**4. Pharmaceutical applications and future prospects of organ‐on‐chip devices**

The field of OOC devices is still in its infancy, although it is a rapidly growing research arena with lots of future potential in biomedicine from understanding the mechanism of complex organ architectures to drug discovery. Earlier studies revealed that while 3D cell cultures were far more superior planar than conventional 2D models due to their better control over cell differentiation, ECM mechanical compliance and a much better response was obtained in terms of tissue‐ and organ‐level functionality by combining microengineering with cell biology. Fortunately, with the recent advances in microfabrication strategies and microfluidics, precise dynamic control of structure, mechanics and chemical delivery at the cellular size scale can be achieved. Microengineered 3D cell culture models, and particularly more sophisticated OOC microdevices, have many potential applications, including disease research and drug discovery, but in this section, we mainly focus on OOC application relevant to pharmaceutical industry.

The pharmaceutical industry is under intense pressure economically, ethically and scientifi‐ cally to find ways to accelerate the drug‐development process, and to develop drugs that are safer and more effective in humans at a lower cost. Traditional animal testing approaches are expensive and often fail to predict human toxicity or efficacy of drugs; in fact, nowadays, questions are arising with regard to the significance of animals testing if they cannot reliably predict clinical outcomes [4, 143]. As correctly suggested by Dr. Ingber, Founder Director‐ Harvard's Wyss Institute for Biologically Inspired Engineering that chips respond to drugs like human organs do—–and have the potential to replace animal testing for safety and efficacy early in the drug‐development process.

#### **4.1. Bottlenecks in drug discovery process**

#### *4.1.1. High cost of compound testing*

Although complete functional body‐on‐chip devices are still far from reach but the latest development in this field has given a glimpse of promising future of this revolutionary field

**Figure 22.** Schematic of a body on chip system. (a) A microdevice containing interconnected cell culture microcham‐ bers integrated with microfluidic culture of intestinal epithelial, hepatocytes and breast cancer cells. (b) A micro cell

**4. Pharmaceutical applications and future prospects of organ‐on‐chip**

The field of OOC devices is still in its infancy, although it is a rapidly growing research arena with lots of future potential in biomedicine from understanding the mechanism of complex organ architectures to drug discovery. Earlier studies revealed that while 3D cell cultures were far more superior planar than conventional 2D models due to their better control over cell differentiation, ECM mechanical compliance and a much better response was obtained in terms of tissue‐ and organ‐level functionality by combining microengineering with cell biology. Fortunately, with the recent advances in microfabrication strategies and microfluidics, precise dynamic control of structure, mechanics and chemical delivery at the cellular size scale can be achieved. Microengineered 3D cell culture models, and particularly more sophisticated OOC microdevices, have many potential applications, including disease research and drug

culture analog (μCCA) representing a colon tumor, the bone marrow and liver [4].

**devices**

of biomedicine. **Figure 22** shows the concept of body‐on‐chip microsystem [4].

108 Lab-on-a-Chip Fabrication and Application

Modern drug development requires implementation of extensive preclinical testing and validation protocols before getting the formal approval to progress to clinical evaluation of the compound. This process is tedious and costly and a single compound can cost more than \$2 million. Moreover, every 10 drugs entering clinical trials, generally only one or two would be licensed for eventual use in humans [2].

#### *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*

patients has led to clinical situations where treatments have proved futile or even detrimental

*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

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

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

to patient well‐being and recovery [3, 144].

110 Lab-on-a-Chip Fabrication and Application

monitored [3, 145].

*4.2.1. Reduction in cost*

testing.

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

clinical trials that limit costs and unrealistic expectations.

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 cham‐ bers. 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 [147–149].

#### *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 con‐ taining 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 combi‐ nation 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 combina‐ tion 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. Research‐ ers 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 absorp‐ tion, 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**

of its pro drug Tegafur and uracil‐a competitive inhebitor of 5‐Fu for the dose response and

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

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

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

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

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 combi‐

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

and to decide for advanced trails, a major step toward drug discovery.

bioavailability.

112 Lab-on-a-Chip Fabrication and Application

*4.2.4. Drug screening*

to predict the bioavailability of ingested drugs [152].

instrumental in addressing these existing imitations [4].

direct visualization of the cancer spheroids.

*4.2.5. Response of combination of drugs*

*4.2.6. Pharmacokinetics and body on‐chip systems*

nation can be predicted.

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, biochem‐ ical 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 com‐ plexity 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 require‐ ments, 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 partner‐ ships 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.
