Human organs-on-chips for disease modelling, drug development and personalized medicine

Lung

The human lung alveolus chip with two parallel microchannels separated by a microporous membrane (Fig. 1a) garnered great attention because it was the first organ chip to replicate complex integrated organ-level physiological and pathophysiological responses, rather than to simply demonstrate retention of cell- or tissue-level differentiated functions¹¹. This chip utilized an established human lung alveolar epithelial cell line (NCI-H441 or A549) interfaced with human umbilical vein endothelial cells. The study revealed that introduction of either living bacteria to mimic infection or nanoparticles to simulate inhalation of airborne smog nanoparticulates into the airspace of the epithelial channel of the two-channel chip resulted in recruitment of circulating immune cells and secretion of inflammatory cytokines, as occurs in vivo, and these processes could be visualized in real-time at high resolution. Perfusion of the cancer drug interleukin-2 (IL-2) at a clinically relevant dose through the endothelium-lined channel of this chip also led to vascular leakage and filling of the air channel with interstitial fluid and thrombi, replicating the pulmonary oedema toxicity it causes in some patients⁵². Moreover, the ability to replicate physiological mechanical cues revealed that breathing motions enhance inflammatory reactions and increase nanoparticle absorption across the alveolar–capillary interface; these findings were not observed in static transwell cultures¹¹. Moreover, mechanical stimulation was required for IL2-induced pulmonary oedema as well⁵². Importantly, in vivo studies carried out in parallel confirmed that all of these responses are also seen in whole lung in vivo. Another lung alveolus chip lined with human lung alveolar A549 epithelial cells was used to assess cytotoxicity responses to zinc oxide nanoparticles, which are used in biopharmaceutical preparations, drug delivery and biomedical imaging⁵³. This study revealed that these nanoparticles exhibited lower toxicity when administered under dynamic flow in the lung chip compared to static conditions.

A lung alveolus chip lined with primary human lung alveolar epithelium interfaced with primary pulmonary microvascular endothelial cells has been used to model pulmonary thrombosis by flowing human whole blood through its vascular channel. This approach enabled quantitative analysis of organ-level contributions to inflammation-induced thrombosis, and recapitulated complex responses, including platelet–endothelial dynamics within individual thrombi that were nearly identical to those visualized within thrombi that formed in vivo⁵⁴. Analysis of thrombosis formation on-chip induced by lipopolysaccharide endotoxin also revealed that it acts indirectly by stimulating the alveolar epithelium to produce other inflammatory molecules that induce endothelium activation, such as IL-6, rather than acting directly on endothelium.

Healthy and diseased human lung airways have been modelled by populating chips with primary bronchial or bronchiolar epithelial cells obtained from healthy donors or patients with chronic obstructive pulmonary disease (COPD) and culturing them under an air–liquid interface^39,55. Chips lined with COPD epithelial cells exhibited selective cytokine hypersecretion, increased neutrophil recruitment and mimicked clinical exacerbation by exposure to viral and bacterial infections as well as cigarette smoke^39,55. Transcriptomic profiles of healthy airway chips exposed to cigarette smoke closely resembled those obtained in past clinical studies. In addition, models of the asthmatic lung airway were developed by exposing healthy airway chips to IL-13, which induced goblet cell hyperplasia, inflammatory cytokine secretion and endothelial activation, while reducing cilia beating frequency, all of which are observed in asthmatic patients^39,56. The IL-13-stimulated airway chips also recruited increased numbers of circulating neutrophils under flow, which could be inhibited by administering an anti-inflammatory drug (bromodomain-containing protein 4 inhibitor), and this recruitment response was greater than observed in a static transwell microphysiological system³⁹.

More recently, lung airway chips have been used to model the cystic fibrosis airway by incorporating primary epithelium isolated from patients with cystic fibrosis⁴⁰. These chips accurately replicate many features of the human cystic fibrosis airway, including increases in cilia density, mucus accumulation, ciliary beating frequency and IL-8 secretion, leading to greater adhesion of circulating immune cells to the endothelium and their transmigration into the airway compartment compared to healthy chips. The cystic fibrosis chips also provided a more favourable environment for growth of Pseudomonas aeruginosa bacteria and an enhanced inflammatory response, both of which are major contributors to morbidity in patients with cystic fibrosis.

The ability of lung alveolus and airway chips lined by primary cells to support growth of human non-small-cell lung adenocarcinoma cells has also been studied⁵⁷. The cancer cells grew much more rapidly in the alveolus chip, which faithfully recapitulated the preferential growth at this site seen in patients with this type of cancer. Interestingly, the ability to control mechanical cues revealed that breathing motions suppress cancer growth and invasion in the lung, and that they alter the efficacy of a first-line anticancer drug (rociletinib). A more sophisticated version of this device, containing three parallel channels with an ECM gel lined with endothelial cells and fibroblasts that supports formation of 3D branching microvascular networks, enabled analysis of the response of human lung adenocarcinoma cells to clinically relevant concentrations of the chemotherapy drug paclitaxel and demonstrated both tumour and endothelial cell toxicities⁵⁸.

A human immunocompetent lung alveolus chip containing primary lung cells and peripheral blood mononuclear cells was used to assess the safety of T cell bispecific antibody cancer therapies³⁷. These chips effectively reproduced non-human primate (NHP) toxicities of T cell bispecific antibodies targeting folate receptor 1 that are currently under clinical development. Importantly, when a lower-affinity T cell bispecific antibody that was found to exhibit lower toxicity in the lung chip was tested in NHPs, none of the animals experienced the lung inflammation observed with the original higher-affinity antibody, thus confirming the safer profile predicted by this human organ chip. Currently, one of the most clinically relevant uses of human lung chips is in the study of respiratory virus infections. For example, infection of a lung alveolus chip lined by an established alveolar epithelial cell (HPAEpiC) line interfaced with a lung endothelial cell (HULEC‐5a) line with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) revealed preferential infection of the epithelium and stimulation of distinct innate immune responses in these two tissues, as well as recruitment of circulating immune cells, which led to endothelium detachment and further increased inflammatory cytokine release⁴¹. Treatment with remdesivir inhibited viral replication and alleviated barrier compromise on-chip. Human lung airway chips containing primary bronchial epithelium and pulmonary endothelium also recapitulated viral infection, cytokine production and recruitment of circulating immune cells, as well as virus strain-dependent virulence observed in human patients when infected with various influenza A virus strains⁴². Use of the model led to the discovery that co-administration of the anticoagulant drug nafamostat with the first-line antiviral drug oseltamivir doubled oseltamivir’s treatment-time window. Moreover, when these chips were infected with a pseudotyped SARS-CoV-2 virus and perfused with clinically relevant doses of the antimalarial drugs chloroquine or hydroxychloroquine, these drugs were found not to be active, thus predicting the negative results seen in clinical studies. By contrast, a related drug, amodiaquine, was shown to actively inhibit SARS-CoV-2 pseudovirus virus entry on-chip, as well as infection by native infectious SARS-CoV-2 in a hamster model. Importantly, these results contributed to the repurposing of this drug and its entry into clinical trials for COVID-19 in Africa, where this drug is used widely for antimalarial prophylaxis.

Another recent example of drug repurposing comes from use of the mechanical control capabilities of the primary lung alveolus chip, which revealed that physical forces associated with cyclic breathing motions influence lung innate immune responses to viral infection⁵⁹. This work led to the discovery that receptor for advanced glycation end products (RAGE), an inflammatory mediator that is most highly expressed in the lung alveolus in vivo, has a central role in this response. Moreover, when a RAGE inhibitor drug (azeliragon) was tested, it potently suppressed production of inflammatory cytokines and synergized with the antiviral drug molnupiravir in this model. These results were included in a recent submission of a pre-IND (Investigational New Drug) meeting application to the FDA to request initiation of clinical trials to test whether azeliragon can suppress the cytokine storm in patients with COVID-19. Use of lung alveolus and airway chips also led to 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 strains⁶⁰.

Virus evolution through both mutation and gene reassortment as seen in patients with influenza has also been reconstituted in vitro by sequentially passaging infected mucus droplets between multiple human lung airway chips infected with influenza H1N1 in the continued presence of the antiviral drugs amantadine or oseltamivir (also known as Tamiflu)⁶¹. This selective pressure led to the spontaneous emergence of clinically prevalent resistance mutations as well as previously unobserved strains. Interestingly, strains that were resistant to both drugs also emerged; however, studies revealed that they were still sensitive to the anticoagulant nafamostat, which targets host serine proteases.

Liver

Multiple liver chip designs have been developed and used to model drug metabolism, drug–drug interactions, hepatotoxicity, inflammation and infection (Table 1). A microfluidic liver chip with the multiwell bioreactor design (Fig. 1e) lined by primary human hepatocytes and Küpffer cells replicated breakdown of the glucocorticoid hydrocortisone to phase I and phase II metabolites, and the intrinsic clearance values measured on-chip correlated with human data⁶². A similar liver chip system was used to show that sustained stimulation of inflammation by IL-6 suppresses cytochrome P450 3A4 isoform (CYP3A4) activity, increases C-reactive protein secretion and decreases shedding of soluble IL-6 receptor⁶³. Treatment with a therapeutic anti-IL-6 receptor monoclonal antibody (tocilizumab) that is used to treat rheumatoid arthritis modulated CYP3A4 enzyme activities and altered metabolism of the small-molecule CYP3A4 substrate simvastatin hydroxy acid in this model, thus replicating the drug–drug interactions observed in patients, which was not possible using static 2D culture models.

Another interesting study explored the effects of human population variability on hepatic drug metabolism using multiple liver chips each lined by hepatocytes from a different donor⁶⁴. Analysis of metabolic depletion profiles of six drugs confirmed the existence of substantial inter-donor variability with respect to gene expression levels, drug metabolism and other hepatocyte functions. Importantly, clearance values predicted on-chip correlated well with those observed in vivo, and a physiologically based pharmacokinetics model developed for lidocaine successfully predicted the observed clinical concentration–time profiles and associated population variability.

One of the most valuable uses of liver chips has been to model human-specific hepatotoxicities (Table 1), which are often missed in preclinical animal models such as rodents or dogs. An early liver chip lined only by primary human hepatocytes but separated from flow by a microengineered porous barrier (to mimic the endothelial permeability barrier) exhibited mass transport properties similar to the liver sinusoid and reproduced the metabolism-specific hepatotoxicity of the anti-inflammatory drug diclofenac⁶⁵. Various cross-species liver toxicities (such as hepatocellular injury, steatosis, cholestasis and fibrosis) were also successfully reproduced in vitro by perfusing drugs with known species-specific toxicities through liver chips lined with multiple primary liver cell types (such as hepatocytes, liver sinusoidal endothelium, Küpffer cells and hepatic stellate cells) isolated from humans, dog or rat⁶⁶. The ability to visualize these responses at high resolution yielded insights into a novel toxicity mechanism in which one drug was found to unexpectedly target the endothelium, rather than a parenchymal cell.

As in the case of the lung, liver chips have been used to model viral infection in vitro. For example, infection of a liver chip lined by human hepatocytes with hepatitis B virus (HBV) reproduced all steps of the HBV life cycle, including replication of patient-derived HBV and maintenance of HBV covalently closed circular DNA⁶⁷. Moreover, innate immune and cytokine responses detected on-chip mimicked those observed in HBV-infected patients.

Heart

A heart chip was developed by culturing human iPS-cell-derived cardiomyocytes on flexible ECM gels overlaid on multi-electrode arrays in a single channel microfluidic device, which supported laminar cardiac tissue formation and enabled recording of tissue-level electrophysiological responses in real time⁵¹. This chip mimicked the difference in safety profiles between the cardiotoxic pro-drug terfenadine and its non-toxic metabolite fexofenadine seen in patients. Another heart chip created by combining microfabrication and 3D printing that contains human iPS-cell-derived cardiomyocytes interfaced with endothelial cells reproduced the toxic effects of the cancer drug doxorubicin on heart myocardium observed clinically²⁴.

Intestine

Numerous organ chip models of the small and large intestine lined by intestinal epithelial cells with or without underlying endothelium have been created to model various diseases as well as to study drug metabolism and toxicities (Table 1). The presence of dynamic fluid flow has been found to be necessary and sufficient to promote villi formation in small intestine chips^36,37,68 as well as production of high numbers of goblet cells and accumulation of a thick mucus bilayer in colon chips⁶⁹, which closely resemble those in living intestine using the two-channel chip design (Fig. 1a); however, additional application of peristalsis-like mechanical motions is required for optimal differentiation⁶⁸. Moreover, when lined with duodenal or colon organoid-derived epithelial cells, these mechanically active microfluidic chips more closely resemble human small and large intestine than organoids cultured in static microphysiological systems (either 3D ECM gel or transwell cultures) based on transcriptomic and histological characterization^36,37,68. In a study on infection with an enteric coronavirus (NL63), organoid-derived intestinal epithelial cells were found to also dramatically increase their ACE2 receptor levels when cultured under flow in the presence of peristalsis-like mechanical deformations in two-channel microfluidic intestine chips compared to when cultured statically as organoids in 3D gels or in transwell inserts⁷⁰. A two-channel human intestine chip lined by Caco-2 intestinal epithelial cells interfaced with endothelium was used to study SARS-CoV-2 infection and to reconstitute the morphological, structural and inflammatory changes consistent with gastrointestinal symptoms observed in patients with COVID-19 (ref.⁷¹).

The human intestine–microorganism interface was modelled in a two-channel microfluidic chip lined by established intestinal epithelial cell lines (Caco-2 or CRL-1459 cells), which recreates an oxygen gradient supporting the growth of a single commensal obligate anaerobe (Lactobacillus rhamnosus GG); this co-culture modified transcriptional, metabolic and immunological host responses in a manner similar to that previously observed in vivo⁷². However, the microorganisms had to be separated from the host epithelium by a nanoporous membrane in this chip and so only the effects of transported soluble mediators could be studied.

More recently, a living complex gut microbiome containing more than 200 different living bacterial species with similar complexity to a human stool microbiome was co-cultured in direct contact with human intestinal epithelium and its naturally secreted mucus layer for 5 days within two-channel intestine chips lined by either Caco-2 cells or primary human organoid-derived epithelium interfaced with primary intestinal microvascular endothelium⁴⁷. This was accomplished by establishing a hypoxia gradient across the epithelial–endothelial interface that mimics the one existing in vivo, which was confirmed using integrated on-chip oxygen sensors, and intestinal barrier function was either maintained or enhanced by the presence of living bacteria. By contrast, exposure of small intestine chips lined by Caco-2 intestinal cells interfaced with primary microvascular endothelium to pathogens can induce inflammation, cause cell death and disrupt the intestinal permeability barrier, as observed clinically in patients with inflammatory bowel disease⁴³. Use of a human colon chip lined by organoid-derived epithelium in combination with metabolomics was also used to identify specific microbial metabolites that mediate species-specific (human versus mouse) differences in sensitivity to enterohaemorrhagic Escherichia coli infection⁷³. Interestingly, cessation of cyclic mechanical deformations results in bacterial overgrowth and epithelial injury in the small intestine chip, hence mimicking the clinic disorder known as ileus, which is caused by intestinal paralysis (for example, due to prolonged anaesthesia)⁴³. By contrast, the presence of dynamic fluid flow and active peristalsis-like cyclic deformations enhance Shigella infection and invasion in a colon chip⁷⁴. It is important to note that none of these mechanobiological behaviours can be detected in static microphysiological systems, and bacteria cannot be co-cultured in these static models for more than about 24 h without inducing cell death^10,48.

An intestine chip model of acute radiation injury also replicated the same radiation dose sensitivity observed in humans, which differs from that observed in animal models⁷⁵. Radiation exposure induced reactive oxygen species production primarily by the endothelium, which resulted in epithelial cytotoxicity, apoptosis and DNA fragmentation, as well as disruption of tight junctions, villus blunting and compromise of the intestinal barrier; pre-treatment with a potential prophylactic radiation countermeasure drug (dimethyloxaloylglycine) suppressed these responses in this model.

An inflammatory condition of the intestine that is endemic in children in low-resource nations, known as environmental enteric dysfunction (EED), was recently modelled in human intestine chips⁷⁶. Key features of this disease, including villus blunting, compromised intestinal barrier function and reduced nutrient absorption, as well as impairment of fatty acid uptake and amino acid transport, were reconstituted in two-channel chips lined by intestinal epithelial cells isolated from EED patient-derived organoids when the cells were cultured in nutrient-deficient medium. These culture conditions led to transcriptional changes similar to those observed in clinical EED patient samples from multiple low-resource nations. Human intestine chips lined by organoid-derived cells and perfused with peripheral blood mononuclear cells also have been used to reproduce and predict clinical toxicities of a T cell bispecific antibody currently under clinical development that targets human carcinoembryonic antigen so specifically that neither its safety nor efficacy can be evaluated in animal models³⁷.

Kidney

Kidney chips lined by human renal tubular or glomerular cells have been used for studies on drug and molecule transport, reabsorption and toxicity as well as disease modelling (Table 1). For example, a two-channel kidney chip lined by primary proximal tubular epithelium that expressed high P-glycoprotein efflux transporter activity replicated a transporter-specific cisplatin toxicity that is observed in patients but not in static 2D cultures or animal models⁷⁷. Albumin reabsorption and cyclosporine toxicity were also recapitulated in a 3D-printed kidney chip containing human proximal tubular epithelium deposited within tiny cylindrical structures surrounded by a thick ECM gel²². This approach was extended further by printing closely apposed kidney tubules and vessels lined by proximal tubular epithelium and kidney endothelium within an ECM gel, which exhibited active reabsorption via tubular–vascular exchange of solutes similar to that observed in vivo²³. Hyperglycaemia-induced endothelial cell dysfunction was replicated in this model, as well as its reversal by administration of a glucose transport inhibitor drug. Moreover, a kidney distal tubule chip was used to explore pathogenesis of Pseudorabies virus-induced renal dysfunctions⁷⁸. Virus infection resulted in altered sodium reabsorption, disruption of the reabsorption barrier and changes in microvilli, which may contribute to the serum electrolyte abnormalities observed in virus-infected patients.

Importantly, a kidney proximal tubule chip also replicated phenotypic features of two rare X-linked monogenetic diseases (Lowe syndrome and Dent II disease), which are characterized by renal reabsorption defects caused by mutations in the OCRL gene⁷⁹. Knocking down OCRL expression in the cells revealed that proximal tubule cells lacking OCRL upregulate collagen deposition, which can contribute to the interstitial fibrosis and disease progression seen in these disorders.

Use of a human kidney glomerulus chip containing closely opposed layers of immortalized kidney podocytes and glomerular endothelial cells revealed that glomerular mechanical forces play a crucial part in cell damage that leads to increased glomerular leakage, as observed in patients with hypertensive nephropathy⁸⁰. Another glomerulus chip lined by human iPS-cell-derived podocytes interfaced with glomerular endothelium reconstituted in vivo levels of urinary clearance and mimicked the toxic effects of the anticancer drug adriamycin on kidney podocytes⁸¹. More recently, a personalized version of this chip was developed using both human iPS-cell-derived kidney glomerular endothelial cells and podocytes from a single patient⁸². In addition, the renal effects of autoimmunity have been studied using a human kidney glomerulus chip that recapitulates the permselectivity of the glomerulus⁸³. When exposed to patient sera containing anti-podocyte autoantibodies, the chips developed albuminuria proportional to patients’ proteinuria, and this phenomenon was not seen using sera from healthy controls or individuals with primary podocyte defects.

Brain

Parkinson disease is a disorder of the substantia nigra region of the brain that is characterized by abnormal accumulation of α-synuclein aggregates, loss of dopaminergic neurons and proliferation of glial cells. A two-channel human brain chip representative of this region was created containing iPS-cell-derived dopaminergic neurons, astrocytes, microglia and pericytes interfaced with iPS cell-derived microvascular brain endothelium⁸⁴. Transcriptomic analysis revealed that gene expression levels on-chip were much closer to those seen in the mature adult substantia nigra than in conventional culture systems. Importantly, introduction of α-synuclein fibrils, key components of Lewy bodies, resulted in their accumulation and phosphorylation, which is also observed in vivo. This phenotype was accompanied by an increase in reactive oxygen species production, mitochondrial dysfunction, cell death and neuroinflammation, which are all key features of Parkinson disease.

Many human blood–brain barrier (BBB) chip models of the neurovascular unit have been developed (Table 1), because the BBB serves as a key barrier to the delivery of many neuroactive therapeutics and plays a central part in many neurological diseases. These two-channel chips contain a brain endothelium interfaced with either brain pericytes and astrocytes, or astrocytes and neurons; some are lined with one or more cell types derived from iPS cells^84,85,86, whereas others use primary cells⁸⁷. BBB chips have been shown to reconstitute a human in vivo-like permeability barrier (for example, when analysed using transepithelial electrical resistance), which is critical for modelling healthy and diseased functions of neurovascular units^84,85,86. They have also been used to model neuroinflammation^84,87 and protection of neurons from injury when the vascular channel is perfused with human whole blood⁸⁶, as well as shuttling of drugs, hormones, monoclonal antibodies, nanocarriers and tumour extracellular vesicles across the BBB^{85,86,88,89,90}. Moreover, when BBB chips were created using brain endothelium from iPS cells derived from a patient with Huntingdon disease, inter-individual variability in BBB permeability could be detected, whereas this was not observed in chips made with cells from healthy donors⁸⁶. This study used a similar approach to model monocarboxylate transporter 8 (MCT8) deficiency, a severe form of psychomotor retardation associated with a reduction in thyroid hormone transport across the brain endothelium, and this clinical phenotype could be replicated on-chip.

In a recent study, the human neurovascular unit was modelled in a microfluidic chip containing a perfused channel lined by endothelium interfaced with pericytes adjacent to a 3D ECM gel containing astrocytes and neurons derived by in situ differentiation of human neural stem cells⁹¹. This chip was used to model brain infection by the fungus Cryptococcus neoformans and revealed that clusters of the fungal cells penetrate the BBB without altering tight junctions, suggesting a transcytosis-mediated mechanism as well as providing a testbed in which to develop novel therapeutics.

Eye

An eye chip that reconstituted the outer retinal–choroid barrier containing human retinal pigmented epithelium adjacent to a perfusable 3D blood vessel network was able to recreate the choroidal neovascularization associated with wet macular degeneration by demonstrating penetration of the retinal pigmented epithelial monolayer by angiogenic sprouts that extended from pre-existing choroidal vessels⁹². This pathological angiogenesis was inhibited by the monoclonal therapeutic antibody bevacizumab, which is used clinically for this condition.

Furthermore, a retina chip was developed containing more than seven different retinal cell types, all derived from human iPS cells, which provided vascular perfusion and recapitulated interactions of mature photoreceptor segments with retinal pigmented epithelium⁹³. In addition to mimicking the formation of outer segment-like structures and establishing in vivo-like physiological processes of the eye (for example, outer segment phagocytosis and calcium dynamics), this organ chip reproduced the clinical retinopathic toxicities of chloroquine and gentamicin.

Bone

A bone chip containing human mesenchymal stromal cells, osteoblasts and bone marrow mononuclear cells embedded within human de-cellularized bone was used to model the release of dissolved metals (cobalt and chromium) from arthroplasty implants, which can lead to peri-implant bone loss⁴⁶. Indeed, both metals were found to integrate into bone matrix in this 3D model in a manner also seen in patient bone samples.

A bone marrow chip was created by incorporating human donor-derived CD34⁺ haematopoietic cells from marrow or blood and bone-marrow-derived stromal cells in a 3D ECM gel separated from a neighbouring endothelium-lined channel⁴⁵. This chip reconstituted differentiation and maturation of multiple blood cell lineages over 1 month in culture and replicated myeloerythroid toxicities in response to clinically relevant exposures to the cancer therapeutic 5-fluorouracil as well as to γ-radiation. Even more impressively, it was able to replicate regimen-specific toxicities of a cancer drug observed in human clinical trials when the drug exposure (pharmacokinetics) profiles previously measured in patients were recreated on-chip. And again, these responses were not observed when the same haematopoietic cells were placed in static cultures. In addition, personalized bone marrow chip models of a rare genetic disorder, Shwachman–Diamond syndrome (SDS), were created in this study using CD34⁺ cells obtained from patients with SDS. These chips replicated key haematopoietic defects seen in patients and led to the discovery of a previously unknown neutrophil maturation abnormality in a subpopulation of these patients.

Reproductive organs

A mammary gland chip lined by mammary duct epithelium cultured in an ECM gel in close proximity to a perfused endothelium-lined vessel underwent branching morphogenesis on-chip⁹⁴. Impressively, this chip was able to reproduce ductal changes associated with invasive cancer progression by amplifying HER2 (also known as ERBB2) receptor expression or expressing constitutively active PI3Kα in non-tumorigenic mammary epithelium using retroviral vectors.

A placenta chip lined by trophoblast cells interfaced with endothelial cells was used to screen drugs for their ability to cross the placenta⁹⁵. This model reconstituted the efflux transporter-mediated active transport function of the human placental barrier, which serves to limit fetal exposure to maternally administered drugs, using the gestational diabetes mellitus drug glyburide.

Blood vessels

Organ chip models of large and small blood vessels have been used to study various vascular disorders, including inflammation, thrombosis and atherosclerosis, as well as to model vascular contributions to numerous diseases (Table 1). Notably, a blood vessel chip lined by vascular endothelium and perfused with human whole blood reproduced the clinical thrombotic toxicity of a therapeutic monoclonal antibody drug (Hu5c8) that had been previously withdrawn from clinical trials owing to unexpected life-threatening complications that were not detected during preclinical animal testing³. This model was also used to validate the lower thrombotic risk of an improved version of a related antibody that incorporates an Fc domain that does not bind the FcγRIIa receptor.

Hutchinson–Gilford progeria syndrome is a premature ageing disease characterized by accelerated death due to cardiovascular disease, particularly involving mechanically active blood vessels. When a progeria blood vessel chip was developed using vascular smooth muscle cells derived from iPS cells of patients with progeria, it exhibited exacerbated inflammation and DNA damage in response to mechanical strain compared to chips lined with cells from healthy donors⁹⁶. Interestingly, this physical stress-induced injury could be reversed by administering two drugs, lovastatin and lonafarnib, which are often used clinically for this disease.

An aorta chip was used to study the development of the more common congenital disorder aortic valve disease⁹⁷. Chips lined by aortic vascular smooth muscle cells isolated from patients exhibited suppressed NOTCH1 expression and impaired mitochondrial dynamics, and genetic knockdown of NOTCH1 in healthy smooth muscle cells suppressed mitochondrial fusion and reduced contractility. Use of this model revealed that the mitochondrial fusion activator drugs leflunomide and teriflunomide can partially rescue the mitochondrial dynamics disorder in these diseased cells.

Some of the vascular applications that have been most pursued using human organ chip technology focus on the study of angiogenesis, inflammation and the role of neovascularization in a wide range of diseases, as well as transendothelial transport and delivery of nanomaterials (Table 1). For example, by incorporating 3D ECM gels into one microfluidic channel, nearby endothelium can be stimulated to extend capillary sprouts in a directional manner; if endothelia are cultured in two parallel channels separated by ECM, the invasive sprouts will meet, differentiate into hollow tubes and form a fully perfused 3D capillary network^15,58. Recently, this approach was adapted to demonstrate endothelial sprouting induced by primary human patient-derived renal cell carcinoma cells from multiple donors that exhibited patient-specific patterns of angiogenic factor production¹⁸. A similarly vascularized tumour chip revealed that gene expression, tumour heterogeneity and therapeutic responses observed on-chip more closely model colorectal tumour clinicopathology than do current standard drug screening modalities, including 2D cultures and 3D spheroids⁹⁸. A three-channel microfluidic device that mimics the vascular–tissue–lymphatic interface has also been developed to study lymphangiogenesis associated with cancer metastasis¹⁹.

Lymphoid organs

Inflammatory reactions including recruitment and activation of immune cells, release of pro-inflammatory cytokines, tissue barrier disruption and cell injury have been reproduced in many organ chips (Table 1). However, it is possible to model more complex human immune responses using organ chip technology as well. A lymphoid follicle chip was recently developed that cultures peripheral blood-derived primary human blood B and T lymphocytes within a 3D ECM gel in one channel while flowing medium through a parallel channel⁴⁴. Superfusion in this manner prevents the lymphocyte autoactivation observed in conventional cultures and promotes the formation of germinal centre-like structures that contain B cells that undergo antibody class switching and plasma cell differentiation upon activation with antigens. When autologous monocyte-derived dendritic cells were integrated into the gel and the chips were inoculated with a commercial influenza vaccine, increases in follicle size and number, plasma cell formation, production of anti-haemagglutinin IgG antibodies and secretion of cytokines similar to those observed in vaccinated humans were detected over clinically relevant timescales.

Organ chips have also been used to evaluate immunotherapies, including personalized therapies when used with patient-derived cells. For example, small-molecule inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) were identified as potential novel immune checkpoint inhibitors using a chip in which patient-derived tumour cell spheroids containing autologous infiltrating immune cells were cultured within 3D ECM gels surrounded by microfluidic channels⁹⁹. These CDK4/6 inhibitors augmented the response to PD-1 blockade, thus providing a rationale for combining these agents with other immunotherapies to enhance therapeutic efficacy.

The tumour microenvironment imposes significant constraints on the anticancer efficacy of adoptive T cell immunotherapy, which cannot easily be modelled in conventional cultures. To address this challenge, human liver cancer cells were cultured in an organ chip inside a 3D ECM gel, and human T cells engineered to express tumour-specific T cell receptors (TCRs) T cells were flowed through an adjacent channel¹⁰⁰. The TCR T cell ability to migrate and kill tumour targets was analysed, as well as the influence of varying levels of oxygen and inflammatory cytokines. This approach could be used in the future to design and evaluate personalized cellular immunotherapies, for example, using 3D-printed organ chips containing patient-derived tumour biopsy samples that enable real-time monitoring of the responses of resident lymphocyte populations¹⁰¹.

Multi-organ physiological coupling and drug PK/PD modelling

One of the challenges in drug development that most pharmaceutical scientists assume is likely not to be solved in the near term is the need for animal testing to assess drug disposition (for example, ADME) in the whole body and to quantify PK/PD parameters that help to guide clinical trial design. Although animal models may still be required for these purposes for some years to come, recent work suggests that many of these complex whole-body physiological responses may be modelled in vitro using fluidically coupled multi-organ human body-on-chips systems. Multi-organ chip models developed so far, which may have from 2 to 10 different organ chips, have provided impressive mimicry of complex physiological and pathophysiological responses in addition to providing new in vitro tools with which to assess drug ADME and PK/PD (Table 2).

In one impressive example of complex physiological mimicry, dynamic inter-organ hormonal coupling and the 28-day menstrual cycle were reconstituted in vitro by fluidically linking human organ chip models of uterus, cervix, fallopian tube and liver to a mouse ovary chip and recirculating the medium²⁵. Coupled organ chip systems have also been used to model multi-organ regulation of insulin secretion^30,102; metastatic spread of cancer between different organs¹⁰³; targeted immune responses to organ-specific damage¹⁰⁴; and the toxic effects of environmental chemicals that required hepatic bioactivation¹⁰⁵, including activation of germ cell toxins in linked liver and testis chips³¹.

The most common application of multi-organ chip systems has been to replicate the absorption (for example, oral or transdermal), distribution and clearance of drugs through metabolism or excretion that naturally occur in the organs of our body, but which are absent in single organ chips or other in vitro models (Table 1). Small-molecule and biologic drug efficacies, as well as both on- and off-target toxicities that can be modified by inter-organ interactions (via hepatic activation or metabolism), have been demonstrated too, and the results generated generally agree with published human data^{32,34,35,106,107,108,109}. In addition, organ chips have been coupled to assess drug efficacy and safety simultaneously. For example, by measuring the effects of anti-EGFR therapeutic monoclonal antibodies in a lung cancer chip while analysing toxicity in a fluidically coupled skin chip, it was possible to replicate the inhibition of keratinocyte growth and altered expression of CXCL8 and CXCL10 seen in patients¹¹⁰. As another example, a clinically relevant drug–drug interaction between the arrhythmogenic gastroprokinetic drug cisapride and the fungicide ketoconazole was reproduced using a liver chip linked to a heart chip when both were lined by cells derived from the same human iPS cell line¹¹¹, which shows the potential of using organ chips to optimize therapeutics selection in a personalized manner.

Microfluidic systems composed of multiple culture chambers in which medium is flowed directly from one parenchymal cell compartment to another via a single channel have been used to carry out PK/PD modelling in vitro for many years, although established cell lines were usually used in early studies³³. Later versions separated the flow chamber from the culture chambers with an intervening porous membrane or used transwell inserts as culture chambers^{26,27,28,29,30} (Fig. 2a). A body-on-chip system of this type composed of linked liver, kidney, intestine, skeletal muscle and BBB chips generated predictions of drug metabolism and barrier penetrance that were consistent with clinical data¹¹²_. But it is only recently that this type of modelling has been carried out in organ chips lined by primary human cells that are fluidically linked by flowing or transferring a common blood substitute medium through endothelium-lined vascular channels that are separated from parenchymal channels or chambers, as occurs in our bodies^{13,31,32,34,35} (Fig. 2b,c). This more physiologically relevant approach has been used in combination with computational modelling to generate predictions of drug PK/PD parameters (Fig. 3) and toxicities in many studies (Table 2). For example, it has been possible to quantitatively predict human clinical pharmacokinetics parameters using a multi-organ chip first-pass model composed of linked two-channel human liver, kidney and intestine chips along with an AV reservoir in combination with computational physiologically based pharmacokinetics modelling³⁵ (Fig. 3b). Successful in vitro to in vivo translation of pharmacokinetics parameters closely mimicked results observed clinically (Fig. 3c) and this was carried out using two drugs (cisplatin and nicotine) administered via two different routes (intravenous versus oral). In addition, when the intestine chip was replaced by a bone marrow chip, predictions of cisplatin pharmacodynamics also matched previously reported patient data³⁵. As these body-on-chip systems are modular, the chips can be linked either in series or in parallel, and the flow can be either unidirectional^{13,30,34,35,102,103,105,111,112} or recirculated^{25,27,28,31,32,104,106,107,108,109,110,113}.