Using Organoids in the Study of Infectious Diseases
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Organoid cell culture has transformed cell-based assays in drug discovery and basic biology by conferring physiologic relevance to in vitro cell-based biological models. When provided with a suitable growth environment, including appropriate cultureware, growth factors, extracellular matrix, nutrients, and culture media, organ-derived progenitor cells harvested from patients grow and assemble into three-dimensional structures – organoids – which incorporate all cell types normally found in the original tissue, and allow physical and chemical interactions between and among cells. By providing greater physiologic relevance and a species- or patient-specific test platform, organoids overcome many limitations of conventional 2D cultures and even live-animal disease models.
Organoids arise from organ-derived adult pluripotent stem cells, organ stem cells, or cancer stem cells which possess the innate capacity to expand and differentiate into multiple cell types. Organoids generated from dozens of tissues and organs available commercially, or accessible through published protocols include patient-derived models of liver, heart, pancreas, brain, GI tract, kidney, and recently, of human airways suitable for drug and vaccine development and for studying infectious human respiratory diseases.
Industry collaborations help advance organoid studies
Hubrecht Organoid Technology (HUB), a foundation located in Utrecht, The Netherlands, commercializes and industrializes advanced organoid technology and distributes human-derived organoids worldwide. HUB's intellectual property is based on pioneering work by Professor Hans Clevers, who in 2009 first described methods for growing and expanding human adult stem cell-derived human epithelial organoids. HUB has produced and validated organoids representing cancers of the colon, breast, lung, liver, ovary, bladder, and pancreas. In addition HUB generated models for other diseases such as Inflammatory Bowel Disease (IBD), Chronic Obstructive Pulmonary Disease (COPD) and genetic diseases such as cystic fibrosis (CF). The company has also introduced adult stem cell-derived kidney organoids, and continues to investigate the molecular mechanisms of tissue development and cancer using organoids generated from adult Lgr5 stem cells.
Corning Life Sciences has collaborated with HUB since 2014 to provide advanced organoids and related technology.
Dr Clevers’ technology allowed, for the first time, the expansion of adult stem cell-derived organoids in genetically stable form and ultimately, the generation of in vitro models of any epithelial disease from any patient.
A second key benefit was indefinite expansion similar to that of transformed cells, but without the genetic abnormalities inherent in cancer cells. Previously, organoids were generated from embryonic or induced pluripotent stem cells, or from tumor cells which by necessity are genetically modified and therefore unrepresentative of the patient.
Under HUB’s commercial development, organoid technology also provides standardization and consistency which is difficult to match, especially with primary cell cultures. Biopsies from the same patient collect differing quantities of cells at widely varying stages of cell lifecycle. When cultured under identical HUB protocols adult progenitor cells give rise to organoids with exactly the same cells in the same proportions, physical configuration, and genetics, every time, and with broad expansion capabilities.
Starting cells make a difference
3D cell cultures have been grown from various cells, including transformed cell lines and primary cells. Conventional primary cell culture methods expand only for several generations if at all, thus limiting their suitability to biobanking or large-scale manufacturing. Furthermore, conventional 3D primary-cell culture methods lack consistency and suffer from poor manufacturability conferring a “one-off” character to studies employing them. Moreover, expression analysis experiments on aggregated primary cell cultures are unreliable since the analysis must occur within the limited timeframe before the aggregate loses integrity. Only methods that allow long term stabilized expansion generate morphology and associated expression patterns that have regained their in vivo characteristics.
Similarly, transformed cells grown on plastic have modified their gene expression to adapt to tissue culture conditions. Studies with such cells can be useful, provided investigators recognize that the patient’s original genetics have not been preserved. In HUB organoids the patient’s molecular footprint is maintained.
One field where this has been particularly useful is infectious diseases. Viruses have evolved to infect and replicate in cells in their normal physiological states. For example, respiratory syncytial virus (RSV) readily grows in organoids but will not infect transformed cells because the cells lack the relevant receptors.
Cell-based studies of airway diseases – topical in light of the current COVID-19 pandemic – were hampered for years for this reason, and technology for expanding primary cell cultures sufficiently for large-scale studies did not exist. By preserving critical cell surface receptors for infectious agents, the HUB method allows the study of such pathogens as RSV, human papillomavirus, norovirus, coronavirus, influenza, malaria and many others.
Organoids in infectious diseases
Even before the COVID-19 pandemic, lower respiratory tract infections were among the leading causes of death worldwide. Rodent models for human barrier immunity are inadequate, and the complex barrier functions that normally clear viruses from airways have been difficult to replicate in vitro, particularly in immortalized cells.
Epithelial cells are the first point of contact for pathogenic microbes in the respiratory tract, and fortuitously the cell types most easily grown as organoids. Receptors on airway epithelia and alveolar cells sense infection, which initiates mucosal barrier immunity through club, ciliated, basal, goblet and neuroendocrine cells, which together clear inhaled pathogens.
In a recent Science paper, researchers from the Hubrecht Institute and Erasmus Medical Center reported on how gut organoids helped them to uncover two potential avenues for treating or preventing infection with SARS-CoV-2, the coronavirus responsible for the current pandemic. SARS-CoV-2 is known to infect the lungs, but clinical evidence suggests intestinal involvement in both symptomatology and transmission. For example, rectal swabs contain viral RNA for a time after nasal swabs indicate the infection has resolved, suggesting gastro-intestinal infection and possibly fecal–oral transmission.
Differentiated enterocytes strongly express the SARS-CoV-2 angiotensin converting enzyme 2 (ACE2) receptor through which the virus enters cells, with the highest receptor levels found in the brush border of intestinal enterocytes. Surprisingly, virus infected both high- and low expressors of ACE2, and infectivity of organoids was not greatly affected by culture conditions.
SARS-CoV-2 rapidly infected a subset of cells within the organoid, and infection increased over time. Using electron microscopy to visualize cellular components, the researchers found virus particles inside and outside the organoids’ constituent cells. Infection induced release of interferon, an endogenous antiviral whose activation could serve as the target for potential therapies.
The researchers concluded that “intestinal epithelium supports SARS-CoV-2 replication,” that human small intestinal organoids “serve as an experimental model for coronavirus infection and biology,” and “that human organoids represent faithful experimental models to study the biology of coronaviruses.”
In addition to drug screening and toxicology studies, airway organoids have been utilized to study the basic biology of infectious diseases. In an application note, Corning scientists reported that Corning® Matrigel® extracellular matrix facilitated the expansion of patient-derived bronchial epithelial cells into airway organoids suitable for high throughput analysis. Organoids streamlined the usual sample preparation protocol to a single operation – cell lysis – eliminating the normal steps of gene amplification, cDNA conversion, and library preparation.
Comparing normal and asthmatic airway organoids, investigators observed increased expression of genes coding for pro-inflammatory chemokines, receptors, and other proteins associated with inflammation in asthmatic airway cells. They also found that the genes upregulated in organoids derived from healthy cells were the same as those downregulated in organoids from asthmatic cells, and vice-versa. Application of the anti-inflammatory steroid dexamethasone induced up- or downregulation to a greater degree in asthmatic organoids compared with normal organoids.
The Corning study illustrates the versatility of organoids for studying airway diseases in the presence of comorbidities, as well as the ability to respond rapidly with suitable models for infectious diseases.
The future of organoids in infectious diseases
In a recent interview Evan Snyder, M.D., Ph.D., director of Sanford Burnham Prebys’ Center for Stem Cells and Regenerative Medicine, described the potential role of airway organoids for studying the natural history of the COVID-19 infection, including how the virus moves from one cell to another, and potentially the ability to predict individual patient outcomes. “We can use this model to determine why some people fare worse than others,” Snyder said. “We can compare organoids created from men and women; younger or older people; people exposed to various environmental toxins from smoking or vaping; people with diabetes, heart or kidney disease; and even people of different racial backgrounds or individuals who have genetic variations that affect their ability to fight infection. If we figure out why the virus affects some people differently, we can potentially create tailored treatments.”
HUB Organoids derived from adult stem cells harvested from cystic fibrosis patients have proved valuable in the study of CF pathology, and have permitted patient-centered drug testing, which was the first use of HUB Organoids in personalized medicine. The CF patient derived organoids are tested to identify drug treatments for CF patients and in treated accordingly.
Recent studies on interleukin-17 receptors on lung epithelia have uncovered a role for this cytokine in acute and chronic inflammation, and demonstrated that IL-17 receptors participate in the innate immune defense against pulmonary fungal infections. In vivo, IL-17 expression and immune function requires polarized epithelial cells. In a paper appearing in 2019 in Frontiers in Immunology, a group at the University of Perugia, in Italy, wrote that because lung organoids recapitulate tissue polarity, they “provide an exciting possibility of using lung organoids to comprehensively investigate IL-17R signaling in the lung,” which is “likely to offer new opportunities to develop and test therapeutics for inflammatory diseases and identify new molecular targets to improve resistance to infections.”
As a scientific discipline, organoids will continue evolving towards greater ease of use, consistency, assay parallelism capabilities, and manufacturability. Organoids and organ-on-a-chip have already been combined in a complex, multi-tissue retina model, while systems consisting of organoids from two or more organs, discussed earlier, are already used routinely.
If organoid research continues at its current pace there is reason to expect significant streamlining of early-stage drug development, specifically around the preclinical and phase 1 stages. Organoids could eliminate some if not all animal testing, but this will require a leap of faith on the part of regulators already accustomed to reviewing animal data and its inherent caveats. At some point organoids might completely eliminate “live” preclinical screens, allowing drug developers to recruit patients directly into phase 2 based entirely on organoid-based screening.
While organoid investigations inevitably lead to systems of greater complexity, investigators should keep in mind that validation is the key to patient relevant models. HUB Organoids for the first time allow researchers to develop a model and directly test if and how it resembles the patient from which the tissue originated. With increasing complexity, the validation step should remain a focus of model developers and users. Complexity is good, but only up to a point.
Advancing organoids towards these lofty goals, including greater manufacturability, will require cell culture tools up to the task. Industry collaborations assure that tools for 3D cell culture will continue to advance, both for general research and to meet the challenges of emerging infectious diseases.
Dr Robert Vries, Chief Executive Officer, Hubrecht Organoid Technology (HUB)
Elizabeth Abraham, Senior Product Manager, Corning Incorporated