Microfluidic cell culture systems for drug research

Transcription

1 TUTORIAL REVIEW Lab on a Chip Microfluidic cell culture systems for drug research Min-Hsien Wu, a Song-Bin Huang b and Gwo-Bin Lee* b Received 16th October 2009, Accepted 9th December 2009 First published as an Advance Article on the web 21st January 2010 DOI: /b921695b In pharmaceutical research, an adequate cell-based assay scheme to efficiently screen and to validate potential drug candidates in the initial stage of drug discovery is crucial. In order to better predict the clinical response to drug compounds, a cell culture model that is faithful to in vivo behavior is required. With the recent advances in microfluidic technology, the utilization of a microfluidic-based cell culture has several advantages, making it a promising alternative to the conventional cell culture methods. This review starts with a comprehensive discussion on the general process for drug discovery and development, the role of cell culture in drug research, and the characteristics of the cell culture formats commonly used in current microfluidic-based, cell-culture practices. Due to the significant differences in several physical phenomena between microscale and macroscale devices, microfluidic technology provides unique functionality, which is not previously possible by using traditional techniques. In a subsequent section, the niches for using microfluidic-based cell culture systems for drug research are discussed. Moreover, some critical issues such as cell immobilization, medium pumping or gradient generation in microfluidic-based, cell-culture systems are also reviewed. Finally, some practical applications of microfluidic-based, cell-culture systems in drug research particularly those pertaining to drug toxicity testing and those with a high-throughput capability are highlighted. 1. Introduction The process from the discovery of a new drug to its commercialization is a complex, lengthy and costly endeavor. It usually requires around years to take one new drug from its discovery stage to Food and Drug Administration (FDA) approval. It is estimated to cost about US$ 800 million to US$ 1 a Graduate Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan, Taiwan b Department of Engineering Science, National Cheng Kung University, Tainan, Taiwan. [email protected]; Fax: ; Tel: Ext billion for the research and development (R&D) of a successful new drug. 1 On average, only five lead compounds from a potential drug compounds enter clinical trials and only one of those will be finally be approved by the FDA. 1 Recently, the rapid progress in genomics, proteomics, combinatorial chemistry and bioinformatics continues to produce considerable numbers of pharmaceutically valuable compounds. This generates an urgent need for an adequate assay scheme to efficiently screen and to validate these potential drug candidates in the initial stage of drug discovery. In pharmaceutical research, cell culture-based assays are increasingly being exploited in drug testing to bridge the gap between the molecular-level assays and Min-Hsien Wu received his BS and MS degrees from the Department of Food Science at Tung-Hai University, Taiwan in 1994 and in Applied Biomolecular Technology from the University of Nottingham, UK in 2002, respectively. He received his PhD from the Department of Engineering Science at the University of Oxford, UK in He is currently an Assistant Professor Min-Hsien Wu in the Graduate Institute of Biochemical and Biomedical Engineering at Chang Gung University, Taiwan. His research has been mainly focused on tissue engineering, micro-bioreactor technology and bio-sensing. Song-Bin Huang Song-Bin Huang received his BS and MS degrees from the Department of Engineering Science at National Cheng Kung University in 2005 and 2007, respectively. He is currently a PhD student at National Cheng Kung University. His research interests lie in microfluidics, bio-sensing, and its biomedical applications. This journal is ª The Royal Society of Chemistry 2010 Lab Chip, 2010, 10,

2 animal tests. This is mainly because cell-based assays may provide more representative and meaningful responses to drug compounds than simplified biochemical assays, and also have the potential to be carried out in a more high-throughput manner than costly animal tests. With the aid of recent advances in laboratory equipment automation and high-throughput screening (HTS) techniques, the throughput of cell-based drug screening has been dramatically improved. In order to better predict the clinical response to drug compounds, however, a cell culture model that is faithful to its in vivo behavior is required. Although cell-based drug screening based on HTS technology has played an important role in pharmaceutical research, the current, simplified cell culture format may not be able to probe the real cellular response to drug compounds due to its inability to control and to mimic extracellular conditions and environments. During the past decade, there have been tremendous advances in microfluidics. Microfluidic devices and systems have been progressively used as versatile tools in different stages throughout drug discovery and development processes, which has been reviewed elsewhere. 2 6 The use of microfluidic-based, cell-culture platforms has found several niches as promising alternatives to the conventional cell culture methods. This review aims to provide experts in microfluidics an overview of microfluidic, cell-culture systems for drug research. It starts with a comprehensive discussion of the general process for drug discovery and development. There is an emphasis on the role of cell culture in drug research and the characteristics of the cell culture formats commonly used in current microfluidic-based, cell-culture practices. Due to scaling effects, microfluidic technology provides unique functionality that is not previously possible by traditional techniques. In the subsequent sections, the niches for using microfluidic cell culture systems for drug research, as well as some design considerations, are discussed. Moreover, in designing a microfluidic cell culture system for drug research, some mechanisms such as cell immobilization, medium pumping and the generation of a concentration gradient are important. The fundamental considerations associated with current progress on these technical issues are also discussed. Finally, some practical applications of microfluidic, cell-culture Gwo-Bin Lee received his BS and MS degrees from the Department of Mechanical Engineering at National Taiwan University in 1989 and 1991, respectively. He received his PhD from the Department of Mechanical & Aerospace Engineering at the University of California, Los Angeles, USA in He is currently a Distinguished Professor in the Department of Engineering Gwo-Bin Lee Science at National Cheng Kung University. His research interests lie in microfluidics, bio-sensing, nanobiotechnology and its biomedical applications. systems in drug research, particularly those for drug toxicity testing, and those requiring a high-throughput capability, are highlighted. 2. Drug discovery and development processes Bringing a new drug to market is a lengthy and costly process, which primarily involves drug discovery, drug development, clinical trials and targeted marketing. 7 In this section, the drug discovery, drug development processes, and clinical trials are discussed. Based on an understanding of the mechanisms for a particular disease, the first step in the drug discovery stage is to identify a specific drug target, which responds to the disease. A drug target can be a receptor or ion channel in living cells, an enzyme, hormone/factor, DNA, RNA, a nuclear receptor, or other unrecognized biological entities. 7 Target validation is the next step in this phase to confirm the role of the discovered drug target on the disease through complicated experiments both in living cells and in animal models. Once a drug target is identified and validated, it is then exposed to thousands of compounds in a HTS mode to search for the lead compound that may act on the confirmed drug target. In the subsequent drug development stage, a series of preclinical studies encompassing pharmacodynamic and pharmacokinetic evaluations as well as toxicity testing are performed on the lead compounds in vitro or in vivo to determine if the potential drugs are safe and effective enough for the subsequent human trials. Pharmacodynamics deals with the actions of the drug on the target, whereas pharmacokinetics is about the actions of the body on the drug (e.g. absorption, distribution, metabolism and excretion (ADME)). An evaluation of toxicity in pre-clinical studies is important to provide crucial information with respect to dosing and the safety of the potential drug. Only compounds that satisfy certain performance and safety criteria may proceed to the next stage of clinical trials. In the following clinical trials (e.g. the US FDA Phase I, II and III), the promising drug is tested in human subjects to determine if the drug is safe and is effective for the disease in question. Once the potential drug successfully goes through the three phases of clinical trials the pharmaceutical company can then file a new drug application with the US FDA. 3. Role of cell culture in drug research Drug discovery strategies vary between pharmaceutical companies. 8 Biochemical assays are commonly used in the earliest stage to quickly identify lead compounds. 9 Nevertheless, the data obtained from such molecule-based assays may not represent actual interactions between the drug compound and the target molecule within an extremely complicated biological system. Therefore, the general role of a cell culture is to perform drug research at a cellular level in order to bridge the gap between the simplified biochemical assays and animal testing. In this manner, cytotoxic or nonfunctional drug compounds can be reasonably ruled out. 9 In addition, under the guiding principle of the socalled three Rs (reduction, refinement and replacement) with respect to the use of animals in research, 10 there is a clear trend to minimize the use of animal testing in drug research. Apart from economic and ethical considerations, in vivo animal models are, 940 Lab Chip, 2010, 10, This journal is ª The Royal Society of Chemistry 2010

3 in fact, progressively revealing their limits to adequately represent the human responses to drug compounds because of speciesspecific variation between human beings and experimental animals. For example, a leading cause for the failure of a new drug in clinical trial is liver toxicity, which may not be well predicted by experimental animals. 11 The development and validation of a reliable in vitro methods as an alternative to conventional in vivo investigations in experimental animal models is a well-recognized research priority in the field of drug discovery. 12 In vitro cell-based assays have been regarded as a promising substitute to in vivo animal testing in drug research. Ideally, a cell culture model that is faithful to its in vivo behavior offers significant advantages in saving time and cost in drug research. However, conventional cell culture models may not meet this requirement due to the use of certain cell types and the cell culture techniques. In general, the use of human cell culture systems can eliminate problems associated with species specificity. Nevertheless, the utilization of human cells for drug research is subject to their availability and phenotypic characteristics. For example, many human primary cell types such as cardiomyocytes or neuronal cells are not easy to obtain. 13 Besides, living cells rapidly lose their phenotypic properties under many conventional cell culture schemes (e.g. monolayer cell culture), in which the inherent cellular microenvironments are changed. 14 Furthermore, with recent progress in stem cell technology, human stem cells, especially embryonic stem cells (ESC), hold great promises to become a large-scale source of specialized human cells for use in cell-based assays for pharmaceutical R&D. More detailed reviews can be found in the literature. 13,15 Besides, in order to faithfully and precisely investigate the cellular response to specific drug compounds or conditions, a biologically-relevant and well-defined cellular microenvironment is needed in order to maintain the phenotypic properties of the tested cells. In order to achieve this, the integration of biomaterials science, tissue engineering and the cell-culture system design are mandatory prerequisites for the development of novel cell culture platforms for drug research. Particularly, due to recent progress in microfabrication and microfluidic technology, several inherent cellular microenvironments can be elegantly mimicked in a microfluidic system. This enables cellbased drug research work to be performed in a micro-scale, highthroughput, and physiologically-meaningful manner. 4. Cell culture systems 4.1 Perfusion culture versus static culture The simplest and most widely used cell culture system nowadays is a static cell culture system (e.g. the use of a multi-well microplate or a Petri dish as a cell culture vessel), where the culture medium is supplied in a batch-wise manner. Although static culture systems are simple to operate and economical to manufacture, in terms of long-term cell culture, they may not be appropriate due to the risk of contamination caused by repeated manual intervention. Most importantly, the culture environment in such a system may fluctuate due to the intermittent medium replacement processes. 16,17 Furthermore, under such improperly controlled conditions, the cellular response to the investigated drug conditions may become more complex. In order to faithfully explore the cellular response to the tested drug conditions in a quantitative format, a more steady and quantifiable environment is essentially required because even minor alterations in extracellular conditions may greatly influence the cell physiology. 18 Cell culture using well-established multi-well microplate technology for HTS-based assays is a typical static cell culture practice. This approach has become a standard operation for many drug researchers partially because of the rapid development of the associated hardware such as automatic plate readers and liquid handling equipment. Although further miniaturization of these systems holds promise to increase assay throughput, the miniaturized scale of multi-well microplates (e.g well plates) is confronted with the problem of uncontrolled liquid evaporation from such tiny wells due to the relatively higher surface area to volume (SAV) ratio. This could accordingly result in poorly defined cell culture conditions. More recently, some modified versions of multi-well, microplatebased, cell culture systems have been proposed. For example, the integration of microfluidic systems with traditional multi-well microplates has been reported for high-throughput, cell-based, drug screening 19 and cytotoxicity evaluation of anticancer drugs. 20 In such systems, a perfusion cell culture is achieved which compensates for liquid evaporation and also maintains a long-term cell culture for drug testing. Compared with a static cell culture, a perfusion cell culture not only can keep the culture system sterile during the entire culture period, but, more importantly, can also continuously provide a system for nutrient supply and waste removal and hence keep the culture environment more stable. 16,17 This contributes to steadier and more quantifiable extracellular conditions, which is particularly meaningful for drug research. Nevertheless, perfusion cell cultures may hamper cell-to-cell communication through intrinsic and extrinsic growth factors owing to the continuous washing away of these biomolecules. To the best of our knowledge, most microfluidic cell culture systems for drug research exploit a perfusion cell culture format, in which medium flows are not only used to continuously feed the cultured cells but also to provide additional functionalities such as generating gradients of drug concentrations, creating a specific physical microenvironment (e.g. shear stress or interstitial fluid flow), and constructing a circulatory system 32 to better mimic the in vivo conditions. Another issue worth addressing herein is the relevance of the cell physiology in the perfusion and the static cell culture systems. In cell-based drug research, one cannot merely extrapolate the data obtained from one cell culture format to another format because the biochemical or biophysical microenvironments in the two situations may not be perfectly identical. Some fundamental investigations with respect to a comparison of the cell physiology in these two cell culture formats have been explored. For example, previous studies 33,34 demonstrated that perfusion increases cell content and matrix synthesis in a chondrocyte three-dimensional (3-D) culture when compared with the use of a static culture. Furthermore, a microfluidic system for the culture of a human hepatocarcinoma cell line in a perfusion manner was reported. 35 In this work, the cellular response to the perfusion and static conditions were also compared. Results This journal is ª The Royal Society of Chemistry 2010 Lab Chip, 2010, 10,

4 demonstrated that there is a time window wherein the cell physiology is comparable; however, outside this time, the cultured cells would behave differently. The reasons contributing to these discrepancies may be due to the uncontrolled modifications of biochemical or biophysical factors in the two culture environments. Consequently, in establishing a cell culture model for drug research, it may be advisable to fundamentally understand the physiological differences of the cultured cells in the two culture systems to avoid any misinterpretation of data D cell culture versus 3-D cell culture The main advantage of a cell culture over traditional tissue or organ cultures is that it is easy to handle, as well as to maintain consistency and reproducibility in cell growth. 36 Nevertheless, one may question whether the cultured cell will behave in the same manner in vitro as it does in vivo. In animal tissues, cells inhabit environments with very specific 3-D features. 37 In their native environment, mammalian cells are subject to various biological cues such as soluble signaling molecules (e.g. growth factors, hormones or cytokines), cell-to-cell interactions, and mechanics and dynamics of the surrounding extracellular matrix (ECM). 38 All these signals may determine the fate of cells as they undergo proliferation, differentiation or apoptosis. Alternatively, in a two-dimensional (2-D) cell culture, in which cells are cultured as a monolayer on a substrate surface, it is generally believed to be simpler than a 3-D cell culture in terms of operation and direct detection of cellular behavior. For example, a 2-D cell culture can be directly observed under a conventional microscope at a single focal plane in contrast to its 3-D counterpart, in which such detection is not straightforward due to the 3-D architecture and the optical properties of cell scaffold materials. Thus, the features of a 2-D cell culture greatly facilitate the manipulation of large quantities of cell samples for highthroughput drug screening in pharmaceutical research. Although cellular assays based on a 2-D culture are widely utilized for drug research, their value for predicting the clinical response to drug compounds is limited. 7 This is attributed to the fact that the conditions for a standard 2-D cell culture are poor models of the in vivo conditions since many of the aforementioned microenvironments in a native tissue are changed in such a culture format. 37 In addition, a 2-D culture normally exposes the cultured cells to a more uniform extracellular condition, which may be different from in vivo 3-D environments where any physiologically-meaningful chemical gradients may occur. 39 For example, it is reported that the cellular heterogeneity within a tumor model which is caused by mass transfer limitations resembles the multiple phenotypes found in solid tumors. This is far more realistic than the cellular homogeneity found in a monolayer culture. 40 In order to faithfully understand the cellular responses to drug compounds, a more biologically relevant culture environment is urgently required. 3-D culture models, where cells are seeded in a 3-D scaffold material, are recognized to provide a better approximation of the in vivo conditions than 2-D surfaces Besides, a great number of recent investigations have revealed the differences in the phenotypic characteristics or cellular response to drug compounds when the same cells are cultured under 2-D versus 3-D environments. For example, it has been demonstrated that chondrocytes could de-differentiate and lose their phenotypic natures to synthesize ECM when they are cultured in a 2-D environment, whereas the phenotype can be restored when the cells are transferred to a 3-D culture. 14 Moreover, for anticancer drug testing, it has been concluded that the cancer cells growing in a 3-D culture are more resistant to cytotoxic agents than in a 2-D culture. 44 It is believed that 3-D cell culture models are more promising because they provide a more physiologically-meaningful culture condition for cellbased assays, and therefore promote the quantitative modeling of biological systems from cells to living organisms. 45 For example, in vitro 3-D culture systems have been demonstrated to replicate the drug sensitivity patterns of tumor cells in vivo. 46 Although there is growing evidence demonstrating that 3-D microenvironments may mimic physiological context better than their 2-D counterparts, 2-D cell cultures are widely favored in industrial settings, mainly due to their aforementioned simplicity and lower operating cost. Likewise, to the best of our knowledge, the vast majority of microfluidic cell culture systems used for drug research have adopted the 2-D cell culture format for investigations of concentration-dependent cellular response to drugs, 20 22,47 49 for toxicity testing, for evaluation of the therapeutic effects of drugs, 20,32,55 60 or for other drug research works. 49,61 63 More recently, 3-D-based microfluidic cell culture systems 17,26,64 70 have gained more attention mainly because of their unique ability to provide more in vivo-like cell culture conditions. 5. Microfluidics as a niche technology 5.1 Characteristics of a microfluidic world: advantages and disadvantages Size and surface area-to-volume ratio. Microfluidics refers to the science and technology that allows one to manipulate tiny (10 9 to liters) amounts of fluids using microstructures with characteristic dimensions on the order of tens to hundreds of micrometers. 71 Within this size scale, microfluidic devices are especially suitable for biological applications particularly at the cellular level, because the scale of microchannels corresponds well with the native cellular microenvironment, in which the cell volume-to-extracellular fluid volume is usually greater than one. 72 This paves the way to create a more in vivolike cellular microenvironment in vitro. Besides, due to the small dimensions in typical microfluidic systems, a microfluidic-based cell culture system consumes relatively less research resources. This is particularly meaningful for drug research since the quantities of tested drug compounds or cells are normally very limited in pharmaceutical R&D. In order to illustrate the extent in which the number of cells is reduced in a miniaturized cell culture system, two 3-D cell culture systems are exemplified. For a 96-well, microplate-based, 3-D, cell culture with the dimensions of culture construct, for example, of 1 mm and 7 mm in height and diameter, respectively, the total volume required for a 3-D culture construct is calculated to be 38.5 ml. Conversely, for a microfluidic-based, 3-D, cell culture platform proposed in our earlier work, 64,65 the dimensions of the microbioreactor chamber are 220 mm and 900 mm in height and diameter, respectively, corresponding to 0.14 ml in total volume. On the basis of equal 942 Lab Chip, 2010, 10, This journal is ª The Royal Society of Chemistry 2010

5 cell density, the previous sample volume of 38.5 ml in the microplate-based culture system can provide 275 samples to the microfluidic-based 3-D cell culture platform. The elegant efficiency of a miniaturized scale cell culture system is demonstrated in this comparison by the fact that cell cultures in microfluidic systems, not only can greatly reduce the needed number of cells, but also makes high-throughput cell-based drug research work more feasible. Another key physical feature in the microfluidic world is related to the surface area-to-volume (SAV) ratio. In a microfluidic system, this ratio increases and thus renders any surface phenomenon to become more dominant than the volume factor. In a microfluidic-based cell culture system, a high SAV ratio gives rise to more efficient gas supply through diffusion. The gas supply is an important design consideration in a microfluidicbased cell culture system because living cells require, for example, oxygen for maintaining metabolism. The oxygen flux rate by diffusion through a microfluidic structure is proportional to its surface area. Thanks to the high SAV ratio in a microfluidic world, the gas can be supplied in an efficient manner, which could possibly eliminate the need of an external oxygenation system. 73 More detailed discussions on the oxygen supply issue in a microfluidic-based cell culture system can be found in the literature. 17,35 Similarly, temperature control plays a critical role in a cell culture as it has been reported that the thermal environment in a cell culture system can have an influential impact on cell physiology. 74,75 As a result of, again, a high SAV ratio, a uniform thermal field and precise temperature control can be easily achieved in a microfluidic-based cell culture system due to the excellent heat transfer characteristics. 76,77 Conversely, due to the high SAV ratio, there may be some shortcomings mainly regarding protein adsorption and liquid evaporation in a micro-scale cell culture system. Most of the microfluidic cell culture systems are constructed with polydimethylsiloxane (PDMS) which is biocompatible and non-toxic, 78 optically transparent, and highly gas permeable. 79 However, due to its hydrophobic nature, the PDMS surface is prone to non-specifically protein adsorption. This is of particular concern with respect to cell culture applications since the culture medium contains diverse proteins, which may be physiologically important to the cultured cells; the proteins arise either from supplemented serum or are secreted by the cells themselves. It has been reported 80 that PDMS adsorbs plasma proteins rapidly and the composition of proteins adsorbed varies dynamically due to the Vorman effect : 81 a dynamic protein adsorption process where proteins compete to adsorb onto a surface. Non-specific protein adsorption onto PDMS can deplete or change protein levels within the medium which may subsequently affect the consistency of the culture conditions and cellular behavior. This problem becomes more significant particularly in a microscale cell culture due to the high SAV ratio. 72 This is thus an important technical issue for designing a microfluidic-based cell culture system for precise drug research. Nevertheless, this issue has been generally ignored in the majority of microfluidic-based cell culture systems. It is commonly believed that hydrophilic surfaces have lower levels of protein adsorption when compared to hydrophobic surfaces. Modifications to a PDMS surface to render it hydrophilic have been realized by use of an oxygen plasma, 82,83 UV/ozone 84 treatments, or surface treatment with poly(ethylene oxide) (PEO) The first two treatments require the use of specialized equipment that is usually not available in many biomedical laboratories. Besides, not only does oxygen-plasma treated PDMS undergo hydrophobic recovery but it also affects the oxygen permeability of the PDMS polymer. 88 These factors may cause instability in handling and may hinder its utilization in microfluidic cell culture devices, respectively. Alternatively, surface modification with PEO, a water soluble nontoxic polymer, has been widely used to provide hydrophilic coatings for improving biocompatibility of biomaterials. The incorporation of PEO polymers onto a surface can be achieved through physical adsorption, 85 covalent coupling 86 and synthetic procedures. 87 Although the physical adsorption approach provides a simpler route than the latter two, surface treatment based on this method gives a surface with unstable properties. Another physical approach to produce a PEO surface is to use PEO-terminated triblock polymers, such as a Pluronic Ò surfactant, which is able to form a more stable adsorbed layer on a hydrophobic surface. It has been reported that Pluronic Ò surfactants can considerably reduce protein adsorption. 89,90 Besides, it has been previously reported that treating a PDMS surface with 3% (w/v) of a Pluronic Ò F68 surfactant solution can reduce serum protein adsorption by 85% over the native PDMS. 91 Also, its long-term capability to prevent serum protein adsorption has been observed by our previous study. 92 This provides a fairly simple but effective route to treat a PDMS surface to prevent nonspecific protein adsorption, which has also been adopted in some previous microfluidic-based cell culture systems. 17,64,65 Another technical hurdle caused by the high SAV ratio is liquid evaporation. A commonly-used material for microfluidicbased cell culture system is gas permeable PDMS polymer. Under a normal thermal condition for cell culture (37 C), the tiny volume of liquid in a microfluidic system is prone to loss by evaporation and this phenomenon, again, becomes more prominent particularly in a microscale cell culture due to its high SAV ratio. The uncontrolled evaporation of liquid in a microfluidicbased cell culture system may lead to changes in the cellular microenvironment, 93 which could in turn complicate the precise quantification of cellular responses to tested drug conditions. Nevertheless, little attention has been paid to this fundamental technical issue. The approach of continuous medium perfusion or submerging a microfluidic system in water 94 has been adopted to solve the problem. However, there remains a need to continue to search for other more effective routes to address evaporation effects Flow patterns and mass transfer. The Reynolds number (Re) is one of the many dimensionless parameters used in characterizing fluids, representing the ratio of inertial to viscous forces on a fluid. Microfluidic systems often operate in a low-re domain (e.g. 10 > Re > 0.001) due to the small dimensional features. 95 At low Re numbers, the fluid flow is laminar and turbulence is not likely to occur. In the laminar flow regimen in microfluidc systems, mass transfer relies mainly on diffusion. 95 Leveraging the micro-scale physical phenomena of a laminar flow pattern and diffusion-dominated mass transfer, this allows us to manipulate solubility factors temporally and spatially in a microfluidic system beyond what is currently possible in This journal is ª The Royal Society of Chemistry 2010 Lab Chip, 2010, 10,

6 macroscale devices. 96 For example, the generation of a concentration gradient can be realized in a microfluidic system, enabling the effective production of different cellular microenvironments in a single device. This allows us to stimulate an array of cells with a controllable and definable gradient of drug conditions, which is found to be especially useful for drug research. 5.2 Mimicking in vivo microenvironments Drug testing models based on conventional cell culture techniques continue to give misleading and non-predictive data for in vivo response. 97 This failure may result from our lack of ability to culture cells mimetically to their in vivo conditions so that their phenotypic characteristics are preserved. A cell culture model that is faithful to its in vivo behavior is ideal for cell-based drug research. In order to achieve this, the imitation of specific in vivo extracellular microenvironments in cell culture settings is pivotal for designing a practical drug testing system. A functional tissue in vivo normally possesses heterogeneous but well-organized 3-D micro-architectures encompassing tissuespecific components such as cells, ECM and soluble signaling molecules and is subject to various tissue-specific physical signals (e.g. interstitial fluid flow, shear stress, pressure or compression) under normal physiological conditions. To re-create an in vitro cell culture system representative to its in vivo counterparts for drug research, cell scaffolding biomaterials play a crucial role, not only to provide a 3-D template for a cell culture, but also to exert biological signals through complicated cell-matrix interactions that mimics multiple characteristics of the natural ECM. Biomaterials have emerged as powerful regulators of the cellular microenvironment for drug discovery 98 and thus adequate consideration of biomaterial cell interactions can enable more realistic cell-based drug assays. With the recent advances in functional tissue engineering and biomaterials science, several biomimetic materials have been proposed for cell-based drug testing applications. 99 In this section, the applications of microfluidic technology to possibly mimic in vivo cellular microenvironment are highlighted. Compared with their macroscale counterparts, microfluidic systems that mimic the in vivo extracellular conditions include a higher ratio of cell volume-to-extracellular fluid volume and a smaller effective culture volume. This later parameter is an indicator of cellular control over the microenvironment in the culture system. 72 Also, the medium flow driven in a microfluidic system can achieve a perfusion cell culture by providing a continuous nutrient supply and waste removal. These features are naturally occurring in many in vivo tissues (e.g. liver). Besides, with the unique features of the laminar flow pattern and diffusion-dominated mass transfer occurring in the microfluidic systems, naturally-occurring gradients of diffusing chemicals that guide the differentiation, proliferation 100 and migration of various cell types in vivo can be imitated in vitro in a governable manner. 101 In addition to these characteristics, through specific geometric and structural designs, the continuous flow feature in a microfluidic-based cell culture system can provide extra functionality by generating fluidic shear stress or interstitial fluid flow 31 to create specific in vivo-like microenvironments. Moreover, taking into account the interactions between organs in complex biological systems, microscale cell culture analog (mcca), also called animal-on-a-chip, is a physical representation of the physiologically-based pharmacokinetic model. Here microchannels are used to connect several compartmentalized chambers containing organ-specific cultured cells to form a circulatory system with continuous medium perfusion. 102 The rationale for the development of CCA is to provide an in vitro alternative to an animal model to predict human response to chemicals or pharmaceuticals in a low-cost and simplified manner. 103 With microfluidic technology, mcca 32 can be used not only to reduce resource consumption in experiments but, more importantly, to properly mimic the physiological scale. This is in contrast to the macroscale CCA, 104 in which the physiologically relevant features (e.g. cell volume-toextracellular fluid volume or flow behavior) are difficult to engineer. 103 Additionally, a functional tissue in vivo is normally heterogeneous, in which one or multiple types of cells are organized in a unique 3-D micro-architecture to perform their physiological role. This is one of the main technical hurdles in mimicking such inherently complicated tissue interactions in vitro. This interaction is not only a challenge for the development of a cell/tissue culture model for drug testing but also for the engineering of functional tissues. The traditional methodology for tissue engineering generally treats the engineered tissue as an intact construct and the relevant cell/tissue techniques developed are usually from the bulk point of view. For example, a 3-D scaffold material is seeded with cells and then is cultured in a bioreactor. Tissue/cell cultures based on this method make it not only difficult to control the distribution of the seeded cells in the cultured construct, but also make it hard to adequately engineer the structure or composition of such a construct. Fortunately, the recently proposed concept of a so-called bottom-up tissue engineering, has opened up a new path to deal with the problem. This strategy refers to the fabrication of a tissue-engineered, 3-D construct by the assembly of small modular building blocks, 105 which can be cell-entrapped, microengineered, hydrogel particles 106 or cell aggregates. 107 Using this approach, a tissue/cell culture system can be established in a structurally controllable manner to better mimic the inherent nature of a native tissue. Although several techniques 108,109 based on bottom-up tissue engineering have emerged as a potentially powerful tool to tailor the structure and composition of a 3-D engineered culture construct, these approaches (basically macroscale techniques with dimensions in millimeters or larger) may not be able to fine-tune the cellular microenvironment precisely to closely mimic its native properties. Conversely, microfluidic techniques may overcome this bottleneck due to their operation at the proper physiological scale. 6. Critical issues in microfluidic-based cell culture systems for drug research 6.1 Cell immobilization In microfluidic-based, cell-culture platforms for drug research, one of the major challenges is efficient, quantifiable and reproducible immobilization of cell samples in a defined area or compartment for the purposes of exposure to the tested drug, analysis or observation. Cell immobilization schemes broadly fall 944 Lab Chip, 2010, 10, This journal is ª The Royal Society of Chemistry 2010

7 into one of three categories: (1) adhesion of cells on a substrate surface, (2) entrapment of cells in porous polymeric materials, and (3) capture of cells behind membrane materials. In general cell/tissue culture practice, the first two approaches are the most commonly used techniques to immobilize living cells for 2-D and 3-D cell culture, respectively. On the other hand, the aforementioned cell samples may range from 2-D cultured cells from an identical species or in a co-cultured manner, to 3-D cultured cells with the same cell type or with multiple cell types. The immobilization of cells on a substrate surface for a 2-D cell culture can be simply realized by locating suspended cells onto a surface treated for enhancing cell adhesion. It is well recognized that the adhesion and proliferation of living cells on a substrate surface depends on many surface characteristics, such as the surface charge, 110 wettability, 111 chemistry, 112 microstructure 113 and surface roughness. 114 Thus, it is of great importance to adequately control surface properties so as to manipulate cell behavior on a surface. This is critical for 2-D cell culture-based drug research. Most of the microfluidic-based cell culture platforms for drug research were fabricated using a PDMS material. PDMS surface modifications to enhance cell adhesion can be achieved by coating the surface with ECM proteins such as collagen, 22,27,115 fibronectin, 21,61 or laminin. 116,117 This mediates the specific interaction of cells to protein-coated surfaces via cell integrin receptors. Besides, a cationic poly-lysine treatment 56,118 is also a popular method to enhance cell adhesion on a PDMS surface. All cells from vertebrate species possess unevenly distributed negative surface charges and the presence of electric charges on a substrate surface has also been shown to play a role in the cell adhesion process. 119 It is worth pointing out herein that the performance of the treatments mentioned above, to enhance cell adhesion on a PDMS surface, may vary. For example, our previous study demonstrated that a poly-lysine treated PDMS surface significantly increases fibroblast adhesion by 32%, over the untreated PDMS; whereas, fibronectin does not have such an effect. 92 Therefore, it may be advisable to test both the treatment methods and conditions to ensure an optimal result for cell adhesion for practical applications. Alternatively, micropatterning cell adhesion on a substrate surface in a microfluidic-based cell culture system has been reported to specifically organize cells on a defined area for drug testing, observation, 21,22,24,27,48,49,52,57,120 or to create a cell co-culture. 50,51 For the latter purpose, a cell co-culture technique is a crucial tool to create a more in vivo-like cell culture model for drug research because a functional organ normally possesses multiple cell types that collectively perform a specific physiological role. Most cell micropatterning approaches fall into two main categories: (1) seeding of cells on a chemically patterned surface of different cell adhesiveness and (2) seeding of cells on a topographically or structurally patterned surface. More detailed reviews for the two strategies can be found in the literature. 121,122 For the first strategy, various techniques such as microcontact printing (mcp), 117,123 microfluidic patterning, 69 photolithography, 124 UV laser irradiation, 125 stencil-based patterning 51 have been reported to perform cell micropatterning on a substrate surface. Two examples regarding mcp and stencilbased cell patterning for drug research are discussed herein. mcp is a high-resolution patterning technique to transfer materials (e.g. proteins) from an elastomeric stamp to a substrate surface. The elastomeric stamp can be simply fabricated by replica molding of a PDMS polymer against a microfabricated master. In practice, the fabricated stamp with patterned microstructures is immersed into a solution of the material of interest (e.g. protein solution) and this is followed by stamping onto a substrate surface through conformal contact. 121,122 Due to its simplicity, flexibility, and low cost, mcp has become one of the most popular methods for the micropatterning of cells. For example, using mcp to pattern cells in a microfluidic system for drug testing was demonstrated. 117 In that work, a PDMS microstamp with parallel line structures was used to pattern laminin on a PDMS substrate and was followed by contacting with a neuronal cell suspension. Those cells were ordered in lines following laminin traces on the PDMS surface, organizing cells on a specific area for drug testing. The cell patterned substrate was then assembled with an eight-line, micro-injection array and a base flow channel to form a microfluidic cell culture system (Fig. 1(a)). 117 Using a micro-injection array, the administration of different pharmaceutical compounds to the patterned cells can be realized. Alternatively, stencil-based cell patterning was reported also for cell patterning. For example, a microscale functionallyimproved tissue model for a liver toxicity evaluation of drug candidates was reported. 51 In this work (Fig. 1(b)), 51 soft lithography techniques were used to fabricate elastomeric stencils. A stencil is a membrane that is structured with through-holes of the desired size and geometry. When the stencil is brought in close contact with the substrate it can be used as a template to locally modify the surface for cell adhesion while the area outside the holes remains protected by the stencil. 122 Using this arrangement, hepatocytes were micropatterned on the wells of a 24-well microplate, which were subsequently surrounded by fibroblasts to form a cell co-culture. By mimicking the hierarchical structure of liver tissue, the phenotypic functions of liver cells can be maintained for several weeks. This more in vivo like cellular microenvironment by means of a cell co-culture can contribute to more realistic cell-based drug research. 51 Most of the microfluidic-based cell culture platforms for drug research widely adopted 2-D cell culture models mainly because of their simplicity for cell handling and immobilization. More recently, it has been recognized that these systems are more biological or clinically relevant if the cells are cultured in 3-D microenvironments The most commonly used technique for trapping cells into a 3-D network is to use hydrogel cell immobilization. Hydrogels are highly hydrated polymers ranging from natural biomaterials to synthetic materials. The basic preparation for a 3-D cell immobilization is to mix cells and hydrogel suspensions, followed by loading the mixture to the desired compartment in a microfluidic system for gelation. A hydrogel suspension can be gelled by a variety of mechanisms including ultraviolet (UV) photopolymerization (e.g. PEG derivatives), 126 temperature change (e.g. agarose, 64,65 collagen or Matrigel 66,67 ), or ionic cross-linking (e.g. alginate). 69 Compared with 2-D cell immobilization, the loading of a hydrogel/cell suspension into a microfluidic system is more technically demanding primarily due to the viscous nature of such a suspension, which may be troublesome to deliver and to handle in a microchannel. For example, the use of a photo-sensitive PEG hydrogel to trap microsomes, tiny vesicular fragments of endoplasmic reticulum This journal is ª The Royal Society of Chemistry 2010 Lab Chip, 2010, 10,

8 Fig. 1 (a) Schematic illustration of the microfluidic-based cell culture platform incorporating the patterned cells with a micro-injection array for drug testing. (Reprinted with permission from ref Copyright 2002 Elsevier Science B.V.) (b) Schematics of the process flow of stencil-based cell patterning to fabricate microscale liver hepatocyte cultures in a multi-well format. (Reprinted with permission from ref. 51. Copyright 2008 Nature Publishing Group.) (c) An illustration of a high-throughput cell/agarose loading mechanism in a micro 3-D cell culture platform with 15 microbioreactor chambers (a cross-sectional view). (Reprinted with permission from ref. 65. Copyright 2008 Elsevier Science B.V.) produced after disruption of cells, in a microfluidic system for testing metabolic properties of drug candidates was reported. 53 However, hydrogel polymerization involving UV radiation remains a concern since such treatment could adversely affect biological substances, which might in turn complicate the drug testing. In contrast to a UV polymerized hydrogel, the temperature- or ion-dependent gelling process is more cell-friendly. For the temperature-induced gel formation, collagen and Matrigel have ideal temperature windows for operation (suspended and gelled at 4 C and 37 C, respectively) than an agarose gel. For an agarose gel, a high temperature is normally required to prepare an agarose suspension and this is followed by cooling to 37 Cin order to mix with a cell suspension. Soon after the mixing process, the suspension starts to gel at a temperature around C depending on the type of agarose used, which could in turn cause problems when loading and delivery in microchannels. The use of collagen and Matrigel to immobilize cells in a 3-D hydrogel in microfluidic-based cell culture systems for drug research has been demonstrated. 66,67 For an agarose gel, a perfusion-based, 3-D, cell culture platform with an integrated cell/agarose loading mechanism has been developed in our previous works (Fig. 1(c)). 64,65 The proposed sample loading mechanism allows loading of the cell/agarose suspension in a simple, precise, volume-adjustable and particularly highthroughput manner (simultaneously to 15 microbioreactor chambers). These features are found to be useful for highprecision and high-throughput 3-D, cell culture-based, drug testing. Similar to an agarose gel, an alginate gel is widely used for cell immobilization and encapsulation in life science-related areas. Different from the above temperature-driven gelation, the formation of an alginate hydrogel relies on the cross-linking of the alginate polymer under the presence of divalent cations such as Ca +2 (e.g. calcium chloride solution). Practically, controllable manipulation of the mixing of the cell-containing alginate 946 Lab Chip, 2010, 10, This journal is ª The Royal Society of Chemistry 2010

9 suspension with a calcium chloride solution at the right timing and location in a microfluidic system is the key to a successful in situ 3-D cell immobilization. Otherwise, it would clog up the microchannels and cause the failure of the microfluidic systems. The use of the laminar flow characteristics in a microfluidic system to mix an alginate suspension and a calcium chloride solution at the interface of two streams in a T-shaped microchannel was reported (Fig. 2(a)). 69 Successful 3-D cell immobilization has been demonstrated. In addition to the utilization of a hydrogel, cell immobilization can also be achieved by spatially restricting cells in a microfluidic system via specific structural features. For example, a micropillar array has been demonstrated for physical immobilization of one or more cell types in a main microchannel with two adjacent microchannels parallel to it for medium perfusion. 54 On the other hand, for establishing a 3-D cell co-culture to better mimic native tissue characteristics in a microfluidic system, the concept of so-called bottom-up tissue engineering is technically promising. Borrowing from this strategy, it is feasible to create a 3-D Fig. 2 (a) Demonstration of an alginate, hydrogel-based, 3-D cell immobilization in microchannels. (Reprinted with permission from ref. 69. Copyright 2005 The Royal Society of Chemistry.) (b) Illustration of the assembly of cell-entrapped hydrogel modules to create a 3-D cell co-culture in a microfluidic-based, cell-culture system. (Reprinted with permission from ref. 70. Copyright 2008 The Royal Society of Chemistry.) cell co-culture in a controllable manner in a microfluidic cell culture system, as demonstrated (Fig. 2(b)). 70 Besides, a multilayer structure for a biomimetic 3-D co-culture was also achieved using layer-by-layer microfluidic technology to mimic the structure and composition of blood vessel walls. 127 Additionally, in an effort to realize vital liver functions, hepatocytes and an endothelial cell co-culture has also been achieved using a photosensitive polymer in a microfluidic-based cell culture system Pumping mechanisms The ability to precisely transport and to manipulate a tiny amount of fluid in a microfluidic-based cell culture system is important for medium perfusion, delivery of drug compounds, generation of drug gradient, cell patterning, creation of specific microenvironments and connection of different cell culture units. Medium delivery in most microfluidic-based cell culture systems is normally achieved by the use of commercially available syringe or peristaltic pumps. Nevertheless, these lab-scale pumps are, to some extent, costly and bulky which could hamper their application for high-throughput drug research tasks and their integration with microscale cell culture systems. Alternatively, with the rapid advances in MEMS and microfluidic technology, microscale devices with various actuation mechanisms for pumping fluids have being extensively investigated since the 1980 s. 129 Briefly, microscale pumping devices can generally be classified into two groups: mechanical (with moving parts) and non-mechanical (without moving parts) micropumps. For the mechanical micropumps, they generally consist of a chamber bounded by a flexible diaphragm or membrane. The reciprocal displacements of the diaphragm or membrane, actuated by piezoelectric, 130 electrostatic, 131 electromagnetic 132 or thermopneumatic 133 forces, can cause a change in the chamber volume and hence drive the fluid flow. However, not only are the fabrication processes for these micropumps highly complicated but also the integration of such pumping devices with microfluidicbased cell culture systems remains a technical challenge. For example, a stirrer-based mechanical micropump has been integrated into a microfluidic system (Fig. 3(a)) for a long-term perfusion cell culture. 27 In such design, a stirring bar (3 mm long, 200 mm wide) has been enclosed in a pumping chamber, which is actuated by an external magnetic field. In this arrangement, a unidirectional continuous flow is generated as the rotational motion of the stirring bar drives the liquid from the pumping chamber inlet to the outlet. Although its feasibility has been proved, the fabrication and assembly process is complicated and its operation is not robust. On the other hand, the micropump actuated by an electroosmotic mechanism 134 requires no mechanically moving parts since it makes use of the electrokinetic phenomenon of electroosmosis for pumping electrically conductive solutions, which not only largely simplifies the design and fabrication steps, but also provides a longer lifetime over other types of micropumps. Nevertheless, such pumps are restricted to pumping fluids with a certain range of electrical conductivity. Moreover, a high electrical field may be generated in these types of micropumps due to the applied electrical voltage. This may restrict its utility for medium delivery in a microfluidic-based cell culture system since the generated electrical field may affect the physiology of This journal is ª The Royal Society of Chemistry 2010 Lab Chip, 2010, 10,

10 Fig. 3 (a) Photograph of microfluidic perfusion cell culture system with an integrated stirrer-based micropump. (Reprinted with permission from ref. 27. Copyright 2008 The Royal Society of Chemistry.) (b) (I) Schematic illustration of a Horizontally-Oriented Mini-Reservoirs pumping scheme (HORM pump) and (II) a photograph of the parallelized HORM pumps. (Reprinted with permission from ref Copyright 2004 The Royal Society of Chemistry.) (c) A 3-D diagram of an elastomeric peristaltic pneumatic micropump. (Reprinted with permission from ref Copyright 2000 American Association for the Advancement of Science.) (d) The top-view layout of a perfusion-based micro, 3-D, cell-culture platform with integrated spider-web-like pneumatic micropumps. (Reprinted with permission from ref. 65. Copyright 2008 Elsevier Science B.V.) the cultured cells, which might in turn complicate the subsequent cell-based drug testing. Another non-mechanical pumping scheme used in microfluidic-based cell culture systems is to exploit the pressure drop caused by the difference in gravitational potential energy to drive the liquid flow. 20,31 The pressure drop in a microfluidic system is generated by the difference in the height of a liquid in the inlet and the outlet. By carefully adjusting the liquid levels, the liquid flow rate can be regulated. However, precise control of the liquid levels in such systems is quite difficult and demanding. In order to address this issue, a pumping system using horizontally-orientated mini-reservoirs (HOMR) (Fig. 3(b)) for a microfluidic-based perfusion cell culture has been developed. 120 In this work, two reservoirs connecting to the inlet and outlet of the liquid microchannels, respectively, were horizontally orientated to maintain a constant hydraulic pressure drop across microfluidic channels even as the volume of liquid within the reservoirs change over time. Using this design, a steady pulse-free medium flow can be generated for a perfusion cell culture. In addition to the above liquid pumping schemes, multiplexed liquid delivery is the key to a microfluidic-based, cell-culture platform for high-throughput drug research. The development of a pneumatic micropump holds great promise for this purpose. Pneumatic micropumps fabricated from an elastic polymer (e.g. PDMS) and a standard soft-lithography process were first reported by Unger and co-workers. 135 The proposed pumping mechanism is based on the pulsations of elastic membranes actuated by the pneumatic chambers above to generate a continuous peristaltic-like activation effect for driving the fluid forward (Fig. 3(c)). Its key features are lower cost, is simpler to fabricate and operate, and is easier to incorporate into a microfluidic-based, cell-culture system compared with the aforementioned micropumps. Borrowing from the concept of peristaltic pneumatic micropumps, some previous studies have successfully demonstrated the integration of multiple (15 or 30 units) pneumatic micropumps 64,65 in a microfluidic-based, 3-D, cell-culture platform for pumping medium for high-throughput anticancer drug testing. Briefly, one of the earlier studies configured 15 pneumatic micropumps into a spider-web layout to achieve uniform medium delivery (Fig. 3(d)), 65 where the conventional linear pneumatic micropumps are in parallel, requiring only three external pneumatic sources to concurrently actuate 15 micropumps. However, the throughput for medium delivery is technically limited in such a design. This is because when the 948 Lab Chip, 2010, 10, This journal is ª The Royal Society of Chemistry 2010

11 number of micropumps connected in parallel exceeds a certain limit, the pneumatic force cannot actuate each micropump without a time delay caused by fluidic resistance inside the pneumatic channel. As a result, the uniformity of the pumping rate for each micropump may be affected. To address this issue, the idea of S-shaped pneumatic micropumps, was first presented by our group (Fig. 4(a)), 136 as a solution. The basic layout is that an S-shaped pneumatic microchannel intersects a liquid flow microchannel underneath at various points along its length, with a thin elastic membrane interfacing the pneumatic and liquid flow channels at each intersection, to form a sequential, membrane-based pneumatic micropump. The working principle of this type of pneumatic micropump is based on the use of the fluidic resistance of air in the S-shaped pneumatic channel to generate the sequential rise of air pressure in the pneumatic channel. This time-phased increase in the air pressure in the pneumatic channel correspondingly leads to peristaltic-like pulsations of the elastic membranes located at the intersections of the liquid flow microchannel and the S-shaped pneumatic channel. Fluid pumping in this device is driven by the hydrodynamic pressure generated by these peristaltic actions. 136 In order to achieve multiplexed medium pumping (e.g. 30 integrated micropumps) in a microfluidic-based, cell-culture platform, the design of a pneumatic micropump with an S-shape layout has been further optimized 137 and also has been demonstrated for a high-throughput, microfluidic-based, 3-D, cell culture (Fig. 4(b)). 64 All of these aforementioned pneumaticbased micropump designs have been found to be useful for medium delivery in a microfluidic-based cell culture system particularly for high-throughput and long-term perfusion cell cultures, where both uniform and simultaneous medium delivery are required. 6.3 Gradient generation Fig. 4 (a) (I) Schematic illustration of a pneumatic micropump with an S-shape layout and (II) photographs showing the actions of an S-shape pneumatic micropump to drive fluid forward. (Reprinted with permission from ref Copyright 2006 IOP Publishing Ltd.) (b) The top-view layout of a high-throughput perfusion microfluidic cell culture platform with 30 integrated S-shape pneumatic micropumps. (Reprinted with permission from ref. 64. Copyright 2007 Springer Science + Business Media, LLC.) The significant difference in dominant physical phenomena between microscale and the macroscale fluid systems has been exploited to provide a variety of new types of functions such as gradient generation, based on the inherent laminar flow and diffusion-dominated mixing occurring in microfluidic systems. 96 This function has been utilized as a versatile tool to generate various biomolecular gradients involving biological signaling phenomena (e.g. immune response, 138 cancer metastasis, 24 and differentiation of cells 116 ) in microfluidic systems to mimic in vivo microenvironments for a variety of research purposes. Besides, the generation of a controllable gradient provided by the microfluidic systems can be incorporated into cell-based drug testing schemes to investigate the concentration-dependent cellular responses to drug conditions. 22,48 47,52,120,139 Traditionally, high-throughput drug research based on commonly-used, multi-well, microplate technology is limited by assay throughput. With this gradient-generation function, various controllable and definable drug conditions can be effectively created on a single device allowing the stimulation of a field of cells in a highthroughput manner. This thus holds great promise as a better substitute for current cell-based high-throughput drug testing schemes. 48,52 Gradient generation can be conventionally achieved with numerous techniques, for example, hydrogel based methods, 140 Transwell assays 141 or Dunn chambers, 142 in which gradients are simply generated by the diffusion of chemical species driven by the initial concentration differences in the devices. Although these approaches are feasible, these devices are relatively bulky and thus require more experimental resources, which could hinder its utility for drug testing with limited resources. Most importantly, gradient generation based on the diffusion of molecules from a source to a sink compartment in these conventional approaches is unable to maintain a stable gradient profile for a long period of time. Besides, the generated gradient lacks the flexibility to be modified and controlled. Conversely, microfluidic-based gradient generation devices, 143 have advantages over these conventional methods including (1) increased throughput, 47 49,120 (2) reduced experimental operating costs, 48,66 (3) flexible and controllable gradient generation with various This journal is ª The Royal Society of Chemistry 2010 Lab Chip, 2010, 10,

12 profiles, 23,25,144 (4) stable gradient generation that can be maintained for a long period of time, 21 (5) gradient generation with a proper concentration resolution for cellular assays 145 and (6) the ability to observe in real-time the cellular responses to these concentration gradients. 22,49,52 Microfluidic-based gradient generation devices can be broadly classified into two categories, including (1) a steady-state and (2) a time-evolving gradient generator. 145 The first type makes use of the laminar-flow in microfluidic systems to mix chemical species at the interfacial regions between continuous parallel fluid streams solely by diffusion to generate gradients. With this approach, chemical gradients with various concentrations ranging from linear to non-linear 24,25,52,144 profiles can be generated in a controllable and quantifiable manner through the design of microchannels with specific geometric layouts 22,23,48,52,144 or structural features, 21,25 or through the manipulation of flow rates. Although these microfluidic-based gradient generators have been proved feasible in providing various stable gradient profiles to meet the needs of specific cellbased drug research, 22,47,48,52,120,139 successful steady-state gradient generation in these cases substantially relies on the ability to maintain perfectly continuous flow patterns. However, this parameter is generally unachievable while using conventional syringe or peristaltic pumps. This problem can be solved by the utilization of pulse-free fluid flows driven by gravity, as described earlier (refer to Fig. 3(b)). 120 Besides, maintaining a stable gradient profile in this type of gradient generator requires constant fluid flow, which could in turn lead to high reagent consumption, particularly for long-term investigations. Moreover, a continuous fluid flow could also wash away autocrine or paracrine signaling factors secreted by cells and subsequently hinder physiologically-meaningful cell-to-cell communication, which could therefore complicate the cell-based drug testing. Furthermore, mammalian cells do not have a cell wall and are, consequently, highly susceptible to the effects of shear stress. The fluid flow-induced shear stress that these microfluidic-based gradient generators exerted on the tested cells may be lethal, or adversely affect cellular behavior and thus cause experimental bias. 52 In order to generate stable chemical gradients without also simultaneously generating fluidic shear forces, several microfluidic-based gradient generators have recently been proposed, in which membranes (Fig. 5(a)), 146 hydrogels 147 or microcapillaries (Fig. 5 (b)) 21 are exploited to minimize or eliminate fluid flows into a cell culture area but still allow for diffusion-based mass transfer to maintain a desirable chemical gradient in the cell culture area or chamber. In addition to their relatively complicated fabrication steps and experimental setups, these devices, again, need constant fluid flows to generate and to maintain a steady gradient profile. Another type of gradient generator is usually referred to as a time-evolving static gradient generator, 145 in which a chemical gradient is passively developed by diffusion along a channel in between a source and sink chamber under a static condition. 148 Without a fluid flow, this type of gradient generator not only can create a more shear-free environment, but also can retain more signaling factors secreted by cells when compared with the previously mentioned constant flow-based, steady-gradient generators. Nevertheless, similar to the traditional gradient Fig. 5 (a) Schematic view of a microfluidic-based cell culture device capable of generating steep, shear-free gradients of small molecules. (Reprinted with permission from ref Copyright 2008 Springer Science + Business Media, LLC.) (b) (I) The top view showing the positions of source and sink (reagent) channels and a cell culture chamber. The microcapillaries connect a reagent channel to the culture chamber. (II) The cross-sectional view. (Reprinted with permission from ref. 21. Copyright 2008 The Royal Society of Chemistry.) generation mechanisms, the long-term stability of gradient profiles in such designs still remains a technical issue. The majority of the microfluidic-based gradient generators for cell-based drug testing 22,47,48,52,120,139 are designed for a 2-D cell culture only. In order to investigate the dose-dependent cellular responses to chemicals in 3-D cell culture conditions, several microfluidic-based, cell-culture platforms incorporating gradient generation functions have been demonstrated. 21,26,66,147 For example, a microfluidic device 147 was reported, in which stable gradients of soluble factors were generated across a cell-entrapped, 3-D, collagen gel using a two-compartment diffusion chamber. Additionally, a cell culture chamber with its top and bottom connected to source and sink channels through multiple microcapillary tubes was also reported (Fig. 5(b)) 21 to produce 950 Lab Chip, 2010, 10, This journal is ª The Royal Society of Chemistry 2010

13 a stable chemical gradient across a cell-entrapped, 3-D, hydrogel matrix in the cell culture chamber. Although these devices have been shown to be promising, the loading of cells/hydrogel in such microscale devices remains technically demanding. 7. Practical applications for drug research Throughout the drug discovery and development processes, a microfluidic-based cell culture or cellular assay systems can play critical roles in, for example, target validation, 62,139 lead compound identification, 49,68,149,150 pre-clinical studies including toxicity testing, 51,52,54,66,151 efficacy study 20,22,44,48,57 60 and the study of drug delivery mechanisms. 31,61,63,152 Herein, some microfluidic-based, cell-culture systems designed for in vitro toxicity testing and those with a high-throughput capability are highlighted. 7.1 Drug toxicity testing Toxicity information in pre-clinical studies provides us with confidence in the safety aspects of the potential drug candidates. The development of an in vitro cell culture system capable of modeling the response of native liver tissues to drug toxicity is a priority in the field of drug discovery. 153 In order to successfully develop a liver cell culture in a microfluidic system, usually referred to as a liver on a chip, one must take into account many technical issues related to transport phenomena, biomaterials, cell biology and tissue engineering. 5 It is recognized that primary hepatocytes rapidly lose liver-specific functions when they are cultured based on conventional cell culture techniques 154 due to the loss of the inherent extracellular microenvironments. To address this issue, one of the microfluidic-based approaches is to culture primary hepatocytes in a membrane-based PDMS microbioreactor, 115 in which cells attached to a porous PDMS membrane is sandwiched between two medium perfusion chambers to mimic an in vivo liver architecture and perfusion conditions. Another similar 2-D perfusion hepatocyte culture was carried out in a so-called NanoLiterBioReactor, 73 in which cells were trapped by the sieve-like structures in this microfluidic device. Although it is simple to maintain a cell culture in these novel devices, the throughput is limited. Furthermore, there are issues with creating a viable cell co-culture using these approaches. Cell co-culturing techniques have been adopted for maintaining hepatocyte viability and to support many liverspecific functions. 155 For example, a microscale co-culture of liver cells with fibroblasts (Fig. 1(b)) 51 capable of maintaining phenotypic functions for several weeks for high-throughput drug research has been reported (refer to the cell immobilization section). Nevertheless, such a system is basically a static cell culture, in which the naturally occurring perfusion condition in liver tissues is not replicated. This can be solved by incorporating medium perfusion into such a co-culture system. 50 In this work, a perfusion microfluidic array (8 8 elements) of a hepatocyte fibroblast co-culture with stable liver-specific functions was demonstrated (Fig. 6(a)). On the other hand, in order to better mimic in vivo 3-D architectures, microfluidic systems to immobilize cells in a 3-D hydrogel and to confine the 3-D culture constructs to the middle of microchannels by the mechanisms of hydrodynamic focusing 43 and microvalving 66 have been demonstrated. They allow for medium perfusion on both sides of these constructs. In such systems, the tested chemicals were added to the medium and administrated to the cultured cells by diffusion, forming a linear concentration gradient across the hydrogel construct. With this approach, the effect of chemicals on the cell viability can be monitored online via microscopic observation. Furthermore, a perfusion, microfluidic, 3-D, cellculture system capable of characterizing drug metabolites and drug toxicity simultaneously has been reported 55 (Fig. 6(b)). In this work, a sol gel was used to confine the cultured cells in a chamber for performing a cytotoxicity assay and for allowing the drug metabolites to be assayed separately in another part of the microfluidic system. Most of the aforementioned microfluidic hepatocyte culture systems have demonstrated their capability to maintain phenotypic functions of the liver 43,50,51,55,73,115 and have also demonstrated their capability for drug toxicity testing. 43,51 Nevertheless, what seems to be lacking is corresponding data validated from living organisms. A microfluidic, 3-D, hepatocyte chip (3D Hepa Tox Chip) integrating a linear concentration gradient generator and a multiplexed cell culture chip has been reported 54 (Fig. 6(c)) for drug toxicity testing. In this design, hepatocytes are immobilized on the middle passageway of the microchannels with the aid of the designed micropillar arrays, which confines cells in the central culture compartment to form 3-D microenvironments, allowing for medium perfusion on the both sides of the culture compartment. With such a precise biomimetic design, this device has been proven to maintain the phenotypic functionality of liver. More importantly, the results from drug toxicity testing correlated well with similar testing in an animal model. Moreover, to further mimic the multi-organ interactions in a complex biological system, a microfluidic-based mcca was reported (Fig. 6(d)), 32 in which cells from various species are cultured in a 3-D hydrogel located in three separate chambers connected by a microchannel to create tissue analogs for drug toxicity test. The result of a drug toxicity evaluation is reported to be consistent with that of clinical trial, showing that the developed system is promising for drug testing. As a whole, the reviewed cases discussed here demonstrate that a microfluidic-based, liver cell-culture system holds great promise to take the role of conventional cell culture techniques, showing better results in terms of maintaining the phenotypic characteristics of liver tissues and in more realistic cellular responses to drug conditions. These results are attributed to the fact that microfluidic technology provides us with versatile tools to adequately engineer extracellular microenvironments. However, there is also a clear trend that with increased complexity, the assay throughput of a microfluidic-based, cell-culture system may be compromised. 7.2 Microfluidic-based, cell-culture systems for highthroughput drug research Successful realization of microfluidic-based, cell-culture systems in drug research substantially relies on their high-throughput capability. In microfluidic-based, cell-culture systems, this issue has technically to do with efficient medium delivery and sample loading (e.g. the addition of chemicals). Commercially available pumps have limitations for high-throughput drug research tasks. Alternatively, with the recent advent of microfluidic technology, This journal is ª The Royal Society of Chemistry 2010 Lab Chip, 2010, 10,

14 Fig. 6 (a) (I) Perspective-view photograph of the gas and medium perfusion channels within a microfluidic array. The cell-culture medium channels allow for the sequential perfusion of individual wells starting from the upper-left to the lower-right positions in the image. The gas perfusion channels are aligned orthogonally to the medium perfusion channels. (II) A phase-contrast micrograph of a co-culture within a well array. Hepatocytes adhering to the collagen micropatterns are shown and (III) on post-seeding day 3 surrounded by 3T3-J2 fibroblasts. (Reprinted with permission from ref. 50. Copyright 2006 American Chemistry Society.) (b) (I) Schematics of a microfluidic device for the simultaneous characterization of drug metabolites and a cytotoxicity assay. (II) The device is composed of three layers, a quartz substrate embedded within separation microchannels and a three-micro-well array filled with human liver microsome in a sol gel sandwiched between two PDMS substrates. (III) A magnified schematic of one sol gel bioreactor on a microfluidic device. (Reprinted with permission from ref. 55. Copyright 2009 The Royal Society of Chemistry.) (c) 3D HepaTox Chip for the simultaneous administration of multiple drug concentrations. (I) Microfluidic design and assembly of a linear concentration gradient generator and 952 Lab Chip, 2010, 10, This journal is ª The Royal Society of Chemistry 2010

15 efficient medium delivery in microfluidic-based, cell-culture systems can be achieved by integrating high-throughput medium pumping schemes (e.g. membrane-based pneumatic 64,65,137 or gravity-driven 120 micropumps) (refer to the pumping mechanism section) or the specific manifold designs for liquid division. 60 For example, our previous studies have incorporated 15 (Fig. 7(a)) 65 and 30 micropumps 64 within a single perfusion-based, micro 3-D, cell-culture platform for chemosensitivity testing of an anticancer drug, in which the culture medium with varied drug concentrations was individually delivered to the microbioreactors where the tested cancer cells were cultured. With a membrane-based pneumatic pumping mechanism, 137 this developed system (refer to Fig. 4 (b)) 64 holds great promise to achieve even more multiplexed medium pumping (e.g. over 30 micropumps) for more high-throughput cellular assays. Moreover, high-throughput cellular assays can also be realized by a proper manifold design for even medium distribution, namely dividing a medium flow into several sub-microchannels, into which the cell culture units for an array layout are connected. For example, by fine-tuning the pressure drop in a pressure-driven flow, an 8 5 array of cell culture microchambers, which utilized a liquid division mechanism (Fig. 7(b)) for the study of the cytotoxic effects of seven different anticancer drugs in a high-throughput manner has been successfully demonstrated. 60 The use of the liquid division strategy can largely reduce the need for additional pumping devices and is thus found to be particularly useful for designing a high-throughput microfluidic-based, cell-culture platform. Although this is a straightforward concept to demonstrate, the practical realization is difficult because the variations in the surface roughness of each microchannel can affect the fluidic resistance, and in turn the flow distribution. Although the feasibility for high-precision and highthroughput drug research has been demonstrated based on the aforementioned devices, these systems require manual preparation and loading of the reagents for the various drug conditions. Alternatively, this issue can be solved by using the gradient generation function as described earlier. For example, eight gravity-driven micropumps (called the HOMR pumping scheme; see the pumping mechanism section) (also refer to Fig. 3(b)) and the laminar flow phenomenon were used to generate multiple drug concentration gradients in a microfluidic, cell-culture platform (Fig. 7(c)) to simultaneously investigate the cellular response to the administered drug concentrations. 120 By controlling the gradient profile, gradients of drug conditions can be effectively produced in a single microfluidic device, allowing for the stimulation of a field of cells in a high-throughput manner. Besides, the variation due to differences in the cell culture conditions can be possibly eliminated using the same group of cells for multiple assays in a microfluidic-based cell culture system with a controlled gradient generation. 120 These traits are found to be useful in the design of high-throughput cell-based drug testing schemes. 48 Microfluidic cellular arrays based on this principle have been actively pursued for various drug research purposes. 22,48,139,143 One typical example is an integrated microfluidic device consisting of multiple drug gradient generators and parallel cell culture chambers (Fig. 7(d)). 48 With such a design, multi-parametric measurement of the cellular responses to drug conditions can be carried out in a single device. As a whole, the gradient-generation function in a microfluidic device enables high-throughput assays to be performed in different formats. However, cell-level drug research based on this approach requires continuous flows to generate and maintain specific gradient profiles and thus may consume more reagents. Apart from this, the manipulation of liquid flow in such systems is usually affected by small perturbations and technically demanding. 8. Concluding remarks The significant differences in several physical phenomena between microscale and the macroscale devices have been exploited to provide various innovative microfluidic-based assays for cell study. Compared with conventional cell culture techniques, a microfluidic-based cell culture possesses several inherent advantages and can provide versatile approaches to regenerate more in vivo-like extracellular conditions, for more realistic cell-based drug research. Although microfluidic-based, cell-culture systems hold immense promise as a platform for drug research, the utilization of such emerging tools has not yet set in motion an evolutionary shift from conventional methods of drug research, either academically or commercially. Microfluidicbased, cell-culture systems are basically at an early stage of development and most of the published literature in this area are still only proof-of-concept demonstrations. With the ultimate goal of becoming a high-throughput, in vitro, drug-testing platform capable of better predicting the human cellular responses to new drugs, there are still many hurdles to overcome before it can move from a demonstration tool to practical applications. The challenges mainly include several technical issues regarding the interface between designers and end-users, biological validation and detection schemes. From the application point-of-view, firstly, the successful development of microfluidic-based, cellculture systems for drug research substantially relies on the cooperation of design engineers and the biologists or pharmacists who use these devices. Namely, the device design should avoid too many operational barriers for the end-users to perform the protocol and should enable the biologists to obtain data in an immediately meaningful format. Secondly, when a novel research tool is utilized, the interpretation of the resulting data is challenging in terms of reconciling differences with data acquired through the use of common conventional methods. These factors inevitably require more fundamental research to bridge the gap a multiplexed cell culture chip to construct the 3D HepaTox Chip. The bottom panel is a sample of the 3D HepaTox Chip. (II) Magnified view of a single cell culture channel of the multiplexed cell culture chip. An array of mm 2 micropillars separate the channel into three compartments: a central cell culture compartment and two side media perfusion compartments. (Reprinted with permission from ref. 54. Copyright 2009 The Royal Society of Chemistry.) (d) (I) Schematic diagram showing how the mcca is assembled. (II) A picture of an assembled mcca with red dye for visualization of channels and chambers. (III) Schematic diagram of the operating principle behind a single mcca with medium recirculation. (IV) Picture of the mcca system with multiple chips. (Reprinted with permission from ref. 32. Copyright 2009 The Royal Society of Chemistry.) This journal is ª The Royal Society of Chemistry 2010 Lab Chip, 2010, 10,

16 Fig. 7 (a) (I) Photographs of a perfusion-based, micro, 3-D, cell-culture platform and (II) the entire experimental setup (the platform is integrated with a hand-held controller). (Reprinted with permission from ref. 65. Copyright 2007 Elsevier Science B.V.) (b) Schematics of the cell culture microchamber array on the perfusion chip. (I) The chip design with the entire microchannel network. (II) Structure of each cell culture microchamber. (Reprinted with permission from ref. 60. Copyright 2008 John Wiley & Sons, Inc.) (c) Photograph showing alternating streams of different colored dyes in a microchannel. Bar: 200 mm. (Reprinted with permission from ref Copyright 2004 The Royal Society of Chemistry.) (d) Schematic of the integrated microfluidic device for cell-based, high-throughput screening. (I) The device consists of eight uniform structure units and each unit is connected by a common reservoir in the center of the device. (II) Magnified section of the single structural unit containing an upstream concentration-gradient generator and downstream parallel cell culture chambers. (Reprinted with permission from ref. 48. Copyright 2007 The Royal Society of Chemistry.) between validation and verification. The third technical hurdle is related to the detection schemes capable of reading out the results of an assay in an efficient and high-throughput manner, as the commercial readers today do for multi-well microplate-based HTS. However, this part of the development, to some extent, lags behind the progress in microfluidic-based, cell-culture techniques. It is envisioned that the development of adequate and user-friendly detection schemes would accelerate the widespread application of microfluidic-based, cell-culture systems in drug research in the near future. Acknowledgements This work is sponsored by the National Science Council (NSC) in Taiwan (NSC E MY3; NSC E MY2). References 1 Pharmaceutical Research and Manufacturers of America, Pharmaceutical Industry Profile 2009, (Washington, DC: PhRMAApril 2009), [accessed Sep 07, 2009]. 954 Lab Chip, 2010, 10, This journal is ª The Royal Society of Chemistry 2010

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