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In this Emulate guest blog, Vivek Thacker, PhD, Group Leader at University Hospital Heidelberg, explains how he and his team at his previous organization, École Polytechnique Fédérale de Lausanne, used the Emulate Lung-Chip to study how bacterial “cords” contribute to the pathogenesis of Mycobacterium tuberculosis.

What is tuberculosis, and what makes it so interesting to study?

Tuberculosis (TB), an infectious bacterial disease, is humankind’s oldest and deadliest foe, still responsible for roughly 1.5 million deaths per year globally. The causative bacterium, Mycobacterium tuberculosis (Mtb), has co-evolved with humans over thousands of years, making TB a chronic disease with a wide spectrum of manifestations. Today, the most common treatment is a multi-drug antibiotic regimen that needs to be administered for several months, leading to poor treatment compliance and an increasing profile of drug resistance.

Interestingly, many individuals in high-risk countries are frequently exposed to Mtb but do not go on to develop TB or adaptive immune responses to Mtb antigens. This suggests that it is important to understand how the human innate immune system effectively halts disease progression. As such, my team and I wanted to characterize Mtb’s earliest interactions with epithelial cells and macrophages in the lung, study how Mtb adapts to the lung microenvironment, and investigate what causes tissue resident immune cells to succeed or fail in their quest to contain infection.

What makes tuberculosis so challenging to study?

TB is spread by aerosols, meaning that the earliest host-pathogen interactions should be single-cell interactions in the vast alveolar spaces of the lung. However, early TB activity in these alveolar spaces is relatively understudied, as it is difficult to isolate a few hundred bacteria in the lungs of any animal model, and it is even more challenging to study the spatiotemporal dynamics of host-pathogen interactions, particularly in BSL-3 conditions.

How did Organ-Chips allow you to overcome these challenges?

These challenges are perfectly suited to Organ-Chips, which fit into the large space in the “middle”—that is, they recapitulate complex cellular interactions that do not occur in simple model systems, allowing us to study the mechanisms of early-stage bacterial exposure and response with high spatiotemporal resolution. As you can imagine, this opens many new research avenues.

Organ-Chips are invaluable for discovery research, allowing us to identify new phenomena or roles for certain cell types in specific host niches. On top of this, the modular nature of this technology allows us to perturb only specific cells in a co-culture for targeted studies. Many “traditional” assays can also be made more precise and quantitative—e.g., fast and accurate measurements of bacterial killing and regrowth after antibiotic treatment. Organ-Chips also enable new kinds of measurements, such as in situ electron microscopy.

Can you describe the model you developed and the insights it yielded?

The Lung-Chip recreates the physiological air-liquid interface seen in alveolar biology while being accessible to long-term live-cell imaging. Using this model, we found that pulmonary surfactant secreted by epithelial cells protected the host from Mtb infection, rendering many Mtb unable to grow in host cells. However, exposure to surfactant in turn stimulated the bacteria to produce lipids that survived this first interaction, leading to the formation of biofilm-like cords in host cells that help Mtb bacteria evade clearance by antibiotics.

The consistent growth of Mtb cords in the Lung-Chip led us to search for these structures in the early stages of infection. To do this, we designed an experimental strategy wherein we imaged thick tissue slices with confocal microscopy in order to search for these cords in situ. We were then able to identify cords in an animal model, not only providing crucial validation but also expanding the predictive reach of the Organ-Chips. This provides a roadmap for future iterative development of the Lung-Chip to explore more aspects of Mtb pathogenesis.

How have these findings changed your research, and what does the future look like for you?

Clearly demonstrating that cords play a role in Mtb pathogenesis opens several new research avenues. In future studies, we will seek to understand how the mechanical rigidity of these structures affects immune function as well as focus on dissecting this complex host-pathogen interaction, with the aim to develop better protective strategies. We will also focus on gaining a better understanding of antibiotic responses within specific tissue microenvironments. Using Organ-Chips, we can study how specific tissue niches and barriers change the “effective” antibiotic concentrations that bacteria may experience and how these antibiotics may be processed differently within these bacteria (e.g., restricted access due to tight packing in cords).

Learn more in the full article published in Cell.

The Origins of Modern Cell Culture

The tone of Robert Hooke’s writing in 1665 was one of unmistakable glee. Hooke, having recently constructed a rudimentary but powerful microscope, was now able to document in detail the underlying components that afforded plants, rocks, and animals their functional properties. To Hooke, this newfound world held the key to understanding the nature of life.  

“And could we so easily and certainly discover the [design] and texture even of these films and of several other bodies…we might as readily render the true reason of all their phenomena.”  

Driven by this potential, Hooke proceeded to catalogue the microscopic world around him. His work, documented in Micrographia, not only introduced the term ‘cell’, but also demonstrated the benefits of meticulously studying the cellular world, thus laying the groundwork for modern cell biology.  

Today, cell culture, which involves growing cells outside of their native organism, is a foundational tool in the molecular sciences. Cells that have been isolated from a patient or immortalized long ago allow researchers to study various aspects of the human body, using the cells as proxies for the larger organs they hail from. However, deriving valuable information about an organism from its cells is far from simple. Cells isolated from the human body (in vitro) are unlikely to behave naturally, and those within the body (in vivo) are often difficult to observe. There have been significant strides towards understanding the mechanics of life and disease at the cellular level, and recent advances in the technology used to study cells are opening up even newer possibilities.  

In vitro cell culture involves growing living cells in a highly controlled, non-living environment. In vitro is Latin for “in the glass,” and as the name suggests, this kind of cell culture is most often carried out by growing cells in a two-dimensional (2D) plane on glass or plastic petri dishes. These cells may be sourced directly from patients (known as patient-derived cells) or collected from a patient long ago and subsequently engineered to enable long-term propagation (so called immortalized cell lines).  

In Vitro Cell Culture Advantages and Disadvantages

Advantages 

• Growing and maintaining cells is relatively inexpensive. 

• Researchers gain a high degree of control over the cell’s environment, maintaining control over the nutrients, temperature, and other variables that cells are exposed to.  
• These cells are often far easier to observe through a microscope, enabling high-content studies of the cell’s behavior. 
• This approach is often amenable to high-throughput applications, such as drug or functional screening. 

Disadvantages 

• The in vitro environment is far removed from the cell’s natural environment in the human body, where cells experience three-dimensional contact with proteins and other cells, biomechanical forces, as well as dynamic nutrient and waste gradients. Each of these factors can influence how the cell behaves. By removing cells from a complex environment to a far more simplified one, the translational value of in vitro cell culture diminishes.  
• In vitro cell culture may result in artificial mutations that cause cells to behave abnormally. 

In Vivo Studies: Advantages and Disadvantages

In vivo, meaning “within the living,” involves studying cells within their native organism. This method provides the most accurate representation of how cells behave in their physiological context. In vivo studies have long been viewed as the gold standard for understanding complex interactions within tissues, organs, and systems, as well as for assessing the real-world effects of drugs and treatments. This is largely because of the aforementioned drawbacks of traditional in vitro cell culture systems.  

However, in vivo studies come with a significant cost. The in vivo environment is inherently more complex, making it difficult to isolate the effects of specific variables on cell and organismal behavior. Additionally, many in vivo studies are performed in model organisms, such as rats and dogs, to predict how the human body will respond to disease or therapeutics. The genetic and physiological differences between animals and humans can erode the accuracy of these models. This is particularly hazardous in preclinical drug safety testing, where inaccurate predictions can lead to dangerous compounds advancing into the clinic.  

Bridging the gap with more advanced in vitro systems  

Recent advances to in vitro cell culture are helping to alleviate the challenges researchers have faced historically, particularly by incorporating more complex, in vivo-relevant environments. The most powerful example of this comes from Organ-on-a-Chip technology, also known as “Organ-Chips.” 

Organ-Chips are advanced, three-dimensional in vitro culture systems that closely mimic the natural environment of a cell. Specifically, these systems expose cells to biomechanical forces, dynamic fluid flow, and heterogenous cell populations while providing three-dimensional contact with proteins or other cells. Collectively, these features encourage the cells to behave as they would in a living organism, thus greatly improving researchers’ ability to accurately study the behavior of cells in vitro. And, unlike many in vivo model systems, Organ-Chips can be constructed with human cells, thus circumventing the interspecies differences that plague many in vivo systems. As such, Organ-Chips are now being used in a variety of applications to help researchers study cell behavior with increasing accuracy. 

Cell biology has evolved considerably since Hooke’s discovery, progressing in ways that allow researchers to study the cellular world with ever greater accuracy. That progression has often been catalyzed by technological development, from Hooke’s microscope to the advent of Organ-on-a-Chip technology. 

Commonly Asked Questions About Modern Cell Culture 

What is Ex Vivo Cell Culture?

Ex vivo cell culture is a form of in vitro cell culture that uses cells or tissue freshly collected from a living organism. Ex vivo cell culture may be particularly valuable when studying patient-specific conditions or when working with mature, terminally differentiated cell types that are difficult to produce with stem cells.  

Are in vitro studies reliable? 

Yes, to an extent. In vitro studies can be extremely valuable tools for studying cell biology and human physiology. While the traditional in vitro cell culture environment is very different from an in vivo environment—and thus cells are less likely to behave as they would in vivo—many cellular phenotypes are quite robust. This means that some cell behaviors in vitro do accurately represent how cells in vivo would behave. However, the reliability of in vitro studies can be greatly improved by using more complex culture systems, such as Organ-Chips, that faithfully emulate the cells’ natural environment.  

What is a typical in vitro study design? 

In vitro studies can be vastly different in design and scale; therefore, there is no single template for an in vitro study. However, there are common elements within subdisciplines. For example, in vitro studies for pharmaceutical development often use human cell lines (for larger scale studies, immortalized cells are common early in the drug development process). These cells may be grown in 96-well plates that allow for automated handling. Candidate drugs may then be added, one to each well of cells, followed by a period of observation. The cell’s ability to survive, proliferate, or release various signaling factors may be measured (among many potential response mechanisms).  

In March 2020, I was fortunate to join Emulate, intrigued by one of those rare opportunities to bring truly game-changing technology to healthcare. 

Five days later, the world shut down.  

Every day since has been motivated by a vital mission to leverage human biology and technology to ignite a new era in human health, driven by a team of people with an unwavering commitment to bring a human-centric approach to biological innovation.

As we were coming to terms with our new normal, our team’s focus and action were nothing short of miraculous. Undaunted, and united under a single vision, the team persevered. We found new ways to move forward with a collective strength to overcome challenges. We embraced new processes to shatter unexpected roadblocks. And we responded to the needs of our customers to ensure their science could keep moving forward.  

We grew our team, adding deep commercial and functional expertise to implement structure and processes in order to rapidly scale. The scientific team has grown to include researchers focused on our application roadmap areas such as immunology and microbiome. We expanded our leadership team with Donald Ingber, MD, PhD, a scientific founder of Emulate and Chairman of its Scientific Advisory Board – and arguably one of the great pioneers in the field of biologically inspired engineering – joining our Board of Directors. Also, Scott Kantor accepted the role of Chief Financial Officer and Larry Weiss came on as Chief Legal Officer.  

Over the past 18 months, we announced our roadmap for expanding Organ-Chip applications with a focus on developing targeted and advanced biologics. Additionally, we released a series of breakthrough applications and organ-models, such as the Brain-Chip and the Colon Intestine-Chip, deepening our portfolio of the human-relevant Organ-Chips that helped solidify our commitment to scientific excellence. 

We have bolstered our leadership team, accelerated product development goals, and seen healthy growth in demand for our products. Several leading indicators validate our belief that organ-on-a-chip technology will dramatically transform the entire drug discovery and development pipeline and ultimately eliminate unnecessary animal testing.

Jim Corbett, Emulate CEO

Aside from our product and commercial developments, we have taken a stronger stance against animal testing, as there is scientific proof that it has significant  limitations in effectiveness – particularly for today’s novel drugs and vaccines. In response to a New York Times article, we championed legislation reducing the use of primates for drug testing. More recently, the FDA Modernization Act and the Humane Research Act have been introduced to Congress to rally support for alternatives to animal testing, citing scientific inadequacies and ethical concerns associated with it. Our team also joined the North American 3Rs collaborative and believe that moving in the right direction means encouraging the use of more accurate and humane technologies. We truly believe that organ-chips will become an integral part of drug development and has the potential to ultimately eliminate animal testing.

This level of achievement would be astonishing in normal times and no doubt helped propel our fundraising efforts. Today, we announced the close of an $82 million dollar round of financing, which will enable Emulate to expand research and development efforts to new heights. We will scale-up R&D activities to facilitate the creation of new human-relevant organ-on-a-chip models in immunology, neuroinflammation, tumor modeling, and more. To help meet growing global demand, we will extend operations in the Asia Pacific region with new distributor relationships. 

I am excited to see what this team will accomplish as we set out into a changed world, continuing to scale and meet global demand as well as allow researchers to predict human response better than conventional cell culture and animal-based experimental testing. This biological revolution is just getting started. Let’s go! 

A summary of the paper published in Nature Biomedical Engineering: On-chip recapitulation of clinical bone marrow toxicities and patient-specific pathophysiology.

Human bone marrow is the body’s major site of hematopoiesis, the proliferation and differentiation of all blood cellular components from hematopoietic stem cells. These actively dividing and maturing cells are highly sensitive to toxic stimuli and, thus, bone marrow is one of the major targets of unwanted toxicity caused by drugs or therapeutic agents. Predicting drug-related hematotoxicity with clinically relevant pharmacokinetics would be extremely valuable for designing safety assessments and human trials. 

Studying bone marrow tissue is particularly difficult due to its inaccessibility and the limited number of physiologically relevant in vitro models that can support growth and maintenance of bone marrow cultures. Current hematopoietic toxicity studies are performed using methylcellulose colony cultures in which cells are suspended and can be exposed to higher-than-usual concentrations of drugs for extended periods of time. To address the unmet need for a predictive model to study hematopoietic dysfunction and pathophysiology of bone marrow, the authors created a Bone Marrow-Chip (BM-Chip) to recapitulate relevant hematopoietic cell types in a three-dimensional (3D), mechanically active next-generation in vitro model. This model possesses relevant levels of complexity to closely mimic the in vivo environment to examine the effects of known myelotoxic stressors and to investigate a genetic condition resulting in bone marrow failure.

• Research Area: Drug development, Toxicity testing, Rare Disease
• Organisms: Human
• Sample Types: Human bone marrow-derived mesenchymal stromal cells, hematopoietic stem/progenitor (CD34+) cells, umbilical cord vascular endothelial cells
• Research Question: Can a Bone Marrow-Chip recapitulate the features of human hematopoiesis and dysfunction resulting from genetic mutations and exposure to drugs and radiation?

 

Experimental Overview

Characterizing the Bone Marrow-Chip

Researchers created the BM-Chip using human bone marrow-derived stromal cells (BMSC) and CD34+ cells co-cultured in a fibrin gel in the upper channel of the chip. The lower channel of the chip contained human umbilical vein endothelial cells (HUVEC) with unidirectional continuous flow of culture medium containing cytokines that support cell growth (stem cell factor, Flt3 ligand and thrombopoietin) as well as factors that encourage multilineage differentiation (granulocyte colony stimulating factor and erythropoietin). Compared to the same cells grown in static models in suspension and in 3D gel, the BM-Chip increased the numbers of proliferating myeloerythroid and neutrophil progenitors, demonstrating increased cell survival and robust hematopoiesis in vitro over prolonged culture time (28 days).  

Schematic of bone and insert micrograph showing normal human bone marrow histology. Left middle: schematic of the cross-sectional view of the human BM-Chip on day 0 after seeding, showing singly dispersed CD34+ progenitors and BMSCs in a gel in the top channel and an incomplete vascular lining (seeded on either day 0 or day 8) in the bottom channel. Right middle: within 2 weeks of culture initiation, endothelial cells grow to cover all four sides of the lower channel and create a vascular lumen, while CD34+ cells undergo expansion and multilineage differentiation. Right: immunofluorescence image of a vertical cross-section through the gel in the upper channel of the BM-Chip taken at day 14 (magenta: erythroid lineage; yellow: megakaryocyte lineage; blue: neutrophil and other hematopoietic lineages). 

Modeling Drug-Induced Hematotoxicity in the Bone Marrow-Chip

To determine if the BM-Chip could be used to model human bone marrow pathophysiology, the researchers exposed the chips to 5-fluorouracil (5-FU) at dosage-appropriate plasma concentrations to observe toxic effects. BM-Chips from six different CD34+ donors infused with 5-FU through the vascular (bottom) channel consistently showed hematotoxicity at clinically relevant concentrations, whereas suspension and static gel co-cultures did not.

In the next set of experiments, the researchers challenged the BM-Chip with AZD2811, a small-molecule inhibitor of Aurora B kinase that is critical for proper cell division and is currently in phase II clinical development as a cancer therapeutic drug. In earlier phases of clinical trials, AZD2811 has been shown to selectively target dividing neutrophil and erythroid cells with varying effects in a time and dose-dependent manner. The authors used the BM-Chip to explore these effects on both mature and progenitor cell types and provide insight into the drug’s mechanisms of action. 2- and 48-hour AZD2811 infusions resulted in dose-dependent toxicities of the erythroid lineage, with much greater effect following the 2-hour infusion regimen than the 48-hour regimen. In the neutrophil lineage, dose-dependent toxicity in the BM-Chip was greater with the 48-hour infusion. Importantly, both regimens revealed maturation-dependent sensitivity to the treatment via selective decrease in immature cells of both lineages compared to mature cells.

Suspension cultures of CD34+ cells failed to replicate the dose-dependent erythroid toxicity that was successfully modeled by the human BM-Chip, suggesting that it can more accurately predict hematological effects at clinically relevant drug exposures than static culture models.

Radiation and Bone Marrow Recovery

Building on the observation that immature, proliferating cell types were selectively affected by AZD2811 treatment, the authors tested maturation-dependent toxicity caused by ionizing radiation. Radiation treatments are widely used as a cancer therapy, and they commonly cause bone marrow depletion – a reduced production of blood cells from all lineages. The authors noted that the BM-Chips showed decreased numbers in all cell types studied that correlated with increasing doses of radiation. These experiments show that the BM-Chip is a useful model of human-specific radiosensitivity, inviting more detailed analyses of radiation-associated cell death.

The researchers went on to demonstrate that the BM-Chip can also be used as an in vitro model to examine bone marrow recovery and response to potential therapeutics using human-relevant pharmacokinetic profiles during preclinical drug evaluation, development, and regulatory assessment, which cannot be done using animal models.

Modeling Rare Conditions: Shwachman-Diamond Syndrome

Shwachman–Diamond syndrome (SDS) is a genetic condition that causes bone marrow depletion and failure, resulting in neutropenia. The extent of hematological dysfunction may involve other cell lineages as well, leading to myeloid malignancies in some SDS patients. Understanding SDS has been limited by a lack of animal models that faithfully recapitulate the condition. In this study, the authors cultured CD34+ cells from two patients with SDS alongside normal BMSCs and endothelial cells to understand the clinical manifestations of the syndrome using this disease model.

The researchers noted that the cells in the SDS BM-Chips exhibited defective hematopoiesis, with fewer numbers of neutrophils and erythroid cells, as well as impaired maturation of neutrophils. The blunted maturation of neutrophils, denoted by their pattern of CD16/CD13 expression, matched retrospective patterns found in bone marrow aspirates of other SDS patients, suggesting that the BM-Chip can replicate the phenotype and some features of the disease. Further studies on the mechanisms of bone marrow failure, anemia, and neutropenia should be possible using the SDS BM-Chip.

Conclusions

This study provides evidence that the human BM-Chip is an effective preclinical model of human BM pathophysiology, enabling analysis of human responses to clinically relevant drug pharmacokinetics profiles and radiation dose exposures. The data show that the BM-Chip supports erythroid differentiation as well as myeloid development and mobilization over four weeks of culture while improving the maintenance of CD34+ progenitors compared to traditional culture methods. Additionally, the BM-Chips were able to better recapitulate the toxicity responses of human bone marrow to clinically relevant dose exposures of AZD2811 and 5-FU and showed an improved ability to recover after injury. 

BM-Chips derived from patients with Shwachman-Diamond syndrome showed dysfunctions that parallel key hematological abnormalities observed in vivo and led to the discovery of a neutrophil maturation abnormality that exists in a significant subset of SDS patients. Taken together, next-generation in vitro modeling of human bone marrow using Emulate’s microfluidic organ-chips has great potential to facilitate drug development, disease modeling, and translational studies for a range of hematopoietic disorders and toxicities – all while offering a human-specific alternative to animal testing for regulatory assessment. 

Testing new vaccines against seasonally emerging influenza strains is a necessary process, due to frequent changes in the virus’s immunogenic surface proteins. Vaccines and other drugs, such as immunotherapies, are evaluated for efficacy and safety in vitro using cultured human immune cells and in animal models. However, these assessments often fail to fully predict human responses, because they lack the biologically relevant microenvironment in which to develop an adaptive immune response. 

Modeling a true human immune response is a complex undertaking. Primary lymphoid organs, like the thymus and bone marrow, allow immune stem cells to proliferate, differentiate, and mature. Secondary lymphoid organs like the lymph nodes, spleen, and tonsils maintain mature but naïve cells ready to be activated by contact with antigen presenting cells, leading to clonal expansion and affinity maturation for the adaptive immune response. Lymphoid follicles (LFs) that are present within lymph nodes or form ectopically within other secondary lymphoid organs contain germinal centers that support the development of plasma cells that secrete high affinity antibodies. 

A human model of the LF would provide a better understanding as to why these organ structures are critical for developing adaptive immune responses and would be extremely useful for preclinical assessment of vaccine efficacy. This study represents the first-of-its-kind human LF-Chip containing B, T, plasma and antigen-presenting cells to test functional immunization responses to vaccines and adjuvants in vitro. 

• Research Area: Vaccine and adjuvant testing
• Organisms: Human
• Sample Types: Peripheral blood-derived immune cells
• Research Question: Can the LF-Chip be a useful human-relevant preclinical tool for assessing the efficacy and safety of influenza vaccines and adjuvants?

 

 

Experimental Overview

Characterizing the LF-Chip

Researchers created the LF Organ-Chip by using peripheral blood mononuclear cells (PBMC) isolated from donor blood and culturing the cells at a high density within the extracellular matrix (ECM) in the lower chamber of the chip, with culture medium flow supplied in the upper chamber. The researchers observed self-aggregation of lymphocytes within the ECM resembling which resembled germinal centers. They did not see autoactivation of B cells (determined by levels of IgG or IgM in the Organ-Chip cultures), overcoming a key challenge seen in static 2D cultures. 

Within the LF-like structures formed in the chip, the researchers detected molecules that indicate immune reaction, including the chemokine CXCL13, which recruits T cells, and activation-induced cytidine deaminase (AID) expression, which is required for the critical antibody class switching step. Class switching and plasma cell formation were observed, and, when exposed to an antigen that mimics the presence of dead bacteria, a robust IgG response was generated, suggesting that the model faithfully recapitulated a functional adaptive immune response.

Vaccination Testing on the LF-Chip 

Protective immunity induced by vaccination requires plasma cell production of antigen-specific antibodies. To see if the model could simulate an immune response to vaccination with viral antigens with or without an immune stimulating adjuvant, the authors added patient monocyte-derived dendritic cells in a 3D ECM gel within the Organ-Chip and tested it with influenza virions and adjuvant. The germinal centers showed plasma cell formation, expression of AID by resident B cells, and production of cytokines, and robust production of antibodies to influenza virus hemagglutinin – similar to vaccinated humans. Notably, these results were not replicated within a 2D static cell culture model. A commercial influenza vaccine was used to induce a clinically relevant immune response in the chip, showing important features like cytokine response and donor variability. 

Towards a Better Preclinical Assessment of Vaccines and Adjuvants

This is the first reported in vitro model that supports formation of human LFs with functional germinal centers similar to those found in secondary lymphoid organs in vivo using cells isolated from peripheral blood. Within the LF-Chip, B and T cells support plasma cell differentiation and antibody secretion while spontaneously forming LFs that express the correct cytokines and chemokines. These Organ-Chip microphysiological systems are also patient-specific and can recapitulate donor variability in response to vaccination. 

Previous attempts at recreating cell culture models of the lymph node for preclinical vaccine testing are not as useful for modeling the human adaptive immune response. These experimental models do not result in the appropriate biomarkers, de novo lymphoid follicle formation, or survival of plasma cells. In addition to ethical concerns and shortages of non-human primates due to the current COVID-19 pandemic, animal models do not usually replicate an intact human immune response. Taken together, these findings suggest that the LF-Chip may be a useful human-relevant tool for assessing the efficacy and safety of vaccines and adjuvants in a patient-specific manner. 

Future Uses for the LF-Chip 

Using primary human blood cells collected non-invasively, the LF-Chip can also be used to study the basic biology of immune organ formation as well as antigen-induced immunological responses. Specifically, results obtained in this study suggest that the presence of dynamic fluid flow promotes LF formation and reduces autoactivation of B cells, possibly facilitated by the ECM to promote proper cell interactions. Future work with the chip could explore how mechanical forces work to support organ formation at the cellular level. The LF-Chip may also be used to understand control of genes encoding cytokines and chemokines that govern the microenvironment critical for formation of germinal centers and immune cell activation. Using high-resolution imaging, many of the cellular and molecular processes occurring in the LF-Chip can be observed over time, enabling valuable longitudinal studies of human immune responses. 

Drug candidates are usually discovered or developed with a specific purpose in mind: to stop infections, to target and kill cancer cells, or to repair a faulty cellular pathway. Sometimes, once these approved treatments are taken by large groups of people, clinicians may observe unexpected and exciting benefits. For example, the drug Raloxifene was developed to prevent and treat osteoporosis by mimicking the effects of estrogen to increase bone density. Through retrospective clinical analysis, the drug was also shown to decrease the risk of developing invasive breast cancer by blocking estrogen’s effect on breast tissue.  

Drugs that inhibit viral infection have been successfully developed and in use for decades, and with the current SARS-CoV-2 crisis, speeding up testing of potential preventive therapies is an urgent priority. One of the fastest ways to respond to a global challenge like a pandemic is to repurpose existing, approved drugs as treatments for emerging pathogens. 

Organ-Chips Identify Potential Prophylactic Treatment for COVID-19

During the past year, many research groups around the world have focused on drug repurposing for COVID-19 using established cell lines, primary tissue-derived human cells, organoids, and explanted human lung tissue cultures. However, it is well known that cells grown in a two-dimensional dish do not behave like the cells in a living human body, and many drugs that appear effective in pre-clinical lab studies do not work well in patients. 

In a collaborative effort to test whether existing drug candidates block infection from respiratory pathogens like SARS-CoV-2, researchers from the Wyss Institute for Biologically Inspired Engineering at Harvard University, the University of Maryland School of Medicine, the Icahn School of Medicine at Mount Sinai and others used Emulate technology to develop an Airway Lung-Chip to examine eight existing candidate therapeutics for SARS-CoV-2, including hydroxychloroquine and chloroquine, that they and others had previously observed in conventional cell culture assays. The study is reported in Nature Biomedical Engineering.  

The Emulate Airway Lung-Chip used for these studies is a small microfluidic culture system device with two parallel channels separated by a porous membrane. Human lung airway cells are grown in one channel exposed to air, while human blood vessel cells are grown in the other channel exposed to cell culture medium to mimic blood flow. Cells in the Airway Lung-Chip differentiate into specific types similar to those in the human airway and develop lung-specific traits such as cilia and the ability to produce and move mucus. The cells also express angiotensin-converting enzyme-2 (ACE2) receptors on their surface, which is co-opted by the virus to gain entry into cells. 

When the cells in the Airway Lung-Chip were infected with a non-infectious SARS-CoV-2 virus, most of these drugs, including hydroxychloroquine and chloroquine, were not effective at stopping infection. However, one antimalarial drug called amodiaquine reduced infection by 60 percent by preventing viral entry. 

Subsequent Research Suggests Drug’s Potential Therapeutic Effects 

Despite the promise of amodiaquine, the team still needed to demonstrate that it worked against the infectious SARS-CoV-2 virus. Virologists Matthew Frieman, PhD at the University of Maryland School of Medicine and Benjamin tenOever, PhD at the Icahn School of Medicine at Mount Sinai, had both the expertise and laboratories designed to study infectious pathogens and were eager to collaborate with the Wyss Institute. The Frieman lab tested amodiaquine and its active metabolite, desethylamodiaquine, against native SARS-CoV-2 via high-throughput assays in cells in vitro and confirmed that the drug inhibited viral infection. 

In parallel, the tenOever lab tested amodiaquine and hydroxychloroquine against native SARS-CoV-2 in a head-to-head comparison in a small animal COVID-19 model and saw that prophylactic treatment with amodiaquine resulted in an approximately 70 percent reduction in viral load upon exposure, while hydroxychloroquine was ineffective. They also saw that amodiaquine prevented the transmission of the virus more than 90 percent of the time, and that it was also effective in reducing viral load when administered after introduction of the virus. Thus, their results suggest that amodiaquine could work in both treatment and prevention modes. 

“Seeing how beautifully amodiaquine inhibited infection in the Airway Lung-Chip was extremely exciting,” said Frieman. “The fact that it seems to work both before and after exposure to SARS-CoV-2 means that it could potentially be effective in a wide variety of settings.” 

“This collaboration has allowed us to do things that we never would have had the resources to do otherwise, including recently setting up Organ-Chips in our own lab so that we can now use them to study the interactions between infectious viruses and their hosts. While we’re proud of what we’ve accomplished so far for COVID-19, we’re also looking forward to studying additional virus-host dynamics using the Organ-Chips in the hopes that we’ll be able to prevent or address future pandemics,” said tenOever, who is a Professor of Microbiology. 

The Organ-Chip-based testing ecosystem greatly streamlines the process of evaluating the safety and efficacy of existing drugs for new medical applications and provides a proof-of-concept for the use of Organ-Chips to rapidly repurpose existing drugs for new medical applications, including future pandemics. Amodiaquine is now in clinical trials for COVID-19 at multiple sites in Africa, where this drug is inexpensive and widely available. 

“The collaboration with the Wyss Institute and the Frieman and tenOever labs has created a preclinical drug development pipeline that produced clinically significant results with incredible speed,” said Lorna Ewart, EVP of Science at Emulate. “With customizable Organ-Chip technology in the hands of researchers, preclinical testing of new drug candidates or repurposed drugs can be further accelerated with more human-relevant models, allowing us to be better prepared to confront this and future pandemics.” 

For more information on how Emulate products are being used to study COVID-19, read our blog post titled How Organs-on-Chips Technology is being used to Provide Scientific Insights into COVID-19. 

A webinar, sponsored by Emulate and Genetic Engineering & Biotechnology News, took a hard look at the models academia, government, and pharmaceutical entities are using to study drug toxicity and how Emulate Organ-Chips, also called microphysiological systems (MPS), are producing promising results. 

While two-dimensional cell studies and animal models provide useful information, they fail to fully replicate the complexities of human biology. In rare cases, they even miss lethal toxicities. One horrific example is fialuridine, a hepatitis B medication that killed five study participants in 1993. Preclinical animal models failed to catch the drug’s effects on human livers. 

“The real problem is animal models don’t predict human response,” said Donald Ingber, MD, PhD, Founding Director of the Wyss Institute for Biologically Inspired Engineering and an Emulate co-founder and board member. 

In addition to the risk to patients, poor models have helped create a slow and expensive drug development process, in which new medicines can take 15 years and $2 billion to develop and 90% of new drug applications ultimately fail. 

For these and other reasons, researchers and drug developers are exploring how to make pre-clinical drug testing more human-relevant and believe Organ-Chips are necessary complements for other cell and animal models to provide more accurate safety and efficacy readouts. 

Emulate Organ-Chips are designed to better replicate specific biological environments. They can include tissue-tissue interfaces, blood flow, immune cell interactions, microbes and mechanical forces. 

This mechanical stimulation is a critical element, helping organize various tissues, noted Nathalie Sauvonnet, PhD, Research Director at Institut Pasteur. While studying the shigella pathogen, Dr. Sauvonnet was having trouble replicating the bacteria’s activity in a dish. However, an intestine-chip gave her team new insights. 

“We could reproduce the activity of shigella as seen in the human patient,” said Dr. Sauvonnet. 

William Proctor, PhD, Senior Director for Predictive Toxicology and Safety Assessment at Genentech, discussed how Organ-Chips have the potential to reduce the need for animal models. In addition to their inability to replicate drug toxicities in humans, animals are expensive to nurture. The industry is eager to find alternatives. 

Genentech has developed its own core facility (the Complex In Vitro Systems Laboratory) to advance Organ-Chips. “We know this is transformative technology,” said Dr. Proctor. “How we get there in drug discovery and development is tailored to each organization, but also how quickly we can get comfortable doing this internally.” 

Tyler Goralski, PhD, is testing MPS for chemical and biological defense. Dr. Goralski is a research biologist at the US Army’s DEVCOM Chemical Biological Center. He believes MPS can help him assess chemical agents before going into in vivo models. 

Speakers highlighted a wide variety of other MPS applications. Luke Dimasi, Senior Director of Product Management at Emulate, outlined their work using the Emulate Colon Intestine-Chip to study inflammatory bowel disease.  

Jordan Kerns, Director of Biological Science at Emulate, noted how Organ-Chips can provide better safety and efficacy data for T-cell bispecific antibodies – an exciting but potentially risky therapy.  

Another session described how neural chips can help dissect the mechanisms that make the brain a fertile place for Alzheimer’s and other neurodegenerative diseases. Anna Herland, PhD, Associate Professor, Biohybrid Systems at Kungliga Tekniska Högskolan and Karolinska Institutet, discussed how combining neural chips with induced pluripotent stem cell-derived endothelial cells, pericytes, astrocytes and neurons provided new insights into the crosstalk between brain cells and the blood brain barrier. 

Overall, the symposium painted a bright picture of how Organ-Chips can provide better safety and efficacy data to drive more efficient drug development. In particular, detecting toxicities that might go undiscovered using more traditional models. 

To drive that point home, Lorna Ewart, Executive Vice President of Science at Emulate, noted that current MPS technology would have caught fialuridine toxicity before it was administered to patients, avoiding a tragic outcome.  In case you missed the must-view event, you can watch it here.

As many as 90 percent of new drug compounds fall short in clinical trials. Many of them fail because the compounds are toxic in humans, particularly to the liver and kidneys. Though pharmaceutical companies use cell cultures and animal models to assess toxicity issues before their drugs go to human trials, those approaches have shown mixed success.  

This established preclinical workflow is wasteful on many levels. Drug companies expend precious resources on new drug applications that are doomed to failure. Clinical trial participants are often exposed to toxic compounds, and some have paid dearly. Patients hoping for more effective medicines must continue to wait. 

In an ideal world, pharma companies would fully understand a drug’s potential toxicities long before it goes to human trials. If the compound is found to have toxicity, medicinal chemists could alter the molecule to mitigate the problem. In other cases, resources could be shifted to drugs that have fewer toxic effects. 

To get there, we need to augment the preclinical workflow, leverage new models, and provide better data on drug toxicity with in vitro toxicology testing at the early stages of research and development. Organ-Chips are a promising option. 

The Advantages of Organ-Chips  

Emulate has developed liver and kidney chips that can provide truer readouts on drug toxicity. These small microfluidic chips, about the size of an AA battery, are designed to better replicate the forces and signals cells encounter in vivo. 

Kidney toxicity is a major reason therapeutic compounds fail in human trials. Toxicity often shows up in the renal proximal tubules, which are responsible for filtering the blood. The Emulate human Proximal Tubule Kidney-Chip was created to model this behavior. The chip contains both tubular and vascular endothelium to replicate mechanical and physiological forces in the body, such as cellular signaling and shear stress, and provide a model that will more accurately predict how these cells respond to medicines or other compounds.  

In addition, because organ-chips are made from a clear polymer, they can be visualized. For example, researchers can easily examine cellular morphology under a microscope.  

Emulate recently tested the Kidney-Chip against cisplatin and gentamycin, which are known nephrotoxic agents. The cells in the chip reacted to these toxins much like cells in the body, showing obvious injuries. In addition, by studying gene expression and other factors, Emulate researchers showed the chips reproduced cellular transporters, which play a significant role in toxicity, and other important kidney mechanisms. Ultimately, the study showed the chip offers a physiologically relevant way to assess drug safety. 

A Dynamic Preclinical Model

Instead of studying compounds on cell cultures, followed by animal models, followed by human participants, researchers can layer in this new model, which more closely replicates how cells behave in the body. Organ-Chips will not replace cell culture or animal models in the immediate future. Rather, they provide a vehicle to inform preclinical data and ultimately predict a drug’s potential toxicity. 

The goal is to catch toxicity much earlier in the process, a refinement that could reduce waste, giving pharma companies better data to decide which compounds move forward into new drug applications and which ones should be reexamined.   

Having this extra layer of data could ultimately make clinical trials more efficient, increasing success rates, reducing the dangers to trial participants and accelerating the drug discovery process. 

On March 10th, 2021 Rep. Alcee L. Hastings (D-Fla.) and Citizens for Alternatives to Animal Research (CAARE) hosted the “21st Century Alternatives to Animals in Biomedical Research” virtual briefing in support of the Humane Research and Testing Act, re-introduced by representatives Hastings and Vern Buchanan (R-Fla.). Attending staffers had the opportunity to hear from a variety of scientific and medical experts and renowned primatologist Jane Goodall about the need to explore alternatives to animal models for biomedical research. Don Ingber, Scientific Founder of Emulate, spoke about how organ-on-a-chip technology can overcome many of the scientific inadequacies and ethical concerns associated with animal models. Together, the speakers highlighted many opportunities to improve drug discovery that will come from creating a National Center for Alternatives to Research and Testing as part of NIH. The message was clear — the use of animal studies and testing is not only inhumane and expensive, but it also doesn’t produce human-relevant data. More compelling human-relevant testing solutions, like Emulate Organ-Chips, are now available.  

Dr. Jane Goodall, founder of the Jane Goodall Institute, began the briefing by recounting one of the “worst days of [her] life:” when she visited a laboratory in the 1980s that used chimpanzees as test subjects. Dr. Goodall stressed that animal testing foundationally violates the rights of animals by subjecting them to a life of captivity and testing that could induce pain and fear in them. “Animal models really aren’t the answer,” she said. 

In addition to their ethical implications, animal studies have proven themselves insufficient in addressing today’s complex public health concerns, according to Paul Locke, JD, DrPH, Associate Professor at the Johns Hopkins Bloomberg School of Public Health and affiliate of the Center for Alternatives to Animals. Locke discussed how more sophisticated testing techniques can better imitate human biological processes and will facilitate the development of novel drugs and therapies with improved accuracy and efficiency. He emphasized that “research has shown us that non-animal methods are definitely the way to go.” 

Emulate Scientific Founder and Board Member Don Ingber presented about the benefits of organ-on-a-chip technology in his capacity as the Director of the Wyss Institute for Biologically Inspired Engineering at Harvard University. He discussed how Organs-on-Chips can be a solution to the demonstrated need for sophisticated, human-focused modeling in biomedical research. He showed attendees animated models of the organ-on-a-chip models and videos of experiment data to demonstrate how organ-on-a-chip technology can replicate and illustrate human biological functions. 

One field that could especially benefit from non-animal research methods is cancer research. “Cancer treatment has remained paleolithic,” noted oncologist Anza Raza, MD, Chan Soon-Shiong Professor of Medicine and Director of the MDS Center at Columbia University and author of the book The First Cell: And the Human Costs of Pursuing Cancer to the Last. Dr. Raza described how cancer treatment has seen little change in approach since the 1970s with the rudimentary tools of surgery, chemotherapy and radiation. Expressing disgust for new cancer therapies which only work for a subset of patients and often only for a short time, she noted “95 percent of experimental drugs fail, and the other 5 percent should have failed.” Anza views inapplicable animal models as a primary reason for cancer treatment’s stunted progress. She stressed that human-oriented models need to be used to understand cancer and other human diseases.  

Finally, Thomas Hartung, MD, PhD, Professor at the Johns Hopkins Bloomberg School of Public Health and Director of the Center for Alternatives to Animals, closed the briefing by stating the importance of the NIH’s role in normalizing new research techniques, and funding research to spur their development and use. He also described the importance of training the next generation of biomedical researchers on the most effective and cutting-edge tools and research methods, which he saw as necessary to making animal models a thing of the past.   Please visit house.gov to find your Congressional representative and encourage him or her to support the Humane Research and Testing Act.

In developing new medicines, the pharmaceutical industry has relied on antiquated research paradigms, established in a different era when the collective human understanding of animal biology was very different. The limitations of animal models in research were somewhat known, but at the time, they were the best available tools for predicting drug safety and response before human trials. In the intervening years, research conducted, and data revealed have given us reason to question the status quo.   

Today, more human-relevant models, such as new organ-on-a-chip technologies, exist which can help researchers understand mechanisms of disease and drug action. Newer pharmaceutical advances which harness the human immune system, or human-specific biological factors, represent roughly 40 percent of the pharmaceutical pipeline.  Animal models simply don’t work in predicting human responses to many biologic medicines and vaccines, and they present a myriad of ethical concerns and geopolitical challenges.  

To improve drug discovery, a paradigm shift is needed. Governments need to invest in future-focused solutions and create pathways for regulators to approve safe and effective drugs based on a wider variety of human-relevant modeling methods. At Emulate, we believe that the Humane Research and Testing Act (HR 1744), recently introduced, is the first step toward that paradigm shift the pharmaceutical industry so desperately needs, and we are proud to give it our endorsement.  

The bipartisan legislation introduced into the U.S. House of Representatives by Rep. Alcee Hastings (D-Fla.) and Rep. Vern Buchanan (R-Fla.) would establish the National Center for Alternatives to Research and Testing under the National Institutes of Health. The new Center would be dedicated to increasing transparency and understanding regarding the use of animals in research and testing and would be focused on ultimately reducing the number of animals used in such practices. It would require the NIH to develop, fund and execute a plan to enumerate the number of animals used in biomedical research, and incentivize companies to use non-animal methods by educating and training scientists on human-relevant research methods.  

Emulate is proud to endorse this legislation as we believe it is an essential first step toward ensuring that human-relevant models play a larger role in biomedical research.  We applaud Rep. Hastings and Rep. Buchanan for their leadership on this issue, as well as Representatives Joyce Beatty (D-Ohio), Julia Brownley (D-Calif.), and Lucille Roybal-Allard (D-Calif.) who are original co-sponsors of the bill.