Organ-on-a-chip: Engineered Microenvironments for Safety and Efficacy Testing

By Julia Hoeng and Manuel C. Peitsch

Organ-on-a-Chip: Engineered Microenvironments for Safety and Efficacy Testing contains chapters from world-leading researchers in the field of organ on a chip development and applications, with perspectives from life sciences, medicine, physiology and engineering. The book contains an overview of the field, with sections covering the major organ systems and currently available technologies, platforms and methods. As readers may also be interested in creating biochips, materials and engineering best practice, these topics are also described.

Users will learn about the limitations of 2D in-vitro models and the available 3D in-vitro models (what benefits they offer and some examples). Finally, the MOC section shows how the organ on a chip technology can be adapted to improve the physiology of in-vitro models.

• Includes case studies of other organs on a chip that have been developed and successfully used
• Provides insights into functional microphysiological organ on a chip platforms for toxicity and efficacy testing, along with opportunities for translational medicine
• Presented fields (PK/PD, physiology, medicine, safety) are given a definition followed by the challenges and potential of organs on a chip

• Language: English
• Publisher: Academic Press
• Release date: Nov 9, 2019
• ISBN: 9780128172032

Book preview

Organ-on-a-Chip

Engineered Microenvironments for Safety and Efficacy Testing

Edited by

Julia Hoeng

PMI R&D, Philip Morris Products S.A., Neuchâtel, Switzerland

David Bovard

PMI R&D, Philip Morris Products S.A., Neuchâtel, Switzerland

Manuel C. Peitsch

PMI R&D, Philip Morris Products S.A., Neuchâtel, Switzerland

Table of Contents

Cover image

Title page

Copyright

List of contributors

Preface

Chapter 1. Need for alternative testing methods and opportunities for organ-on-a-chip systems

Abstract

Introduction

Why we need alternative and improved methods

Requirements for in vitro alternatives to animal testing

Remaining technical challenges

Conclusions

Outlook: stimulating the adoption of new technologies to replace animal testing

References

Chapter 2. Cell sources and methods for producing organotypic in vitro human tissue models

Abstract

Introduction

Human tissue/cell sources and isolation methods

Methods for producing three-dimensional organotypic tissue cultures

Summary/Outlook

References

Further reading

Chapter 3. Organs-on-a-chip engineering

Abstracts

Introduction

Microengineering

Engineering fluid control for organ-on-chips

Stimulation and sensing

References

Part I: Organ-on-a-chip platforms to model disease pathogenesis

Chapter 4. Lung-on-a-chip platforms for modeling disease pathogenesis

Abstract

Introduction

In vitro and in vivo models of respiratory disease pathogenesis

Current lung-on-a-chip systems

Organ-on-a-chip systems for modeling pathological conditions

Improvements needed in lung-on-a-chip platforms for disease modeling and lung regeneration

Conclusions

Acknowledgments

References

Chapter 5. Requirements for designing organ-on-a-chip platforms to model the pathogenesis of liver disease

Abstract

Introduction

Liver function and structure

Drug-induced liver injury

Liver fibrosis

Organs-on-a-chip

Conclusion

References

Chapter 6. Brain-on-a-chip systems for modeling disease pathogenesis

Abstract

Introduction

State-of-the-art brains-on-chips

Higher-order system-on-a-chip functionality

Manufacturing approaches

Future outlook

References

Chapter 7. Kidney-on-a-chip

Abstract

Introduction

Kidney-on-a-chip models

Kidney-on-a-chip: future perspectives

References

Chapter 8. Heart-on-a-chip

Abstract

Introduction

Anatomy and physiology of the heart

Dimension, location, and envelope

Heart wall

Heart cavities

The cardiac valves

Microscopic anatomy of the heart muscle

Mechanism of cardiac contraction

Physiology of the heart muscle

Spontaneous production of action potential

Excitation and action potential propagation

Heart–nerve connections

Electrocardiography

In vitro models of the heart

Traditional two-dimensional cell culture heart models

3D cell culture models of the heart

Limitations of traditional two-dimensional model and 3D heart models

Organ-on-a-chip models of the heart: a new opportunity to mimic cardiac physiology, pathology, and toxicity

Effects of heart-on-a-chip models on cell differentiation

Final remarks and future directions

Acknowledgments

References

Chapter 9. Gut-on-a-chip microphysiological systems for the recapitulation of the gut microenvironment

Abstract

Introduction

Gut models with 3D structures mimicking intestinal epithelial layer topology

Microfluidics-based gut models for mimicking the dynamic environment

First-pass metabolism models

Gut microbe coculture models

Conclusion

Acknowledgments

References

Chapter 10. Computational pharmacokinetic modeling of organ-on-chip devices and microphysiological systems

Abstract

Introduction

Computational models for designing organ-on-a-chip devices

Models of drug pharmacokinetics in organ-on-a-chip devices

Models of drug pharmacokinetics in microphysiological systems

Models based on in vitro-to-in vivo translation

Conclusions and future perspectives

Acknowledgments

References

Further reading

Chapter 11. Caenorhabditis elegans-on-a-chip: microfluidic platforms for high-resolution imaging and phenotyping

Abstract

Introduction

Caenorhabditis elegans as a whole-animal model in scientific research

The use of microfluidic platforms for Caenorhabditis elegans research

Large-scale microfluidics for phenotyping multiple populations of Caenorhabditis elegans nematodes

Future directions

Acknowledgments

References

Part II: Multi-organs-on-a-chip platforms to mimic humans physiology

Chapter 12. Design and engineering of multiorgan systems

Abstract

Motivation for in vitro multiorgan systems

Scope of the chapter

Concepts of multiorgan systems

Building blocks for multiorgan systems

Discussion

Conclusion

References

Chapter 13. Human body-on-a-chip systems

Abstracts

Introduction

Why we need organismal models on a chip

Design principles

Opportunities

Challenges

Conclusion

References

Chapter 14. Automation and opportunities for industry scale-up of microphysiological systems

Abstract

High-throughput versus high-content systems

History of laboratory automation

The purpose of microphysiological system and their suitability for automation

Automation of device production

Automation of tissue preculture and loading

Automation of system operation

Automation of monitoring and sensing

Summary and outlook

References

Further reading

Chapter 15. How to build your multiorgan-on-a-chip system: a case study

Abstract

Introduction

Selecting the appropriate models and coculture medium

Multi-organ-on-a-chip development project

Testing the multiorgan-on-a-chip system

Conclusion

Acknowledgments

References

Index

Copyright

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List of contributors

Adela Ben-Yakar,     Department of Mechanical Engineering, The University of Texas at Austin, Austin, TX, United States

David Bovard,     PMI R&D, Philip Morris Products S.A., Neuchâtel, Switzerland

Eva-Maria Dehne,     TissUse GmbH, Berlin, Germany

Alessandra Dellaquila

Elvesys Microfluidic Innovation Center, Paris, France

Biomolecular Photonics, Department of Physics, University of Bielefeld, Bielefeld, Germany

Hendrik Erfurth,     TissUse GmbH, Berlin, Germany

Dario Fassini,     Cherry Biotech SAS, Rennes, France

Olivier Frey,     InSphero AG, Schlieren, Switzerland

Pierre Gaudriault,     Cherry Biotech SAS, Rennes, France

Erika Györvary,     Centre Suisse d’Electronique et de Microtechnique SA, Neuchâtel, Switzerland

Alexander P. Haring

Department of Industrial and Systems Engineering, Virginia Polytechnic Institute and State University, Blacksburg, VA, United States

Macromolecules Innovation Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA, United States

Patrick J. Hayden,     MatTek Corporation, Ashland, MA, United States

Sarah Heub,     Centre Suisse d’Electronique et de Microtechnique SA, Neuchâtel, Switzerland

Antoni Homs-Corbera,     Cherry Biotech SAS, Rennes, France

Seiichi Ishida,     Division of Pharmacology, National Institute of Health Sciences, Kawasaki, Japan

Blake N. Johnson

Department of Industrial and Systems Engineering, Virginia Polytechnic Institute and State University, Blacksburg, VA, United States

Macromolecules Innovation Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA, United States

School of Neuroscience, Virginia Polytechnic Institute and State University, Blacksburg, VA, United States

Felix Kurth,     Centre Suisse d’Electronique et de Microtechnique SA, Neuchâtel, Switzerland

Diane Ledroit,     Centre Suisse d’Electronique et de Microtechnique SA, Neuchâtel, Switzerland

Seung Hwan Lee

Department of Bionano Engineering, Hanyang University, Ansan, Republic of Korea

Nanosensor Research Institute, Hanyang University, Ansan, Republic of Korea

Department of Bionanotechnology, Hanyang University, Ansan, Republic of Korea

Sasha Cai Lesher-Pérez,     Elvesys Microfluidic Innovation Center, Paris, France

Frédéric Loizeau,     Centre Suisse d’Electronique et de Microtechnique SA, Neuchâtel, Switzerland

Uwe Marx,     TissUse GmbH, Berlin, Germany

Alexander H. McMillan

Elvesys Microfluidic Innovation Center, Paris, France

Department of Microbial and Molecular Systems, Centre for Surface Chemistry and Catalysis (COK), KU Leuven, Leuven, Belgium

Sudip Mondal,     Department of Mechanical Engineering, The University of Texas at Austin, Austin, TX, United States

Ann-Kristin Muhsmann,     Technische Universität Berlin, Medical Biotechnology, Berlin, Germany

Samantha Paoletti,     Centre Suisse d’Electronique et de Microtechnique SA, Neuchâtel, Switzerland

Andrzej Przekwas,     CFD Research Corporation, Huntsville, AL, United States

Kasper Renggli,     ETH Zürich, Department of Biosystems Science and Engineering, Basel, Switzerland

Vincent Revol,     Centre Suisse d’Electronique et de Microtechnique SA, Neuchâtel, Switzerland

Antonin Sandoz,     PMI R&D, Philip Morris Products S.A., Neuchâtel, Switzerland

Mahadevabharath R. Somayaji,     CFD Research Corporation, Huntsville, AL, United States

Jong Hwan Sung,     Department of Chemical Engineering, Hongik University, Seoul, Republic of Korea

Emma K. Thomée

Elvesys Microfluidic Innovation Center, Paris, France

University of Strasbourg, Strasbourg, France

Marine Verhulsel,     Fluigent SAS, Le Kremlin-Bicêtre, France

Gilles Weder,     Centre Suisse d’Electronique et de Microtechnique SA, Neuchâtel, Switzerland

J. Malcolm Wilkinson,     Technology For Industry Ltd., Chesterfield, United Kingdom

Filippo Zanetti,     PMI R&D, Philip Morris Products S.A., Neuchâtel, Switzerland

Preface

Forward-thinking scientists are developing alternative methods to animal testing that are relevant to human health. These sophisticated tests include not only advanced computer-modeling techniques (often termed in silico models) and advanced methodologies for clinical trials with human volunteers, but also the use of human cells and tissues in vitro. The book focuses on in vitro testing that is revolutionized by a new technology: organs-on-a-chip. The potential of this technology is remarkable as it should enhance drug discovery and development by increasing efficiency, reducing costs, and bringing safer drugs with fewer side effects to the market. The results obtained so far have shown the advantages of these chips, and it is anticipated that their value could increase from $5 million in 2016 to as much as $170 million in 2023.

Organ-on-a-chip platforms have emerged at the interface between engineering and in vitro research. Indeed, advances in the miniaturization of electronic systems, three-dimensional printing, and the development of three-dimensional tissue cultures are key enablers of this new technology. In recent years, it has become possible to cultivate tissues not only in simple plastic plates but also in devices capable of recreating the dynamic environment surrounding cells in vivo. This evolution of in vitro test systems led, in most cases, to improved model lifespan, functionality, and overall resemblance to the in vivo context. These organ-on-a-chip models open the door to advances in personalized medicine, for example, by modeling in vitro the principal tissues of an individual using cells from a specific person ultimately aiming for clinical trials in chips.

Academics and entrepreneurs have shown great interest in the potential and versatility of this technology. The organ-on-a-chip platforms hold considerable promise to more accurately predict adverse and nonadverse effects of new drug candidates. The pharmaceutical industry has also embarked on the development or use of such systems in drug discovery and development. This book was compiled by experts in the field to highlight technology advances, current status, and promises. It was developed as an educational resource for anyone already working with these systems or planning to do so.

The book first discusses the different in vitro models available, with their respective advantages and limitations, followed by detailed discussions of the various technical choices available to scientists when creating organ-on-a-chip systems. As many devices are designed to recreate the environment for a particular organ, several organ-specific chip development chapters were included. One chapter is dedicated to showcasing how the evaluation of drug safety and efficacy has been improved by the use of organ-on-a-chip devices. Another chapter discusses the suitability of the technology to model drug pharmacokinetics and pharmacodynamics. The book also presents in detail several multiorgan-on-a-chip platforms, all aimed at creating a human-on-a-chip model system. To assist researchers wishing to create a multiorgan-on-a-chip system, one chapter describes the steps to create a lung–liver-on-a-chip platform. This chapter reinforces that collaboration between biologists and engineers is essential for the successful development of this research tool. Finally, it is highlighted how microfluidic chips can be used to investigate entire organisms, such as Caenorhabditis elegans, following exposure to chemical compounds.

Chapter 1

Need for alternative testing methods and opportunities for organ-on-a-chip systems

J. Malcolm Wilkinson,    Technology For Industry Ltd., Chesterfield, United Kingdom

Abstract

Two-dimensional (2D) cell culture systems are a poor representation of human physiology and cannot replace animals in biomedical research. Cell–cell crosstalk and signals from mechanical stimuli are missing from 2D static cultures. However, recreating the three-dimensional environment in vitro requires adequate supplies of nutrients and oxygen, which can be provided by perfusing the cells and tissues with enriched media. Multiple microfluidic chambers can then be coupled to enable crosstalk between tissues. Chamber systems built at millimeter scale are termed organ-on-a-chip devices and present practical challenges, such as blockages, air bubbles, and difficulties loading cells. For scientists to embrace the new physiologically relevant culture methods, the devices must be affordable and easy to use, leveraging existing protocols wherever possible. The widespread use will be a prerequisite for the technology to become an effective replacement for animal testing in biological research.

Keywords

Animal replacement; organ-on-a-plate; microphysiological systems; homeostasis; disease model; organoid; perfusion; coculture

Introduction

Two-dimensional (2D) in vitro cell culture systems are a poor representation of human or animal physiology (Kirkpatrick et al., 2007), because they fail to replicate the complexity of the physiological environment in Petri dishes or microplates (Zhang, 2004). Cells are sensitive to their microenvironments, which are rich in molecular signals from the extracellular matrix, other cells, and mechanical stimuli induced by flow, concentration gradients, and movement. These mechanical and biochemical signals are almost completely absent from static cultures in well plates. One method for recreating the three-dimensional (3D) environment is to seed cells at a higher density on scaffolds. However, at this higher cell density, the supply of nutrient and oxygen becomes critical, particularly for culture experiments that last several days. Media flow can be introduced to overcome this limitation but renders the design of the cell culture chamber far more complex to predict and control flow-induced stress. With flow systems, practical issues, such as avoiding leakage and blockages, must also be overcome. Once the flow is introduced, multiple chambers can be coupled to enable the construction of more sophisticated coculture models and studies of crosstalk between various tissues (Mazzei et al., 2010).

The interest in flow and coculture has developed parallelly with the concept of organ-on-a-chip (OOC) devices that incorporate microfluidics. Because of the widespread industrial use of 96 and 384 well plates or microtiter plates, it was considered that a worthwhile goal would be to scale the cell culture chambers to similar small dimensions. Although there are intense research-and-development efforts in this direction, it has proved difficult to translate experimental methods from the millimeter to the micrometer scale because of practical problems such as blockages, air bubbles, and loading cells into microscopic chambers. Since OOC devices do not actually aim to recapitulate a complete organ, an alternative description, microphysiological systems, is coming into use.

For biologists and laboratory technicians to embrace these new, physiologically more relevant culture methods, the transition from current wells and dishes to other tools must be simple and inexpensive. Ideally, the use of existing protocols and equipment should be maximized to allow third-party laboratories or academic laboratories to adopt microscale devices. Some organ-on-a-plate approaches, scaled slightly larger than OOC systems, are being developed by TissUse GmbH in Germany (Dehne et al., 2017) and Kirkstall Ltd. in the United Kingdom (Ahluwalia et al., 2011). Multiple cell types have been successfully cultured in these devices, including hepatocytes (Vinci et al., 2011), Caco-2 gut cells (Ucciferri et al., 2013), adipocytes, and endothelial cells (Vinci et al., 2012). Current work is extending the range of applications and cell types to skin, kidney, respiratory epithelium, and the blood–brain barrier. The companies and laboratories developing smaller scale OOC devices are also making rapid progress in widening the range of cell models used in-house, but with less success in transferring these developments to the third parties.

Why we need alternative and improved methods

The justification for change stems from economic, ethical, and scientific arguments. There is a clear market need for improvements in the drug discovery and development process in the pharmaceutical industry. Although the development of a drug takes, on average, 13.5 years and costs $2.5 billion, 92% of drugs fail in human-clinical trials and never reach the market (Maschmeyer, 2019). Systemic, human cell-based models that better reflect human physiology are therefore urgently needed, and organ-on-a-plate and OOC devices may save hundreds of millions of dollars.

The ethical arguments relate to the use of animals, a large number being sacrificed in experiments. This involves not only discomfort and suffering for the animals but also stress for the human researchers carrying out these experiments. The animal experiments have an enormous economic cost invested (Bottini and Hartung, 2009) in breeding, housing, and disposal burdens.

The scientific arguments arise from the recognition that there are differences between human and animal biology, even for primates. Hence, the findings obtained from animal tests do not translate into the clinic (Pistollato et al., 2014). The years of wasted research also exert an economic cost. Many animals are bred in sterile conditions and neither do they develop an immune response that is necessary to model disease (Landhuis, 2016) nor do they possess a balanced gut microbiome, affecting drug metabolism (Simon et al., 2019).

In vitro testing of the activity (toxicity or efficacy) of chemical compounds needs to accurately predict what will happen in the clinic. Problems arise when a test gives a false positive (toxic effect where the compound is actually safe) or false negative (no adverse reaction detected where the compound is toxic). Since many compounds are safe at low dose but toxic at high dose, the sensitivity of the test is critical. These issues have been reviewed by Proctor et al. (2017) for the particular case of liver toxicity. Few of the in vitro models contain the full complement and functionality of metabolic enzymes and transporters present in human hepatocytes in vivo. 2D cultures of plated primary human hepatocytes rapidly lose liver phenotype and CYP450 activity in traditional monolayer cultures. These factors significantly limit the ability of these platforms to detect metabolite-induced cytotoxicity as well as the effects of the parent drug and its metabolites on bile-acid homeostasis/intrahepatic cholestasis and mitochondrial impairment.

Several improvements in in vitro methods have been identified to yield more physiologically relevant results. These include the transform to 3D cultures, the use of human primary cells, the introduction of flow and mechanical stimulation, and coculturing multiple cell types. 3D in vitro methods are now more widely adopted (Gaskell et al., 2016) and have been shown to be more effective as toxicity screens than simple 2D cultures. Better methods for testing drugs, nutraceuticals, and cosmetics are still needed, however, and the shift to patient-specific medicines and individually tailored therapies will demand new methodologies as well.

Requirements for in vitro alternatives to animal testing

An alternative method must meet the following requirements:

• Fulfilling the required function

• Exhibiting correct and physiologically relevant human biology

• Robust and repeatable

• Ability to scale to the required throughput (e.g., the number of compounds that can be tested at a given time and at a given cost)

• Low startup and recurring consumable costs, to justify the change to a new methodology

Meeting functional requirements for improved in vitro methods

A growing body of evidence shows that the use of animal cells in vitro contributes to the poor performance of the current methods. Even the use of whole animal models does not replicate the in vivo human situation, so it is not surprising that animal cells in an in vitro environment yield misleading results (Zeeshan et al., 2018). The choice to use animal cells is often driven by convenience rather than scientific reasons. Human cells are difficult to obtain, are often derived from a single-diseased patient, and are not representative of a larger pool of donors. Cell lines derived from human cells are more readily available, but the cell lineage may be problematic. Tumor-derived cell lines proliferate readily, but their functionality may differ from that of healthy tissue, and their robustness may undermine a sensitivity test for toxicity of a chemical or drug. Even when a representative supply of cells has been secured, the models may be inadequate. Current research indicates that 2D static cell cultures with no medium flow are not as good at predicting toxicity as 3D cell cultures. Perfusion (flow) of media over or through the cells has been shown to produce a better prediction of the half-maximal inhibitory concentration of a drug than static immersion in medium (Davidge and Bishop, 2017). Building on this research, we can set out a list of requirements for any advanced in vitro method, including OOC devices.

Correct and physiologically relevant human biology

Animal cells may be easier to obtain and maintain than human primary cells, but in no way can they advance our understanding of human disease and toxicity mechanisms. Human-tumor-derived cell lines are easy to culture but are not representative of healthy tissue. Human-induced pluripotent stem cells appear promising but are currently expensive to culture and require long, complex protocols to derive the differentiated cells needed for organ models. Human-donor tissue could be considered the gold standard, but cryopreservation is required to store such tissues prior to experiments and can compromise the cellular function. A review of the cell types used in OOC models is available elsewhere (Esch et al., 2015).

Once the appropriate cells have been selected, they must be cultured under conditions that produce physiologically relevant organoids. Cells under static conditions (no flow) grown on plastic rapidly change their shape and function and no longer represent human tissue (Maltman et al., 2010). 3D cultures on scaffolds or with an extracellular matrix exhibit better performance. In the body, cells do not exist in isolation but exchange molecular signals with cells from other organs. An ideal model of human biology would then need to include connected organoids, so that the system models the whole organism. There is a clear trade-off in OOC platforms between complexity and accuracy. High-throughput screening (HTS) typically relies on short-term culturing and exposure to the compounds of interest. Long-term culturing and homeostasis are important in repeat-dose testing or testing low-clearance compounds. Models for studying cancer and lung and neurological diseases must support longer testing periods and ideally be fully immunocompetent.

Robust and repeatable methods

For any new technology to achieve regulatory acceptance, it must demonstrate robustness and repeatability. Many OOC methods are a long way from this goal, being complex to set up and operate and therefore precluding widespread adoption. Kirkstall Ltd. has designed its Quasi Vivo organ-on-a-plate platform to be easy to use and fast to set up in the laboratory. This system is targeted at the academic market and has already shown that results are repeatable across multiple laboratories, with a current academic user base of more than 70 universities.

Mundane practical issues can occasionally derail sophisticated equipment. In microfluidic systems connected by capillaries or channels smaller than 100  μm in diameter, air bubbles can disrupt the flow, and cellular material can cause blockages.

Ability to scale

Fig. 1.1 shows the stages in the drug discovery and development process where OOC devices could be leveraged. There is a clear divergence between the requirements for HTS and the animal replacement. The former application needs to screen thousands of compounds and improves accuracy (fewer false positives and false negatives). The latter needs to test tens of compounds in depth and replace hundreds of animals used in preclinical screening. Most of the current OOC developments indicate that screening large numbers of compounds is their commercial goal. In contrast, TissUse and Kirkstall have opted for 24-well plate-size chambers that should be more suited to in-depth studies and a focus on animal replacement.

Figure 1.1 Drug discovery and development process. The different stages in the process that are being targeted by OOC developers. Developed from an original by Clerk, S., Villien, M., 2017. Organs-On-Chips 2017, Market & Technology. Report available from Yole Developpement, Le Quartz, 75 Cours Emile Zola, 69100 Lyon-Villeurbanne, France. Yole Developpement.

Low capital and consumable costs

Since so few of the OOC projects have reached the market, it is difficult to assess the costs likely involved. Many could be scaled to volume production and so, in theory, should meet customer expectations on cost. The economics of animal replacement have been thoroughly researched and summarized (Bottini and Hartung, 2009). The acceptable capital and consumable costs for a product to replace animal testing are likely higher than costs to replace HTS. However, the throughput (number of compounds that can be tested at a given time) is considerably higher, suggesting that the market for advanced cell cultures and OOC devices could become segmented into high-throughput approaches and high-content (lower throughput) methods.

Remaining technical challenges

Scaling the biology down to the microlevel

Individual cells can be seeded into the micrometer-sized space in an OOC device, but there is not much scope for growing functional organoids or tissue slices. Once the cells form a 3D structure resembling tissue, insufficient oxygen and nutrient levels will influence cellular function and can even lead to a necrotic core at the center of the cell mass (Berger et al., 2018). Perfusing with a medium can largely overcome this but raises other questions for which data are not necessarily available yet: what flow stresses, velocities, and pressures are actually experienced by cells and tissue in vivo? Gut and kidney models are being used to investigate these questions on the ranges of flow characteristics (Lennon et al., 2014). Microscale devices do limit the amount of biological material available for measuring simple endpoints; this may be acceptable in HTS, which has long used 96-well plates, but other techniques, such as Western blot, require more cellular material.

Coculturing cells

Coculturing cells or tissues is essential to constructing in vitro models that are representative of the in vivo situation, as cells communicate to induce and modulate functions and metabolism in other parts of the body. A simple way to culture multiple cell types together is to place them in a single chamber, but this is less representative of the in vivo situation than placing cells in separate chambers connected by perfusion (flowing media). Perfusion also introduces other aspects, such as nutrient supply and laminar flow stimulation that better mimic and recapitulate the in vivo environment in vitro.

Physiological relevance

A workshop held in the United Kingdom addressed the progress of OOC devices toward more physiologically relevant models (National Centre for the Replacement, Refinement & Reduction of Animals in Research and Medical Research Council Centre for Drug Safety Science, 2018) and included an overview of OOC technology and its utility presented by Gianni Dal Negro. Producing a representative, validated, and qualified 3D cell model is challenging, but this holds the promise of a positive impact across the drug-discovery pipeline from target identification and validation through efficacy and safety assessment. The model must be relevant to the specific biological question under study, and one model cannot answer all questions. Currently available models typically lack integrated physiology and longitudinal (time course) measurement capacity. The challenges to overcome include physically relevant cell interactions, scaling ratios between organs, incorporation of immune or endocrine systems, and the requirement for a common signal-carrying medium flowed appropriately between the components.

Malcolm Haddrick reviewed the progress toward connected systems. Although 4-, 7-, and 10-organ cultures have been connected in a microfluidic setup involving subcircuits and tunable flow rates, prolonged viability has only been demonstrated thus far for individual organs (Esch et al., 2015). Cell models must be specialized and incorporate mixed populations of cells capable of some functions of the organ they represent. Primary tissue is accessible, but reproducibility and scalability are problematic. Induced pluripotent stem-cell-derived cells often exhibit an immature phenotype, limiting their potential as alternatives to primary tissue. To monitor the health of the cell models and interpret their biological responses will require on-chip label-free, real-time biosensors.

Regulatory view and validation

At the same workshop, a regulatory view of OOC technologies was outlined by David Jones of the UK Medicines and Healthcare products Regulatory Agency. In the future, human-based OOC devices may be applied in human-clinical trials with improved safety and efficacy profiles. OOC technology is promising but requires validation and improved translational understanding; the systems do not yet fully mimic human-organ physiology because they lack endocrine and immune responses. Moreover, in vivo toxicity and human-disease processes are not fully understood.

David Hughes, director of the OOC company CN Bio Innovations, commented that validation of new methods will involve testing appropriate numbers of relevant annotated compounds. In the United States, the National Institutes of Health established tissue-chip testing centers to independently generate data from various platforms to corroborate manufacturer claims and stimulate wider acceptance and use of these devices. Regulatory agencies such as the US Food and Drug Administration have also begun to evaluate these technologies.

Choice of materials for organ-on-a-chip systems

Historically, most OOC systems have used the silicon-based polydimethylsiloxane, as it is easy to mold. However, this material can interact with both the biological sample and quite a number of drugs. It is also gas-permeable, which precludes experiments where oxygen tension should be controlled, as the medium in the chamber will equilibrate with ambient gas in the incubator within seconds. Varying the oxygen tension to create aerobic or anaerobic conditions is a key requirement for a zonal liver model, for example (Tomlinson et al., 2019). The choice of materials for OOC is extensively covered in the Chapter 3 of this book.

Conclusions

It is apparent that the OOC technology is developing rapidly fueled by the significant government grants and commercial venture capital funding. There are real but potentially diverging requirements between the need for incremental improvements for in vitro methods and the potentially disruptive shift away from the use of animals. In common with many earlier technology-driven developments, there is a clear need for standards to emerge. These will facilitate the adoption of the technology and ensure that users have a choice between competing methods to meet the specific application needs. Much of the current interest and excitement about OOC technology has been fueled by marketing hype and may soon be replaced by disillusionment unless practical working systems are developed.

Outlook: stimulating the adoption of new technologies to replace animal testing

A possible divergence in the market for OOC systems was mentioned earlier in the chapter. Here, we focus on the animal-replacement opportunity in the academic community. Although there is a clear need in the industrial sector for improvements in the field of drug discovery and development process, the situation and motivation in universities is different. There is a surprising level of inertia in the academic world; many careers have been built on the use of animal models, and it is possible that only a new generation of researchers will adopt different methods. The career paths of academics are driven by their ability to attract grants and to publish their work. Hence, researchers depend on the availability of grants and willingness of reviewers to consider alternatives to animal testing. The peer-review system for awarding grants and controlling approval for publication will take time to change. It is only over the past decade or so that academic centers of excellence have emerged to support animal-replacement technologies. The Center for Alternatives to Animal Testing at Johns Hopkins University in the United States was one of the first, and now has a satellite location at the University of Konstanz in Germany. The United Kingdom has the Animal Replacement Centre of Excellence at Queen Mary University, London, and Canada launched the Canadian Centre for Alternatives to Animal Methods at the University of Windsor. These centers act as nuclei for creating further awareness, funding research, evaluating technology, and supporting industry. Their most effective contribution may be to train a new generation of researchers in this transformative technology as agents for change.

Activities in academia and industry can synergize to support the shift to nonanimal methods. Fig. 1.2 describes an innovative approach to synthesizing a transformative change using a number of small, incremental steps. The foundation begins with good science from a few opinion leaders in academia and continues with the creation of centers of excellence that eventually drive widespread adoption of the new paradigm. Of note, academic research accounts for 49% of the total number of animals used in the United Kingdom (UK Government, 2017). Industry adoption follows, but it is slower at first because of the need for extensive evidence to support claims of superiority for the new technology. The early evidence comes from academia and is followed by the development of robust protocols by contract research organizations. The pharmaceutical industry is increasingly using these organizations to perform validation and development work that may previously have been done in-house.

Figure 1.2 A roadmap and strategy for accelerating the adoption of alternative methods, emphasizing the important role of the academic community and the incremental steps in the paradigm shift.

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Chapter 2

Cell sources and methods for producing organotypic in vitro human tissue models

Patrick J. Hayden,    MatTek Corporation, Ashland, MA, United States

Abstract

Organ-on-a-chip (OoC) technology aims to reproduce key organ systems with miniaturized in vitro cultures that replicate the smallest functional unit of each organ. OoC technologies are expected to facilitate the transition from animal-based models to human-based models, lead to improved prediction of human toxicity, and faster development of effective human therapeutics. A requisite for development and widespread use of human OoC technologies is reliable access to human tissues and cells. This chapter provides a detailed survey of human tissue/cell sources and isolation methods that are currently available to researchers. State-of-the-art culture devices and techniques that can be applied to OoC systems—such as nanopatterned substrates, tunable elastic substrates, air–liquid interface cultures, spheroid/organoid culture techniques, and three-dimensional bioprinting—are also described.

Keywords

Immortal cell line; primary cell; induced pluripotent stem cell; organotypic culture; nanopatterned substrate; air–liquid interface; organoid; 3D bioprinting

Introduction

The goal of organ-on-a-chip (OoC) technology is to reproduce key human organ systems using miniaturized in vitro cultures that are equivalent to at least the smallest functional unit of each organ (Dehne et al., 2017; Ronaldson-Bouchard and and Vunjak-Novakovic, 2018; Huh et al., 2011). The organ equivalents incorporated into the chips do not necessarily resemble their in vivo counterparts in a visual sense, but will reproduce the essential functions of the organ, and will ideally also incorporate any relevant physical/mechanical features [e.g., three-dimensional (3D) extracellular environment/architecture, stretching, contraction, fluid flow, and shear forces] that contribute to organotypic differentiation and organ functions such as breathing, cardiac beating, and blood flow. OoC platforms should also incorporate sensors or features that allow measurement of relevant functional parameters (e.g., compatibility with imaging devices, sensors for measuring real-time conditions, and ports for removal of media/tissues for downstream analysis). A wide variety of OoC platforms have been developed, ranging from systems that incorporate multiple repetitions of a single organ for high-throughput screening applications to systems that incorporate several interacting organs (Fig. 2.1). The ultimate vision for OoC platforms is to create a human-on-a-chip device that will replicate all key organ systems and physiologically relevant interactions of the human body (Fig. 2.2). OoC technologies are expected to facilitate the transition from animal-based models to human-based models, provide faster and more predictive human toxicity assessments, and lead to faster development of effective human therapeutics.

Figure 2.1 (A) OrganoPlate 3-lane microfluidic tissue culture device containing 40 independent microfluidic culture chips. (B) TissUse Multi-Organ-Chip platform containing 4 interacting organ models. (A) (Courtesy MIMETAS); (B) Reproduced with permission from Maschmeyer I., Lorenz A.K., Schimek K., Hasenberg T., Ramme A.P., Hübner J., et al., A four-organ-chip for interconnected long-term co-culture of human intestine, liver, skin and kidney equivalents, Lab Chip 15(12), 2015a, 2688–2699. https://doi.org/10.1039/c5lc00392j.

Figure 2.2 Human-on-a-chip concept: in vitro platform reproducing all key organ tissues and physiological interactions. (Courtesy TissUse, Gmbh)

A requisite for development and widespread use of human OoC technologies is reliable access to human tissues and cells. This chapter provides a detailed survey of human tissue/cell sources and isolation methods, including methods that use induced pluripotent stem cells (iPSCs) currently available to researchers. State-of-the-art culturing devices and techniques that can be applied to OoC systems—such as the use of nanopatterned substrates, tunable elastic substrates, air–liquid interface cultures, spheroid/organoid culture techniques, and 3D bioprinting—are also described.

Human tissue/cell sources and isolation methods

Continuous (immortal) cell lines

Human somatic cells are in general capable of only a limited number (40–60) of cell divisions in culture before they become senescent and lose their ability to divide (Hayflick and Moorhead, 1961). However, immortal somatic cell lines with a capacity for unlimited division potential can be obtained by a number of processes. Genetically mutated cells derived from cancerous tissues are a common source for establishing immortal cell lines. In rare cases, cultured normal (noncancer-derived) cells may spontaneously acquire genetic mutations that provide the ability for unlimited growth. Normal cells may also be transformed into immortalized cells by the introduction of viral oncogenes such as EBV, SV40LT, HPV16 E6/E7, and Ad5 E1A (Honegger, 2001; Freshney, 2016). Induction of telomerase activity by transduction of human Telomerase reverse transcriptase (hTERT) into cells can induce immortal transformation while retaining more normal cell phenotypes than viral-induced transformations (Freshney, 2016).

The development of methods for establishing and maintaining continuous cell lines in culture marked a revolutionary advancement in biology. Since the establishment of HeLa cells as the first immortal human cell line in 1952 (Gey et al., 1952), continuous cell lines have become widely used as indispensable and inexpensive tools for basic biological research, chemical metabolism and toxicity tests, and production of biological compounds such as vaccines, antibodies, and therapeutic proteins. Numerous immortal cell lines derived from a wide variety of tissues are now readily available. Key advantages of immortal cell lines are that they are affordable, well characterized, and easy to culture. However, immortal cell lines generally exhibit significant genotypic and phenotypic abnormalities that may limit their ability to reproduce normal cell behavior and may undergo additional genotypic or phenotypic drift with continued long-term passaging. Furthermore, many continuous cell lines have been misidentified or have become contaminated with mycoplasma or other cell lines over time (Geraghty et al., 2014; Lorsch et al., 2014). Authentication of cell lines is now recommended or required for publication or submission of research results to regulatory authorities (Geraghty et al., 2014).

Despite their shortcomings, immortal cell lines remain in widespread use and will continue to be important biological tools that may be suitable or advantageous for specific OoC applications. Table 2.1 presents a list of commonly used immortal human cell lines derived from a variety of organs. These cell lines, as well as others, are also available as authenticated and quality-controlled resources from a number of nonprofit repositories (Table 2.2).

Table 2.1

Adapted from Freshney, R.I. (2016). Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (7th ed.). NJ: Wiley-Blackwell, with additions.

Table 2.2

ATCC, American Type Culture Collection; ECACC, The European Collection of Authenticated Cell Cultures; JCRB, Japanese Collection of Research Bioresources.

Primary cell cultures and early-passage cell lines with finite lifespan

Cells obtained directly from fresh tissue are commonly termed as primary cells. Advancements in the development of defined culture media, culture conditions, and matrix requirements have led to an increasing ability to culture many types of normal (nonimmortal) primary cells. With the exception of hematopoiesis-derived cells, which may be cultured as cell suspensions, most primary cells require attachment to a substrate to survive and proliferate. Adherence-dependent primary cell cultures may be initiated by explanting small pieces of tissue into a culture plate with the appropriate medium and allowing cells to migrate and proliferate as monolayer cultures. Alternate methods of initiating primary adherent cell cultures involve mechanical and/or enzymatic disaggregation of tissue to form a cell suspension, following by plating the suspension at a low density onto cell culture plates or flasks. Coating the culture plate with various forms of extracellular matrix material or the presence of an established feeder cell layer may be required to support culture establishment when using either the explant or disaggregation methods (Honegger, 2001; Freshney, 2016).

Primary cultures obtained by seeding cells or explanted tissue fragments directly after isolation from fresh tissue will consist of those cells that are capable of attachment and survival under the culturing conditions (e.g., culture medium and extracellular matrix coating). The primary culture will consist of a mixed population of cells at various stages of differentiation and, for certain tissue types, tissue-specific stem cells with proliferative potential. Cells that are already committed to terminal differentiation may attach and remain viable but will not proliferate further in culture. The proliferation of tissue-specific stem cells will continue until space in the culture vessel becomes limited, at which point the cells may be harvested and passaged into fresh culture vessels.

Cells are typically passaged while still in log-phase growth and before reaching confluence, to avoid loss of proliferative capacity owing to contact inhibition that may occur when the cultures become too densely packed. Adherent cells are generally harvested from culture using enzymatic reagents (trypsin or Accutase) or by manual scraping. Primary cells that have been passaged are thereafter termed primary cell lines. The passaged lines will be enriched in cells that have adapted well to the specific culture conditions and those that retain proliferative capacity, while differentiated/nonproliferative cells will die off. With continued culture and successive passaging, the cell lines will at first continue to adapt and become further enriched in cells with proliferative capacity. However, the cells will eventually exhaust their ability to proliferate and become senescent. If the culture conditions are not specifically tuned to promote only the growth of the desired cell type, the cultures may become overgrown with unwanted cell types such as fibroblasts.

Conditions that allow in vitro proliferation of many types of primary human cells [e.g., epithelial cells, stromal cells (fibroblasts, stellate cells, pericytes, astrocytes), and endothelial cells] have been successfully developed. However, certain types of cells from key organ systems (e.g., cardiomyocytes, hepatocytes, neurons, islet cells, and monocytes) can only be maintained for a limited time as primary cultures and do not have significant proliferative capacity in vitro.

A number of comprehensive texts on the subject of animal and human cell isolation and cell culture techniques are available (Honegger, 2001; Randell and Fulcher, 2013; Picot, 2005; Mitry and Hughes., 2012). Table 2.3 lists protocols in the literature for the isolation and culture of organ-specific human cell types.

Table 2.3

Human tissue sources

While protocols for isolation of many organ-specific human cell types have been developed, availability and access to fresh human tissue samples may be a significant limitation for researchers outside of clinical research university or hospital settings. Access to fresh human tissues for research requires informed consent of the tissue donor and institutional review board approval (Pirnay et al., 2015). Even for researchers with access to fresh human tissues, cell isolation protocols require specialized techniques that may be difficult to master and are a time-consuming endeavor that may not be feasible or desirable for many laboratories. As an alternative, a number of vendors offer primary human cells (Table 2.4).

Table 2.4

ATCC, American Type Culture Collection.

Induced pluripotent stem cells

A seminal advancement in cell culture and regenerative medicine occurred in 2006 with the development of methods for generating iPSCs from differentiated somatic cells via induced expression of four transcription factors (Takahashi et al., 2007a,b; Yu et al., 2007; Sayed et al., 2016). Because adult cells are used, iPSCs avoid the restrictions and controversy surrounding human embryonic stem cells. Human somatic tissues, fluids, and cell types such as fibroblasts, blood cells, and urine have been used to generate iPSCs. The initial iPSC protocols used retroviral and lentiviral systems to integrate transcription factors into the host genome. Recently developed protocols allow the use of nonintegrating systems, including Sendai virus, episomal reprogramming factors, and microRNAs, to generate iPSCs without integrating the reprograming factors into the genome (Fusaki et al., 2009; Warren et al., 2010).

Once generated, iPSCs are theoretically capable of differentiation into any cell type; iPSCs are therefore a valuable source for generation of large numbers of cells that normally do not proliferate in vitro (e.g., cardiomyocytes, hepatocytes, and neuronal cells) (McKernan and Watt, 2013). In addition, iPSCs allow researchers to recreate in vitro models of inherited genetic human diseases and enable the derivation of multiple types of organ models from the same donor (body-on-a-chip or you-on-a-chip devices). Development of protocols to drive differentiation of iPSCs into various tissue-specific lineages is an area of current intense effort. The growing list of organ-specific cell types that have been generated to date from iPSCs includes hepatic, cardiac, neuronal, endothelial (including blood–brain barrier), pancreatic, lung, renal, and intestinal cells (Table 2.5). iPSC technology currently requires significant expertise and is a time-consuming process. The science is still developing, and current protocols do not yet reproduce fully differentiated organ-specific cell phenotypes.

Table 2.5

A growing number of iPSC sources currently exist (McKernan and Watt, 2013; De Sousa et al., 2017; Kim et al., 2017; Ntai et al., 2017). The California Institute for Regenerative Medicine (CIRM) hPSC Repository is the world’s largest, containing iPSCs from over 3000 individuals. The CIRM iPSC lines are produced by nonintegrating episomal reprogramming. Demographic and clinical data are available for a variety of diseases or conditions affecting the brain, heart, lung, liver, and eyes. The Coriell Institute for Medical Research (Camden, NJ, United States) offers dozens of iPSC lines related to Parkinson’s disease, amyotrophic lateral sclerosis, and Huntington’s disease, and supplies iPSCs to other repositories. European-based cell banks include the European Bank for Induced Pluripotent Stem Cells (De Sousa et al., 2017), the Human Induced Pluripotent Stem Cell Initiative, and StemBANCC (Table 2.6).

Table 2.6

Methods for producing three-dimensional organotypic tissue cultures

Traditional cell isolation techniques and culture methods for adherence-dependent cells typically involve submersion cultures of cell monolayers on two-dimensional (2D) plastic substrates. These methods were developed to promote proliferation of cells and generally lead to loss of differentiated cellular functions. Moreover, traditional 2D culture environments lack the important cell–cell, cell–matrix, 3D architecture, and mechanical cues (e.g., stretch, strain, shear forces, nanotopography, and substrate compliance) that are found in the in vivo environment of the cells and that are essential for functional differentiation (Huh et al., 2011; Schmeichel and Bissell, 2003; Alhaque et al., 2018).

Recognition of the inherent limitations of 2D culture environments has motivated efforts to develop 3D cell culture conditions that better replicate in vivo tissue architectures and provide more physiologically relevant organotypic in vitro tissue models of normal function and disease. A