Cell-based Assays on Microfluidics for Drug Screening - ACS Sensors

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Cell-based Assays on Microfluidics for Drug Screening Xiaoyan Liu, Wenfu Zheng, and Xingyu Jiang ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.9b00479 • Publication Date (Web): 10 May 2019 Downloaded from http://pubs.acs.org on May 10, 2019

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Cell-based Assays on Microfluidics for Drug Screening Xiaoyan Liu,†,‡,∥ Wenfu Zheng,*,†,∥ and Xingyu Jiang*,†,§,∥ †Beijing

Engineering Research Center for BioNanotechnology and CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for NanoScience and Technology, No. 11 Zhongguancun Beiyitiao, Beijing 100190, P. R. China. ‡Academy

for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, P. R. China

§Department

of Biomedical Engineering, Southern University of Science and Technology, No. 1088 Xueyuan Rd, Nanshan District, Shenzhen, Guangdong 518055, P. R. China ∥University

of Chinese Academy of Sciences, 19 A Yuquan Road, Shijingshan District, Beijing, 100049, P. R. China

KEYWORDS: microfluidics, drug screening, high-throughput, biomimetics, cell assay.

ABSTRACT: Microfluidics is an appealing platform for drug screening and discovery. Compared with the conventional drug screening methods based on Petri dishes and experimental animals, microfluidic devices have many advantages including miniaturized size, ease-to-use, high sensitivity and high throughput. More importantly, bio-assays on microfluidics can avoid ethical issues which can be a big obstacle hindering the performance of the experiments on animals or human being. Furthermore, three dimensional (3D) microchips can recapitulate various biochemical and biophysical conditions in vivo and mimic the natural microenvironment of the tissues/organs, providing versatile in vitro models for biomedical applications. In this review, we will focus on the cell-based microfluidic assays for drug screening. Meanwhile, we also propose potential solutions for the difficulties in this field and discuss the prospects of microfluidics-based technologies for drug screening.

With the development of genetic and proteomic sequencing techniques, new doors are opened for understanding various biological processes and the mechanism of diseases. However, in the field of drug screening, conventional methods based on Petri dish and animal experiments face many challenges. The time for the complete evaluation of a drug candidate, on average, is more than 10 years before it is developed successfully into a new drug. Apart from the disadvantages of high costs in time and labor, ethical issue also is an unavoidable problem, which usually hinders the transformation of drug candidates to clinical medications. Even more seriously, a large number of the screened drug candidates failed in the clinical trials. Thus, an efficient platform for drug screening and discovery is highly anticipated. High throughput screening (HTS) refers to a group of advanced methods for screening drug candidates. The preliminary methods of HTS are performed on multi-well plates, such as 384- and 1536-well plates. However, the conditions on the plates are too simple to mimic the in vivo microenvironment. Microfluidics, which can manipulate materials or cells in liquids in microscale fluidic channels, has made enormous progress.1-2 Microfluidic technologies have the potential to revolutionize the drug screening,3 from the synthesis of nanoparticles to clinical

medications.4 In addition, microfluidic systems are economical, easy-to-modify and can be combined with other technologies. Compared with conventional systems for drug screening, microfluidic platforms can dramatically reduce the volumes of high-cost compounds and save the costs of drug discovery. Concentration gradient generator on microchip provides an excellent platform to evaluate the toxicity and the optimal concentration of different drugs in a large scale and quantity (Figure 1A).5-6 Single-cell chips have great significance to monitor the single cell responses to chemical compounds, producing greater statistical information than conventional methods by small numbers of cells (Figure 1B). For the evaluation of drug candidates in vitro, the microenvironment in Petri dish is far from physiological conditions, while microfluidic devices can construct microenvironment which is closer to the in vivo physiological conditions by co-culturing different cells in a natural way (Figure 1C).7-8 Presently, many research rely on animal experiments. However, the experiments on animals are expensive and concern ethical issues. Three-dimensional (3D) organ-on-chips provide the opportunity to partly or completely replace animal experiments to investigate the pharmacology of drug candidates, providing a platform to track the distribution and toxicity of drugs in real time for predicting the in vivo

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behavior (Figure 1D).9 Furthermore, microfluidic system has the potential capability for high-throughput screening, and it can be used for screening a great deal of drugs at different concentrations.

Figure 1. Microfluidic technologies for drug discovery. (A) Two concentration gradient generators on a microfluidic chip. Gradient concentration of the drug is achieved by precise control of distance between the cells and the drug, and multi-step dilution at the branched structure. Reprinted with permission from ref 5. Copyright 2013 Royal Society of Chemistry. (B) Single-cell arrays on a microfluidic chip. The arrays can passively capture individual cells for kinetic analysis of the responses of single cells to drugs. (C) Reconstituted cell-cell co-culture conditions in microfluidic systems for in vitro evaluation. The nerve-cancer cell co-culture microchip can provide a tool to investigate the interaction between neuron and tumor and mimic the tumor microenvironment. Reprinted with permission from ref 8. Copyright 2017 Nature Publishing Group. (D) 3D organ-on-chip produces a natural microenvironment for in vivo evaluation, promoting the study of nanomedicine.

To illustrate the microfluidics-based drug screening platform, in this review, we introduce the advantages of microfluidic systems and the on-chip assays for drug screening and discovery. The future research directions of the microfluidics-based drug screening are also discussed. MAIN ADVANTAGES OF MICROFLUIDIC CELL-BASED PLATFORMS FOR DRUG SCREENING Concentration gradients on microfluidics.

Due to the excellent performance in spatial and temporal control, microfluidic devices have been widely applied in cell-based microengineering： the manipulation of cell-cell and cell-microenvironment interactions. Furthermore, microfluidic systems can robustly reduce the volume of chemical reagents and precious drugs because of the microscale size of microfluidic model. Microfluidic chips can provide an excellent platform to precisely control the concentration gradients of drug candidates, for studying how cellular phenotypes respond to the chemical and physical signals in the simulative extracellular microenvironment.6, 10-11 In the past few years, biochemical concentration gradients generated and precisely controlled by microfluidic systems have been widely used in the field of drug screening, including bacterial chemotaxis,12 cancer cell migration, and axon growth.13-15

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To achieve serial dilution of compounds, we developed a miniaturized and effective microfluidic chip for analyzing multiple antibodies.16 The micro-dilutor network (μDN) consists of branching structures which can dilute one stream into two streams. In the hybrid module, we integrated chaotic advective mixers similar to “herringbone” into the microchannels to ensure perfect mixing. Samples of human serum containing antibodies and BSA (bovine serum albumin) were first introduced into the microchannels and the streams are repeatedly mixed and generate multiple streams. At the end of μDN, gradients of antibodies can be produced and bind to antigens absorbed on the substrate. The gradient generator replaces manual serial dilutions operated in microwells. This miniaturized serial dilution system has great potential in investigating the response of cells to temporal and spatial stimuli. To evaluate the cell metabolism and cytotoxicity of drug candidates, researchers presented a Christmas tree-shaped gradient generator integrating with mass spectrometer (MS) for high-throughput screening.17-18 The chip-MS platform consisted of three parts: a concentration gradient generator, a cell cultivation chamber, and a MS detection module (Figure 2A). They investigated the difference of lactate concentration from cancer cells and normal cells at hypoxia condition. On the other hand, the different inhibitory effects on the same type of cancer cells appeared in a dose-dependent manner with different concentration gradients of α-cyano-4-hydroxycinnamate. Compared with conventional screening systems, the microchip platform has great superiority including high-throughput, minimal compound consumption and the ease of operation. Furthermore, this system has huge potential for investigating pharmacological effects of compounds on living cells, and promoting the progression of drug discovery as well as evaluating more effective drugs. Recently, a circular concentration gradient microfluidic device was developed for screening anti-diabetic drugs.19 This platform consists of a top concentration gradient generator and a bottom 3D insulinoma cells culture chamber. The 3D microfluidic platform can screen drugs which stimulate insulin secretion and achieve high-throughput screening of diabetes-related drugs. Five concentrations and four parallel of anti-diabetic drugs can be successfully evaluated on this device. In addition, researchers presented a high-throughput microfluidic device with multichannel networks to generate concentration gradients for high content screening. By integrating a multiple anti-cancer drug gradient generator with a cell-based chamber, this device has huge potentials including drug dilution and diffusion, cell cultivation and cell-based assay.20 In this system, researchers investigated the apoptosis of human liver carcinoma cells (HepG2) at the presence of several anti-cancer drugs with discrete concentration gradients, they also evaluated other cellular responses such as nuclear size and membrane permeability in a simple way. Applying the same idea of concentration gradient, researchers developed a scalable and high-throughput drug-combination screening device.21 In this design, the Christmas tree-shaped network can produce a serial concentrations gradient between different


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drug pairs. Furthermore, this module connects with 1032 cancer spheroid units in cell culture chambers, integrating with custom software for fast readout and data analysis. This system was used to develop a logarithmic gradient concentration and evaluate the response of different cancer cells lines to these different drug combinations. In addition, this platform constructs 3D tumor spheroid to simulate tumor microenvironment. This study has great significance for precision medicine and high throughput drug screening due to the advantages including low sample consumption and rapid readout. Besides drug evaluation, concentration gradient-integrated microchip systems also have huge potential in toxicology studies. Recently, to investigate the lung inflammatory and cytotoxic responses of benzopyrene, researchers developed a chemical gradient generator on a bronchial epithelium microchip which can monitor the cell morphology and inflammatory signals.22 With the precise control of the concentration gradient of benzopyrene for 48 hours in this system, researchers successfully evaluated benzopyrene-induced inflammatory and cytotoxicity by integrating with bronchial epithelial cell chambers in real-time. On the whole, concentration gradient generators on microfluidics promote the development of drug screening and toxicology evaluation. Most researches have been focusing on serial dilution of small molecules on microfluidics. However, the existing concentration gradient generators on microfluidics have difficulty in diluting nanoparticles, since nanoparticles are easy to aggregate when flowing in microfluidic channels.23 We previously designed a double spiral-shaped microfluidic chip for synthesizing lipid nanoparticles, which has the capacities of precise dispersing nanoparticles in microchannels.4 To achieve gradient dilution of nanoparticles, a possible way is to develop a microchip with the spiral-shape similar channels to prevent the aggregation of nanoparticles. This may make it possible to extent high-throughput drug screening to the field of nanomedicine, such as lipid nanoparticles and inorganic nanoparticles. In the field of neurobiology, many studies suggested that chemical signal molecules (such as cAMP, cGMP) and biochemical signal molecules (such as laminin, Slit) play vital roles in the guidance of axons.24-25 To establish a diffused gradient of the Slit protein for studying axon response, we presented a strategy by co-culturing hippocampal neurons and Slit-producing cells in a microfluidic chip (Figure 2B).5 This design can control distance between neurons and Slit-producing cells precisely, to produce a concentration gradient of Slit protein. We assessed axon fasciculation induced by Slit protein and the expression of L1 cell adhesion molecule on this platform. In recent years, accumulating literatures reported that many guidance molecules, such as netrin, Slit proteins and nerve growth factors, play important roles in regulating neurite chemotaxis.26 Our microchip provides a convenient tool for investigating chemotactic assays of neuronal sensation by precise control of distance between neurons and guidance molecules. However, the 2D chemotactic assays cannot fully reflect the in vivo scenario since the chemotaxis usually forms in a 3D way. To address

this, researchers developed a 3D microfluidic platform on which a series of hydrogel cylinders can generate a concentration gradient of molecule for studying neuronal chemotactic assay (Figure 2C).27 A clear limitation of this platform is how to keep a stable concentration gradient over a long period, due to molecular diffusion. The combination of distance control microchip and 3D neuron culture is a possible way to solve this problem.

Figure 2. Generation of concentration gradients on microfluidics. (A) Schematic drawing of a multi-channel microfluidic chip containing gradient generator (reagent preparation), cell chamber (cell culture) and detection module (paper spray ionization-MS). Reprinted with permission from ref 18. Copyright 2016 American Chemical Society. (B) Schematics of a Slit gradient generation device. This microchip can generate a concentration gradient of Slit protein by precise control of distance between the neurons and the Slit-producing cells. Reprinted with permission from ref 5. Copyright 2013 Royal Society of Chemistry. (C) Schematic of a high-throughput 3D neuronal chemotactic device. The microfluidic chip can generate molecular gradients in different hydrogel cylinders. Reprinted with permission from ref 27. Copyright 2017 Nature Publishing Group. Cell arrays on microfluidics

Single-cell assays help biologists and chemists investigate the heterogeneous behavior of different cells. Monitoring single-cell signaling dynamics is necessary to understand the various biological functions, as well as the variability of single-cell signaling mechanisms.28-29 Single-cell analysis provides vast quantities of information for the development of personalized therapeutics such as the dose of drugs and the combinations of therapeutic strategies for individual patient and individual disease. The limitation of conventional analytical methods such as microplate and flow cytometry includes the lack of the information of the individual cells responding to the therapeutics. Microfluidic platforms have huge potential in the dynamic monitoring of individual cells, which can promote the understanding of cellular behavior of a specific cell. Recently, to study the temporal effect of external chemical signaling molecules on the signaling pathways of single-cell, researchers developed a microfluidic device consisting of an upstream linear gradient generator and a downstream single-cell trapping microwell.30 In this device, they evaluated the dynamic responses of intracellular signaling pathways to chemical stimuli in single-cell resolution in real time.


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Microfluidics can not only analyze single cell response to environmental factors, but also investigate cell-cell interactions. Due to the vital role of cellular interactions in immune responses, researchers developed an innovative single-cell level microchip with microarrays to investigate the lymphocyte interactions (Figure 3A).31 In this design, U-shaped traps could passively capture two different types of immune cells by using a four-step protocol and obtain cell-cell pairing. By monitoring the dynamics of CD8+ T cells in this microchip, researchers evaluated the early activation of each pair of lymphocytes. As a result, this platform can not only capture the dynamic information of lymphocytes but also obtain the static parameters of each pair. Using this microfluidic cell pairing system, they further explored the interaction of natural killer cells and tumor cells by monitoring the calcium signaling and IFN-γ production.32 This device has high efficiency in capturing and pairing two types of cells (such as tumor cell/macrophage). Previous work has reported cancer cell/macrophage pairing has effect on various cellular parameters such as cell protrusion, cancer cell migration.33 The cell-pairing platform has potential to assess complex immune responses between immune/immune cells and/or tumor/immune cells at long-term and may open opportunities for the evaluation of immune drug and/or antitumor drug libraries. The most prominent feature of microfluidics is its capability to carry out high-throughput drug screening. Recently, researchers reported a microfluidic single-cell array to screen anti-cancer drugs in real time.34 This microdevice consists of many micromechanical traps, which can be used for passively capture individual nonadherent cells. In the single-cell level platform, researchers investigated kinetic influence of anticancer compounds on cancer cells, as well as the quantification of anticancer drugs-induced tumor apoptosis. This platform provides an excellent method for high throughput screening of anticancer drugs. Furthermore, this platform can not only simplify the process of single-cell preparation, but also reduce cell and drug consumption. Being similar to this design, another group developed a pneumatic microfluidic platform with micromechanical traps to culture 3D tumor samples with uniform sizes (Figure 3B).35 Researchers successfully investigated tumor response to different anticancer drugs by precisely controlling the formation of single spheroid in micromechanical traps. Utilizing this device, different apoptotic signals of 3D tumors and tumor behavior are monitored during chemotherapy. Compared with conventional 3D tumor culture system, this design provides a reusable and high throughput tool to produce similarly sized tumor samples. Collectively, cell arrays on microfluidics provide an excellent platform that help scientists to further understand the interaction between cells and drug candidates in single cell resolution, and also promote the development of microfluidics for drug screening with high throughput capabilities. A dramatic challenge of current individual cell and single tumor spheroid analysis techniques is how to combine singe-cell culturing, physical/chemical stimulation, imaging, data analysis into an individual system. Recently, researchers established an ultra-multiplexed microfluidic chip that

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integrates different cell culture modes including singe cell and 3D neurosphere, different dynamic chemical stimuli, as well as 1500 culture chambers on a single chip.36 On the chip, different signal molecules/drugs with different compositions and concentrations can be evaluated. This platform adopted a customized MATLAB procedure which can extract individual cells information including migration, variation of the nucleus and the cytoplasm. Integrating single-cell device and data analysis module is a tendency in high-throughput screening. By doing this, researchers can not only understand a population by measuring enough individual cells, but also identify some special cell functions in a cell population, to benefit high-throughput drug screening. In addition to this, future directions of the design of microfluidic chips may mainly focus on constructing an independent microenvironment between single cells, and separately retrieving single cells for further study.

Figure 3. Microfluidic single-cell arrays. (A) Microfluidic cell pairing at single-cell level. The U-shaped traps can passively capture two different cells to investigate the immune cell interactions. Reprinted with permission from ref 31. Copyright 2015 Nature Publishing Group. (B) Schematic of a pneumatic microfluidic platform for 3D tumor culture. The micromechanical traps can precisely control the formation of 3D tumor for drug screening. Reprinted with permission from ref 35. Copyright 2015 American Chemical Society. Microfluidic cell co-culture models

Microfluidics is a promising platform for investigating the cell-cell or cell-microenvironment interactions.37 Microfluidic cell co-culture models have been widely applied in studying cellular communication and tumor progression. Our group explored a microfluidic platform to co-culture three types of cells for simulating cell-cell interactions and studying cell migration.38 To mimic the natural microenvironment of endometriosis, we constructed a microfluidic system by co-culturing human endometrial stromal cells and peritoneal mesothelial cells, which could be used for monitoring the dynamic of endometriosis.39 Another important kind of cell-cell interaction takes place during the cancer cells. To investigate tumor metastasis during cancer progression, researchers designed a microfluidic system that can mimic mild, moderate and severe tumor models by co-culturing breast cancer cell and endothelial cell (Figure 4A).40 They verified that the migration of breast cancer cells was relevant to the secretion of interleukin-6. Based on the microfluidic platform, the anti-cancer effect of paclitaxel and tamoxifen was successfully evaluated by monitoring cancer cell migration. For neurobiologists, it would be great if the individual function of neuron soma and neurites can be separately


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studied. A recently reported microchip can separate the somas and the axons in different chambers for different treatments, allowing the isolation and studies of the two different parts of the same neuron.41 Due to the absence of in vitro models for mimicking the brain physiology, there is a bottleneck hindering the development of brain-related new drugs. To evaluate toxicity and therapeutic efficacy of a compound library, a microfluidic model was developed for investigating the networks of neurons and glial cells.42 The microfluidic model has the capability to analyze a wide range of neural parameters, such as cell viability, electrophysiological activity and neurite outgrowth. There is a great challenge in efficient delivery of nanomedicine across the blood-brain barrier (BBB), which prevents the development of drugs addressing disorders in the central nervous system. In order to achieve in-vivo like BBB microenvironment on a microfluidic chip, it must take into account flow-induced shear stress. Researchers developed a microfluidic BBB model for co-culturing brain microvascular endothelial cells with rat primary astrocytes, which can boost future growth of brain drug screening and promote the development of neurotherapeutics (Figure 4B).43 Natural brain is composed of highly compartmentalized, layer-by-layer structure where different cells are connected via the axons and neurites. To simulate this structure, a microfluidic platform named “Compartmentalized Neuron Arraying” that can precisely control the neuron networks was developed.44 Accumulating research literatures implicate

that the neural microenvironment plays a vital role in the development and metastasis of cancers, especially for pancreatic cancer and prostate cancer. The study of the signaling pathway and molecular mechanism between neurons and cancer cells can provide the novel diagnostic and therapeutic strategies for malignant tumors. Recently, we developed a microfluidic model for simulating microenvironment of pancreatic cancer and investigated cancer-neuron interaction and screened neuron-related drugs for inhibiting tumor progression (Figure 4C).45 Compared with the traditional in-vitro drug screening system, the high-throughput microfluidic chip provides a good platform to investigate the crosstalk of nerves and cancer cells. Based on the microfluidic platform, we successfully screened the candidate siRNAs targeting nerve growth factor (NGF) and candidate nanocarriers for delivering the siRNAs.8 The most effective drug-nanocarrier combination, gold nanocluster assisted NGF-siRNA, was proven by the in vitro and in vivo experiments to be effective in breaking the neuron-cancer interactions and inhibiting pancreatic cancer progression. This high-throughput microfluidic chip provides a versatile platform to screen drugs that can influence cell-cell interactions. However, a possible limitation of present cell co-culture models is the relatively low level of physiological microenvironment, which may explain the discrepancies of drug screening results between cell co-culture models and clinical trials.

Figure 4. Microfluidic cell co-culture platforms for drug screening. (A) Microfluidics co-culture model for mimicking the metastatic breast tumor. Reprinted with permission from ref 40. Copyright 2016 Nature Publishing Group. (B) Schematic of Blood-Brain Barrier model on a chip. The brain-on-chip model can mimic blood-brain barrier characteristics to evaluate drug permeability. Reprinted with permission from ref 43. Copyright 2017 Wiley. (C) 2D microfluidic platform for investigating neuron-cancer cell interaction and nerve-related anti-cancer drug screening. This platform including neuron chamber, cancer cell chamber and microgrooves. Reprinted with permission from ref 45. Copyright 2016 Royal Society of Chemistry. Mimicking 3D tissues on microfluidics

The natural tissues or organs are composed of various types of cells organized in a highly ordered and heterogeneous way. Conventional methods can hardly recapitulate these highly complicated tissues. By contrast, microfluidic techniques facilitate the construction of complex tissue-level constructions to mimic physiological

microenvironments in vitro, providing excellent platform for the studies of disease progression and drug screening. Natural blood vessels are featured by multilayered 3D structure composed of different cell types. To simulate the structure of the native blood vessels, our group explored a strategy to construct multilayered tubular model by microfluidics.9, 46 We first established a stress-induced


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rolling membrane (SIRM) by bonding a piece of stretched polydimethylsiloxane (PDMS) membrane and a relaxed PDMS membrane together. Next, we sealed a microfluidic chip containing 3 microchannels to the SIRM membrane, and seeded different types of cells in the microchannels. When the SIRM was released, it can roll up automatically, which can form a 3D multilayered tube by the internal stress of SIRM (Figure 5A). Based on this design, we further developed a layer-by-layer 3D tubular structure using two kinds of biocompatible polymers that can keep the shape of the vessel during biodegradation.47 This multilayered structure balanced the degradation of the tubular materials and the construction of biomimetic microenvironment, which has huge significance in the field of biomedicine. To simulate anti-cancer drug delivery in blood circulation, researchers reported a 3D microﬂuidic cell array for reconstructing tumor microenvironment combining microvascular endothelial cells and cancer cells.48 In this design, to mimic micro-vessels and the infiltration of nonendothelial cells into extracellular matrix, they built three PDMS layers in the microfluidic array. This platform is a high throughput screening tool that may predict the response of anti-cancer drugs in the simulated blood circulation. The brain is another typical 3D structure which is highly compartmentalized. To recapitulate the functional brain structure, we developed a 3D neuronal network on a microchip by culturing neurons, astrocytes on layer-by-layer assembled microbeads in chambers which were connected by multilayered microchannels that only allow the growth of neurites (Figure 5B). The neurites-connected, functional 3D neuronal network recapitulated the generation and transmission of the spontaneous activities that takes place in the natural brain.49 The aforementioned microfluidic cell co-culture systems provide the possibility for high-throughput drug screening in vitro. Tumor microenvironment which includes cancer cells, stromal cells and immune cells deserves extensive investigations on 3D models.50 Recently, microfluidics has been developed as a valuable tool to investigate the tumor microenvironment and tumor progression in vitro.51-52 The 3D microfluidic models promote the development of anti-cancer drugs by constructing the complex interaction of tumors and their microenvironments. An increasing number of microfluidic models with 3D tumor spheroids are used for mimicking tumor microenvironment and drug screening. To mimic the morphology of microvasculature of tumor, researchers developed a 3D tumor-microvascular microchip models for investigating the effect of antioxidants on glioma cancer cells.53 In this design, they co-cultured tumor cells and endothelial cells on the 3D hydrogel microfluidic device to simulate tumor-microvascular microenvironment. The effect of three antioxidants on glioma cells was successfully evaluated on this platform. To simulate the role of interstitial flow during the tissue vascularization and angiogenesis, a microfluidic 3D network was designed to co-culture human endothelial vascular cells.54 On this platform, biochemical and mechanical stimuli were investigated by stimulating fibroblast-secreted factors and

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flow-mediated stresses. To reconstitute a versatile in vitro platform for observation of tumorigenesis, a biomimetic microfluidic model was constructed (Figure 5C).55 Different types of cancer cells were co-cultured with primary fibroblasts within gel in different microchambers, and the gel allowed cancer cells to interact with fibroblasts by paracrine pathway. Using this design, tumor and stroma interaction as well as the angiogenesis of tumor tissues can be evaluated. With the advantages of ease-to-operate and high-throughput, this kind of systems has huge potential to screen anti-cancer drugs. Although 3D models have successfully recapitulated the fundamental function of organs/tissues, the huge challenges for drug screening on 3D models still exist. One of the challenges is how to construct standardized 3D models as a personalized drug screening platform for clinical trial.56 Future directions should be mainly focused on the construction of microvasculature for continuous perfusion, the reproduction of shear stress for mimicking in vivo microenvironment, the generation of chemical and oxygen gradient, and the delivery of nutrients through vascular network.57 Solutions should be proposed to promote the development of 3D models. Through the establishment of 3D in vitro models to accurately recapitulate the in vivo physiological microenvironment of tissues/tumors, it will not only help scientists further understand the absorption and metabolism of drugs, but also promote personalized cancer therapy.

Figure 5. Microfluidic models for mimicking physiological microenvironment. (A) Schematic of multilayered blood vessel scaffolds on microfluidics. Reprinted with permission from ref 46. Copyright 2012 Wiley. (B) 3D neuron network on microfluidic model. This system can culture neurons and astrocytes on layer-by-layer assembled microbeads in different chambers. Reprinted with permission from ref 49. Copyright 2014 Wiley. (C) Microfluidic chip model of tumor microenvironment. Reprinted with permission from ref 55. Copyright 2017 Wiley. ADVANCED SCREENING

MICROFLUIDIC

MODELS

FOR

DRUG

Drug screening by organ-on-chips

The emergence of organ-chip technology has also provided a new method to explore the differences between animal models and human body in response to drugs.58-59 During the animal experiments, human cancer cells are


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usually transplanted into mice, rabbits or moneys for investigating human diseases. However, because of the different physiological conditions between humans and animals, the drug screening results of animal models cannot guarantee that the drug candidates are successful in the clinical trials. At least 30% of drug candidates have no chance to be approved by the Food and Drug Administration, because of the toxicity of drug candidates or the inability of the human liver to metabolize. This is the main reason why many drug candidates have failed in the clinical phase I trial or clinical phase II trial.60 Hence, it is necessary to develop new strategies for drug discovery and drug screening. Recently, researchers are aiming to develop microfluidic platforms to construct the organ-level functions, which have potential capability to decrease the costs for developing new drugs. Researchers can construct basic structure of human organs on the microfluidic chip systems and mimic the functions of different organs. By utilizing organ-on-chip systems, researchers can assess and analyze the bio-effects of the drug candidates and their biodistribution, metabolism, as well as discharge.61-62 Furthermore, organ-on-chip systems can predict the in vivo behavior of the drug candidates. By culturing human cells on the microchips which can mimic physical and chemical microenvironment of human body, the in vitro organ-on-chip system will bridge the gap between the animal experiments and clinical trials and provide a platform for driving the discovery and development of new drugs. So far, researchers have successfully developed a series of microfluidic models of liver, lung, kidney, heart, gut, and blood vessels.63-66 In 2010, a microfluidic chip that can reconstitute the functions of human lungs was reported. The chip is fabricated by co-culturing the microvascular endothelial cells and the alveolar epithelial cells on both sides of a porous and flexible PDMS membrane (Figure 6A).67 By exerting vacuum to the side chambers, the membrane can be mechanically stretched. Accordingly, the perforated membrane can mimic the expansion and contraction of pulmonary alveoli due to breathing. By using the lung-on-chip model, some pulmonary bacteria and inflammation cytokines can be studied. The researchers found that, by stimulating the epithelial cells with tumor necrosis factor-α, the expression of intercellular adhesion molecule-1 is up-regulated on the endothelium. Besides, they found on the chip that the cyclic stretching can increase the toxic and inflammatory responses of the lung when the lung is exposed to silica nanoparticles. This phenomenon has not been observed in other in vitro models. However, the current lung-on-chip mostly generated the structures of fetal airway. 68 Future research on biomaterials will provide a method for construction adult-like hung organoids. Besides, to evaluate intestinal drug metabolism in vitro, researchers developed a gut-on-chip platform with two layers separated by a porous nitrocellulose membrane and lined by collagen and caco-2 cells (Figure 6B).69 This model successfully recapitulated human intestinal physiological microenvironment that is important for the intestinal functions. Researchers successfully investigated the metabolism of verapamil and ifosfamide on the chip.

The gut-on-chip system has huge potential to study the gut functions, including fluid flow and intestinal drug metabolism. Conventional liver models are hard to simulate the physiological or pathological microenvironment of the liver through co-culturing liver sinusoid endothelial cells with hepatic stellate cells. Recently, a 3D liver fibrosis model were developed, which was also called fibrotic microniches (FμNs) and can study the influence of the biomechanics and hepatic vascularization on the generation and progression of fibrosis (Figure 6C).70 The researchers miniaturized the FμNs to a 384-format chip for drug screening and toxicity tests. Utilizing these FμNs, they evaluated the response of liver fibrosis to anti-angiogenic drugs (such as sorafenib and vascular endothelial growth factor antibodies). It demonstrated that these anti-angiogenic drugs were effective only in the treatment of early-stage liver fibrosis. However, the phenotype of late-stage liver fibrosis was not reversed after treatment with these drugs. Furthermore, it suggested that inhibitors of collagen condensation could delay the progression of late-stage liver fibrosis. Based on this 3D biomimetic liver fibrosis platform, researchers obtained precise intervention strategies for different stages of the liver fibrosis. It has potential applications in understanding fibrosis progression and liver fibrosis-related drug screening. Apart from the organ-on-chips systems for specific organs, tumor models also deserve extensive investigations at an organ level.71 To recapitulate the pathological information of glioblastoma, a bio-printed glioblastoma-on-a-chip model was constructed (Figure 6D).72 Different bioinks laden with different cell lines (glioblastoma cells, endothelial cells) and extracellular matrix were co-cultured on a concentric-ring chip. This platform can form a radial oxygen gradient, which is an important pathological feature of glioma cancer. The researchers examined the resistance of glioblastoma patients to chemoradiation and temozolomide treatment. Furthermore, to identify the optimal therapeutic strategies for specific patients, the researchers evaluated different combinations of drug candidates on the glioblastoma-on-a-chip model. Compared with the conventional tumor-on-chip models, this platform recapitulates the pathological condition of glioblastoma, including heterogeneity by surrounding tumor with micro-vessels, as well as central hypoxia. The glioblastoma model will be beneficial for the construction of other solid tumor models and guide personalized cancer therapy. Organ-on-chip platforms have the potential to accelerate the process of preclinical testing. However, a potential drawback of the existing organ-on-chip models is that commercially available immortalized cell lines are commonly used in the construction of organoids. With issues of mutation and contamination, using immortalized cells for constructing healthy tissue/organ is questionable. Thus, the selection of cell lines is a big challenge for the construction of organ-on-chip. Human embryonic stem cells and induced pluripotent stem cells serving as cell sources may provide huge opportunities in the future. Moreover, current detection methods mainly rely on cell


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proliferation and apoptosis. It is obviously not comprehensive enough to assess drug effects and predict drug toxicity. For heart-related or nerve-related drugs, it is necessary to track frequency/amplitude variation of the time-varying signals. The combination of organ-on-chip and microelectrode array (MEA) sensor will be capable of recording the signals.73 Besides, the integration of multiorgan-on-chip systems can simulate the physiological environment of human body and predict the absorption, metabolism and elimination of new drugs. However, there are challenges associated with replicating the relative size of each organ to accurately reflect physiological

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interactions between organs.74 Further research on the combination of multiple organ systems on a chip will focus on considering various physiological parameters, including surface area of organ barriers, organ size, blood circulation time in each organ.59 In addition, integrated multiorgan-on-chip systems with computational modelling will further promote researchers understand the interplay of different organs. If organ-on-chip system could consistently and accurately predict the pharmacology of drug candidates, organ-on-chip models will replace animal tests in the future.

Figure 6. Organ-on-a-chip models. (A) Schematic of a biologically inspired design of human lung-on-a-chip microdevice. Reprinted with permission from ref 67. Copyright 2010 Science. (B) Schematic of a human gut-on-a-chip device. Reprinted with permission from ref 69. Copyright 2018 Wiley. (C) Conceptual design of a biomimetic liver fibrosis 3D pathological model. Reprinted with permission from ref 70. Copyright 2017 Nature Publishing Group. (D) Schematic of bio-printed glioblastoma-on-a-chip for the evaluation of drug combination for glioblastoma patient. Reprinted with permission from ref 72. Copyright 2019 Nature Publishing Group. Combination of microfluidics throughput systems.

with

other

high

Recently, to improve the throughput of drug screening, more and more scientists focused on how to integrate the microfluidic platforms with other automated high-throughput devices to maximize their function. Although there are many advances of microfluidic systems for drug screening, there is a great need to resort to some routine devices that possesses the capability of fast and continuous imaging or fast readout, to broaden the applicability of the microfluidic system. Microfluidic system has been integrated with mass spectrometry for qualitative and quantitative analysis of cell metabolites and other biomarkers.75 Recently, researchers developed a system that could integrate microfluidic chip with automated high-content imaging technique, to assess the

barrier integrity of perfused intestinal epithelium tubes.76 Due to pathological states of the intestinal epithelium tubes and the drug toxicity, the function of epithelial barriers is easily to be impaired which limits drug development in clinical trials. In general, the study of para-cellular permeability of epithelial barriers is achieved by culturing intestinal epithelium cells on rigid membranes, such as Transwell system.77 However, the Transwell system is unsuitable for high throughput readouts as well as difficult to evaluate the changes of protein expression and the cell-microenvironment interaction. Microfluidic techniques have the capability to obtain vast amounts of readouts, including different compounds, dilutions and controls. Thus, more information rather than optical information can be recorded, and the limitation of fluorescence can be solved.


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Moreover, the integrated system can also achieve user-friendly operation, multiplexed cellular analysis and good repeatability in routine laboratories. To build up a more powerful drug screening platform, usually there is a need to integrate microfluidics with data analysis system. Recently, researchers provided a microfluidic platform for live-cell phenotypic-biomarker assay.78 This system includes a microfluidic device with collagen-I/fibronectin extracellular-matrix, and machine-vision analysis is combined with machine-learning algorithms for subsequent analysis (Figure 7A). Utilizing live-cell phenotypic-biomarker microfluidic system, they evaluated single-cell behavior (including cell-ECM interaction) and cellular heterogeneity (such as functional protein-complex formation and signaling dynamic) which implicated in the invasiveness and metastasis of tumors. This platform has great significance in assessment of the efficacy of drug candidates for tumors. Moreover, microfluidic cell-based platforms can also integrate with other detection techniques for pharmacokinetics evaluation. Recently, researchers designed an integrated electrode to evaluate worm larvae viability by impedance-based method. This system consists of two pairs of electrodes and a small channel for counting and behavior detection of single larvae (Figure 7B).79 The researchers successfully evaluated the viability of larvae and drug toxicity at different concentrations of mefloquine. Compared with conventional evaluation approach, this platform shows great potential in drug discovery because it achieves fast-readout and overcomes the subjectivity of the lab worker evaluation. Besides, the microfluidic cell-based platforms can also be combined with surface-enhanced Raman scattering (SERS) technique to obtain pharmacokinetic parameters. Researchers designed a microfluidic platform with SERS bio-probe to monitor the action of living cells at different concentrations of dual-drug (6-mercaptopurine and methimazole).80 On the whole, integrated microfluidic systems promote the development of drug screening owing to their fast-readout, high-throughput, and high-resolution.

with automated high-content imaging technique and machine-vision analysis system. Reprinted with permission from ref 78. Copyright 2018 Nature Publishing Group. (B) Photograph of a microfluidic platform integrated with electrodes and output signal of electrodes. This system can evaluate worm larvae viability by impedance-based method, and evaluate drug toxicity at different concentrations of mefloquine. Reprinted with permission from ref 79. Copyright 2018 American Chemical Society. CONCLUSION AND FUTURE OUTLOOK

In this perspective, we introduced recently developed microfluidic technologies for drug discovery and screening. We first summarized the indispensable advantages of microfluidic cell-based platforms for drug screening including the precise control of compound concentration gradients, single cell analysis, cell co-culture, and 3D tissue mimicking. We also discussed the applications of the microfluidic chips for cell-based drug screening. Although there are great advances in microfluidics-based drug screening, we are facing challenges such as the lack of highly biocompatible biomaterials, personalized program, and low throughput. To address these issues, the first task is to develop new biocompatible materials for the fabrication of microfluidic chip, allowing more bio-friendly fabrication methods to ensure the modification of the systems with bioactive molecules or living cells. Bio-functionalized hydrogel microfluidic systems have the potential to provide the platform with wider applications for drug research and development. Furthermore, the growing interest in construction of biomimetic physiological environment suggests the development of novel microfluidic models for personalized medication. A suitable treatment for any patient could be identifiable through constituting microfluidic drug screening system with biopsy samples, and this technology can promote the development of personalized and precision medicine. Finally, the combination of microfluidic chips and fast readouts can be considered, including the balance among simplifying the design of microfluidic chip and achieving complicated functions for mimicking the physiological environment as well as incorporating machine-learning algorithms. Taken together, rapid development in high throughput drug screening will be achieved by addressing these issues. Efficient progress will be favored by collaboration between multidisciplinary teams including chemists, material scientists, and biologists.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. *E-mail: [email protected].

ORCID Xingyu Jiang: 0000-0002-5008-4703.

Notes All authors admit the manuscript and declare no competing financial interest. Figure 7. Microfluidics combined with high-content analysis platform. (A) Workflow of live-cell phenotypic-biomarker assay on microfluidic platform. This microchip can integrate

ACKNOWLEDGMENTS


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We would like to acknowledge the National Key R&D Program of China (2017YFA0205901), the National Natural Science Foundation of China (21535001, 81730051, 21761142006, and 81673039) the Chinese Academy of Sciences (QYZDJ-SSW-SLH039, 121D11KYSB20170026) and for financial support.

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W. R.; Joshi, N.; Steinke, N.; Min, A.; Berger, D.; Saphirstein, R. J.; Dixit, G.; Meyyappan, T.; Chu, H.-M.; Knopf, K. B.; Albala, D. M.; Sant, G. R.; Chander, A. C., Live-cell phenotypic-biomarker microfluidic assay for the risk stratification of cancer patients via machine learning. Nat. Biomed. Eng. 2018, 2 (10), 761-772. 79. Chawla, K.; Modena, M. M.; Ravaynia, P. S.; Lombardo, F. C.; Leonhardt, M.; Panic, G.; Burgel, S. C.; Keiser, J.; Hierlemann, A., Impedance-Based Microfluidic Assay for Automated Antischistosomal Drug Screening. ACS Sensors 2018, 3 (12), 2613-2620. 80. Fei, J. Y.; Wu, L.; Zhang, Y. Z.; Zong, S. F.; Wang, Z. Y.; Cui, Y. P., Pharmacokinetics-on-a-Chip Using Label-Free SERS Technique for Programmable Dual-Drug Analysis. ACS Sensors 2017, 2 (6), 773-780.

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In this perspective, we first summarized the indispensable advantages of cell-based microfluidic platforms (including concentration gradient generator, single-cell array, 2D chip for cell co-culture and 3D chip for mimicking organs/tissues) for drug screening. Meanwhile, we highlighted some advanced drug screening applications (including organ-on-chip and integrated microfluidics with other high throughput systems) based on microfluidics. Besides, the potential solutions for the difficulties in the field and the future research directions of microfluidics-based technologies for drug screening are also discussed.


Cell-based Assays on Microfluidics for Drug Screening - ACS Sensors

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