Organ-on-a-chip

Definition

Organs-on-a-chip are small devices designed to replicate as closely as possible the microenvironment and physiological conditions that cells live in within their native tissues and organs to provide more realistic responses than traditional culture experiments. Also known as ‘In Vitro Cell Culture Technology’ and ‘Micro Physiological Systems’, the chips are often made from synthetic transparent materials and contain minute hollow tubes and chambers coated with cells as well as mechanical components to enable more precise simulation of the natural cellular microenvironment. Furthermore the transparency and integration of biosensors can provide more detailed and accurate insights into the molecular and cellular activities that underpin the function of human organs and particular disease states.

Organ-on-a-chip. Credit: Wyss Institute.

Importance

Organ-on-a-chip (Organ Chip) technology promises to be a major tool to address the rising cost and declining efficiency of drug research and development in addition to providing a new approach to personalised medicine. One of the main attractions of Organ Chips is that they provide a better predictive model of drug responses in real human tissues and organs than animal experiments. This is sorely needed because more than 70 percent of all drugs that appear promising in preclinical animal studies go on to fail in human clinical trials because the animal model is often not applicable to humans and the drug proves to toxic and ineffective when tested in humans (Tagle). Many approved drug products also get withdrawn from the market because of unanticipated adverse reactions (Oleaga, Bernabini, Smith).

One of the drivers of this problem is the difficulty of translating safety, toxicity and efficacy data from animal studies to humans. This can have tragic consequences, as shown by the example of TGN1412, a monoclonal antibody drug developed to direct the immune system to destroy cancer cells. Initially found to be safe and effective in rodents and monkeys, it proved catastrophic when first tested in humans in phase I clinical trials because it prompted an adverse immune response caused by a cytokine storm. Subsequent research indicated that this might have been due to a slight difference in the amino acid sequence of the CD28 receptor between the animals and humans (Oleaga, Bernabini, Smith). In a case like this, testing the drug in chips containing human cells prior to the clinical trials would have negated such a problem.

Furthermore, Organ Chips can be loaded with cells unique to an individual patient, providing more accurate predictions of toxicity and efficacy specific to them, thereby opening up a more effective means of personalised medicine. Organ chips could also prove useful for validating potential drug targets identified by genomic and proteomics so they can enhance the drug discovery process. This is particularly important because of the great variability found between different ethnic and genetic groups in their responses to therapy (Ingber 2022). Overall, Organ Chips have the potential to streamline the clinical trial process, which can be one of the most time-consuming and expensive aspects of drug development.

Aside from promising to improve the speed and accuracy of drug testing, Organ Chips, potentially integrated with biosensors to generate large quantities of data, offer researchers a cost-effective mini-laboratory to study disease processes at the cellular level in real-time. Such devices, for example, can be constructed to mimic conditions like cancer (Sontheimer-Phelps Hassell Ingber) or asthma (Nesmith, Agarwal, McCain, Parker) to enable investigators to study the effects of certain immune cells or drugs. Creating separate chips coated with cells taken from the gut of a human or an animal, like a cow or an insect, also enables the possibility of comparing the impact of products like pesticides on the digestive systems of different species, something which would be difficult to do otherwise.

Organ Chip technology has yet to realise its full potential, but research in this area is growing rapidly. It is rooted in the concept of the 3Rs (Reduce, Refine and Replace animal testing) first outlined in 1959 (Russell, Burch), which the European Council implemented in its directive in the use of experimental animals in November 1986 and then more firmly in a new directive passed in September 2010 (Marx, Walles, Hoffmann). The new directive specified that animal experiments should only be conducted when results could not be gathered by any other means. Providing a cheaper and more accurate model of humans, the wider adoption of Organ Chips should help to meet this directive.

Just how quickly the field is moving can be judged by the number of academic publications that have appeared on the topic in recent years. A search of the term 'organ-on-a-chip' on Google scholar reveals that the number of publications in this area rose from just 4 in 2000 to 2,660 in 2021. The number rose particularly strongly from 2016 onwards. Similar growth patterns appeared for the term 'microphysiological systems' (MPS). These numbers are smaller than the number of publications returned for the term 'microfluidics' but are representative of the growth of the field as a whole (see figure 1 and 2). Similar growth trends can be seen from the number of patents issued each year for Organ Chips which grew from just 4 in 2000 to 94 in 2015 (ALTEX). The number of companies being set up around the technology has also been increasing. At least 28 companies were set up between 2011 and 2018 (Zhang, Korolj, Fook, Radisic).

The importance of the technology can also be seen from the partnership established in 2011 between the US National Institutes of Health's National Center for Advancing Translational Sciences (NACT), the Food and Drug Administration (FDA) and the Defense Advanced Research Projects Agency (DARPA) to support the development of Organ Chips to improve the screening process for safe and effective medicines. In January 2016 the European Union's Horizon 2020 research and innovation programme also awarded over €30 million for a six year project, called EU-ToxRisk, to help deliver reliable animal-free systems using advances in cell biology, omics technologies, systems biology and computational modelling to aid the testing of drugs, cosmetics, pesticides and chemicals (EU-ToxRisk). Similarly, in 2017, the Japanese Agency for Medical Research and Development (AMED) launched a project to encourage the development of MPS devices (Ishida).

Further evidence of the importance of the field can be seen by the FDA Modernisation Act of 2021 and the Human Research Testing Act (HR 744) currently passing through the US Congress which questions the over reliance on genetically engineered mice and other animal models for basic scientific research and drug development (Ingber 2022).

How far the field has travelled can also be seen by the fact that in 2021 an Organ Chip platform developed by CN Bio, a British cell culture company founded in 2008 in Cambridge, was ranked second in the 10 top innovations listed by The Scientist, a highly respected magazine which has conferred awards for top innovations in science technology since 2008. CN Bio's platform allows scientists to connect different individual Organ Chips to create a model or a multi-organ system enabling more accurate research into disease conditions and for drug development (Brackley; The Scientist).

Discovery

The development of Organ Chip technology was founded on the back of the long history of cell culture research. Scientists first began finding ways to cultivate eukaryotic cells outside of an organism in the late nineteenth century, and by the early twentieth century it had become possible to maintain such cells for several months (Yao, Asayama). As early as 1876 John Syer Bristowe, a British physician, coined the term ‘organoid’ to denote ‘the smallest functional organ or tissue unit’ (Marx, Andersson, Bahinski). Efforts to create artificial organs were inspired by the work of Henry Van Peters Wilson, a professor of biology and zoology at the University of North Carolina, Chapel Hill. In the early twentieth century, he discovered that if he kept individual tissue cells that he had mechanically separated from Microciona prolifera (a red oyster sponge) in sea water, they naturally clumped together and fused to become new sponges (Costello). Following this, a number of other scientists generated different types of organs using organ tissues taken from different sources, including embryonic chicks (Weiss, Taylor) and frogs (Richter Piwocka Musielak).

Cell culture was greatly aided by the work of Ross Granville Harrison, an American biologist and anatomist at Yale University. In the early 20th century, he managed to grow frog nerve cells using a ‘hanging drop technique’ used in microbiology. His method involved immersing frog nerve cells in droplets of lymph solution on the cover side of a conventional petri dish and then turning the plate upside down. The advantage of his method was the cells could grow without being constricted by the flat plane of a conventional petri dish. Harrison’s three-dimensional cell culture method was subsequently used for growing other cell types (Navis; Linder; Richter Piwocka Musielak).

For a long time living mammalian cells were ‘primarily cultured in nutrient medium under static conditions on 2D substrates coated with serum or extracellular matrix’ optimised to stimulate cell growth. But this often resulted in a loss of tissue-specific functions which made many question the ‘physiological relevance of results from in vitro experiments’. Because of these difficulties scientists began looking for ways to develop MPSs that could better sustain tissue functionality for lengthy periods (Ingber 2022).

New possibilities opened up for cultivating organs as a result of advances in knowledge about stem cells, which became the subject of intensive investigation from the 1960s because of their ability to turn into multiple types of cell and organise into structures found in different tissues. In part they were of interest because of their important role in the process of repair, maintenance and regeneration of tissue within the body. The availability of such cells was limited, however, by the fact that, with the exception of the bone marrow, most organs in the body only produce a small amount of stem cells that have the capacity to differentiate into different cell types. Embryos offer a more malleable source of stem cells, but ethical and political controversies make them highly unattractive for regular use in research. One alternative is to take stem cells from a patient and convert them into the desired cell type for a particular chip. This approach greatly benefited from the discovery in 2007 that adult somatic cells could be genetically reprogrammed into an embryonic, stem-cell-like state. Known as ‘induced pluripotent stem cells’ (IPSCs), whilst the clinical application of these cells has been limited so far due to their tendencies to become malignant, they have made the development of Organ Chips much easier in recent years (Marx, Andersson, Bahinski).

Another critical factor that helped propel Organ Chip technology forward has been the emergence of microfluidics, enabling the manipulation of fluids within microscale channels that are moulded or engraved into the tiny chips. The field was galvanised by a number of different forces. The first was the invention of Integrated Circuits technology and photolithography in late 1940s and early 1950s which opened the door for creating microelectronic chips upon which the semiconductor and computer industry was built. The second was the development of new fluid-handling devices with interconnected microchannels, mixers, valves, pumps, pipettes and flowsensors. A key pioneer in this field was Stephen Terry at Stanford University. In 1979 he and his team created a miniaturised gas chromatograph integrated on a silicon wafer. This laid the foundation for the creation of the first 'laboratory-on-a- chip'. Integrating several laboratory processes on a tiny single integrated circuit, this chip provided a means for automation and high -throughput screening (Terry, Jerman, Angell). The rise of Organ Chips was also greatly helped by a DARPA initiative launched in 1994 to fund the development of portable microfluidic systems to detect chemical and biological weapons. A fourth stimulus came from the explosion of genomics in the 1980s, which required tools with much higher sensitivity and greater throughput than previously needed in biology, such as capilliary electrophoresis, PCR and DNA microarrays (Whitesides). Advances in this area were also helped by advances in new fabrication methods like 3D printing from the 1980s.

Development

One of the key pioneers in the Organ Chip field has been Michael Shuler, a chemical engineer at Cornell University, who began pondering the difficulties of testing new drugs as a result of caring for his youngest daughter, who has Down syndrome. As he explains, ‘People with Down syndrome are at much higher risk of certain diseases, such as Alzheimer’s disease and a wide variety of heart defects. In addition, it is difficult to test drugs on this population and their unique genetic makeup increases the chance that a drug may work differently for them than for others'. He was also keen to find a way to help speed up the drug development process so that patients could get the treatments they needed without waiting the many years it took to approve them (Hartman; Kacapyr).

Shuler was not the only one active in the field. So too was Andre Kleber and his colleagues at the University of Bern, Switzerland. In 1991 they developed the first in vitro cardiac model by growing myocytes - a type of heart muscle cell - from neonatal rats in a particular pattern within linear channels on conventional glass coverslips (Rohr, Scholly, Kleber). The model helped pave the way for the first biophysical explanation for how electrical impulses can sometimes block the heart beat (Zhang, Korolj, Fook, Radisic).

From 1989 Shuler began developing what he called ‘cell culture analogue’ (CCA) devices to mimic the physiological and toxicological responses of animals and humans to drugs (Shuler Interview). Using tissue cultures of different organ systems in animals, Shuler built the devices based on physiologically based pharmacokinetic models for drug development. Pharmacokinetics is the study of how a drug gets absorbed, distributed, metabolised and eliminated from the body. The CCA device had the advantage that it could operate for over 24 hours and sustain live tissue culture for that period of time. It proved useful for validating toxicology and pharmacology tests (Shuler, Ghanem, Quick).

By the late 1990s Shuler’s team had begun looking for ways to build microscale systems that linked pseudo-organs (artificial organs) together in a way that they appear in the body. They did this in collaboration with Greg Baxter, a specialist in microfluidics, based at Cornell's nanofabrication facility. As Shuler explains ‘Greg and I realised that it was fairly expensive to do this with large cell cultures and flasks and stuff like that and we needed to make it much more efficient in terms of space and cost. By going to the microscale we could get by with cell cultures which are much smaller.' To build their new system they looked to the physiologically based pharmacokinetic computer models that had been developed in the 1960s which made it possible to look at how different organs respond to different doses of drugs (Shuler Interview).

Shuler’s group first developed a prototype of an animal-on-a-chip. First reported in 2001, the chip was created by etching groves into silicon wafers to create tiny compartments to ‘hold gut, liver and fat cells’ to act like organs, ‘all linked by microfluidic channels’ to act like blood vessels (Sin, Baxter, Shuler; Baker). A solution replicating the composition of blood serum could be circulated around the chip, with the help of a pump, together with a test compound to mimic what happens when a drug goes through the body. One of the first experiments the two scientists conducted with the chip was with naphthalene. Often used in mothballs because it has a strong smell, naphthalene is usually harmless in its natural state, but toxic to mammals when metabolised in the liver. Pumped first through the lung chamber and then split between the liver and another cell-less compartment, Shuler and Baxter found that naphthalene was converted in the liver compartment to naphthalene epoxide which circulated to the lung compartment where it caused lung cell death (Anon 2015; Shuler Interview).

The team then moved on to integrating cell culture and microfabrication technology to build human models. These were used for functional characterisation and detection of drugs, pathogens, odorants and toxicants. The advantage of the small size of the systems was that they only required small amounts of samples and reagents. This was helpful because cell culture can be the most expensive part of running toxicology and drug tests (Park, Shuler; Shuler Interview).

In 2008 Shuler and his colleagues began to create a gastrointestinal tract (GIT) microscale cell culture analogue for predicting drug transport within the gut. Within two years, they had successfully developed an in vitro platform that combined microfabrication, microfluidics, and cell culture to help predict human responses to drugs. Their system had multiple compartments, each of which represented a different organ or tissue type (Shuler, Esch).

Shuler soon joined forces with James J. Hickman, an expert in nanoscience and the developer of functional cell constructs that mimic the electrical and mechanical responses of cardiac, neuronal, and muscle tissues to chemicals (Oleaga, Bernabini, Smith) to design multi-tissue, physiologically realistic devices. called 'body-on-a-chip' or 'human-on-a-chip' devices (Sung, Esch, Prot; Tagle). Their aim was to connect chips, each representing individual organs, like the liver or heart, to evaluate the toxicological and efficacy responses of drugs in a similar manner to the use of animal models in the field (Hartman). Hickman brought to the project his expertise in measuring functional responses to stimuli, particularly electrical activity in tissues such as neuronal and cardiac tissues along with measurements of force generation by cells such as muscle. His ability is unusual in the field. Shuler and Hickman were helped in this work by funding from a Common Fund set up by the National Institutes of Health (NIH), in 2010, to support the development of a heart-lung chip model to test the safety and efficacy of drugs (Tagle).

In 2015, Shuler and Hickman co-founded a start-up company in Orlando, called ‘Hesperos’, to commercialise their technology. The company’s work was supported by the NCAT’s Small Business Innovation Research Programme. In the last few years, a number of pharmaceutical companies, including AstraZeneca, Roche, and Sanofi, have partnered with Hesperos to test out its Organ Chip for drug testing (Keown).

Another prominent figure in the organ chip arena is Donald Ingber, a cell biologist and bioengineer based at Harvard University. He originally set out to explore the role of mechanical forces in biology in the 1980s, believing that they were as important as chemicals and genes for health and biological development. This involved investigating how different cell shapes affect cellular activity (Ingber Interview; Ingber 1993). By the early 1990s, Ingber was working with the chemist George Whitesides, also at Harvard, to adapt an inexpensive ‘Soft Lithography’ method Whitesides had originally developed to manufacture computer microchips to ‘print little adhesive islands coated with extracellular matrix’ to help make these models. Their method involves moulding a clear flexible silicone rubber, called poly (dimethylsiloxane) (PDMS), on top of a silicon chip etched on the nano- to micro-scale using photolithographic techniques commonly used to manufacture computer microchips. The rubber stamp is then peeled off and used to pattern chemicals on other substrates. The advantage of this technique was that it enabled precise control at the nanometre to micrometre scale of the environment in which cells live (Ingber Interview). Ingber and Whitesides published their approach in two articles in Science in 1994 and 1997 showing that a cell’s shape governs ‘whether individual cells grow, differentiate, or die, regardless of the type of matrix’ used to mediate adhesion (Chen, Mrksich, Huang).

Whitesides subsequently adapted the method to fabricate microfluidic devices containing little hollow channels with which they can control the flow of solutions through the chip, which he published in 1998 (Duffy, McDonald, Schueller). Industry was already using this strategy to miniaturise instrumentation for diagnostics and analytical systems. Ingber and Whitesides first reported the culture of cells inside the channels of these devices in 1999 (Kane, Takayama, Ostuni). The introduction of PDMS into the field greatly accelerated experimentation in this field because the polymer is non-toxic, and more flexible and much easier to manufacture into microfluidic devices than glass or silicon. It also allows for the incorporation of valves and pumps and does not require expensive clean room facilities (Convery, Gadegaard).

Having worked for years on vascular networks and systems, Ingber wondered if microfluidics could be leveraged to do more than pass fluids between different cell types, and to actually replicate organ-level structures and functions in vitro. His starting point for this was a lung-on-a-chip created by the bioengineer Shuichi Takayama who had been postdoctoral fellow with both Whitesides and Ingber. Takayama’s chip had little channels simulating the microenvironment of the small airway of the lung. Ingber was impressed that Takayama had demonstrated that it was possible to recreate the crackling sound associated with certain lung diseases by passing a little droplet of fluid through the channels of the chip. Ingber was amazed by the sound it made. As he recalls, ‘That noise was exactly the noise I was taught to listen for through a stethoscope when I went to medical school'. For years, nobody had known what caused such a noise, and Takayama ‘basically showed that it was due to little mucus plugs going through bronchioles, small airway-size conduits’ (Ingber Interview). Inspired by Takayama’s result, Ingber launched a project to further develop a living, breathing human lung on a microchip, together with Dan Dongeun Huh, a South Korean postdoctoral researcher who joined his laboratory in 2007 after working with Takayama at the University of Michigan (SLAS; Huh, Fujioka, Tung).

By 2010, they had engineered a chip with a pair of microchannels separated by a very thin flexible permeable membrane coated with natural extracellular matrix adhesive molecules. One channel was lined with a layer of primary human cells isolated from the lining of the lung air sac, or alveolar tissue, which after a couple of weeks multiply and differentiate into the cells native to alveolar tissue. These secrete mucus when cultured on the top of the permeable membrane and exposed to air introduced into the upper channel. Human capillary blood vessel cells from the lung were used to line the lower surface of the same membrane with a liquid medium flowing through the lower channel to provide nutrients to both the alveolar and capillary cells as they would in the body. Two hollow chambers were placed on either side of the two microchannels which allowed for cyclic suction to be applied, thereby enabling the rhythmical stretch and relaxation of the engineered 'alveolar-capillary interface' to model the mechanics of breathing. The device provides a means to simulate the effects of air pollution and bacterial infections on the lungs, and to investigate what happens when lungs become inflamed (Huh, Matthews, Mammoto). The complex breathing action of the engineered lung also subsequently opened up new insights into the cause of pulmonary oedema, a serious condition caused by excess fluid in the lungs, and helped predict the activity and toxicity of a new drug developed by GlaxoSmithKline for the disease (Huh, Leslie, Matthews; Kusek).

Being the first microfluidic device to capture organ-level functionality, this lung-on-a-chip marked a major milestone. Heralded as one of the most important research advances in biological and medical science in 2010, the chip provided a prototype for creating other types of organ chips (Anon 2011). Fabricating the chip out of a soft and stretchy material, Ingber and Huh showed it was possible to build a working model of an organ chip and they ‘recorded beautiful movies of immune cells crawling over the surface of the vascular cells, crossing the membrane through the matrix-filled holes in the membrane, passing through the alveolar lining cells, and tracking down a bacterium’ (Borfitz).

In October 2010, the National Institutes of Health and US Food and Drug Administration awarded Ingber and Kevin Kit Parker at Harvard University $3 million to develop a ‘Heart-Lung Micromachine’. Combining two different organ systems within a single microsystem for the first time, the aim of the device was to help speed up drug safety and efficacy testing (Anon 2011). Ingber’s laboratory subsequently received a further $37 million in funding in 2012 from DARPA for a five-year project to develop a human body-on-chips model with 10 integrated Organ Chips to predict drug pharmacokinetics and accelerate the process of drug discovery and testing the toxicity of new products. Two years later, Ingber founded a spin-out company called ‘Emulate’ to commercialise the technology. Over the course of the next four years the company raised $57 million and went on to raise more than this in subsequent years to fund its work on different Organ Chips, and established partnerships with several pharmaceutical companies including Merck and Johnson & Johnson to test different drugs (Woods; Fassbender).

According to Ingber, he and his collaborators have been able to make a chip designed to replicate any organ they have taken on as a challenge so far. As he puts it, ‘there were some that we couldn't do because we couldn't get the cells for years, and then we developed a way to get cells using IPSC technology, and then it worked.’ What is particularly gratifying to Ingber is that they can now create chips that not only replicate normal physiology, but also ‘pathophysiology and disease states with high fidelity.’ The fact that the chips can also simulate ‘flow peristaltic-like motions in the intestine, breathing motions in the lung, and rhythmic distortions of the kidney caused by pulses of blood generated with each beat of the heart' is also very important, because we do not see optimal mimicry of in vivo functions unless we replicate these mechanical forces, which he has dedicated his life’s work to investigating (Ingber Interview).

Uwe Marx, a German physician by training, tissue engineer is another key figure in the organ chip field. Since the early 1990s, he has been pioneering micro-physiological systems to mimic human organs in vitro. His first breakthrough was the development of disposable bioreactor-based antibody manufacturing technologies in the late 1990s, which helped contribute to the ban of the production of monoclonal antibodies within animals in Europe. He then co-developed a microfluidic human artificial lymph node model based on a miniaturised and perfused bioreactor which enabled monitoring of the effects of different substances on the human immune system, thereby opening the way to animal-free immune reactivity-testing of new drugs and other biological entities (Giese, Demmler, Ammer).

In 2015 Marx hosted the first stakeholder workshop for the community developing MPS organised by CAAT (Center of Alternatives to Animal testing) Europe. Held in Berlin, this workshop included 35 experts from academia, industry and regulatory bodies. Its aim was to map out the current status of the technology and provide a roadmap for getting it robust enough to be adopted by industry (Marx, Andersson, Bahinski). He subsequently organised a second stakeholder workshop in 2019. Attended by 46 leading international experts from academia, industry and regulatory bodies, this workshop reported that the main obstacles to industry’s adoption of MPS-based models often lay in the technical immaturity of many of the systems and lack of communication between the providers and the end-users (Marx, Akabane, Andersson). He was responsible for the publication of two comprehensive reports on biology inspired MPS put out by t4 (The Transatlantic Think Tank for Toxicology) (Marx, Andersson, Bahinski).

In addition to being an opinion leader in the field, Marx has founded several biotechnology companies to help advance the technology. In 2010 he founded TissUse GmbH, a company he spun out of his laboratory at the Technical University of Berlin. Around this time he also began to develop the concept and principles of organismoid theory to generate miniature, mindless and emotion-free equivalents of a human individual’s body on chips which he believed could revolutionise drug development and the precision of personalised medicines in the future (Marx, Walles, Hoffmann; Marx, Accastelli, David).

By 2016 he and his colleagues had managed to build 16 human organ chip models, which were 100,000-fold smaller than the original organs. These chips could be used to simulate diseases like diabetes or cancer and to test the efficacy and side effects of new drugs. Initially using tissue donated from different donors to create individual organs, the subsequent models were created with induced stem cell technology (Laska). Such work formed the basis for the creation of the ‘multi-organ chip’ which mimics various human organs on one device enabling the simulation of human processes over a period of two months (Materne, Maschmeyer, Lorenz).

In 2019 Marx and his colleagues reported that they had managed to combine miniaturised human intestine, liver, brain and kidney equivalents into a four-organ-chip. All four organs had been created with pre differentiated iPSCs from the same healthy donor. These were then integrated into the microphysical system (Ramme, Koenig, Hasenberg).

Another multi-organ chip has been created by a team led by Frank Sonntag at the Fraunhofer Institute for Material and Beam Technology in Dresden. The team began working on the chip in 2009, for which they received an Innovation Award from the European Association of Research and Technology Organisations in 2018. Measuring about three by ten centimetres, the chip was assembled with the help of a laser to cut chambers in plastic foils for blood vessels and organ cells, which are stacked on top of each other and connected together with the addition of sensors, valves, pumps, mass exchangers, and electronic controls. Already tested for numerous medical applications, the cells in the chip were found to survive much longer than in classical Petri dishes. Another advantage of the system is that it allows for differentiated regulation of the blood supply in each chamber, which is more representative of what happens in the body where different organs and tissues require different amounts of blood. The brain, for example, receives more blood than the eyes. Small microscopic valves incorporated into the chip also make it possible to use the chip to simulate heart attacks and strokes (Fraunhofer).

Largely galvanised by the work of Shuler's group, scientists have intensified their efforts to develop multi organ chips over the past few years. Known as 'body-on-a-chip' or 'human-on-a-chip' these devices aim to replicate the complex body system which is composed of organs and tissues each of which have multiple physiological roles and interactions with each other. Such chips are not able to reproduce all biological responses in the body. But they can help provide insights into heretofore unknown responses which can only be picked up by observing the interactions between different organs in real-time. This information is important to improving the prediction of pharmacokinetic (PK) processes which can help determine how to administer a drug and the dose to be given. It can also help in predictions of pharmacodynamics (PD) which evaluates the pharmacological effect of a drug on the body which is critical to assessing its efficacy (Sung, Srinivasan, Brigitte;Kimura, Sakai, Fujii).

Application

Where Organ Chips could prove most useful is to reduce the cost and shorten the time needed to develop drugs, which on average take 10 to 12 years each. Much of the drug development cost is related to the clinical trials needed to get the drug approved for use in humans. Organ Chips could also help with personalised medicine through the use of chips for individual patients to help identify which drug is most beneficial and least toxic to them (Knight Interview).

Elsewhere, the technology could help with rare genetic disorders, which are particularly challenging because of the difficulties associated with recruiting sufficient numbers of patients with such diseases to clinical trials. At present, this work is done by replicating the disorders in animals, but it could be done instead with Organ Chips (Shuler Interview). In fact, this was demonstrated by Ingber's team in 2020 when they replicated a rare genetic blood disorder called Shwachman Diamond syndrome by building bone marrow chips starting with blood cells from children with this disorder (Chow, Frismantas, Milton). Ingber and his postdoctoral researcher Ratnakar Potla have also created a chip to mimic cystic fibrosis, a progressive genetic disorder which causes severe damage to the lungs and cells responsible for the production of mucus, sweat and digestive juices. Supported by a grant from the Cystic Fibrosis Foundation, in 2021 they published a human lung airway chip with cells taken from a cystic fibrosis patient which replicated multiple hallmarks of the disease in the lung, 'including enhanced mucus accumulation, increased cilia density, and a higher ciliary beating frequency'. Encouragingly the chip's environment proved conducive to the growth of Pseudomonas aeruginosa bacteria and mimic inflammatory responses that are especially dangerous to patients with cystic fibrosis (Plebani, Potla, Soong).

Organ Chip technology also opens the way to investigating how women and men respond differently to medical treatments. In 2018, for example, Ingber's team received research funding from the FDA to use bone-marrow chips to study 'how male and female bone marrow responds to radiation and drugs known to cause bone marrow damage' (Brownell, Nov 2018). In addition, Organ Chips are now being developed to study specific functions of the female reproductive tract and changes that occur during conception and fertility. A placenta-on-a chip, for example, has been used to study the role of chronic inflammation in poor pregnancy outcomes and also how drugs are transported across the human placenta barrier. Organ Chips also provide a way to screen drugs that cannot normally be done in pregnant women, who are generally excluded from clinical trials because of the risk of harm to a foetus (Blundell Yi Ma; Mancini, Pensabene).

Organ Chip models also provide scope to generate and be mined for large amounts of data, which, when combined with modern computational methods, could yield far greater insights into drug pharmacokinetics and disease mechanisms than animal models can. Such information would help mitigate potential side effects in human clinical trials. It could also provide more precise drug response models to determine appropriate dose administration. Until now this has been done by dose-increase schemes, whereby trial participants are started with low doses which are gradually increased. Organ Chips offer the ability to study controlled environments and simultaneously test different variables, providing better results in cause-and-effect experiments and enabling more specific research questions to be answered. Overall, the more advanced Organ Chip technology gets, the more accurate it will become in replicating real human responses, making the drug development process more effective and giving better predictions of side effects (Ingber Interview).

Another area where Organ Chips could be beneficial is in the development of biological drugs. These medicines are derived from living cells or through biological processes. Such drugs include a diverse selection of compounds with a biological origin, including peptides, nucleic acids, cytokines, replacement enzymes, various replacement proteins, and monoclonal antibodies. Immunotherapies for cancer are one of the most important classes of biologics developed in recent years, spurred on by the improvements they have made to patient survival (Marks, Feb 2018). One of the advantages of biologics is they are highly target-specific, so they are less likely to cause harmful non-target effects. Biologics, however, are difficult, if not impossible, to test in animals because they are highly specific to human biological targets. In addition, only a fraction of patients have so far been found to respond to such drugs. Another problem is that biologics often elicit adverse immune reactions including skin and liver toxicity, colitis, and pneumonitis. In this case, Organ Chips offer an important way forward. Importantly, Organ Chips provide a better simulation of the interactions of a complex multifactorial biological environment than Petri dishes, and offer more accurate human responses to biologics (Shuler Interview; Kerns, Belgur, Petropolis).

Where Organ Chips could also be helpful is to understand the blood-tissue and other biological barriers that play a pivotal role in regulating the transport of molecules within the body, including drugs from the capillaries to targeted organs. The blood-brain barrier is one area where it could provide value. Because it protects the brain from harmful compounds from the blood and ensures a homeostatic environment for the central nervous system, a chip that can model the blood-brain barrier could provide an invaluable key to drug development. Over the last decade a number of researchers have found a way to fabricate some promising chips to study the blood-brain barrier (Zakharova, do Carmo, van der Helm; Shuler Interview). One model published in 2017 by Shuler's team was one of the first to demonstrate in vivo realistic values of permeability in a human model with recirculating flow, and that this was sustained for 10 days. This provided a valuable tool for screening drug candidates (Wang, Abci, Shuler).

Beyond drug testing, Organ Chips could also help eventually eliminate the use of animals in basic scientific research. They are already being investigated for this purpose in cancer research. Until recently, most of the advances in understanding the processes involved in cancer metastasis were learnt by 'taking a piece of a patient's tumour and putting it into an animal, like a mouse, to see how it grows' (Biddle Interview; Yu, Stott, Toner). The advantage of Organ Chips is that they provide a means to study cancer cells taken directly from patients within a human-like environment in the lab.

Furthermore, Organ Chips provide some significant improvements in our modelling of cancers. In the past, researchers relied on growing individual tumours on Petri dishes to understand why each patient responds differently to drugs. Organ Chips offer a way to study cellular interactions at a far higher level of complexity and under much more controlled conditions than animal models. For instance, Peter Friedl, an expert in microscopical imaging of cancer at the University of Nijmegen, has been using engineered in vitro models to study the invasion and migration of cancer cells through human tissues (Beunk, Brown, Nagtegaal; Biddle Interview).

Meanwhile, at Harvard and Massachusetts General Hospital, a team led by the mechanical engineer Shannon Stott has taken advantage of the micro-vortices created by herringbone structures within a chip, observed first by Stroock and colleagues, to enhance the capture of extremely rare cancer cells circulating in the bloodstream of cancer patients. This work could provide earlier diagnosis and a better understanding of how cancer cells spread and kill (Stott, Hsiu, Tsukov).

Similarly Adrian Biddle and his research group at Queen Mary University of London are also investigating microfluidic chip devices to pick out cancer cells from the blood. This research should make it easier to characterise a patient's tumour to help tailor their treatment, thereby enabling more personalised medicine. Organ Chip technology offers a much more sophisticated means of investigating cancer development because it can measure more variables. Furthermore it provides a way to recreate the tumour microenvironment in vitro (Biddle Interview) as well as making it possible to investigate the interactions between cancerous cells in multiorgan systems (Sun, Luo, Lee).

As well as offering a new way to study cancer, Organ Chips are opening a new chapter to investigate complex human-microbiome interactions. Now thought to be pivotal to the process of aging, the immune system, modulation of the central nervous system, and a person's psychological well-being, the human microbiome is now considered one of the essential organs in the human body without which it cannot function (Marks, Sept 2018). In 2019 Ingber and his team reported that they had managed to create an experimental chip with human intestinal cells on which they cultured a complex human gut microbiome containing over 200 different types of microbes (Jalili-Firoozinezhad, Gazzaniga, Calamari).

In addition to the human microbiome, Organ Chip technology could provide a way to better understand infectious diseases. Importantly, it offers a way to model the native tissue microenvironment and mucosal surfaces lining the respiratory, gastrointestinal and urogenital tracts to study the role of the important immune defence system in preventing infection here and a means to understand the host-pathogen interactions (Baddal, Marrazo; Aguilar, da Silva, Saraiva). One such example is a bladder-on-chip which has also shown the technology to be a powerful tool for understanding antibiotic resistance in the case of urinary tract infection recurrences (Free). Such information is vital to improving treatment strategies which will be increasingly important given the rise in antimicrobial resistance (Marks, Nov 2020).

Similarly, in 2018 a group of British scientists led by Marcus Dorner at Imperial College found it was possible to reproduce all the steps of the life cycle of the hepatitis B virus in a liver chip lined with human hepatocytes. Able to be maintained for over 40 days, the chip provides a major step forward for understanding hepatitis B infection which every year claims the lives of more than 900,000 lives worldwide. More infectious than HIV, the hepatitis B virus is particularly worrying because its carriers initially show no symptoms but it can cause serious damage to the liver leading to premature death from cirrhosis or liver cancer many years later (Marks, May 2018). Importantly, the chip replicated similar biological responses to the virus as in a real liver which heretofore has been difficult to do in traditional in vitro models. It therefore provides a new means to dissect the key pathways that the virus uses to replicate and evade the human immune system, thereby opening up new targets for drug development (Ortego-Prieto, Skelton, Wai).

Organ Chip platforms are also now being deployed to better understand COVID-19. This includes the Lung Airway Chip and Lung Alveolus Chip developed by Ingber's group. Already used to study the influenza virus, the chips provided a means to study inflammatory responses within the lung to the SARS-COV-2 virus and confirm the safety and efficacy of drug compounds already approved for other conditions against the virus in animal models. One of these drugs is already in COVID-19 clinical trials across many sites in Africa (Si, Bai, Rodas) and an investigational new drug (IND) application has been submitted to the FDA to initiate COVID-19 trials in the United States for another drug found to suppress the cytokine storm (Bai, Si, Jiang). Having both a fluid channel and an air channel the chip can also be used to study the effect of drugs administered either as an injection or breathed through an inhaler (NIH; Brownell June 2020). Elsewhere, Milica Radisic working with Axel Guenther and Edmond Young at the University of Toronto have created tiny models of the nose, mouth, eyes, and lungs to investigate how COVID-19 invades the human body (University of Toronto). Lung alveolus and airway chips also laid the foundation for the discovery of 'a new class of broad-spectrum RNA therapeutics that induce a potent type I interferon response and inhibit infection by SARS-CoV2, SARS-CoV, MERS-CoV, HCoV-NL63 (a common cold virus) and multiple influenza A virus strains' (Si, Bai, Oh).

Another application Organ Chips offer is in the vaccine arena. A team of scientists at Sanofi Pasteur, for example, have patented an artificial immune system consisting of T cells, B cells, and antigen-primed dendritic cells for testing immune responses to vaccines (Warren, Drake, Moser). Ingber's group has also created a model of the human immune system in a microfluidic chip with which they made the unexpected discovery that B and T cells organise themselves into three-dimensional structures that resemble lymphoid follicles, which play a central role in activating the immune response. Furthermore, they found that the chip faithfully replicates human immune responses when vaccinated with a commercial flu vaccine (Irving). The attraction of the Organ Chip technology is it could potentially replace non-human primates as a more accurate and cheaper model for vaccine development (Ingber Interview; Goyal, Prabhala, Mahajan).

Issues

Organ Chips are often confused with the term 'organoids'. Both have 3D structures grown from stem cells with organ-level structures, but the Organ Chip is more heavily engineered with microfluidic channels lined with specific living human organ cells. While organoids provide a more realistic model of a developing tissue within an organ, its environment is less easy to control than an Organ Chip and it is difficult to study transport, absorption, or interactions among different types of tissues that comprise a living organ or with living microbiome (Salig Penn).

Currently, Organ Chip technology is still primarily being used at various stages in the drug development process or as a form of validating existing data. Shuler believes that true acceptance of the utility of Organ Chips will only happen once the first drug that was pre-clinically tested in an Organ Chip has been approved. One of the pivotal challenges is that regulators still need to see convincing data proving that Organ Chip produces better results compared to animal models or 2D cell culture (Shuler interview). The validation process is in itself highly complex and has yet to be fully standardised. Over the last few years the European Union Reference Laboratory for Alternatives to Animal Testing, established in 2011, has provided funding to help support validation studies for alternative methods to animal testing (European Commission). But the task remains so difficult that many companies, particularly small spin-outs with limited funding, often give up pursuing it. In this context it is the regulatory issues, rather than technical obstacles, that are hampering the innovation of Organ Chip technology (Low, Mummery, Berridge).

While the technology still needs to be improved to be fully reliable, some progress has begun to be made on the regulatory front. In April 2022 the pharmaceutical company, Sanofi Pasteur, announced that it had submitted a FDA Investigational New Drug (IND) application to launch clinical trials for a drug to treat rare neuromuscular diseases with data collected from a tissue chip containing two cell types: motoneurons, which transmit messages from the brain to muscles, and Schwann cells which help the signals to move more quickly. It was designed to mimic the biology of chronic inflammatory demyelinating polyneuropathy and multifocal motor neuropathy. The FDA had already been approved the drug for a different condition. This marked a major milestone as it was the first time a pharmacautical company used data from an Organ Chip to back up an IND. Critically the chip made it possible to see how the drug was working by evaluating how far the drug was slowing down the brain's messages to muscles (NIH 2022).

The development of Organ Chips requires input from a wide range of experts from different disciplines including stem cell biology, microfabrication, microelectronics, computer modelling, and liquid plastics. This can make the process expensive and also poses challenges in terms of standardisation, which can make validation difficult. At the moment, each research group and company uses their own approach and different materials to manufacture their chips. Chambers and plate sizes, for example, can vary, and diverse methods are used to feed the cell culture media into the chambers.

Organ Chips are reliant on having access to live human cells. Most human cell lines used in industrial in vitro assays are derived from adult donors. The advantage of these cell lines are they can be easily expanded, have relative stability, and can be stored for a long time. One of the first such cell lines was derived from a sample of cancer cells taken from Henrietta Lacks, who died from an aggressive form of cervical cancer in 1951. Found to have an extraordinary capacity to survive and reproduce, her cells became known as the HeLa cell line. Shared widely without Henrietta's consent or knowledge, her cells are now the 'workhorse of biological research' (Editorial). Human cancer-derived cell lines have proven useful for studying the biology of cancer and testing the efficacy of anti-cancer drugs. But not all cancer cell lines have equal value as tumour models (Gillet, Varma, Gottesman).

Thousands of human patient-derived cell lines from different organ/tissue types can now be ordered to use in Organ Chips (Marx, Andersson, Bahinski). Another method is to take a few primary cells from a healthy human donor. This can be useful in the case of creating a chip for personalised medicine to tailor a treatment to a specific patient. However, such cells are a limited resource and thus cannot be used to generate chips for multiple experiments over a number of years, or for carrying out drug screening. A more promising source is to use IPSCs. First pioneered in 2007, these cells have made the development of Organ Chips much easier in recent years. However, such cells are expensive and involve long complex protocols to create the differentiated cells needed for organ models, and often these cells do not fully develop into adult specialised cells (Knight Interview). Organoids also serve as an useful source of adult stem cells as they can be dissociated and cultured within Organ Chips, thus taking advantage of the best features of both technologies.

Many considerations go into the material used to create the microfluidic system or chip. It needs to be transparent to allow for real-time monitoring and detection of cell activity. It also has to be amenable to providing an integrated environment for cell maintenance. PDMS has particular attractions for cell culturing because it is non-toxic, transparent, and is permeable to gases. Its flexibility also gives it the capacity for cyclic stretching of cell culture surfaces. But the downside is its high permeability can sometimes lead to the compromising of results due to small molecules, such as tracers, drugs, toxicants, and water diffusion (Knight Interview; Balijepalli, Sivaramakrishan; Allwandt, Ainscough, Viswanathan). A number of new materials are now being developed for fabricating Organ Chips to help get over this problem but have yet to be widely adopted (Zhang, Korolj, Fook, Radisic).

Compared to a simple Petri dish experiment, a chip is quite expensive, but Organ Chips promise to be more cost-effective overall than conventional animal testing. Some idea of the difference in cost is offered by the Liverchip commercialised by C.N. Bioinnovations, which in 2015 cost US$22,000 with potential additional payments to the company depending on the success of drug testing. By comparison, the same year animal testing cost between US$6,500 and US$800,000 depending on the complexity and amount of time required for the testing. The costs associated with animal testing does not include the husbandry costs required for housing and caring for animals, which need to be constantly looked after and monitored by technicians (Knight Interview; Balijepalli, Sivaramakrishan).

Overall, Organ Chips are predicted to help reduce the cost of drug research and development by 10-26 percent (Franzen, van Harten, Retél). While this makes them an attractive option for larger pharmaceutical companies, they largely remain too expensive for smaller companies and academic researchers who have smaller budgets. Blank microfluidic chips offer a slightly cheaper solution. Supplied by various companies, academics and others can put their own cells inside such chips to build their own bespoke models (Knight Interview).

One of the reasons for the high price associated with Organ Chips is that companies have to spend a lot of money to develop the technology, so they need to find a way to recoup that cost. How much is charged for a chip depends on how complex it is. The big question now is working out just how much of the in vivo environment needs to be replicated on a chip. Much depends on what it is being designed to do. Martin Knight, the director of Queen Mary+Emulate Organs-on-chips Centre in London, illustrates the complexity of the issues to consider when designing a chip for researching arthritis. As he argues, 'We need a functioning human knee that moves and is loaded in the same way as our actual knees and that is aged in the same way as our bodies age, because we want to look at ageing as well. To do all of that would be unrealistic. It would take years of development and at the end of that you've only got a knee. What about all the other joints and all the other things?' (Knight Interview).

While development of Organ Chips has come a long way in the last decade, progress remains relatively slow. Knight argues that in part this is because 'it is not particularly fancy science, you're not necessarily going to win a Nobel Prize or publish a top paper if all you do is … work out how to make a model that replicates an aspect of a particular disease'. Another key obstacle is that much more research still needs to be done to demonstrate whether or not Organ Chip models can outperform existing simple in vitro models or animal models in predicting human physiology, disease, and response to therapeutics. Until this is done companies will 'prefer to go on using the existing models.' As Knight puts it, ' Until we can work out how to fund the development and validation of those models, progress will be slow' (Knight Interview).

Authors

This piece was written in April 2022 by Lara Marks, Daniel Power and Nomthandazo Ziba.

Acknowledgements

Many thanks go to Donald Ingber, Lindsay Brownell, Michael Shuler, Uwe Marx, Sebastien Farnaud, Martin Knight and Adrian Biddle for reading earlier drafts of this piece.

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Organ-on-a-chip: timeline of key events

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