Open access peer-reviewed chapter

# Cells and Organs on Chip—A Revolutionary Platform for Biomedicine

By Preeti Nigam Joshi

Submitted: October 19th 2015Reviewed: May 5th 2016Published: June 29th 2016

DOI: 10.5772/64102

## Abstract

Lab‐on‐a‐chip (LOC) and microfluidics are important technologies with numerous applications from drug delivery to tissue engineering. LOC integrates fluidic and electronic components on a single chip and becomes very attractive due to the possibility of their state‐of‐art implementation in personalized devices for the point‐of‐care treatments. Microfluidics is the technique that deals with small (10-9 to 10-18 L) amounts of fluids, using channels with dimensions of 10 to 100 μm. These LOC and microfluidics devices enable the development of next‐generation portable and implantable bioelectronics devices. Superior chip‐based technologies are emerging with the advances in microfluidics and motivating various chip‐based methods for rapid low‐cost analysis as compared to traditional laboratory method.An organ‐on‐chip (OOC) is on‐chip cell culture device created with microfabrication techniques and contains continuously perfused chambers inhabited by living cells that simulate tissue‐ and organ‐level physiology. In vitro models of cells, tissues and organ based on LOC devices are a major breakthrough for research in biologic systems and mechanisms. The recapitulations of cellular events in OOC devices provide them an edge over two‐dimensional (2D) and three‐dimensional (3D) cultures and open a gateway for their newer applications in biomedicine such as tissue engineering, drug discovery and disease modeling. In this chapter, the advancement and potential applications of OOC devices are discussed.

### Keywords

• lab‐on‐chip
• MEMS
• organ‐on‐chip
• 3D cell culture
• drug discovery

## 1. Introduction: why cell and organ on chip?

The field of microfluidics or lab‐on‐chip (LOC) technology aims to advance and broaden the possibilities of bioassays, cell biology and biomedical research based on the idea of miniaturization. Microfluidic systems allow more accurate modeling of physiological situations for both fundamental research and drug development [1].

Drug discovery and research is the prime aspect of any pharmaceutical company. The past 50–60 years have witnessed significant scientific and technological growth in entire field of biotechnology, computational drug design and screening and advances in scientific knowledge, such as an understanding of disease mechanisms, new drug targets and biomarkers discovery. In principal, these advancements should also be reflected in rise of new commercial products and drugs, but unfortunately, the pharmaceutical industry is facing unprecedented challenges owing to rising costs and the declining efficiency of drug research and development. Modern drug development requires implementation of extensive preclinical testing, and validation protocols before potential therapeutic compounds are approved to progress to clinical evaluation. This process is costly and time‐consuming, as well as inefficient as for every 10 drugs entering clinical trials, only one or two will typically be licensed for eventual use in humans [2]. The number of new drugs approved per billion US dollars spent on R&D has halved roughly every 9 years since 1950, falling around 80‐fold in inflation‐adjusted terms.

The failures of drug clinical trial are primarily due to the poor predictive power of existing preclinical models. The existing cell culture techniques often failed to mimic the complexity of living systems and are incapable of modeling situations where organ‐organ or tissue‐tissue communication are important. Moreover, cells maintained in standard in vitro culture conditions often suffer from incomplete maturation or are held in a configuration that prevents their full functional development, making predictions of in vivo tissue function more difficult to extrapolate. Although animal models preserve the intricacy of living systems, due to the inherent complexity of interconnected tissues, elucidation of specific mode of drug action is often difficult that leads to confound observations. Furthermore, animal models have, on multiple occasions, been predicated human responses to drug treatment in a rather harmful way [3, 4]. The drug discovery community has identified the critical need for new testing approaches and an intermediate human in vitro model in the early stage of drug development to generate reliable predictions of drug efficacy and safety in humans that could mitigate the side effects observed in clinical trials and LOC systems can play a pivotal role in this by fulfilling this unmet need by microengineered cell culture models with miniaturized and automated assays that will increase resolution and precision. These models leverage cutting‐edge microfabrication and microfluidics technologies to control the cellular microenvironment with high spatiotemporal precision and to present a variety of extracellular cues to cultured cells in a physiologically relevant context [56].

This chapter deals with the cutting‐age research in the field of microfabrication technologies and multiorgan microdevices that mimic key aspects of human metabolism. We discuss about latest advancements and how this emerging field transforms the face of biomedicine.

### 1.1. Need of microfluidics technologies for global health: applications and limitations

Diagnostic applications for global health have seen a fast pace in recent years. LOC, micro total analysis systems (μ‐TAS) or microfluidics systems are the major breakthrough in this regard and with their state‐of‐art technology, these miniaturized integrated devices have great potential to change the face of healthcare sector globally. Basically, from industrial perspective to develop a high‐throughput diagnosis system, it must utilize small chemical volumes to keep the cost of development at an affordable level. The current trend of miniaturized and automated assays can address these issues directly owing to their better resolution and accuracy. Microfluidics devices are new and promising players in healthcare segments. These devices, which scaled down analytical processes in conjugation with advances in microfluidics technology, are the soul motivation behind various chip‐based methods of lower cost and rapid analysis than the conventional laboratory bench‐scale methods. Although these microelectromechanical systems (MEMS) or miniaturized chip‐based systems have seen a fast pace in other fields, such as electronics, aerospace and computer science, since their inception in early 1990s and have witnessed many innovations based on these techniques, in this chapter, our prime focus is how these technological advancements have been transformed into the face of biomedical sciences with its wide range of biological applications, such as high‐throughput drug screening, single cell or molecule analysis and manipulation, drug delivery and advanced therapeutics, biosensing and point‐of‐care diagnostics, among others. [7]

Extracting new phenomena and elaborated information about the biologically active systems is the basis of all innovations in the field of biomedical sciences. The complex live systems and richness of biological processes are stimulating factors for new LOC approaches, and these emerging technologies are gradually changing the scenario, and now, we can seek experimental answers at the molecular level.

#### 1.1.1. Development of microfluidics technologies for different applications in healthcare segment

In a broader sense, microfluidics can be linked to the development of integrated circuit technology and wafer fabrication facilities. They have unique ability to combine different systems possessing high‐throughput capabilities, new data processing and storage strategies. These miniaturized devices provide new tools for highly parallel, multiplexed assays with better isolation, purification and handling of entities, cells or organisms for a simplified, parallel analysis. Initially, silicon and related materials were the preferred choices to fabricate miniaturized devices but now polymeric materials are also the stake holders for because of ease of manufacturing by embossing or molding [8]. They are attached to other surfaces such as silicon, and the formation of fluid channels and patterns on polymeric devices are relatively easy. Other materials, such as semiconductors and metals, are other necessary components of electrical detection schemes, and earlier reports are there where semiconductor nanowires and carbon nanotubes are being studied as sensor components [9, 10]. Integration of mechanical devices with fluid systems for biological implementation and to fabricate disposable systems has been reported earlier and summarized in many reviews [7, 11, 12]. Figure 1 shows an on‐chip disposable diagnostic card. In this segment, few latest applications of LOC devices are discussed briefly [50].

#### 1.1.1.1. On‐chip DNA hybridization and PCR

An on‐chip deoxyribonucleic acid (DNA) hybridization assay refers to the bioassay conducted on the microfluidic system/device based on the nucleic acid hybridization technique [13]. From its earlier applications in 1980s, it has been evolved as a powerful tool to detect and identify the presence of a specific DNA sequence. On‐chip DNA hybridization systems are amalgamation of advantages of both microfluidics and hybridization.

In the past 20 years, microfluidics devices have been emerged as an important area of research. As a combination, miniaturization eliminates the need of large reagent consumption, time‐consuming labor‐intensive procedures and involvement of bulky or expensive equipment while keeping its distinctive advantages of high sensitivity, selectivity and specificity of conventional techniques. Additionally, these miniaturized devices can play a pivotal role in healthcare sector of the Third World countries, by bringing cheaper and smaller, but still sophisticated analytical tools to rural areas and resource‐poor regions [14]. This section focuses on few recent application of on‐chip polymerase chain reaction (PCR) devices. There are few criteria to be taken care of while designing on‐chip PCR systems such as high‐temperature resolution and acquisition rate for precise thermal cycling in microfluidics. Apart from traditionally embedded thermocouples and thermometers [1517], Wu et al. [18] reported an integrated PCR system with a temperature controller using platinum (Pt) thin film as heater and temperature sensor, an optical detection system and an interchangeable (disposable or modular) PCR chip, which was independent from the two functional systems as shown in Figure 2. In this system, Pt thin‐film sensor was patterned to microsize and integrated to thin‐film heater into the chip to provide rapid response and precise integration.

In another approach, Chia et al. developed fully integrated, portable PCR device that consists of the following four major parts: a disposable chamber chip with microchannels and pumping membranes, a heater chip with microheaters and temperature sensors, a linear array of electromagnetic actuators and a control/sensing circuit. Apart from the small size (67 × 67 × 25 mm3) and less power consumption (5V DC) and reduced volume of DNA solution, this system could effectively reduce the PCR process time into one‐third of the time required by typical commercial PCR system [19]. In another approach, Steinbach et al. [20] came forward with their K‐Ras mutation detection on chip. Figure 3 shows schematic of the on‐chip detection device. They aimed to develop a fast and reliable chip‐based K‐Ras mutation based on existing microfluidic chip platform for visual signal readout of K‐Ras mutation profiling. Successful hybrid formation was monitored by streptavidin horseradish peroxidase binding, followed by an enzymatic silver deposition. Silver spots represented robust endpoint signals that enabled visual detection and grey value analysis. This study has the potential to replace expensive detection devices. These few examples give a gist of microfluidics in DNA detection and PCR. Many reviews are available on this topic [13, 21, 22].

#### 1.1.1.2. On‐chip biosensing and disposable point‐of‐care devices

Over the past decade, on‐chip diagnostic systems observed explosive growth and showed significant potential for clinical diagnostics specifically for diseases, including toxicity. The early, rapid and sensitive detection of the disease state is the prime objective for every on‐chip clinical diagnosis. Initially, this field was focused on developing the concepts of LOC and later evolved to applications in a number of biochemical analysis operations, such as clinical analysis (blood gas analysis, glucose/lactate analysis, etc.) [23].

In on‐chip diagnosis devices, apart from pregnancy detection kit and glucometer, most applications are based on genes and peptides detection for early indicators of disease [2426]. For instance, Dinh et al describe a multifunctional biochip with nucleic acid and antibody probe receptors specific to the gene fragments of Bacillus anthracis and Escherichia coli, respectively [25]. These devices were based on the detection of specific diseases or biological warfare agents by incorporating biomarkers specific to such agents. Monitoring of regular metabolic parameters, such as glucose and lactate, was demonstrated by the I‐Stat analyzer that provides point‐of‐care testing for monitoring a variety of clinically relevant parameters [26]. Immunosensing applications as a part of clinical diagnostics have also been demonstrated [27, 28].

Recent years have witnessed a vast range of applications of LOC due to the significant benefits of small sample and reagent volume utilization, economic and rapid analysis with less wastage and possibility of developing disposable devices. Ahn et al. demonstrated a fully integrated module of wristwatch-sized analyzer that included a smart passive microfluidic manipulation system based on the structurally programmable microfluidic system (sPROMs) technology, for preprogrammed sets of microfluidic sequencing with an on‐chip pressure source for fluid driving, sequencing and biochemical sensors [23]. Point‐of‐care testing (POCT) is one of the most impressive developments of microfluidics in life sciences and can be defined as diagnostic testing at or near the site of patient care to make the test convenient and immediate. In many countries, DNA test kits for HIV are already available [29]. This is a rapidly growing field, and more detailed information can be obtained from various reviews in this area [23, 31, 32].

#### 1.1.1.3. Drug delivery applications

The major objective of drug delivery systems is to localize the pharmacological activity of the drug at the site of action as targeted drug delivery systems directly deliver the payload to the desired site of action with minimum interaction with normal cells. This phenomenon is especially important for anticancer drugs, as their toxicity to healthy cells is a cause of concern to improve therapeutic response and patient compliance. Last decade witnessed tremendous growth in targeted dosage forms for controlled release [3335].

Approximately 10 million people suffer from different kinds of cancer per year and many of them unfortunately die due to lack of better treatment strategies. With the advancement in diagnostic, therapy techniques and nanomedicine, now better understanding of disease onset and treatment is possible, but still more will be offered by state‐of‐art microfluidic technology in terms of control over particle size, composition, encapsulation rate and better performance of nanoformulations, which have a great impact on the cancer survival rate.

In the series of microfluidics‐based delivery systems, a gas‐filled lipospheres was reported by Hettiarachchi et al. for targeted delivery of doxorubicin, using polydimethylsiloxane (PDMS)‐based microfluidic chip that contained two distinct hydrodynamic flow‐focusing regions for local administration into tumor tissues as shown in Figure 4a [36]. Generally, liposomal‐encapsulated doxorubicin suffers from relatively nonspecific biodistribution due to size selection and nontargeted accumulation [37]. As a solution, Hettiarachchi et al. prepared multilayer lipospheres with oil layer of triacetin (capable of carrying bioactive molecule) sandwiched between inner gas‐filled core and outer lipid layer (polyethylene glycol (PEG) lipid conjugate DSPE‐PEG2000‐Biotin) with avidin as targeting moieties based on the fact that multilayer gas‐filled lipospheres for high payload delivery at target sites could overcome the limitations of liposomal preparation. Figure 4b is representation of the modified delivery system.

Another strategy that is gaining importance in diagnosis and treatment of cancer is theranostic nanomedicine that combines imaging, diagnostic agent and antitumor agent. Theranostic lipid complex nanoparticles formed by bulk mixing do not give control over composition and size which can be overcome with a microfluidic setup [37, 38]. A static micromixer‐coaxial electrospray (MCE) for the single‐step synthesis of theranostic‐lipid complex nanoparticles (cationic lipid‐nucleic acid complexes called lipoplexes) was designed by Wu et al. to overcome this limitation. Multicriteria evaluation (MCE) technique produced monodispersed particles with a diameter of ∼194 nm and high encapsulation efficiency compared to a more conventional bulk process; the advantage of this process is shown in Figure 5a. Quantum dots (QD605) and Cy5‐labeled antisense oligodeoxynucleotides (Cy5‐G3139) were encapsulated as the model imaging reagent and therapeutic drug, respectively, with successful cytoplasm to delivery of drug into cytoplasm of A549 cells (nonsmall‐cell lung cancer cell line) leading to 48 ± 6% down regulation of the Bcl‐2 gene expression [37].

A microfluidic gradient generator (MGG) was developed by Abhyankar et al. [39, 40] for testing drug response on a cellular basis. These devices offered unique features of, higher resolution, real‐time observation, tunable drug concentration and reduced costs in comparison with their conventional counterparts, Transwell and Dunn chambers. MGGs are based on two techniques—gradient achievement through time‐evolving diffusion or parallel streams mixing. Figure 5b shows a sink‐source flow‐free gradient generator. The absence of convection flow is the key advantage of this system that eliminates the shear‐stress induced to cells.

Apart from nano‐based drug delivery techniques, administration of drug to the whole body is another application of microfluidics where miniaturized needles can be designed (microneedles) for improved delivery effectiveness and reduce the pain related to drug administering.

Microneedles can be classified into the following four general types: (i) solid microneedles, (ii) drug coated, (iii) polymeric microneedles with encapsulate drug that fully dissolve in the skin and (iv) hollow microneedles for drug infusion into skin.

#### 1.1.1.4. Microarrays technologies

A microarray is an analytical device that comprises an array of molecules (oligonucleotides, cDNAs, clones, PCR products, polypeptides, antibodies and others) or tissue sections immobilized at discrete ordered [41]. In a general microarray device, sample solutions are confined in microfabricated channels and flow through the probe microarray area. Enhanced sensitivity is obtained due to high surface‐to‐volume ratio in microchannels of nanoliter volume and advantages of both fields can be exploited simultaneously by combining DNA microarray with microfluidics [42, 43]. Consumption of small volumes in microfluidic systems is an added advantage to develop low‐cost, compact and portable LOC systems. Secondly, the surface hybridization of target DNA can also be accelerated on microfluidics platform by electrokinetic delivery of negative charged DNA molecules on to the probe area [44].

Lee et al. proposed a recirculating microfluidic device for the hybridization of oligonucleotides to DNA microarray [45]. Peristaltic pump was connected to the both ends of the microchamber to generate circulatory flow as shown in Figure 6a. With this device, hybridization time was also shortened to 2 h and sample volume was 100 μL.

Many companies are involved in designing microfluidic technology for various high‐throughput applications, such as immunoassays, diagnostic devices, single molecule DNA and protein detection as well [42]. Researchers from the University of Chicago, USA, and other laboratories demonstrated the use of two‐phase droplet systems that generate droplets within microfluidic channels to be used as microreactors for high‐throughput screening of compounds and multiple chemical reactions [46, 47]. Recently, Huang et al. presented a microfluidic device integrated with pneumatically controlled microvalves and micropumps for parallel DNA hybridizations to analyze 48 different DNA targets (18‐mer oligonucleotides derived from the Dengue viral genes) simultaneously. A schematic of device is shown in Figure 6b [48].

The commercialization of microarray and microfluidic technologies is evolving very fast as demonstrated by the emergence of many start‐up companies due to its state‐of‐art technology. Affymetrix is an example where they generated a new market based on their GeneChip® technology over a 12‐year period.

#### 1.1.2. Challenges for lab‐on‐chip devices

Apparently, microfluidics devices have the potential to serve different scientific needs of healthcare and biomedical sectors and as we discussed earlier, their several successful applications have already been reported. The major advantages associated with miniaturized systems are faster/more accurate diagnoses; better epidemiological data for disease modeling; vaccine introduction; and utilization of minimally trained healthcare workers and better use of existing therapeutics but still many hurdles are there in broader applications of microfluidics systems.

However, there is always a silver lining and due to vastly increased interest in global health issues, the current funding climate for the development of diagnostics kits is significantly good. Financial support for new and improved diagnostic tools for priority diseases, such as tuberculosis and cancer, is there. The Gates Foundation's Grand Challenges in Global Health initiative is supporting the development of prototypes of a disposable/hand‐held reader system [49]. Thanks to increased attention on the global health issues and the motivation for their better treatment, we are witnessing the beginning of microfluidics diagnostic devices for early detection of these fatal diseases in coming few years.

We started our discussion on the issues of need of miniaturized devices for pharma industry and biomedicine. After a brief overview on impact of existing LOC systems on global health, we discuss how the new emerging cells and OOC techniques will have an everlasting effect on different areas of human health. The latest progress in microfluidics has led to the development of OOC microdevices, which recapitulate the complex structure, microenvironment and physiological functionality of living human organs. The practical implementation of these miniature organ systems is revolutionary for the field of biomedical sciences and will play a pivotal role for drug discovery and will improve our understanding for mode of action of molecules of therapeutic potential—overall, this state‐of‐art technology is expected to be a boon for pharma and healthcare sector.

## 2. Evolution of cells and organ on chip: from 3D culture to organ on chip

The process of growing eukaryotic cells in vitro was put forth by Harrison in 1907 to investigate the origin of nerve fibers [50] and since then its almost 100 years, these 2D cell cultures have greatly advanced our knowledge of cellular biology. They have been routinely and diligently undertaken in thousands of laboratories worldwide. However, the 2D cell cultures are arguably primitive and do not reflect the anatomy or physiology of a cell or tissue microenvironment in true sense. Two‐dimensional (2D) cell cultures oversimplify the extracellular matrix (ECM) and cell microenvironment and the processes, such as drug delivery, toxicological analysis, gene expression and apoptosis, may not be directly taken up for the in vivo experiments from 2D analysis as ECM is completely different in in vitro and in vivo and cannot be adequately mimicked by 2D cell systems [51, 52]. These limitations of 2D cell culture led to the innovation of 3D cell culture methodologies; the concept that gave birth to the idea of OOC devices. In 3D culture, cells are grown in extracellular matrix, that is, hydrogels, scaffolds or on hanging drops. The cells, growing in third dimension, exhibit enhanced expression of differentiated functions and improved tissue organization but require a multidisciplinary approach and expertise [53, 54].

Generally, spheroids, cell aggregates and cell sheets are the common platforms for 3D culturing [5560]. Basic objectives for developing 3D cell culture systems vary from engineering tissues for clinical delivery to the development of models for drug screening. It was observed that certain cellular processes of differentiation and morphogenesis for tissue engineering occurred preferentially in 3D instead of 2D.

In one study by Slamon et al., alteration of cellular architecture between 2D and 3D cells was observed in the growth of SKBR‐3 cells that overexpress HER2, an oncogene found to be overexpressed in approximately 25% of breast tumors [61]. Cells grown as 3D spheroids using p‐HEMA‐coated plates had HER2 homodimers form, while in 2D cultures, HER2 formed heterodimers with HER3 [61]. Recently, Choi et al. [62] also reported that human neural stem cells with familial Alzheimer’s disease mutations when grown in 3D culture recapitulate both amyloid‐β plaques and neurofibrillary tangles. 3D cell culture more accurately simulates normal cell morphology, proliferation, differentiation and migrations. Similarly, in chemotherapy procedures, a difference in sensitivity to drug exposure was observed in cells grown in 2D or 3D microenvironments [63]. A study by Tung et al. indicated that A431.H9 cells grown in 2D and 3D show differences in viability when treated with the same concentrations of 5‐fluorouracil (5‐FU) and tirapazamine (TPZ). In the case of 5‐FU, 2D cultures were reduced to approximately 5% viability following a 96‐h treatment (5‐FU; 10 mM), whereas 3D cells treated with the same concentration and duration, showed 75% viability; indicating that these 3D spheroids were more resistant to the antiproliferative effects of 5‐FU [64].

In recent years, an increasing shift in research focus from 2D cells cultures to 3D cell cultures occurred which in turn translated 2D in vitro research to 3D in vivo animal models.

### 2.1. Advantages and limitations of 3D cell culture

• Flexible synthesis approach in 3D cell culture allows facile manipulations for cellular microenvironment modeling.

• With 3D cell culture systems, study at different states of disease models can be done in a similar tissue microenvironment that may reduce the need of animal testing.

• 3D culturing is more authentic way of monitoring drug metabolism studies instead of 2D. Due to the presence of layers of cells in 3D culture with tightly bind cells as compare to a monolayer in 2D, drug diffusion to cells by blocking or slowing simulate the real barriers for drug action.

• Scaffolds to support 3D cell with simultaneous growth factor, drug or gene delivery can also be synthesized.

• 3D cell culture has direct applications in tissue engineering and regenerative medicine.

Figure 7 is schematic of various methods of synthesis of 3D culture, including hanging drop, forced floating method, etc.

It is an evolving field and requires further research for its optimization, and therefore, it is evident that some clarity is needed in selecting the best method for the generation of 3D cells from individual cell lines. Additionally, the best established 3D culture methods currently available produce avascular tumor models that failed to mimic the full architecture of in vivo tissues and vascularization aspect of tumor development is left out, which is a huge significant part of true tumorigenesis. These limitations are the prime hurdles in the application of 3D culture as potential drug discovery tools.

### 2.2. From 3D culture toorgans on chips: a giant leap toward biomedicine revolution

In previous section, we discussed the role of 3D cell culture and its significant impact on different fields. The next important step of 3D microfabrication is evolution of integrated OOC microsystems with the ability to mimic key structural, functional, biochemical and mechanical features as well as interactional effect of microenvironment on cell and tissues in vivo of living organs in a single device [65]. By definition, OOC devices are microfluidic devices for culturing living cells in continuously perfused, micrometersized chambers in order to model physiological functions of tissues and organs [66].

Cellular behavior and its interaction with in vivo microenvironment is still an unsolved mystery. Advancements in the field of 3D OOC opened entirely new possibilities to create in vitro models that reconstitute more complex, 3D, organ‐level structures, with integrated chemical signals and important dynamic mechanical cues. OOC devices not only mimic the cells biomechanical and biochemical behavior in in vivo tissue but also predict the interactional effects of microenvironment on cells and tissue functions [58]. This unique ability of OOC devices makes them a potential candidate for drug discovery programs and a boon for healthcare segment. Though this state‐of‐art innovation is in its nascent state, preliminary data obtained had shown promising future of OOC devices with wide applications in biomedical sciences. As a proof of concept, researchers have fabricated two stacked PDMS cell culture chambers separated by permeable synthetic membrane to study polarized functions of various epithelial cells of intestine [67, 68], lung [69], kidney [70], heart [71], etc.

### 2.3. Basic microfabrication techniques and material for OOC devices

To mimic in vivo organ‐specific microenvironment, OOC devices required high precision and accuracy. Microfabrication techniques are the preferred methodologies to fabricate OOC devices due to feasibility of constructing tissue‐specific environment at microscale. Typical techniques include replica modeling, soft lithography and microcontact printing [52, 66, 72]. Figure 8 is a schematic representation of these techniques.

Replica molding techniques have been used to replicate complex surface relief patterns to produce biomimetic structures that mimic organ‐specific microarchitecture. Lee et al. designed the replica modeling techniques to recreate the artificial liver sinusoid and natural endothelial barrier layer in liver. [73] This was an important breakthrough that successfully reconstituted a tissue‐tissue interface that was a critical element of whole liver organ structure, and was not possible in conventional 3D ECM gel cultures. In other report by Esch et al [74], photolithography was explored to recreate the key aspects of villi structure on microfluidic chambers covered by 3D shaped, porous membranes for models of the gastrointestinal tract epithelium by two‐exposure step fabrication process. As shown in Figure 9, complete crosslinking was used to fabricate the chamber and partial with SU‐8 to form the porous membrane. This microdevice could create better in vitro models of human barrier tissues, such as the gastrointestinal tract epithelium, the lung epithelium or other barrier tissues with multiorgan “body‐on‐a‐chip” devices for drug‐screening application.

An array of PDMS microchambers interconnected by 1 μm wide channels was similarly used to enable growth and in vivo‐like reorganization of osteocytes in a 3D environment that replicated the lacuna‐canalicular network of bone [76]. In a similar approach, Sudo et al. came up with the idea of a microdevice incorporating ECM gels microinjected between two parallel microchannels to investigate vascularization of liver tissues in 3D culture microenvironments [76], while a compartmentalized microfluidic system for coculturing of neurons and oligodendrocytes to study neuron‐glia communication during development of the central nervous system was developed by Park et al. [77]

From their inception, production of these microdevices relied on silicon microfabrication and micromachining techniques. Although widely explored and applied, silicon micromachining is rather complex, costly with limited accessibility to specialized engineers. To overcome these practical hurdles, researchers developed microfluidic systems made of the silicone rubber, poly(dimethylsiloxane) (PDMS), that are less expensive and easier to fabricate, which opened entirely new avenues of exploration in cell biology. [6]

PDMS has several unique properties that make it a perfect choice for the fabrication of microdevices for the culture of cells and tissues. First, PDMS possesses superior gas permeability and flexibility for adequate oxygen supply to cells in microchannels, which eliminates the need for separate oxygenators, commonly required in silicon, glass and plastic device and is particularly important to maintain differentiated function of primary cells of high metabolic demand [54, 78]. PDMS microfluidic systems enabled the formation of viable and functional human tissues.

Excellent optical transparency is prime advantage of PDMS that enabled real‐time monitoring of nitric oxide production and variation in pulmonary vascular resistance in a microfluidic model and cell morphology, tissue repair and reorganization. [7981]

Moreover, control of cellular parameters is another important phenomenon in designing OOC devices and recent advances in microfabrication techniques have significant contribution toward efficient monitoring and control of cellular responses and study of broad array of physiological factors that wasn't possible with 3D static cultures. Electrical, chemical, mechanical and optical probes for direct visualization and quantitative analysis of cellular biochemistry, gene expression, structure and mechanical responses also can be integrated into virtually any microfabricated cell culture devices and more relevant data can be obtained with these advanced OOC devices. [54, 66]

## 3. Organ‐on‐chip devices: concept to application

In this section, various state‐of‐art existing OOC platforms and their structural features, working principles, potential and feasibility for biomedical application are discussed. OOC devices can be defined as microfluidics systems for living cells culturing in continuously perfused, micrometersized chambers in order to model physiological functions of tissues and organs [66]. The prime objective of this emerging technique is to fabricate minimal functional units of an organ that recapitulate tissue‐ and organ‐level interactions. These devices have great potential for investigating basic mechanisms of organ physiology and are well suited for the study of biological phenomena that depends on tissue microarchitecture and perfusion and last for relatively short span (< I month). These chips often consist of featuring multiple, controllable parallel channels, splitting and merging channels, various pumps, valves and integrated electrical and biochemical sensors. Some kind of microenvironment stimuli derived from organ‐level functions can be applied to cells from certain organ.

### 3.1. Basic working mechanism of OOC devices

OOC systems are basically elaborated microengineered physiological systems that reconstitute the key features of specific human tissues and organs and their interactions as depicted in Figure 10 [82, 83].

Key factors in OOC designing include the following:

• Fabrication of OOC devices start with identifying the key aspects of biochemical, mechanical environment of specific organ, including local factors from neighboring cells or tissues and stretch of organ. [82].

The next step is application of microengineering fundamentals to introduce the key aspects into a working organ‐specific model.

Now, the cells are introduced into the model under designed stimuli and they self‐organize themselves based on the stimuli and display comparatively more realistic function then the conventional 2D in vitro models.

• The final step is to measure the functional output parameters of the cultured cells.

Earlier, with 2D and 3D cell cultures, efforts were taken to control and regulate the cell growth, shape and other cellular events but due to lack of precise 3D environment, these models suffered with inaccuracy and reliability in recapitulating the issue‐ and organ‐specific systems [83]. But with the state‐of‐art OOC technology, new possibilities to create efficient in vitro models with organ‐specific microenvironments, tissue microarchitecture reconstruction, spatio‐temporalchemical gradients, tissue‐specific interfaces, crucial dynamic mechanical cues and biochemical signals [54, 84]. In this section, we describe recent progress in this field and currently reported OOC devices such as liver, kidney, intestine, kidney, heart, skin and blood vessels.

OOC devices can be classified into three broad segments based on the working mechanisms: [82]

1. Membrane‐based penetration and mechanical stimuli—blood‐brain barrier, lung, kidney, gut, heart on chip.

2. Organ function mimicking based on anatomy—arteries and spleen on chip.

3. Perfusion‐based OOC devices—liver, brain and womb on chip.

### 3.2. Membrane‐based organ on‐chip devices

To study the drug response with respect to human biological barriers is a crucial step in drug discovery. Researchers developed 3D compartmentalization with membrane‐based multilayer compartments for mimicking biological barriers such as the blood‐brain barrier [85, 86, 99], the kidney transport barrier [87, 71], and the lung's alveolar‐capillary interface [88, 89] that can be considered a major breakthrough for biomedicine. In this segment, recent discoveries in membrane and muscular thin films to recapitulate the physiochemical interface and mechanical cues are described.

#### 3.2.1. Lung on a chip

Lung is an important organ of respiratory system for the exchange of oxygen and carbon dioxide in blood stream. The elementary tissue unit of the lung is the layer of epithelial and endothelial cells over which the exchange of gases between air and blood takes place. The geometry of the lung tissue contains the epithelial‐endothelial interface, epithelium‐air interface, endothelium‐blood interface and periodic mechanical force with each respiratory cycle. Understanding of cell‐cell interactions, cell‐blood and cell‐gas flow is utmost necessary for drug discoveries and physiochemical research. Complex geometric and compositional structure of lung is the great barrier to enable straightforward manipulation and observation of cells.

Lung‐on‐chip is the microreplica of the lung on a microchip. This is used for nanotoxicology studies of various nanoparticles that are introduced into the air channels and to understand the pulmonary diseases where due to the formation of liquid plug that blocks small airways and obstruct gas flow in alveoli [89]. To understand the mechanism of liquid plug propagation and rupture, Huh et al. designed a microengineered system that consists of two PDMS chambers separated by thin polyester membrane with 400‐nm pores. This system mimicked an in vivo basement membrane for small airway epithelial cells (SAECs) attachment and growth.

Using this system, injurious response of SAECs to propagation and rupture of finite liquid plugs at an air‐liquid interface afflicted with surfactant deficiency was demonstrated [88]. Another report by Huh et al. designed an alveoli‐on‐chip having alveolar and the capillary interface. To mimic the breathing pattern, two chambers were constructed at the side through which air is pumped in at certain required pressure, continuous increase and decrease of the flow is done in order to accomplish the inhalation and exhalation pattern. A thin flexible layer of PDMS was used in the central chamber where coculturing of human alveolar epithelial cells and blood vessel wall cells on the opposite sides is done. The membrane stretches and relaxes according to the flow of air. The culture medium is pumped through the lower microchannel to mimic the blood flow and the sample is injected on the top layer that interacts with the alveolar epithelial cells as shown in Figure 11 [70]. In another model to study alveolar cell complexities, Douville et al. put forth their system consisting of two compartments—alveolar chamber and actunation channel. These chambers were separated by a PDMS thin membrane to create both cyclic stretch and fluid mechanical stresses. This in vitro model successfully demonstrated the difference in morphological changes cells undergo when exposed to combine stresses as compared to cells exposed solely to cyclic stretch [90].

#### 4.1.2. Lack of exact simulation of human systems in static 2D cells culture

The lack of preclinical model systems to provide accurate predictions of human responses to novel therapeutic drugs is another critical limiting factor in drug discovery. The current gold standard for laboratory‐based preclinical evaluation is based on in vitro cell culture assay and in vivo animal model experimentation and assessment. Although cell culture assays have advantage of controlled environments where cellular maturation and activity are easily observed and tested, they lack the complexity of living systems and are incapable of mimicking the conditions of organ‐organ or tissue‐tissue communication. This simplicity is a major drawback in drug‐development studies since drug metabolism and the effect of metabolite activity on nontarget tissues cannot be predicted [3].

#### 4.1.3. Time period of animal studies and loss of numerous animal lives

Another crucial limiting factor is time involved in in vivo studies. Although animal studies can somehow better predict the drug metabolism and response as animal models maintain the intricacy of living systems and assessment of organ‐organ crosstalk and nontarget organ toxicity is possible , these models on multiple occasions, been proven to be wrong predictors of human responses to drug treatment. Human system is more complex and developed than laboratory animals and the response and mechanisms are different for many therapeutic agents. The hypothesis that favorable outcomes observed in animals will translate to human patients has led to clinical situations where treatments have proved futile or even detrimental to patient well‐being and recovery [3, 144].

#### 4.1.4. Lack of accurate prediction of clinical response and diminished number of new drugs for patients

As discussed earlier, due to inadequate in vitro data and practical difficulties of in vivo studies, the clinical response is not always as expected. Eroom's law (Moore's law backwards) states that “the number of new medicines halves every nine years,” despite an “astronomical” increase in research funding from government and industry. This situation exists in large part because the traditional journey from drug discovery to drug development still occurs mostly in 2D static cell cultures and animal studies, which are not the true predictors of response of new compounds in the human body resulting in failure of approximately 85% of therapies in clinical trials and of those that make it to advanced phase III, generally the last step before regulatory approval, only half are actually approved. This data itself ignite are concerns for the pharma industry and how to expedite the current drug discovery scenario [149].

Microengineered cell culture systems that mimic complex organ physiology have the potential to be used for the development of in vitro human‐relevant disease models. These are more predictive of drug efficacy and toxicity in patients and can provide better insight into drug mechanism of action. OOC devices provide compelling advantages over other in vitro cell culture models for the evaluation of drug safety and metabolism. In broader sense, in vitro assays incorporating cultured human cells can act as savior in identifying environmental toxins and providing better understanding of their mechanisms of action, as well as improving our ability to predict risks for specific compounds. In addition, the ability to integrate functional organ mimetics, such as gut, liver, lung and skin‐on‐chips within a “human‐on‐a‐chip,” the interplay of different organs in determining pharmacokinetic properties of compounds can be monitored [3, 145].

### 4.2. Role of organ‐on‐chip devices in drug discovery

#### 4.2.1. Reduction in cost

The drug‐development process is costly in the phases of clinical trials, which can cost millions of dollars. However, despite extensive animal testing of drugs before starting a clinical trial with humans, many drugs fail because of low efficacy or unexpected toxic side effects not predicted with earlier trials. In this regard, the most promising advantage of body‐on‐a‐chip devices is that the devices can mimic both animal and human metabolism and predict differences between them that will allow for a higher level of accuracy when predicting the outcome of clinical trials. Moreover, any toxicity observed before human trial with in vitro on chip systems can prevent unsuitable drug candidates from entering the expensive phase of clinical trials that limit costs and unrealistic expectations.

Body‐on‐a‐chip devices are low‐cost platforms that can substantially reduce the cost of drug testing.

#### 4.2.2. Drug‐target identification

Organs‐on‐chips have the potential to serve as a new enabling platform to identify and validate the effectiveness, safety of potential targets early in the pipeline to increase the likelihood of success in clinical trials [4]. Song et al has recently a microengineered model of vasculature to mechanistically examine chemokine‐mediated interactions between circulating breast cancer cells and the microvascular endothelium that induced site‐specific basal stimulations and activation of the microfluidic endothelium by introducing chemokines into the lower chambers. Through quantitative analysis of cancer cell attachment to the endothelium and the levels of cell surface receptor expression, this system predicted that endothelial recruitment of breast cancer cells induced by a chemokine‐CXC‐chemokine ligand 12 (CXCL12), involved in cancer metastasis, is mediated by the endothelial receptor CXCR4 and this response is independent of the expression of CXCL12 receptors on circulating cancer cells. These findings gave a new insight into critical role of the vascular endothelium in the metastatic behavior of circulating tumor cells and how to control and manipulate a biological target to analyze a functional outcome of target modulation. This discovery related with OOC model was an important breakthrough in indentifying a valid therapeutic target for preventing cancer metastasis [146].

Other studies on OOC platforms for understanding of molecular mechanisms of cell‐cell interactions, mitochondrial cardiomyopathy of Barth syndrome, and drug‐induced toxicities in pulmonary edema have also been successfully performed [147149].

#### 4.2.3. Toxicity and drug efficacy evaluation

This a very important aspect of drug research as toxicity analysis is utmost important for any new therapeutic agent. Liver and kidney tissues are of great interest to drug developers due to their predominant role during the absorption, distribution, metabolism and excretion (ADME) process of a drug [3]. Physiologically, drug is metabolized mainly in the liver while kidney deals with their elimination. These two critical processes make these two organs highly susceptible to drug injury. In a coculture bio‐analytical microplatform of liver‐kidney, toxicity of anticancer drug ifosfamide illustrated the importance of the liver‐kidney interaction. Ifosfamide is a prodrug, activated in body system by CYP450 enzymes in the liver, but some of its metabolites, such as chloracetaldehyde, are nephrotoxic. With this model of highly differentiated liver cells (HepaRG), perturbation of cell proliferation and calcium release in the kidney tissue could be monitored that was not possible with the single culture. Previously, the same group simulated the performance of hepatocytes on‐chip system coupled with NMR for toxicity analysis of flutamide [149, 150].

These contributions signify the role of on‐chip systems for toxicity analysis of drug in vitro that is an important step for clinical trials.

Multiorgan interactions in drug testing and their importance were highlighted by Sung et al. also. They studied the dose response and efficacy of 5‐fluorouracil (5‐FU) on a system containing system that contained liver cells (HepG2/C3A), colon cancer cells (HCT‐116) and myeloblasts (Kasumi‐1) [151]. They monitored the degradation phenomenon of 5‐Fu and effect of its pro drug Tegafur and uracil‐a competitive inhebitor of 5‐Fu for the dose response and bioavailability.

Predicting the bioavailability of a drug accurately can be difficult with animal models. Multiorgan microdevices that contain a combination of the gastrointestinal tract epithelium and the liver at the appropriate sizes and with realistic liquid‐to‐cell ratios have the potential to predict the bioavailability of ingested drugs [152].

#### 4.2.4. Drug screening

The absence of predicted therapeutic effects of a drug or increased dose levels is the major cause of drug toxicity. The failure of existing methods to accurately predict in vivo drug efficacy before clinical trials give rise to the undesirable outcomes. Human OOC models can become instrumental in addressing these existing imitations [4].

The potential of OOC approaches for testing drug efficacy was recently explored by Aref et al. in a microengineered 3D assay of epithelial‐mesenchymal transition (EMT) during cancer progression [153]. By culturing lung cancer spheroids in a 3D matrix gel adjacent to an endothelialized microchannel, this model recapitulated EMT‐induced tumor dispersion and phenotypic changes in cancer cells in an endothelial cell‐dependent manner. Twelve drugs ranging from prospective drugs to US Food and Drug Administration (FDA)‐approved drugs were screened into the vascular channel, and their ability to inhibit EMT was analyzed by direct visualization of the cancer spheroids.

The results obtained for drugs efficacy in cancer treatment by on‐chip systems, significantly varies from 2D static culture and were in close proximity with human clinical trials. This study concluded that such OOC systems will be developed as a more realistic platform for efficacy and to decide for advanced trails, a major step toward drug discovery.

#### 4.2.5. Response of combination of drugs

Since microdevices are relatively inexpensive, and many such devices will be operated in parallel, it is possible to test many drugs and combinations of drugs at different concentrations with devices. Testing combinations of drugs is useful to monitor drug interactions and cross talks. Synergistic interactions are of particular interest. Another benefit of such studies is that the drugs having similar functions, but different side effects could potentially be combined at reduced dosages to achieve the needed tissue response. These multiorgan on‐chip systems can play a major role to design individualized therapy regimen for patients that do not respond to routinely used drug combinations as a synergistic effect and dose of different drug combination can be predicted.

#### 4.2.6. Pharmacokinetics and body on‐chip systems

Physiologically based pharmacokinetic models (PBPKs) are mathematical models that are used to extrapolate data from animal experiments and predict human response to a drug. These models mainly rely on existing understanding and knowledge of a drug's metabolism from traditional 2D static cultures and animal studies and as we discussed, these methods are not the accurate predictors. This is the reason for the equations used in a PBPK are not complete and the models are not accurate. Multiorgan microdevices can be modeled more precisely with PBPKs and divergence between the model's prediction and experimental data obtained with the devices can enhance our understanding of human response to a wide variety of combination of inputs with higher accuracy than before.

To generate a precise PBPK model, for pharmacokinetics and pharmacodynamics studies, recapitulating human physiology at the whole‐body level is the most crucial aspect. Researchers have begun to pursue the development of multi‐organ models, and in one such study, combined models of breast cancer, the intestine and liver were designed to create a network of interconnected microfabricated cell culture chambers that exhibited the sequential absorption, metabolism and efficacy of four anticancer drugs [154]. Shuler et al [155] applied pharmacokinetic and pharmacodynamic modeling (PKPD) principles to micro cell culture analog comprising interconnected microchambers representing a colon tumor, the liver and bone marrow, which imitated the in vivo distribution, retention and recirculation of drug‐containing blood in these organs. Hepatic metabolism‐mediated cytotoxicity of the prodrug tegafur to colon cancer, liver cancer and bone marrow cells was investigated by this system. These multiorgan on‐chip systems are better than the existing models and can expedite the drug discovery process by increasing the efficiency and mitigating the high cost associated with drug‐development process.

### 4.3. Future prospects of organ‐on‐chip devices

As an alternative to conventional cell culture and animal models, human OOC could transform many areas of basic research and drug development. They have wide applications in research on molecular mechanisms of organ development and disease, organ‐organ coupling and the interactions of the body with stimuli, such as drugs, environmental agents, consumer, products and medical devices. Due to complexities involved, OOC have limited or no applications in certain areas of biomedical research, such as chronic diseases, adaptive immune responses or complex system‐level behaviors of the endocrine, skeletal and nervous systems. As described previously, OOC are effective for investigating physiological and disease processes that occur in a relatively short‐time frame (less than ∼1 month) and depend on relative cell positions within an organ‐ or tissue‐specific microarchitecture [66].

OOC technology has certain technical and entrepreneurial challenges also. One of the critical technical challenges is material for fabrication—such as poly(dimethylsiloxane) (PDMS) that have gained widespread use in rapid‐prototyping of OOC microdevices as most of the OOC models rely mostly on synthetic materials (e.g. PDMS, polycarbonate and polyester), the physicochemical properties of which are not appropriate for mimicking extracellular matrices in vivo. It is utmost important to identify new cell culture substrates to produce devices for more accurate predictions. For successful translation of OOC from proof of concept in the laboratory to commercial screening platforms, identification and optimization of new low‐cost materials and fabrication strategies suitable for their mass production and integration into existing infrastructures in the pharmaceutical industry is call of time.

More reliable and sustainable sources of human cells, especially disease‐specific cells that are acquiescent to in vitro culture in OOC and phenotypically are true representative of their in vivo counterparts are required. To overcome this hurdle, human embryonic stem cells and iPS cells can be engineered to suit specific needs in the development of OOC [3, 156]. The OOC models with stem cells can generate and control physiologically relevant structural, biochemical and mechanical cues required for stem‐cell differentiation and maturation.

With the new avenues opened by OOC in drug development, there is a need of fabricating human on‐chip or multiorgan on‐chip devices and to maintain a balance between the complexity and practicality will play an important role in their wide applications. With the improvement in physiological relevance, complexity in the model is obvious that presents major challenges to practical operation and management of the system. Accurate identification of minimal subset of cells and microenvironmental factors will be helpful to create a balance and designing a simplest model possible that recapitulates physiological responses of interest.

Integration of laboratory on‐chip platforms with miniaturized analytical systems is also important for better detection sensitivity despite of low culture volumes and cell numbers [1].

OOCs are not universal solutions, and alternative tools will continue to be better solutions for modeling certain in vivo processes as animal offer whole‐organism toxicity testing and this parallel analysis will be required until the current OOC scenario attains the maturity and refine human on‐chip systems come into existence.

Despite their limitations, OOCs have the potential to play a transformative role across drug discovery and development. Eventually, OOC models may play a pivotal role in streamlining the clinical trial process. Due to the complexities of organ function and regulatory requirements, it is unlikely that OOCs will replace animal testing anytime soon [66].

However, with the scientific advancements, this field is evolving at a fast pace and these hurdles could be surmountable with tri‐lateral partnerships between academic institutions, industry and regulatory agencies. The paradigm‐shifting potential of OOC technology has been recognized by funding agencies integrated microphysiological systems [157, 158]. Pharmaceutical companies are also coming forward to establish industry‐ academia partnerships to jointly explore this emerging research arena and to establish themselves at the forefront of expected OOC advances. In nut shell, it is concluded that despite of several limitations, achievements in this revolutionary field of biomedicine, OOC technology present exciting new avenues for drug discovery and development and a perfect picture of a promising future.

## Acknowledgments

Author is grateful to Department of Science and Technology, Government of India for providing INSPIRE Faculty award to pursue independent research.

## How to cite and reference

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Preeti Nigam Joshi (June 29th 2016). Cells and Organs on Chip—A Revolutionary Platform for Biomedicine, Lab-on-a-Chip Fabrication and Application, Margarita Stoytcheva and Roumen Zlatev, IntechOpen, DOI: 10.5772/64102. Available from:

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