The following article originally appeared on June 1, 2020 in Technology Networks here.
Overcoming the Limitations of 2D Cell Culture
Countless medical breakthroughs over the past century, including the discovery of new drugs, have begun with two-dimensional (2D) cell culture models. Most cell-based assays are still based on 2D culture models but the limitations of 2D monolayers cultured in flasks or plates is immediately obvious – cells, tissues, and organs exist in three dimensions, not two, and tissues of interest almost always consist of more than one cell type.
Three-dimensional (3D) cell culture has changed how biologists and medical researchers approach cell-based assays.1 By providing greater physiologic relevance, 3D cultures narrow the divide between in vitro assays and live-animal2 or human embryonic assays.3 Organoids, in particular, show promise for their physiologic-like complexity and manufacturability. By enabling direct co-culture, or the self-directed differentiation of cultured precursor cells into distinct cell types, organoids capture many of the relevant interactions between test cells and their native niches, including chemical communication with neighboring structural cells and interactions with biological pathways that cells typically experience in vivo.4
Of Organoids and Spheroids
The terms “organoid” and “spheroid” are often used interchangeably but the differences with regard to physiologic relevance are germane to this discussion. Spheroids are typically free-floating cell aggregates generated from single, usually terminally differentiated cell types. Organoids arise from cultured induced pluripotent stem cells, organ-progenitor cells from adults, or cancer stem cells, all of which possess varying capacities for expansion and/or differentiation.5 In addition, current organoid production protocols are compatible with homogeneous assays in which cells are grown and tested in situ.
Industrializing organoid production has been hampered by problems typically encountered with out-scaling living systems (think CHO culture for biomanufacturing), specifically throughput and consistency.6 As we will learn, working with the right tools mitigates most of these issues.
Significance of Airway Organoids
Organoids representing dozens of tissue types and subtypes are now available commercially, or accessible through published protocols. These include 3D models of liver, heart, pancreas, brain, GI tract, kidney,7 and notably, as the world fights a coronavirus pandemic, of human airways.8
Lung and airway organoids are of interest for both drug and vaccine development and are valuable tools for studying infectivity in human respiratory diseases, particularly for challenging viral diseases like COVID-19. Airway organoids, which include multiple cell types, and which must account for cell–air, cell–fluid, and cell–cell interactions are every bit as complex as the physiologic entities they represent.9
Chinese and European researchers recently described a method for producing airway organoids for evaluating the infectivity of novel viruses in humans.10 In addition to goblet, club, and basal epithelial cells, their technique produced organoids that included operational ciliated cells in numbers comparable to those observed in airways. In addition, the organoids secreted serine proteases which are required for influenza viruses to infect cells.
The investigators concluded that differentiated airway organoids “morphologically and functionally simulate human airway epithelium and as a proof of concept can discriminate human-infective influenza viruses from poorly human-infective viruses.” Additionally, their method generates airway organoids that may be expanded “indefinitely” – a boon for industrialization – and display “remarkable phenotypic and genotypic stability.”
A recent presentation by scientists at Corning and NanoString Technologies described novel methods for high-throughput evaluation of gene expression in 3D airway organoids.11 For the last 30 years, researchers have used permeable support materials to design air-liquid interface cultures to study airways. Through this method, airway progenitor cells differentiate into the main airway cell types, which eventually assemble into serviceable models for investigating asthma, cystic fibrosis, and other airway pathologies.
An earlier paper reported that airway organoids derived from primary human cells and grown in Matrigel also form 3D structures consisting of goblet, basal, and ciliated cells, but without the need for a permeable support structure.12 This discovery freed investigators from the 96-well throughput limitations of the permeable support model. Multiplexing of up to 384 wells became feasible for the first time, with even higher throughputs possible.
Additionally, the new assay format permitted the use of a gene expression profile comparison tool, the nCounter® PlexSet™ assay, to compare gene expression in healthy and diseased tissue. PlexSet contributes further to throughput enhancements by requiring only cell lysis, thus eliminating several sample preparation steps associated with standard protocols.
Corning also supports other technology for situations where rapid-enough, low-throughput experimentation suffices. Transwell™ permeable supports in a 24-well format from Corning are cell culture systems consisting of permeable culture inserts in 24-well receiver plates or reservoirs.13
Both methods are useful in evaluating the infectivity of new respiratory viruses, as described by a Taiwanese group studying differentiated human airway organoids to assess infectivity of emerging influenza virus.10 Infectivity, a critical characteristic of pandemic viruses, differs widely among pathogens that cross from animals to humans. To evaluate the ability of airway organoid models to predict this trait, investigators tested strains known to preferentially infect animal vectors or humans in both 2D and 3D cell culture models. As an improvement over conventional 2D methodology researchers generated the 2D airway models from 3D organoids that had previously differentiated into relevant airway component cells, particularly ciliated cells. Both models distinguished between viruses capable of infecting humans and those that were not.
News stories during the COVID-19 pandemic have reported on serious non-airway effects of the virus, even among recovered individuals.14 Whether these conditions are due to the virus or were present before infection remains to be seen. That these conditions exist, however, suggests several interesting possibilities for further utilization of organoids.
We know that the COVID-19 virus affects the digestive tract, as 20% of those infected report at least one gastrointestinal symptom.15 Citing previous work using airway organoids, a European research team confirmed in May, 2020, that SARS-CoV-2, the virus that causes COVID-19, infects human gut enterocytes.16 Their infection model consisted of human gut progenitor cells differentiated under organoid-forming protocols favoring four distinct mature cell types normally found in human gut enterocytes. The human enterocyte organoid model further permitted the study of genomic alterations and expression of virus-related cytokines.
The near-exponential growth in research utilizing 3D cell cultures, and organoids, suggests that organoids are well-established in cell-based assays for drug discovery, toxicology, and basic biology.8 Organoids provide physiologic relevance in a culture format appropriate for in situ assays. Due to the involvement of lungs in so many diseases – not limited to respiratory ailments – airway organoids represent a critical testing platform for the discovery of drugs to combat infection, cancer, and other lung diseases, to quantify infection and pathology, to monitor disease progression, and for vaccine discovery.
Organoid culture protocols based on Corning Life Sciences’ extracellular matrices, reagents and cultureware provide a complete toolbox for exploring the capabilities of organoids in the study of airway diseases. Examples from the recent literature indicate that airway organoids may be produced in numbers sufficient for high-throughput screening, and relatively rapidly in response to imminent or existing threats from infectious diseases.
Written by Elizabeth Abraham, Senior Product Manager and Hilary Sherman, Senior Applications Scientist for Corning Life Sciences.
1. Fang & Eglen. (2017). Three-Dimensional Cell Cultures in Drug Discovery and Development. SLAS Discov. DOI: 10.1177/1087057117696795
2. Yin, et al. (2017). Stem Cell Organoid Engineering. Cell Stem Cell. DOI: 10.1016/j.stem.2015.12.005
3. Bredenoord, et al. (2017). Human Tissues in a Dish: The Research and Ethical Implications of Organoid Technology. Science. DOI: 10.1126/science.aaf9414
4. Baker. (2018). Organoids Provide an Important Window on Inflammation in Cancer. Cancers. DOI: 10.3390/cancers10050151
5. Next-Generation Organoids. (2020). Weill Cornell Medicine. Available at: https://eipm.weill.cornell.edu/2020/01/next-generation-organoids/
6. Lu, et al. (2017). Scalable Production and Cryostorage of Organoids Using Core-Shell Decoupled Hydrogel Capsules. Adv Biosyst. DOI: 10.1002/adbi.201700165
7. Akbari, et al. (2019). Next-Generation Liver Medicine Using Organoid Models. Front. Cell Dev. Biol. DOI: https://doi.org/10.3389/fcell.2019.00345
8. Li, et al (2020). Organoids as a Powerful Model for Respiratory Diseases. Stem Cells International. DOI: https://doi.org/10.1155/2020/5847876
9. Sachs, et al. (2019). Long-term Expanding Human Airway Organoids for Disease Modeling. EMBO J. DOI: 10.15252/embj.2018100300.
10. Zhou, et al. (2018). Differentiated Human Airway Organoids to Assess Infectivity of Emerging Influenza Virus. PNAS. DOI: 10.1073/pnas.1806308115
11. High Throughput Gene Expression Analysis of 3D Airway Organoids. (2019) Corning. Available at: https://www.corning.com/catalog/cls/documents/application-notes/CLS-AN-534.pdf
12. Danahay, et al. (2014). Notch2 is Required for Inflammatory Cytokine-Driven Goblet Cell Metaplasia in the Lung. Cell Reports. DOI: https://doi.org/10.1016/j.celrep.2014.12.017
13. Sample GSM3099451. (2019). NCBI. Available at: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM3099451
14. Mysterious Heart Damage, Not Just Lung Troubles, Befalling COVID-19 Patients. (2020). KHN. Available at: https://khn.org/news/mysterious-heart-damage-not-just-lung-troubles-befalling-covid-19-patients/
15. COVID-19 and Gastrointestinal Symptoms. (2020) WebMD. Available at: https://www.webmd.com/lung/covid19-digestive-symptoms#1
16. Lamers, et al. (2020) SARS-CoV-2 Productively Infects Human Gut Enterocytes. Science. DOI: 10.1126/science.abc1669