Organ-on-a-chip

Engineered Microenvironments for Safety and Efficacy Testing

Julia Hoeng, David Bovard, Manuel C. Peitsch

• Publisher: Academic Press
• Year: 2019
• Print ISBN: 9780128172025
• eBook ISBN: 9780128172032
• Topic: Technology & Engineering
• Subtopic: Engineering General
• Index: Technology & Engineering

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

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 c...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• List of contributors
• Preface
• Chapter 1. Need for alternative testing methods and opportunities for organ-on-a-chip systems
• Chapter 2. Cell sources and methods for producing organotypic in vitro human tissue models
• Chapter 3. Organs-on-a-chip engineering
• Part I: Organ-on-a-chip platforms to model disease pathogenesis
• Part II: Multi-organs-on-a-chip platforms to mimic humans physiology
• Index