances. In a renal pathophysiology study, Zhou et al[34] designed a circular chip containing 12 channels to study the epithelial-to-mesenchymal transition (EMT) induced renal interstitial fibrosis. When human proximal renal tubular epithelial cells were exposed to serum proteins in a constant flow rate, the cells developed to a mesenchymal phenotype and incubating cells with C3a were induced with similar features. But this phenomenon did not appear in heatinactivated serum. This study provided a new perspective to the study of this pathological process. Wei et al[35] designed a single chip with a tubular channel. Monolayers of human renal tubular epithelial cells (HK-2) were cultured in the tube wall by adding CaCl2 and Na3PO4 in HBSS buffer to simulate the formation of calcium phosphate stone. The kidney stone model in microfluidic chip was established for the first time and was proved to have the potential to be used as disease model. In addition to PDMS, hydrogel materials were also successfully applied in the establishment of renal physiology model. Mu et al[36] utilized fibrillogenesis of collagen to splice two piece of hydrogel together and form two parallel three-dimensional micro-vascular networks to establish a physiological model of nephron. In this model, different cells like MDCK cells and human umbilical vein endothelial cells (HUVECs) could be cultured in different channel, and passive diffusion of nephron could be mimicked through the mass transfer of hydrogel. The hydrogel structure could be applied to establish a more complex physiological kidney model on chip, which increased the diversity and complexity of the chip.
Fig.1 Microfluidic endothelial-like barrier model[28] a. SEM micrograph and structure diagram of microfluidic chip, scale bar represents 50 μm; b. primary hepatocyte cultured for 7 days
SUN Wei et al. / Chinese Journal of Analytical Chemistry, 2016, 44(4): 533–541
3.3
Gut-on-a-chip
Intestine is the longest part of digestive tract, and also the most important part for digestive function. Most drugs are taken into human body via oral administration. Oral drugs pass through small intestine into blood circulation, so the study of drug absorption through intestinal cells is very important in drug screening[37]. The villi on the intestine wall render a huge surface area to small intestine and facilitate rapid absorption of nutrients. The building of small intestinal villi on the chip is of great significance to study the role of intestinal function. March’s group[22] used laser ablation, PDMS soft lithography and hydrogel as template to construct the “comb shaped” 3D hydrogel structures on chip, for the first time, to mimic human intestinal villi. The structure was close to the intestinal villi in the shape and distribution density with a length about 450–500 μm. Human intestinal epithelial cells (Caco-2) were seeded onto the structure to measure permeability and trans-epithelial electrical resistance (TEER). The results showed that this model was closer to real situation in human body compared with traditional 2D model. In further experiments, vascular endothelial cells might be introduced into the hydrogel structure to create a more complex physiological model for drug absorption experiments. Intestinal tract has special modes of motion, including segmental contraction, intestinal peristalsis and movement of intestinal villi. Intestinal peristalsis plays an important role in nutrient digestion and absorption. To simulate human intestinal peristalsis, Kim et al[38] designed a dual-layered chip. The chip was separated into two layers by an elastic porous membrane. Stretching the membrane in cycles by a vacuum pump (0.15 Hz) could mimic the expansion and contraction of human intestinal peristalsis. Simultaneously, Caco-2 cells were cultured on the membrane, and the experimental results showed that the cells could differentiate and form intestinal villi structure. More importantly, human intestinal parasitic E. coli and epithelial cells were co-cultured on the chip. The results showed that the co-cultured system could increase barrier function of Caco-2 cell. This study successfully
realized the control of multiple physiological related parameters including cyclic mechanical force, fluid shear stress and microbial co-culture, which closely mimicked the complex physiological condition of human intestinal. In addition, Esch et al[39] fixed a layer of SU-8 porous membrane on different shapes of silicon matrix array. After removing the silicon matrix by xenon difluoride, a 3D membrane was obtained. Caco-2 cells were cultured on the membrane and the fluorescence detection results showed that the cells could form small intestinal villi structure and secrete occludin, indicating that the cells could form tight junctions. Although organs-on-chips technology has made an accurate simulation of the complex intestinal physiology function, the intestinal absorption ability on chip has been rarely studied. Imura et al[40] created a dual-layered chip separated by a porous membrane and measured the absorptivity of cyclophosphamide and fluorescent yellow. Therefore, the determination of drug absorption based on the existing intestinal model should be a development trend. 3.4
Lung-on-a-chip
Lung is the respiratory organ of human, and alveoli are the main site of gas exchange and the functional unit of lung. The alveoli are composed of a single layer of epithelial cells and endothelial cells in the inner layer of the pulmonary capillary. Due to the complex physiological structure of alveoli, traditional in vitro culture modes cannot accurately mimic the physiological models of lung structure. Microfluidic technology can provide a powerful platform for the establishment of lung model and lung pathology research in vitro because of its precise control of fluid flow and chip size, continuous fluid perfusion and gas exchange capacity. Huh et al[41] constructed a double-layered lung-on-a-chip to mimic human respiratory process as shown in Fig.2. The chip was formed of two layers. The upper layer was a gas channel and the lower was a liquid one. The channels were separated by a porous elastic PDMS membrane. Human alveolar epithelial cells were cultured in the gas-liquid interface of the upper side
Fig.2 Structure of microfluidic lung-on-a-chip device[41]
SUN Wei et al. / Chinese Journal of Analytical Chemistry, 2016, 44(4): 533–541
of membrane, and microvascular endothelial cells were introduced into the lower channel in dynamic fluid environment to form an alveolar-capillary barrier. Two adjacent side channels connected to the vacuum pump were on the two sides of the main channel. Through regular vacuum condition changes (0.25 Hz), PDMS membrane was deformed to simulate expansion and contraction of alveolar wall in breathing, which was difficult to be achieved in the traditional in vitro model. More importantly, physiological response could be induced in the system by introducing inflammatory stimuli through the airway and adding neutrophils to the liquid channel. So this chip can be used in studying the toxicity of nanoparticles. In the following work, the researchers successfully established a pulmonary edema pathology model by introduction of interleukin-2 into the system[42]. The alveolar wall is composed of only one layer of epithelial cells, which is vulnerable to injury. The damage models of lung epithelial cells based on microfluidic platform include the shear stress model, mechanical tension model, and the model of the two forces at the same time[43]. For example, Takayama’s group[44] designed an automated chip for studying fluid shear stress on cell injury. The chip was composed of two fluid channels which were separated by a porous polyester membrane. The lower channel was perfused with liquid and small airway epithelial cells were cultured on the upper channel. At the same time, liquid plugs were generated in upper channel to cause flow fluid shear stress in the flow process, which was in favor of the establishment of cell injury model. Based on this model, researchers successfully designed a microfluidic lung airway chip[45] to simulate the lung epithelial cells damage by airway surface liquid (ASL) deposition of liquid plug flow. Studies showed that most cell damage occurred in the propagation of liquid plugs, and surfactant could effectively prevent it, suggesting that surfactant could be used as a potential treatment agent in lung diseases such as acute respiratory distress syndrome and other illnesses. Although the lung-on-a-chips described above have special physiological structure, the existing models are still relatively simple compared with lung structure in the aspects of complexity and specificity. Therefore, how to extensively mimic lung physiology structure and establish a complete lung model will be the research focus in the future. 3.5
Heart-on-a-chip
Mature cardiomyocytes are highly polarized and contractile cells. The contractility of cardiomyocytes is closely related to the physical and chemical environment, such as flow rate, calcium ion concentration, substrate material, electrical stimulation, etc[46]. In recent decades, researchers developed functional heart models and gained many important achievements. But the physiological structure and external support of these models could not be combined with
microfluidic technology, which could manipulate cardiac tissue in microscale to get closer to the physiologically morphological, electrophysiological and contractile function. Grosberg et al[47] covered a thin layer of cardiomyocytes on a piece of elastic thin film surface to study the contraction of cardiomyocytes by electrical stimulation. In addition, researchers used real-time data collection and analysis technology to detect dynamic changes of adrenaline in the device. The detection range was from 10‒12 M to 10‒4 M. Myocardial hypoxia can cause myocardial cell damage, leading to arrhythmia, ischemia and heart failure. Ren et al[48] designed a biomimetic microfluidic chip to mimic hypoxia damage on cardiac muscle cells. The chip utilized two columns of parallel microcolumn to construct capillary endothelial barrier, which could accurately mimic the structure and function of myocardial tissue. When different concentrations of the uncoupler reagent FCCP were added to cardiomyocytes, the apoptosis occurred, which was related with FCCP concentration and action time. The regulation of Ca2+ is one of the main features of cardiomyocytes, and it is affected by acute hypoxia. To study the changes of intracellular Ca2+ concentration in early hypoxia progress, Martewicz et al[49] designed a chip to detect dynamic changes of intracellular Ca2+ concentration online and simulated the acute hypoxia of cells by rapid changing the oxygen concentration in the chip. The experimental results showed that the hypoxia could induce reversible changes of Ca2+ concentration in neonatal rats. Most of the heart models based on microfluidic technology use PDMS material, but PDMS is not conducive to cell adhesion on the chip. Annabi et al[50] covered two kinds of cell compatible hydrogels, GelMA and Metro, on the surface of PDMS channel respectively to promote the adhesion of cardiomyocytes. Compared with the GelMA-covered channels, the primary cardiomyocytes grown on Metro showed better adhesion and contractility. The existing heart-on-a-chip models reflect the contractility of cardiomyocytes well by changing the external parameters. In order to establish more realistic models of heart, more factors such as 3D environment and co-culture mode should become the key points. In addition, mammalian cardiomyocytes are normally used on chip at this stage. So in the further study, more human cells are needed to be used in drug toxicity experiments to obtain more realistic results. 3.6
Vessel-on-a-chip
Blood vessels are series of channels that allow blood passes through. They are the major parts to connect all organs and realize mass exchange among organs. Microvascular networks play an important role in maintaining stability of the metabolism and tissue microenvironment. Establishment of an in vitro model of vascular morphology and physiology can
SUN Wei et al. / Chinese Journal of Analytical Chemistry, 2016, 44(4): 533–541
accelerate the pathophysiology research in complex microvascular networks system[51]. Therefore, the study of the interaction between organs in vitro and even the "human-onchip" construction are of great significance. Kim et al[52] designed a 3D perfused chip which was composed of five parallel channels. The channels were separated by microcolumn arrays, but the material transport was allowed. The center channel and the outside channel on the chip were used to cultivate human umbilical vein endothelial cells (HUVECs) and stromal cells respectively, and the rest was used for perfusion. The stromal cells secreted pro-angiogenic factor which acted on HUVECs, achieving the vasculogenesis and angiogenesis on chip. The main advantage of this chip was that the perfused mode could reflect the morphological characteristics of blood vessels in human body realistically and showed stronger barrier function and long-term stability. Human blood is in a state of continuous flow. Shear force can be produced when the fluid impacts on vascular wall. Investigation of the effect of shear stress on the vessel wall is very important in the pathological study of vascular diseases such as atherosclerosis, thrombosis, inflammatory vascular diseases and tumor metastasis by blood vessels[53]. Nguyen et al[54] designed a microfluidic artificial blood vessel to control the direction and flow rate of fluid flow and found a threshold (10 dyn cm‒2) of capillary shear to cause sprouting angiogenesis, thus putting forward the potential vascular density self-balancing mechanism in living organisms. Zhang et al[55] fabricated a three-dimensional structure of the microvascular network on a chip by using type I collagen as a scaffold to study the interactions and angiogenesis in HUVECs and peripheral vascular cell by the addition of growth factors such as vascular endothelial growth factor (VEGF). In further experiments, researchers introduced chemical stimuli such as phorbol ester to mimic the vascular inflammatory response. The experimental results showed that the chemical stimulation might induce endothelial cells changing into prethrombotic state. In addition, Wang et al[56] synthesized an artificial blood vessel by elastic porous transparent cellulose and implanted it into three-dimensional collagen matrix microchip to mimic tumor adhesion and migration via vascular. Because of the complexity of the microvascular network, researchers also need to consider other factors besides shear stress in building vessel-on-a-chip model, for instance, introducing VEGF, transforming growth factor-β[51] and cholesterol to the system and observing the response of vascular endothelial cells, to increase authenticity of the vessel-on-a-chip. 3.7
Multiple organs-on-a-chip
To predict and evaluate the response of human body to various drugs and establish the "human-on-chip", one of the
important steps is to integrate multiple independent organs on a single chip. The toxins produced by liver metabolism can often cause organ toxicity. Therefore, liver cells or tissues are often investigated together with other organs to study metabolic mechanism of different organs. Groothuis’s group[57] designed a perfusion microfluidic chip with two cell culture chambers in series. Rat intestine and liver slices were placed in the two chambers simultaneously to mimic the first pass situation in vivo. Growth factors were secreted by intestinal fibroblast when adding bile secretory products in the first compartment, which led to the decrease of hepatocytes viability in downstream chamber. But similar phenomenon didn’t appear in the hepatocytes cultured alone. Choucha-Snouber et al[58] used a co-culture chip for liver-kidney cell culture and studied the renal toxicity of an anti-cancer drug ifosfamide. Liver and kidney cells were cultivated on the chip and incubated for 24 h first, then 50 μmol ifosfamide medium was added to the chip and perfused for 72 h. The single culture systems of liver and kidney cells were alone set as controls. Toxicity testing was carried out by determination of cell numbers, amount of calcium released, cellular metabolite and other index. Experimental results showed that in the liver- kidney co-culture system, nephrotoxic chlorine acetaldehyde produced by ifosfamide metabolism in hepatocytes resulted in a 30% reduction of kidney cell numbers, which didn’t occur in the control groups. The complexity of physical structure of human body prompts researchers to build more complex microfluidic multi-organ chip system. Zhang et al[1] designed a multichannel 3D microfluidic chip, on which four different human cell types were cultured in different channels to represent the human liver, lung, kidney and adipose tissue, and the control of TGF-β1 concentration in different channels was realized. Shuler’s group[10,59,60] used multiple organs-on-a-chip in a series study of drug pharmacokinetics- pharmacokinetics (PK-PD). For example, Sung et al[59] designed a multi-layered chip as shown in Fig.3. In their work, three different cell lines were cultured with 3-D hydrogel to simulate the liver, tumor and bone marrow. The experiment was carried out by adding 5-fluorouracil to the system, and the results were consistent with that of the PK-PD computational model of 5-fluorouracil. In a recent study, Maschmeyer et al[61] designed a multiple organs-on-a-chip at a size of 105-fold smaller than counterpart human intestines, liver, skin and kidney, thus establishing a multi-organ simulation system on chip. This co-culture system could maintain active function over 28 days during which the cells could maintain high activity, forming functional structure spontaneously and achieving the self-homeostasis of system. From above we can know that the researches of organ-on-achip involves main organs of human body. In addition, other organs models have been successfully constructed in the microfluidic chip, such as the blood brain barrier[8,62,63], muscle[64], skeletal[65], spleen[66], breast[67] and skin models[68,69].
SUN Wei et al. / Chinese Journal of Analytical Chemistry, 2016, 44(4): 533–541
Fig.3 A multi-organ microfluidic framework[59] a. Schematic of layered multi-organ chip; b. Picture of the assembled device
4
Conclusions
The microfluidic technology has unique practicality and potential in cell culture in vitro because the microchannel size, spatial location and connection type can be accurately controlled through precise micromachining technology, and a variety of new materials can be used as basement of cell survival, thus a continuous perfused culture mode can be provided. With the combination of micromachining technology and three-dimensional cell culture, as a new cell culture platform, organs-on-chips received extensive attention and made rapid development. The organs-on-chips technology aims to establish an artificial bionic microenvironment, and realize simulation of tissue or organ level, then perform human physiological relevant research, drug development and toxicology study on the chips. It can overcome the disadvantages of traditional 2D cell culture models and animal experiments. It also has the potential to establish highly biomimetic in vitro physiological model, which may change the development process of some industries represented by pharmaceutical industry. The ultimate goal of organs-on-chips is to integrate different cells of various organs in a single chip and to build more complicated multiple organs-on-a-chip model even human models to ultimately realize “human-on-chip”. The system will provide a new platform for researches of the human circulatory system, drug pharmacokinetics and pharmacodynamics. Organs-on-chips technology is still in its infancy. There are still a large number of technical and industrial challenges to be solved. The first one is to develop new suitable materials for cell culture. The existing materials on chips are normally based on PDMS, polycarbonate, etc, which have been widely used for cell culture on chip because of good permeability and biological compatibility. However, PDMS was demonstrated to adsorb hydrophobic small molecules, which could result in a reduction of effective concentration and activity of drug and experimental errors. So chemical modification should be done or other substitute materials were needed. The second one is to use more reliable human cells. The research of next generation
organs-on-chips will focus on the use of primary cells and human induced pluripotent stem cells, which have important implications for the study of specific diseases, personalized medicine and new drug development. And for the third one, due to the small size and low cell capacity of the chip, we need to develop highly sensitive detection strategies and devices. Only when the biological markers and cellular processes are accurately detected in real time without the cell viability loses, and the potential of the organs-on-chips can be fully realized. Therefore, developing suitable electrochemical, optical and immunological detection methods on chip, such as using all kinds of sensors and designing more standardized chips for matching with traditional biological detection method, will also become the research focus. We believe that, with the development of technology, organs-on-chip will show extraordinary talents in life science, medical and pharmaceutical research.
References [1]
Zhang C, Zhao Z, Abdul Rahim N A, van Noort D, Yu H. Lab Chip, 2009, 9(22): 3185–3192
[2]
http://www.accessdata.fda.gov/scripts/fdatrack/view/track_proj ect.cfm?program=oc-chief-scientist&id=OCS-Modernizing-To xicology
[3]
Huh D, Hamilton G A, Ingber D E. Trends Cell Biol., 2011, 21(12): 745–754
[4]
Esch E W, Bahinski A, Huh D. Nat. Rev. Drug Discov., 2015, 14(4): 248–260
[5]
Lin B C. Micro/Nano Fluidic Chip Laboratory. Beijing: Science Press, 2013: 2–5
[6]
Ye N, Qin J, Shi W, Liu X, Lin B. Lab Chip, 2008, 7(12): 1696–704
[7]
Galie P A, Nguyen D H, Choi C K, Cohen D M, Janmey P A, Chen C S. Proc. Natl. Acad. Sci. USA, 2014, 111(22): 7968–7973
[8]
Ho C T, Lin R Z, Chen R J, Chin C K, Gong S E, Chang H Y, Peng H L, Hsu L, Yew T R, Chang S F, Liu C H. Lab Chip, 2013, 13(18): 3578–3587
SUN Wei et al. / Chinese Journal of Analytical Chemistry, 2016, 44(4): 533–541
[9]
Booth R, Kim H. Lab Chip, 2012, 12(10): 1784–1792
[10] Sung J H, Shuler M L. Lab Chip, 2009, 9(10): 1385-1394 [11] Sung J H, Esch M B, Prot J M, Long C J, Smith A, Hickman J J, Shuler M L. Lab Chip, 2013, 13(7): 1201–1212 [12] van der Meer A D, van den Berg A. Integr. Biol. (Camb), 2012, 4(5): 461–470 [13] http://mimetas.com/news_files/pharma-funds-kidney-on-a-chip. php [14] http://wyss.harvard.edu/viewpressrelease/204/emulate-announc es-strategic-collaboration-with-johnson-johnson-innovation-touse-organsonchips-platform-to-better-predict-human-response-i n-drug-development-process [15] Liu W M, Li L, Ren L, Wang J C, Tu Q, Wang X Q, Wang J Y. Chinese J. Anal. Chem., 2012, 40(1): 24–31 [16] Bhatia S N, Ingber D E. Nat. Biotechnol., 2014, 32(8): 760–772 [17] Jang K J, Mehr A P, Hamilton G A, McPartlin L A, Chung S, Suh K Y, Ingber D E. Integr. Biol. (Camb), 2013, 5(9): 1119–1129 [18] Wang L H, Liu D Y, Wang B, Sun J, Li L H. Chinese J. Anal. Chem., 2008, 36(2): 143–149 [19] Wang Y, Toh Y C, Li Q, Nugraha B, Zheng B, Lu T B, Gao Y, Ng M M, Yu H. Integr. Biol. (Camb), 2013, 5(2): 390–401 [20] Yum K, Hong S G, Healy K E, Lee L P. Biotechnol. J., 2014, 9(1): 16–27 [21] Keenan T M, Folch A. Lab Chip, 2008, 8(1): 34–57 [22] Sung J H, Yu J, Luo D, Shuler M L, March J C. Lab Chip, 2011, 11(3): 389–392 [23] Zhou J, Khodakov D A, Ellis A V, Voelcker N H. Electrophoresis, 2012, 33(1): 89–104 [24] Ho C T, Lin R Z, Chang W Y, Chang H Y, Liu C H. Lab Chip, 2006, 6(6): 724–734 [25] Takayama S, Ostuni E, LeDuc P, Naruse K, Ingber D E, Whitesides G M. Nature, 2001, 411(6841): 1016 [26] Toh Y C, Lim T C, Tai D, Xiao G, van Noort D, Yu H. Lab Chip, 2009, 9(14): 2026–2035 [27] Iredale J P, Arthur M J. Gut, 1994, 35(6): 729–732 [28] Lee P J, Hung P J, Lee L P. Biotechnol. Bioeng., 2007, 97(5): 1340–1346 [29] Hegde M, Jindal R, Bhushan A, Bale S S, McCarty W J, Golberg I, Usta O B, Yarmush M L. Lab Chip, 2014, 14(12): 2033–2039 [30] Gómez-Lechón M J, Tolosa L, Conde I, Donato M T. Expert Opin. Drug Metab. Toxicol., 2014, 10(11): 1553–1568 [31] Hoffmann D, Adler M, Vaidya V S, Rached E, Mulrane L,
[35] Wei Z, Amponsah P K, Al-Shatti M, Nie Z, Bandyopadhyay B C. Lab Chip, 2012, 12(20): 4037–4040 [36] Mu X, Zheng W, Xiao L, Zhang W, Jiang X. Lab Chip, 2013, 13(8): 1612–1618 [37] Yeon J H, Park J K. Anal. Chem., 2009, 81(5): 1944–1951 [38] Kim H J, Huh D, Hamilton G, Ingber D E. Lab Chip, 2012, 12(12): 2165–2174 [39] Esch M B, Sung J H, Yang J, Yu C, Yu J, March J C, Shuler M L. Biomed. Microdevices, 2012, 14(5): 895–906 [40] Imura Y, Asano Y, Sato K, Yoshimura E. Anal. Sci., 2009, 25(12): 1403–1407 [41] Huh D, Matthews B D, Mammoto A, Montoya-Zavala M, Hsin H Y, Ingber D E. Science, 2010, 328(5986): 1662–1668 [42] Huh D, Leslie D C, Matthews B D, Fraser J P, Jurek S, Hamilton G A, Thorneloe K S, McAlexander M A, Ingber D E. Sci. Transl. Med., 2012, 4(159): 159ra147 [43] Douville N J, Zamankhan P, Tung Y C, Li R, Vaughan B L, Tai C F, White J, Christensen P J, Grotberg J B, Takayama S. Lab Chip, 2011, 11(4): 609–619 [44] Huh D, Fujioka H, Tung Y C, Futai N, Paine R, Grotberg J B, Takayama S. Proc. Natl. Acad. Sci. USA, 2007, 104(48): 18886–18891 [45] Tavana H, Zamankhan P, Christensen P J, Grotberg J B, Takayama S. Biomed. Microdevices, 2011, 13(4): 731–742 [46] Polini A, Prodanov L, Bhise N S, Manoharan V, Dokmeci M R, Khademhosseini A. Expert Opin. Drug Discov., 2014, 9(4): 335–352 [47] Grosberg A, Alford P W, McCain M L, Parker K K. Lab Chip, 2011, 11(24): 4165–4173 [48] Ren L, Liu W, Wang Y, Wang J C, Tu Q, Xu J, Liu R, Shen S F, Wang J. Anal. Chem., 2013, 85(1): 235–244 [49] Martewicz S, Michielin F, Serena E, Zambon A, Mongillo M, Elvassore N. Integr. Biol. (Camb), 2012, 4(2): 153–164 [50] Annabi N, Selimovic S, Acevedo Cox J P, Ribas J, Afshar Bakooshli
M,
Heintze
D,
Weiss
A
S,
Cropek
D,
Khademhosseini A. Lab Chip, 2013, 13(18): 3569–3577 [51] van der Meer A D, Orlova V V, ten Dijke P, van den Berg A, Mummery C L. Lab Chip, 2013, 13(18): 3562–3568. [52] Kim S, Lee H, Chung M, Jeon N L. Lab Chip, 2013, 13(8): 1489–1500 [53] Zheng Y, Chen J, Craven M, Choi N W, Totorica S, Diaz-Santana A, Kermani P, Hempstead B, Fischbach-Teschl C, Lopez J A, Stroock A D. Proc. Natl. Acad. Sci. USA, 2012, 109(24): 9342–9347
Gallagher W M, Callanan J J, Gautier J C, Matheis K, Staedtler
[54] Nguyen D H, Stapleton S C, Yang M T, Cha S S, Choi C K,
F, Dieterle F, Brandenburg A, Sposny A, Hewitt P,
Galie P A, Chen C S. Proc. Natl. Acad. Sci. USA, 2013,
Ellinger-Ziegelbauer H, Bonventre J V, Dekant W, Mally A. Toxicol. Sci., 2010, 116(1): 8–22 [32] Jang K J, Suh K Y. Lab Chip, 2010, 10(1): 36–42 [33] Jang K J, Mehr A P, Hamilton G A, McPartlin L A, Chung S, Suh K Y, Ingber D E. Integr. Biol. (Camb), 2013, 5(9): 1119–1129 [34] Zhou M, Ma H, Lin H, Qin J. Biomaterials, 2014, 35(5): 1390–1401
110(17): 6712–6717 [55] Zhang H Y, Zhang Z, Ji X H, He Z K. Chinese J. Anal. Chem., 2014, 42(9): 1276–1280 [56] Wang X Y, Jin Z H, Gan B W, Lv S W, Xie M, Huang W H. Lab Chip, 2014, 14(15): 2709–2716 [57] van Midwoud P M, Merema M T, Verpoorte E, Groothuis G M. Lab Chip, 2010, 10(20): 2778–2786
SUN Wei et al. / Chinese Journal of Analytical Chemistry, 2016, 44(4): 533–541
[58] Choucha-Snouber L, Aninat C, Grsicom L, Madalinski G, Brochot C, Poleni P E, Razan F, Guillouzo C G, Legallais C, Corlu A, Leclerc E. Biotechnol. Bioeng., 2013, 110(2): 597–608 [59] Sung J H, Kam C, Shuler M L. Lab Chip, 2010, 10(4): 446–455 [60] Esch M B, Mahler G J, Stokor T, Shuler M L. Lab Chip, 2014, 14(16): 3081–3092 [61] Maschmeyer I, Lorenz A K, Schimek K, Hasenberg T, Ramme
Sidoryk-Wegrzynowicz M, Aschner M, Pant K. Lab Chip, 2013, 13(6): 1093–1101 [64] Grosberg A, Nesmith A P, Goss J A, Brigham M D, McCain M L, Parker K K. J. Pharmacol. Toxicol. Methods, 2012, 65(3): 126–135 [65] Altmann B, Lochner A, Swain M, Kohal R J, Giselbrecht S, Gottwald E, Steinberg T, Tomakidi P. Biomaterials, 2014, 35(10): 3208–3219
A P, Hubner J, Lindner M, Drewell C, Bauer S, Thomas A,
[66] Baker M. Nature, 2011, 471(7340): 661–665
Sambo N S, Sonntag F, Lauster R, Marx. Lab Chip, 2015,
[67] Grafton M M, Wang L, Vidi P A, Leary J, Lelievre S A. Integr.
15(12): 2688–2699 [62] Griep L M, Wolbers F, de Wagenaar B, ter Braak P M, Weksler B B, Romero I A, Couraud P O, Vermes I, van der Meer A D, van den Berg A. Biomed. Microdevices, 2013, 15(1): 145–150 [63] Prabhakarpandian B, Shen M C, Nichols J B, Mills I R,
Biol. (Camb), 2011, 3(4): 451–459 [68] Atac B, Wagner I, Horland R, Lauster R, Marx U, Tonevitsky A G, Azar R P, Lindner G. Lab Chip, 2013, 13(18): 3555–3561 [69] Abaci H E, Gledhill K, Guo Z, Christiano A M, Shuler M L. Lab Chip, 2015, 15(3): 882–888
Cite this article as: Chin J Anal Chem, 2016, 44(4), 533–541.
REVIEW
Organs‐on‐chips and Its Applications SUN Wei1, CHEN Yu-Qing1, LUO Guo-An2,3, ZHANG Min3, ZHANG Hong-Yang1, WANG Yue-Rong1, HU Ping1,* 1
Shanghai Key Laboratory of Functional Materials Chemistry, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, China 2 Department of Chemistry, Tsinghua University, Beijing 10084, China 3 Shanghai Key Laboratory of New Drug Design & Modern Engineering Center for TCM, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
Abstract:
Microfluidic chips have been proven to be attractive platforms for cell culture in vitro. Microfluidic chip-based
organs-on-chips technology has received more attention because it can mimic the complex structures and functions of human organs. In this review, the recent advances of organs-on-chips technology in different organs are introduced, including the build of human physiological models, drug discovery, and toxicology research. The development trend of this technology is also proposed. Key Words:
1
Microfluidic chip; Organs-on-chips; Cell; Human organ; Review
Introduction
Since 2011, Barack Obama, the president of the United States of America, announced the official start of the “human-on-chip[1]” special project established by the National Institutes of Health (NIH), Food and Drug Administration (FDA) and United States Department of Defense[2], a worldwide research interest of the “human-on-chip” has been aroused. The so-called “human-on-chip”, as the research level of now or rather, is the organ-on-a-chip systems, also known as organs-on-chips[3], which is a biomimetic system based on the microengineering technology in microfluidic chip to mimic the key functions of human organs[4]. Organs-on-chips not only has the advantages such as miniaturization, integration and low consumption[5], but also it can accurately control the multi-parameters of system such as chemical concentration gradient[6], fluid shear stress[7], cell patterning[8], tissue-tissue interface[9], organ-organ interaction[10] and so on, mimicking the complex structure, microenvironment and physiological function of human organs. The study of human physiology is a significant part of life science research. The conventional two-dimensional cell
culture modes have been extensively used in the human physiological and pathological studies, but these simple modes couldn’t reflect complex physiological functions of human tissues and organs[11]. So the researchers often did animal experiment instead of in vitro culture mode. However, animal experiments also have drawbacks such as long cycle and high cost. What’s more, animal models often fail to predict the human response to various drugs[12]. The organs-on-chips helps solving the problems of animal experiments, establishing more realistic physiological models on chips, and is expected to become a biomimetic, efficient, and energy-saving physiological research and drug development tools. After the rapid development in recent years, researchers have realized the construction of various organs in microfluidic chips, such as liver, lung, intestine, kidney, vessels, heart and multiple organs. The collaboration between well-known research units and pharmaceutical companies has made the organs-on-chips step into the practical stage. According to the reports from websites, the Netherlands biotechnology company Mimetas developed a kidney-on-achip and reached an cooperation agreement with several
________________________ Received 26 October 2015; accepted 23 December 2015 *Corresponding author. Email: [email protected] This work was supported by the National Science and Technology Major Project for Significant New Drugs Development of China (No. 2013ZX09507005). Copyright © 2016, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(16)60920-9
SUN Wei et al. / Chinese Journal of Analytical Chemistry, 2016, 44(4): 533–541
pharmaceutical companies for drug screening[13]. In addition, Johnson & Johnson also planned to use the thrombosis-onchip system, which was developed by Wyss Institute in Harvard University affiliated with Emulate company, for drug experiments and liver-on-a-chip for the test of drug hepatotoxicity[14]. This review summarizes the research progress of organs-onchips in recent years, and the development trend of this technology is also prospected.
2
Design concepts of organs-on-chips
The growth of cells requires the synergetic effect of various complex external and internal environmental factors. Hence, the reality of the external environmental parameters is to be considered in establishment of in vitro physiological models[15]. By combination of microfluidic technology, micromachining and cell biology, environmental parameters can be precisely controlled on organs-on-chips, which may in turn generate fluid shear stress, mechanical stress, biochemical concentration gradient and other physical and chemical stimuli. The cells on the chip can response to these stimuli, develop self-organization and express more realistic physiological functions, exhibiting special advantages of organ-on-chip in the establishment of in vitro physiology model[16]. (1) Generation of fluid shear stress. Fluid shear stress exists in the human body all the time, but is absent in the traditional static culture. Microfluidic technology enables dynamic perfusion in cell culture through micro pumps, which is conducive to give stable nutrients and discharge waste timely. Furthermore, dynamic environment of cells is more similar to that in vivo. In addition, the fluid shear stress in perfusion culture is essential for some physiological functions of the human body such as renal reabsorption[17]. (2) Generation of dynamic mechanical stress. The pressure in human body is related to many life activities, such as blood pressure, pulmonary pressure, skeletal stress, etc. The steady pressure plays an important role in the body's physiological functions including tissue formation, cell differentiation and even tumor formation[18]. Microfluidic technology can produce periodic mechanical stress by utilizing flexible porous membrane on which the cells are cultured. The mechanical stretching of the membrane caused by external force can mimic the partial physiological functions such as lung breathing, intestinal peristalsis and heart contractions, etc. (3) Generation of concentration gradient. The flow of fluid in the microscale is laminar, which is beneficial to producing various types of concentration gradients in the channel[19]. Various concentration gradient-driven biochemical signals play critical roles in many physiological processes such as cell migration, differentiation, immune response and cancer metastasis[20]. Microfluidic technology can be used to achieve a stable three-dimensional biochemical concentration gradient
by transforming the flow rate and channel size and using micro valve, micro pump or unique channel design so as to simulate a variety of complex physiological processes in human body[21]. In addition, the generation of multi-channel concentration gradients makes it possible for high-throughput screening of drugs on chip. (4) Control of cell patterning. Human body is not just cell stacking, but is consisted of a variety of cells constructed elaborately in an orderly arrangement, to form a functional unity through complex interaction. Microfluidic technology shows superior control ability to cell patterning. Template method[22], surface modification[23], electrochemical method[24], laminar flow[25] and micropillar array[26] have contributed to the cell patterning on the chip. This is of great benefit to the construction of an in vitro physiological model with a complex geometric structure. Meanwhile, it provides an ideal platform for studying the cell-cell and cell-matrix interaction.
3 3.1
Research progresses of organs-on-chips Liver-on-a-chip
Liver is the most important organ where drug metabolism occurs. Researches on the hepatotoxicity of drugs is significant important in drug discovery. The hepatocyte cultured in traditional static two-dimensional cell culture model lacks contact with the extracellular matrix and will lose liver-specific function soon[27]. Moreover, due to lacking of continuous perfusion, this model cannot be presented the in vivo microenvironment. Microfluidic and tissue engineering technology can make the hepatocyte self-organize, thereby maintaining the liver function partly. Most liver-on-a-chip researches focused on the establishment of physiological related models of liver on a chip, such as bile duct, hepatic lobule, and hepatic sinusoid model, which are designed for drug screening and toxicology studies. For example, in a early study, Lee et al[28] designed an endothelial-like barrier structure, in which the primary hepatocytes were cultured, and the media was perfused in external channel as shown in Fig. 1. It is the first model of liver sinusoid established on microfluidic chip. The penetrative endothelial-like barrier structure could not only isolate primary hepatocytes and external sinusoid-like region spatially, but also realize mass transport. The cells could survive for seven days without an extracellular matrix coating. Experiment results showed that the drug diclofenac had no hepatotoxicity in 4 h, but the cell would die in a longer period of time (24 h), indicating that hepatocytes in this model had physiological activity. This study laid the foundation for the establishment of liver model on chip. Ho et al[24] used field-induced dielectrophoresis trap to generate radial-pattern electric fields, patterned hepatocytes and epithelial cells in a circular polydimethylsiloxane (PDMS) chip and successfully mimicked the structure of hepatic lobule.
SUN Wei et al. / Chinese Journal of Analytical Chemistry, 2016, 44(4): 533–541
In subsequent studies, this group improved the model and designed the “honeycomb” dielectrophoresis array to mimic hepatic lobule. The results of cell-cell interactions study showed that the structure could effectively enhance the activity of CYP450-1A1 enzyme. Hegde et al[29] developed an on-chip method to culture hepatocytes in a sandwich configuration. They designed a dual-layered chip using the porous PET film to separate the channels. Continuous perfusion flow was in the upper channel to culture rat primary hepatocytes sandwiched by collagen and fibronectin in the lower channel. After more than two weeks of culture, the hepatocytes could form a bile canaliculi structure and some liver function was achieved. Compared with the existing in vitro liver model, the main advantage of liver-on-a-chip is achieving part of the liver function of liver cell clusters in micron scale, thereby establishing a more realistic liver model and maintaining liver specific functions for a long time[30]. Because microfluidic technology has great potential in high content screening, how to realize the multi-parameter, high content and rapid analysis on chip will be the focus in future studies. 3.2
Kidney-on-a-chip
Kidney is an important excretory organ in human body and plays an important role in maintaining osmotic pressure and self-homeostasis in vivo. The basic functional unit of kidney is nephron which can remove metabolites and waste in vivo through production of urine, and retain moisture and other nutrients by reabsorption function[31]. Fluid shear stress is one of the significant influencing factors of nephron function. It can change the function and morphology of nephrocytes, and is closely related with some diseases such as polycystic kidney disease (PKD). Jang et al[32] designed a double-layered chip to simulate the structure of kidney proximal tubule, in which 1 dyn/cm2 of fluid shear stress was introduced. The chip was divided into two layers by an elastic porous membrane, and the primary human renal proximal tubular epithelial cells were cultured in the upper channel. Upon exposure to the fluid shear stress, albumin transport and glucose reabsorption
capacity were 2-fold and 3.5-fold respectively as those of transwell experiment, which fully proved the importance of fluid shear stress on establishment of renal physiology model. These researchers further tested the cytotoxicity of cisplatin and compared with the results from Transwell culture on the renal chip[33] by adding quantitative concentration (100 μmol) of cisplatin solution in the lower channel and perfusing 24 h to cause cell death. Cell viability was detected by lactate dehydrogenase activity and V-FITC/PI staining method. The results showed that under the same conditions, cell viability on chip was higher than that in Transwell cultures in same circumstances. In a renal pathophysiology study, Zhou et al[34] designed a circular chip containing 12 channels to study the epithelial-to-mesenchymal transition (EMT) induced renal interstitial fibrosis. When human proximal renal tubular epithelial cells were exposed to serum proteins in a constant flow rate, the cells developed to a mesenchymal phenotype and incubating cells with C3a were induced with similar features. But this phenomenon did not appear in heatinactivated serum. This study provided a new perspective to the study of this pathological process. Wei et al[35] designed a single chip with a tubular channel. Monolayers of human renal tubular epithelial cells (HK-2) were cultured in the tube wall by adding CaCl2 and Na3PO4 in HBSS buffer to simulate the formation of calcium phosphate stone. The kidney stone model in microfluidic chip was established for the first time and was proved to have the potential to be used as disease model. In addition to PDMS, hydrogel materials were also successfully applied in the establishment of renal physiology model. Mu et al[36] utilized fibrillogenesis of collagen to splice two piece of hydrogel together and form two parallel three-dimensional micro-vascular networks to establish a physiological model of nephron. In this model, different cells like MDCK cells and human umbilical vein endothelial cells (HUVECs) could be cultured in different channel, and passive diffusion of nephron could be mimicked through the mass transfer of hydrogel. The hydrogel structure could be applied to establish a more complex physiological kidney model on chip, which increased the diversity and complexity of the chip.
Fig.1 Microfluidic endothelial-like barrier model[28] a. SEM micrograph and structure diagram of microfluidic chip, scale bar represents 50 μm; b. primary hepatocyte cultured for 7 days
SUN Wei et al. / Chinese Journal of Analytical Chemistry, 2016, 44(4): 533–541
3.3
Gut-on-a-chip
Intestine is the longest part of digestive tract, and also the most important part for digestive function. Most drugs are taken into human body via oral administration. Oral drugs pass through small intestine into blood circulation, so the study of drug absorption through intestinal cells is very important in drug screening[37]. The villi on the intestine wall render a huge surface area to small intestine and facilitate rapid absorption of nutrients. The building of small intestinal villi on the chip is of great significance to study the role of intestinal function. March’s group[22] used laser ablation, PDMS soft lithography and hydrogel as template to construct the “comb shaped” 3D hydrogel structures on chip, for the first time, to mimic human intestinal villi. The structure was close to the intestinal villi in the shape and distribution density with a length about 450–500 μm. Human intestinal epithelial cells (Caco-2) were seeded onto the structure to measure permeability and trans-epithelial electrical resistance (TEER). The results showed that this model was closer to real situation in human body compared with traditional 2D model. In further experiments, vascular endothelial cells might be introduced into the hydrogel structure to create a more complex physiological model for drug absorption experiments. Intestinal tract has special modes of motion, including segmental contraction, intestinal peristalsis and movement of intestinal villi. Intestinal peristalsis plays an important role in nutrient digestion and absorption. To simulate human intestinal peristalsis, Kim et al[38] designed a dual-layered chip. The chip was separated into two layers by an elastic porous membrane. Stretching the membrane in cycles by a vacuum pump (0.15 Hz) could mimic the expansion and contraction of human intestinal peristalsis. Simultaneously, Caco-2 cells were cultured on the membrane, and the experimental results showed that the cells could differentiate and form intestinal villi structure. More importantly, human intestinal parasitic E. coli and epithelial cells were co-cultured on the chip. The results showed that the co-cultured system could increase barrier function of Caco-2 cell. This study successfully
realized the control of multiple physiological related parameters including cyclic mechanical force, fluid shear stress and microbial co-culture, which closely mimicked the complex physiological condition of human intestinal. In addition, Esch et al[39] fixed a layer of SU-8 porous membrane on different shapes of silicon matrix array. After removing the silicon matrix by xenon difluoride, a 3D membrane was obtained. Caco-2 cells were cultured on the membrane and the fluorescence detection results showed that the cells could form small intestinal villi structure and secrete occludin, indicating that the cells could form tight junctions. Although organs-on-chips technology has made an accurate simulation of the complex intestinal physiology function, the intestinal absorption ability on chip has been rarely studied. Imura et al[40] created a dual-layered chip separated by a porous membrane and measured the absorptivity of cyclophosphamide and fluorescent yellow. Therefore, the determination of drug absorption based on the existing intestinal model should be a development trend. 3.4
Lung-on-a-chip
Lung is the respiratory organ of human, and alveoli are the main site of gas exchange and the functional unit of lung. The alveoli are composed of a single layer of epithelial cells and endothelial cells in the inner layer of the pulmonary capillary. Due to the complex physiological structure of alveoli, traditional in vitro culture modes cannot accurately mimic the physiological models of lung structure. Microfluidic technology can provide a powerful platform for the establishment of lung model and lung pathology research in vitro because of its precise control of fluid flow and chip size, continuous fluid perfusion and gas exchange capacity. Huh et al[41] constructed a double-layered lung-on-a-chip to mimic human respiratory process as shown in Fig.2. The chip was formed of two layers. The upper layer was a gas channel and the lower was a liquid one. The channels were separated by a porous elastic PDMS membrane. Human alveolar epithelial cells were cultured in the gas-liquid interface of the upper side
Fig.2 Structure of microfluidic lung-on-a-chip device[41]
SUN Wei et al. / Chinese Journal of Analytical Chemistry, 2016, 44(4): 533–541
of membrane, and microvascular endothelial cells were introduced into the lower channel in dynamic fluid environment to form an alveolar-capillary barrier. Two adjacent side channels connected to the vacuum pump were on the two sides of the main channel. Through regular vacuum condition changes (0.25 Hz), PDMS membrane was deformed to simulate expansion and contraction of alveolar wall in breathing, which was difficult to be achieved in the traditional in vitro model. More importantly, physiological response could be induced in the system by introducing inflammatory stimuli through the airway and adding neutrophils to the liquid channel. So this chip can be used in studying the toxicity of nanoparticles. In the following work, the researchers successfully established a pulmonary edema pathology model by introduction of interleukin-2 into the system[42]. The alveolar wall is composed of only one layer of epithelial cells, which is vulnerable to injury. The damage models of lung epithelial cells based on microfluidic platform include the shear stress model, mechanical tension model, and the model of the two forces at the same time[43]. For example, Takayama’s group[44] designed an automated chip for studying fluid shear stress on cell injury. The chip was composed of two fluid channels which were separated by a porous polyester membrane. The lower channel was perfused with liquid and small airway epithelial cells were cultured on the upper channel. At the same time, liquid plugs were generated in upper channel to cause flow fluid shear stress in the flow process, which was in favor of the establishment of cell injury model. Based on this model, researchers successfully designed a microfluidic lung airway chip[45] to simulate the lung epithelial cells damage by airway surface liquid (ASL) deposition of liquid plug flow. Studies showed that most cell damage occurred in the propagation of liquid plugs, and surfactant could effectively prevent it, suggesting that surfactant could be used as a potential treatment agent in lung diseases such as acute respiratory distress syndrome and other illnesses. Although the lung-on-a-chips described above have special physiological structure, the existing models are still relatively simple compared with lung structure in the aspects of complexity and specificity. Therefore, how to extensively mimic lung physiology structure and establish a complete lung model will be the research focus in the future. 3.5
Heart-on-a-chip
Mature cardiomyocytes are highly polarized and contractile cells. The contractility of cardiomyocytes is closely related to the physical and chemical environment, such as flow rate, calcium ion concentration, substrate material, electrical stimulation, etc[46]. In recent decades, researchers developed functional heart models and gained many important achievements. But the physiological structure and external support of these models could not be combined with
microfluidic technology, which could manipulate cardiac tissue in microscale to get closer to the physiologically morphological, electrophysiological and contractile function. Grosberg et al[47] covered a thin layer of cardiomyocytes on a piece of elastic thin film surface to study the contraction of cardiomyocytes by electrical stimulation. In addition, researchers used real-time data collection and analysis technology to detect dynamic changes of adrenaline in the device. The detection range was from 10‒12 M to 10‒4 M. Myocardial hypoxia can cause myocardial cell damage, leading to arrhythmia, ischemia and heart failure. Ren et al[48] designed a biomimetic microfluidic chip to mimic hypoxia damage on cardiac muscle cells. The chip utilized two columns of parallel microcolumn to construct capillary endothelial barrier, which could accurately mimic the structure and function of myocardial tissue. When different concentrations of the uncoupler reagent FCCP were added to cardiomyocytes, the apoptosis occurred, which was related with FCCP concentration and action time. The regulation of Ca2+ is one of the main features of cardiomyocytes, and it is affected by acute hypoxia. To study the changes of intracellular Ca2+ concentration in early hypoxia progress, Martewicz et al[49] designed a chip to detect dynamic changes of intracellular Ca2+ concentration online and simulated the acute hypoxia of cells by rapid changing the oxygen concentration in the chip. The experimental results showed that the hypoxia could induce reversible changes of Ca2+ concentration in neonatal rats. Most of the heart models based on microfluidic technology use PDMS material, but PDMS is not conducive to cell adhesion on the chip. Annabi et al[50] covered two kinds of cell compatible hydrogels, GelMA and Metro, on the surface of PDMS channel respectively to promote the adhesion of cardiomyocytes. Compared with the GelMA-covered channels, the primary cardiomyocytes grown on Metro showed better adhesion and contractility. The existing heart-on-a-chip models reflect the contractility of cardiomyocytes well by changing the external parameters. In order to establish more realistic models of heart, more factors such as 3D environment and co-culture mode should become the key points. In addition, mammalian cardiomyocytes are normally used on chip at this stage. So in the further study, more human cells are needed to be used in drug toxicity experiments to obtain more realistic results. 3.6
Vessel-on-a-chip
Blood vessels are series of channels that allow blood passes through. They are the major parts to connect all organs and realize mass exchange among organs. Microvascular networks play an important role in maintaining stability of the metabolism and tissue microenvironment. Establishment of an in vitro model of vascular morphology and physiology can
SUN Wei et al. / Chinese Journal of Analytical Chemistry, 2016, 44(4): 533–541
accelerate the pathophysiology research in complex microvascular networks system[51]. Therefore, the study of the interaction between organs in vitro and even the "human-onchip" construction are of great significance. Kim et al[52] designed a 3D perfused chip which was composed of five parallel channels. The channels were separated by microcolumn arrays, but the material transport was allowed. The center channel and the outside channel on the chip were used to cultivate human umbilical vein endothelial cells (HUVECs) and stromal cells respectively, and the rest was used for perfusion. The stromal cells secreted pro-angiogenic factor which acted on HUVECs, achieving the vasculogenesis and angiogenesis on chip. The main advantage of this chip was that the perfused mode could reflect the morphological characteristics of blood vessels in human body realistically and showed stronger barrier function and long-term stability. Human blood is in a state of continuous flow. Shear force can be produced when the fluid impacts on vascular wall. Investigation of the effect of shear stress on the vessel wall is very important in the pathological study of vascular diseases such as atherosclerosis, thrombosis, inflammatory vascular diseases and tumor metastasis by blood vessels[53]. Nguyen et al[54] designed a microfluidic artificial blood vessel to control the direction and flow rate of fluid flow and found a threshold (10 dyn cm‒2) of capillary shear to cause sprouting angiogenesis, thus putting forward the potential vascular density self-balancing mechanism in living organisms. Zhang et al[55] fabricated a three-dimensional structure of the microvascular network on a chip by using type I collagen as a scaffold to study the interactions and angiogenesis in HUVECs and peripheral vascular cell by the addition of growth factors such as vascular endothelial growth factor (VEGF). In further experiments, researchers introduced chemical stimuli such as phorbol ester to mimic the vascular inflammatory response. The experimental results showed that the chemical stimulation might induce endothelial cells changing into prethrombotic state. In addition, Wang et al[56] synthesized an artificial blood vessel by elastic porous transparent cellulose and implanted it into three-dimensional collagen matrix microchip to mimic tumor adhesion and migration via vascular. Because of the complexity of the microvascular network, researchers also need to consider other factors besides shear stress in building vessel-on-a-chip model, for instance, introducing VEGF, transforming growth factor-β[51] and cholesterol to the system and observing the response of vascular endothelial cells, to increase authenticity of the vessel-on-a-chip. 3.7
Multiple organs-on-a-chip
To predict and evaluate the response of human body to various drugs and establish the "human-on-chip", one of the
important steps is to integrate multiple independent organs on a single chip. The toxins produced by liver metabolism can often cause organ toxicity. Therefore, liver cells or tissues are often investigated together with other organs to study metabolic mechanism of different organs. Groothuis’s group[57] designed a perfusion microfluidic chip with two cell culture chambers in series. Rat intestine and liver slices were placed in the two chambers simultaneously to mimic the first pass situation in vivo. Growth factors were secreted by intestinal fibroblast when adding bile secretory products in the first compartment, which led to the decrease of hepatocytes viability in downstream chamber. But similar phenomenon didn’t appear in the hepatocytes cultured alone. Choucha-Snouber et al[58] used a co-culture chip for liver-kidney cell culture and studied the renal toxicity of an anti-cancer drug ifosfamide. Liver and kidney cells were cultivated on the chip and incubated for 24 h first, then 50 μmol ifosfamide medium was added to the chip and perfused for 72 h. The single culture systems of liver and kidney cells were alone set as controls. Toxicity testing was carried out by determination of cell numbers, amount of calcium released, cellular metabolite and other index. Experimental results showed that in the liver- kidney co-culture system, nephrotoxic chlorine acetaldehyde produced by ifosfamide metabolism in hepatocytes resulted in a 30% reduction of kidney cell numbers, which didn’t occur in the control groups. The complexity of physical structure of human body prompts researchers to build more complex microfluidic multi-organ chip system. Zhang et al[1] designed a multichannel 3D microfluidic chip, on which four different human cell types were cultured in different channels to represent the human liver, lung, kidney and adipose tissue, and the control of TGF-β1 concentration in different channels was realized. Shuler’s group[10,59,60] used multiple organs-on-a-chip in a series study of drug pharmacokinetics- pharmacokinetics (PK-PD). For example, Sung et al[59] designed a multi-layered chip as shown in Fig.3. In their work, three different cell lines were cultured with 3-D hydrogel to simulate the liver, tumor and bone marrow. The experiment was carried out by adding 5-fluorouracil to the system, and the results were consistent with that of the PK-PD computational model of 5-fluorouracil. In a recent study, Maschmeyer et al[61] designed a multiple organs-on-a-chip at a size of 105-fold smaller than counterpart human intestines, liver, skin and kidney, thus establishing a multi-organ simulation system on chip. This co-culture system could maintain active function over 28 days during which the cells could maintain high activity, forming functional structure spontaneously and achieving the self-homeostasis of system. From above we can know that the researches of organ-on-achip involves main organs of human body. In addition, other organs models have been successfully constructed in the microfluidic chip, such as the blood brain barrier[8,62,63], muscle[64], skeletal[65], spleen[66], breast[67] and skin models[68,69].
SUN Wei et al. / Chinese Journal of Analytical Chemistry, 2016, 44(4): 533–541
Fig.3 A multi-organ microfluidic framework[59] a. Schematic of layered multi-organ chip; b. Picture of the assembled device
4
Conclusions
The microfluidic technology has unique practicality and potential in cell culture in vitro because the microchannel size, spatial location and connection type can be accurately controlled through precise micromachining technology, and a variety of new materials can be used as basement of cell survival, thus a continuous perfused culture mode can be provided. With the combination of micromachining technology and three-dimensional cell culture, as a new cell culture platform, organs-on-chips received extensive attention and made rapid development. The organs-on-chips technology aims to establish an artificial bionic microenvironment, and realize simulation of tissue or organ level, then perform human physiological relevant research, drug development and toxicology study on the chips. It can overcome the disadvantages of traditional 2D cell culture models and animal experiments. It also has the potential to establish highly biomimetic in vitro physiological model, which may change the development process of some industries represented by pharmaceutical industry. The ultimate goal of organs-on-chips is to integrate different cells of various organs in a single chip and to build more complicated multiple organs-on-a-chip model even human models to ultimately realize “human-on-chip”. The system will provide a new platform for researches of the human circulatory system, drug pharmacokinetics and pharmacodynamics. Organs-on-chips technology is still in its infancy. There are still a large number of technical and industrial challenges to be solved. The first one is to develop new suitable materials for cell culture. The existing materials on chips are normally based on PDMS, polycarbonate, etc, which have been widely used for cell culture on chip because of good permeability and biological compatibility. However, PDMS was demonstrated to adsorb hydrophobic small molecules, which could result in a reduction of effective concentration and activity of drug and experimental errors. So chemical modification should be done or other substitute materials were needed. The second one is to use more reliable human cells. The research of next generation
organs-on-chips will focus on the use of primary cells and human induced pluripotent stem cells, which have important implications for the study of specific diseases, personalized medicine and new drug development. And for the third one, due to the small size and low cell capacity of the chip, we need to develop highly sensitive detection strategies and devices. Only when the biological markers and cellular processes are accurately detected in real time without the cell viability loses, and the potential of the organs-on-chips can be fully realized. Therefore, developing suitable electrochemical, optical and immunological detection methods on chip, such as using all kinds of sensors and designing more standardized chips for matching with traditional biological detection method, will also become the research focus. We believe that, with the development of technology, organs-on-chip will show extraordinary talents in life science, medical and pharmaceutical research.
References [1]
Zhang C, Zhao Z, Abdul Rahim N A, van Noort D, Yu H. Lab Chip, 2009, 9(22): 3185–3192
[2]
http://www.accessdata.fda.gov/scripts/fdatrack/view/track_proj ect.cfm?program=oc-chief-scientist&id=OCS-Modernizing-To xicology
[3]
Huh D, Hamilton G A, Ingber D E. Trends Cell Biol., 2011, 21(12): 745–754
[4]
Esch E W, Bahinski A, Huh D. Nat. Rev. Drug Discov., 2015, 14(4): 248–260
[5]
Lin B C. Micro/Nano Fluidic Chip Laboratory. Beijing: Science Press, 2013: 2–5
[6]
Ye N, Qin J, Shi W, Liu X, Lin B. Lab Chip, 2008, 7(12): 1696–704
[7]
Galie P A, Nguyen D H, Choi C K, Cohen D M, Janmey P A, Chen C S. Proc. Natl. Acad. Sci. USA, 2014, 111(22): 7968–7973
[8]
Ho C T, Lin R Z, Chen R J, Chin C K, Gong S E, Chang H Y, Peng H L, Hsu L, Yew T R, Chang S F, Liu C H. Lab Chip, 2013, 13(18): 3578–3587
SUN Wei et al. / Chinese Journal of Analytical Chemistry, 2016, 44(4): 533–541
[9]
Booth R, Kim H. Lab Chip, 2012, 12(10): 1784–1792
[10] Sung J H, Shuler M L. Lab Chip, 2009, 9(10): 1385-1394 [11] Sung J H, Esch M B, Prot J M, Long C J, Smith A, Hickman J J, Shuler M L. Lab Chip, 2013, 13(7): 1201–1212 [12] van der Meer A D, van den Berg A. Integr. Biol. (Camb), 2012, 4(5): 461–470 [13] http://mimetas.com/news_files/pharma-funds-kidney-on-a-chip. php [14] http://wyss.harvard.edu/viewpressrelease/204/emulate-announc es-strategic-collaboration-with-johnson-johnson-innovation-touse-organsonchips-platform-to-better-predict-human-response-i n-drug-development-process [15] Liu W M, Li L, Ren L, Wang J C, Tu Q, Wang X Q, Wang J Y. Chinese J. Anal. Chem., 2012, 40(1): 24–31 [16] Bhatia S N, Ingber D E. Nat. Biotechnol., 2014, 32(8): 760–772 [17] Jang K J, Mehr A P, Hamilton G A, McPartlin L A, Chung S, Suh K Y, Ingber D E. Integr. Biol. (Camb), 2013, 5(9): 1119–1129 [18] Wang L H, Liu D Y, Wang B, Sun J, Li L H. Chinese J. Anal. Chem., 2008, 36(2): 143–149 [19] Wang Y, Toh Y C, Li Q, Nugraha B, Zheng B, Lu T B, Gao Y, Ng M M, Yu H. Integr. Biol. (Camb), 2013, 5(2): 390–401 [20] Yum K, Hong S G, Healy K E, Lee L P. Biotechnol. J., 2014, 9(1): 16–27 [21] Keenan T M, Folch A. Lab Chip, 2008, 8(1): 34–57 [22] Sung J H, Yu J, Luo D, Shuler M L, March J C. Lab Chip, 2011, 11(3): 389–392 [23] Zhou J, Khodakov D A, Ellis A V, Voelcker N H. Electrophoresis, 2012, 33(1): 89–104 [24] Ho C T, Lin R Z, Chang W Y, Chang H Y, Liu C H. Lab Chip, 2006, 6(6): 724–734 [25] Takayama S, Ostuni E, LeDuc P, Naruse K, Ingber D E, Whitesides G M. Nature, 2001, 411(6841): 1016 [26] Toh Y C, Lim T C, Tai D, Xiao G, van Noort D, Yu H. Lab Chip, 2009, 9(14): 2026–2035 [27] Iredale J P, Arthur M J. Gut, 1994, 35(6): 729–732 [28] Lee P J, Hung P J, Lee L P. Biotechnol. Bioeng., 2007, 97(5): 1340–1346 [29] Hegde M, Jindal R, Bhushan A, Bale S S, McCarty W J, Golberg I, Usta O B, Yarmush M L. Lab Chip, 2014, 14(12): 2033–2039 [30] Gómez-Lechón M J, Tolosa L, Conde I, Donato M T. Expert Opin. Drug Metab. Toxicol., 2014, 10(11): 1553–1568 [31] Hoffmann D, Adler M, Vaidya V S, Rached E, Mulrane L,
[35] Wei Z, Amponsah P K, Al-Shatti M, Nie Z, Bandyopadhyay B C. Lab Chip, 2012, 12(20): 4037–4040 [36] Mu X, Zheng W, Xiao L, Zhang W, Jiang X. Lab Chip, 2013, 13(8): 1612–1618 [37] Yeon J H, Park J K. Anal. Chem., 2009, 81(5): 1944–1951 [38] Kim H J, Huh D, Hamilton G, Ingber D E. Lab Chip, 2012, 12(12): 2165–2174 [39] Esch M B, Sung J H, Yang J, Yu C, Yu J, March J C, Shuler M L. Biomed. Microdevices, 2012, 14(5): 895–906 [40] Imura Y, Asano Y, Sato K, Yoshimura E. Anal. Sci., 2009, 25(12): 1403–1407 [41] Huh D, Matthews B D, Mammoto A, Montoya-Zavala M, Hsin H Y, Ingber D E. Science, 2010, 328(5986): 1662–1668 [42] Huh D, Leslie D C, Matthews B D, Fraser J P, Jurek S, Hamilton G A, Thorneloe K S, McAlexander M A, Ingber D E. Sci. Transl. Med., 2012, 4(159): 159ra147 [43] Douville N J, Zamankhan P, Tung Y C, Li R, Vaughan B L, Tai C F, White J, Christensen P J, Grotberg J B, Takayama S. Lab Chip, 2011, 11(4): 609–619 [44] Huh D, Fujioka H, Tung Y C, Futai N, Paine R, Grotberg J B, Takayama S. Proc. Natl. Acad. Sci. USA, 2007, 104(48): 18886–18891 [45] Tavana H, Zamankhan P, Christensen P J, Grotberg J B, Takayama S. Biomed. Microdevices, 2011, 13(4): 731–742 [46] Polini A, Prodanov L, Bhise N S, Manoharan V, Dokmeci M R, Khademhosseini A. Expert Opin. Drug Discov., 2014, 9(4): 335–352 [47] Grosberg A, Alford P W, McCain M L, Parker K K. Lab Chip, 2011, 11(24): 4165–4173 [48] Ren L, Liu W, Wang Y, Wang J C, Tu Q, Xu J, Liu R, Shen S F, Wang J. Anal. Chem., 2013, 85(1): 235–244 [49] Martewicz S, Michielin F, Serena E, Zambon A, Mongillo M, Elvassore N. Integr. Biol. (Camb), 2012, 4(2): 153–164 [50] Annabi N, Selimovic S, Acevedo Cox J P, Ribas J, Afshar Bakooshli
M,
Heintze
D,
Weiss
A
S,
Cropek
D,
Khademhosseini A. Lab Chip, 2013, 13(18): 3569–3577 [51] van der Meer A D, Orlova V V, ten Dijke P, van den Berg A, Mummery C L. Lab Chip, 2013, 13(18): 3562–3568. [52] Kim S, Lee H, Chung M, Jeon N L. Lab Chip, 2013, 13(8): 1489–1500 [53] Zheng Y, Chen J, Craven M, Choi N W, Totorica S, Diaz-Santana A, Kermani P, Hempstead B, Fischbach-Teschl C, Lopez J A, Stroock A D. Proc. Natl. Acad. Sci. USA, 2012, 109(24): 9342–9347
Gallagher W M, Callanan J J, Gautier J C, Matheis K, Staedtler
[54] Nguyen D H, Stapleton S C, Yang M T, Cha S S, Choi C K,
F, Dieterle F, Brandenburg A, Sposny A, Hewitt P,
Galie P A, Chen C S. Proc. Natl. Acad. Sci. USA, 2013,
Ellinger-Ziegelbauer H, Bonventre J V, Dekant W, Mally A. Toxicol. Sci., 2010, 116(1): 8–22 [32] Jang K J, Suh K Y. Lab Chip, 2010, 10(1): 36–42 [33] Jang K J, Mehr A P, Hamilton G A, McPartlin L A, Chung S, Suh K Y, Ingber D E. Integr. Biol. (Camb), 2013, 5(9): 1119–1129 [34] Zhou M, Ma H, Lin H, Qin J. Biomaterials, 2014, 35(5): 1390–1401
110(17): 6712–6717 [55] Zhang H Y, Zhang Z, Ji X H, He Z K. Chinese J. Anal. Chem., 2014, 42(9): 1276–1280 [56] Wang X Y, Jin Z H, Gan B W, Lv S W, Xie M, Huang W H. Lab Chip, 2014, 14(15): 2709–2716 [57] van Midwoud P M, Merema M T, Verpoorte E, Groothuis G M. Lab Chip, 2010, 10(20): 2778–2786
SUN Wei et al. / Chinese Journal of Analytical Chemistry, 2016, 44(4): 533–541
[58] Choucha-Snouber L, Aninat C, Grsicom L, Madalinski G, Brochot C, Poleni P E, Razan F, Guillouzo C G, Legallais C, Corlu A, Leclerc E. Biotechnol. Bioeng., 2013, 110(2): 597–608 [59] Sung J H, Kam C, Shuler M L. Lab Chip, 2010, 10(4): 446–455 [60] Esch M B, Mahler G J, Stokor T, Shuler M L. Lab Chip, 2014, 14(16): 3081–3092 [61] Maschmeyer I, Lorenz A K, Schimek K, Hasenberg T, Ramme
Sidoryk-Wegrzynowicz M, Aschner M, Pant K. Lab Chip, 2013, 13(6): 1093–1101 [64] Grosberg A, Nesmith A P, Goss J A, Brigham M D, McCain M L, Parker K K. J. Pharmacol. Toxicol. Methods, 2012, 65(3): 126–135 [65] Altmann B, Lochner A, Swain M, Kohal R J, Giselbrecht S, Gottwald E, Steinberg T, Tomakidi P. Biomaterials, 2014, 35(10): 3208–3219
A P, Hubner J, Lindner M, Drewell C, Bauer S, Thomas A,
[66] Baker M. Nature, 2011, 471(7340): 661–665
Sambo N S, Sonntag F, Lauster R, Marx. Lab Chip, 2015,
[67] Grafton M M, Wang L, Vidi P A, Leary J, Lelievre S A. Integr.
15(12): 2688–2699 [62] Griep L M, Wolbers F, de Wagenaar B, ter Braak P M, Weksler B B, Romero I A, Couraud P O, Vermes I, van der Meer A D, van den Berg A. Biomed. Microdevices, 2013, 15(1): 145–150 [63] Prabhakarpandian B, Shen M C, Nichols J B, Mills I R,
Biol. (Camb), 2011, 3(4): 451–459 [68] Atac B, Wagner I, Horland R, Lauster R, Marx U, Tonevitsky A G, Azar R P, Lindner G. Lab Chip, 2013, 13(18): 3555–3561 [69] Abaci H E, Gledhill K, Guo Z, Christiano A M, Shuler M L. Lab Chip, 2015, 15(3): 882–888