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NASA NTRSDate not supplied

BioNutrients-2: Improvements to the BioNutrients-1 Nutrient Production System

The BioNutrients (BN) project aims to develop an on-demand biological production system capable of rapid and safe delivery of multiple nutrients in single-use production packs for long-duration missions. The general concept of this system is to develop edible engineered microorganisms that produce nutrients for human consumption following long-term storage, revival, and growth. BN-1 is a five-year flight project currently on the International Space Station (ISS). It is testing the long-term storage of various microorganisms for the biomanufacturing of space-relevant compounds and validating the performance of the first generation of production packs. The BN-2 project expands the BN-1 flight project scope by introducing novel products, broadening the range of microbial food sources, and improving upon production pack hardware. We are optimizing the biomanufacturing platform for in situ production of yogurt and kefir on the ISS. BN-2 also will test engineered microorganisms, including a probiotic strain, to produce carotenoids or follistatin. To improve the feasibility of this technology for mission use, we transitioned from a hard-shell pack to a second-generation fluorinated ethylene propylene (FEP) bag. The single-use FEP bioreactor facilitates growth of target organisms in a contaminant free environment, while reducing mass and volume compared to the BN-1 hardware. These bioreactors will be employed for production of additional space-relevant products in the BN-2 project. On-orbit testing of FEP production packs will enable optimization of the platform for eventual flight use by the crew.

Natalie Ball↗
NASA NTRSDate not supplied

Precision Fermentation: Relieving the Crabtree Effect in S. cerevisiae through Genetic Engineering

Nutrient degradation in the current NASA pre-packaged food system poses significant challenges to crew health in missions beyond low earth orbit as certain vitamins and nutrients have been shown to degrade during extended storage, and resupply and fresh food items cannot be readily provided. BioNutrients(BN) uses synthetic biology in an effort to supplement the NASA food system by delivering in situ production of nutrients and therapeutics produced by genetically engineered microbes. One yeast S. cerevisiae strain utilized in BN is genetically modified to produce two types of antioxidants: zeaxanthin and β-carotene. Zeaxanthin is a non-provitamin A carotenoid and has been implicated in eye health and vision performance, and β-carotene is a provitamin A carotenoid. Microbial production of high-value nutrients can aid our understanding and address the issue of nutrient degradation and loss in the NASA food system. Although S. cerevisiae is an excellent candidate for a long shelf life, on-demand nutrient production system, its space applications are limited by the Crabtree effect where respiration is shut down in favor of fermentative metabolism when glucose is present in high concentrations, causing the release of unwanted ethanol. Current and likely future life support systems are negatively impacted by ethanol and ethanol release is strictly limited.To improve feasibility of using S. cerevisiae, we explored ways to reduce the Crabtree effect, thereby limiting its ethanol production. Specifically, we used CRISPR-cas9 to genetically modify various genes in this pathway

E Zaroff↗
NASA NTRSDate not supplied

Bionutrients: Microbial Production of on-Demand Nutrients on the International Space Station

Providing adequate nutrition to crew members is essential as deep-space missions cannot rely on consistent Earth-based resupply. The current NASA pre-packaged food system is designed for low-earth-orbit missions with a stated shelf-life of two years and notable vitamin degradation over time. One strategy to mitigate nutrient loss is to implement bioregenerative food sources to supplement the pre-packaged food system. The BioNutrients project is designed to provide targeted production of short shelf-life nutrients in a single-use production pack. BioNutrients-1 includes two strains of edible yeast that have been genetically engineered to produce carotenoids, β-carotene, and zeaxanthin. This five-year mission on the International Space Station (ISS), launched in 2019, tests the long-term storage and nutrient production of the microorganisms of interest. Additional microorganisms are also stored in stasis packs to determine the effects of long-duration storage on the ISS. These organisms may be useful for production of fermented foods, pharmaceuticals, or biomanufacturing processes. Improving on the BioNutrients-1 project, BioNutrients-2 has optimized the production pack hardware by reducing the overall mass and volume of the system. Furthermore, BioNutrients-2 has expanded the projects scope by investigating novel products and microbial food sources. BioNutrients-2 is a six-month mission, launched in 2022, which tests production of fresh foods such as yogurt and kefir. The BioNutrients missions seek to provide advances for in-space biomanufacturing by addressing safe and reliable production of high-value nutrients and on-demand foods for future exploration efforts.

BioNutrients↗
NASA NTRSDate not supplied

Bionutrients: Microbial Production of on-Demand Nutrients on the International Space Station

Providing adequate nutrition to crew members is essential as deep-space missions cannot rely on consistent Earth-based resupply. The current NASA pre-packaged food system is designed for low-earth-orbit missions with a stated shelf-life of two years and notable vitamin degradation over time. One strategy to mitigate nutrient loss is to implement bioregenerative food sources to supplement the pre-packaged food system. The BioNutrients project is designed to provide targeted production of short shelf-life nutrients in a single-use production pack. BioNutrients-1 includes two strains of edible yeast that have been genetically engineered to produce carotenoids, β-carotene, and zeaxanthin. This five-year mission on the International Space Station (ISS), launched in 2019, tests the long-term storage and nutrient production of the microorganisms of interest. Additional microorganisms are also stored in stasis packs to determine the effects of long-duration storage on the ISS. These organisms may be useful for production of fermented foods, pharmaceuticals, or biomanufacturing processes. Improving on the BioNutrients-1 project, BioNutrients-2 has optimized the production pack hardware by reducing the overall mass and volume of the system. Furthermore, BioNutrients-2 has expanded the projects scope by investigating novel products and microbial food sources. BioNutrients-2 is a six-month mission, launched in 2022, which tests production of fresh foods such as yogurt and kefir. The BioNutrients missions seek to provide advances for in-space biomanufacturing by addressing safe and reliable production of high-value nutrients and on-demand foods for future exploration efforts.

BioNutrients↗
NASA NTRS2012

Can Terrestrial Microbes Grow on Mars?

The theme for AbSciCon 2012 is "Exploring Life: Past and Present, Near and Far." The conference will address our current understanding of life - from processes at the molecular level to those which operate at planetary scales. Studying these aspects of life on Earth provides an essential platform from which to examine the potential for life on other worlds, both within our solar system and beyond. Mars exhibits a variety of extreme environments characterized by high UV and ionizing radiation flux, low pressure anoxic atmosphere, scarce or absent liquid water, extreme low temperatures, etc. The ability of terrestrial microorganisms to survive and adapt to the Mars environment has profound implications for astrobiology, planetary protection, and Mars life detection missions. At the NASA Ames Synthetic Biology Initiative, we believe that synthetic biology has the potential to revolutionize human space exploration. As such, the initiative is dedicated to applying the tools and techniques of synthetic biology to space exploration and astrobiology. Biological solutions will be invaluable for space exploration because they are not resource intensive, and they are versatile and self-renewing. An understanding of how to work with DNA in an unfavorable environment is paramount to utilizing biological tools on space missions. Furthermore, the ability to adjust life to the parameters of Mars is vital both to discovering what life on Mars might look like, and to using biological tools under such conditions. As a first step, we need an energy-efficient, low cost means of transporting, storing, and protecting genomic DNA, DNA parts, and whole microbial strains. Our goal is to develop and demonstrate viable and superior alternatives to standard DNA storage methods, which can be optimized to the conditions of space exploration, using synthetic biology as a tool. This includes protocols and kit designs for easy and repeatable DNA and strain recovery from protective storage conditions. We are constructing newly engineered genetic parts for different valuable host organisms, designed to increased long-term survival and functional retention. These methods should be applied for DNA and strain storage and transportation. In parallel, we seek inspiration from natural organisms that have developed means for survival in extreme environmental conditions. We are utilizing novel techniques for analysis of lipid biomarkers in the Antarctic Dry Valleys in order to identify resident microbes in the Antarctic soil and permafrost, as well as biomarker fossils of organisms that survived in the valleys in ages past. Through the identification of these life forms, we hope to understand and draw on new biological tools and strategies for synthetic biological applications on Mars.

Rothschild, Lynn↗
NASA NTRS2019

Bionutrients-1, On-Demand Production of Nutrients in Space

Future long-duration missions face significant challenges maintaining crew health. A critical area is supplying adequate nutrition, as certain vitamins and nutrients in supplied foods and supplements demonstrate substantial degradation during extended storage. To address this issue, we are developing and flight-testing a platform technology that demonstrates in situ microbial production of targeted nutrients over extended mission durations. This 5-year experiment, known as BioNutrients-1, was started on the International Space Station in May 2019. It involves two components: an on-orbit hydration and production experiment; and the development of space-compatible, key bio-manufacturing microorganisms. On-orbit testing utilizes a small “production pack” system that encloses sterile edible growth substrate and desiccated Saccharomyces cerevisiae strains genetically engineered to produce the nutrients beta-carotene or zeaxanthin. On hydration and mixing of the production pack, the organisms revive and grow until limited by the depletion of growth media, hypothetically leading to consistent amounts of biomass and nutrients. In eventual mission applications, the packet contents would be heat treated to inactivate the microorganisms prior to consumption. For these flight experiments, the packet will not be heat treated, but will instead be frozen for return to Earth for analyses. In addition to the production pack trials, 14 different microorganisms/treatments were also delivered to ISS for long-duration storage. These samples will be intermittently returned to Earth and analyzed to determine survival rates and genomics. For this presentation, initial data from returned samples and ground controls will be discussed.

Hindupur, Aditya↗
NASA NTRS2019

BioNutrients-1: On-Demand Production of Nutrients in Space

Future long-duration missions face significant challenges maintaining crew health. A critical area is supplying adequate nutrition, as certain vitamins and nutrients in supplied foods and supplements demonstrate substantial degradation during extended storage. To address this issue, we are developing and flight-testing a platform technology that demonstrates in situ microbial production of targeted nutrients over extended mission durations. This 5-year experiment, known as BioNutrients-1, was started on the International Space Station in May 2019. It involves two components: an on-orbit hydration and production experiment; and the development of space-compatible, key bio-manufacturing microorganisms. On-orbit testing utilizes a small "production pack" system that encloses sterile edible growth substrate and desiccated Saccharomyces cerevisiae strains genetically engineered to produce the nutrients beta-carotene or zeaxanthin. On hydration and mixing of the production pack, the organisms revive and grow until limited by the depletion of growth media, hypothetically leading to consistent amounts of biomass and nutrients. In eventual mission applications, the packet contents would be heat treated to inactivate the microorganisms prior to consumption. For these flight experiments, the packet will not be heat treated, but will instead be frozen for return to Earth for analyses. In addition to the production pack trials, 14 different microorganisms/treatments were also delivered to ISS for long-duration storage. These samples will be intermittently returned to Earth and analyzed to determine survival rates and genomics. For this presentation, initial data from returned samples and ground controls will be discussed.

Hindupur, Aditya↗
NASA NTRS2023

Regulatory response to a hybrid ancestral nitrogenase in Azotobacter vinelandii

Biological nitrogen fixation, the microbial reduction of atmospheric nitrogen to bioavailable ammonia, represents both a major limitation on biological productivity and a highly desirable engineering target for synthetic biology. However, the engineering of nitrogen fixation requires an integrated understanding of how the gene regulatory dynamics of host diazotrophs respond across sequence-function space of its central catalytic metalloenzyme, nitrogenase. Here, we interrogate this relationship by analyzing the transcriptome of Azotobacter vinelandii engineered with a phylogenetically inferred ancestral nitrogenase protein variant. The engineered strain exhibits reduced cellular nitrogenase activity but recovers wild-type growth rates following an extended lag period. We find that expression of genes within the immediate nitrogen fixation network is resilient to the introduced nitrogenase sequence-level perturbations. Rather the sustained physiological compatibility with the ancestral nitrogenase variant is accompanied by reduced expression of genes that support trace metal and electron resource allocation to nitrogenase. Our results spotlight gene expression changes in cellular processes adjacent to nitrogen fixation as productive engineering considerations to improve compatibility between remodeled nitrogenase proteins and engineered host diazotrophs.

nitrogen fixation↗
NASA NTRS2017

Development of Storage Methods for Saccharomyces Strains to be Utilized for In Situ Nutrient Production in Long-Duration Space Missions

Long-duration space missions will benefit from closed-loop life support technologies that minimize mass, volume, and power as well as decrease reliance on Earth-based resupply. A system for In situ production of essential vitamins and nutrients can address the documented problem of degradation of stored food and supplements. Research has shown that the edible yeast Saccharomyces cerevisiae can be used as an on-demand system for the production of various compounds that are beneficial to human health. A critical objective in the development of this approach for long-duration space missions is the effective storage of the selected microorganisms. This research investigates the effects of different storage methods on survival rates of the non-sporulating probiotic S. boulardii, and S. cerevisiae spores and vegetative cells. Dehydration has been shown to increase long-term yeast viability, which also allows increased shelf-life and reduction in mass and volume. The process of dehydration causes detrimental effects on vegetative cells, including oxidative damage and membrane disruption. To maximize cell viability, various dehydration methods are tested here, including lyophilization (freeze-drying), air drying, and dehydration by vacuum. As a potential solution to damage caused by lyophilization, the efficacy of various cryoprotectants was tested. Furthermore, in an attempt to maintain higher survival rates, the effect of temperature during long-term storage was investigated. Data show spores of the wild-type strain to be more resilient to dehydration-related stressors than vegetative cells of either strain, and maintain high viability rates even after one year at room temperature. In the event that engineering the organism to produce targeted nutrient compounds interferes with effective sporulation of S. cerevisiae, a more robust method for improving vegetative cell storage is being sought. Therefore, anhydrobiotic engineering of S. cerevisiae and S. boulardii is being conducted.

life support↗
NASA NTRS2014

Application of Synthetic Biology to Bioregenerative Life Support for Human Spaceflight

The conversion of carbon dioxide into higher value products is a key challenge for the development of closed-loop life support systems for human space flight. Much of the past research on bioregenerative life support systems has focused on plant growth chambers as a solution for CO2 removal and O2 generation, but photosynthetic microorganisms may also have a role to play in these functions. Cyanobacteria have the advantages of relatively high CO2 fixation rates and fairly well-developed molecular biology tools, allowing for genetic engineering approaches to strain improvement. Manned missions to Mars or other targets beyond low Earth orbit will require advances in the nutritional systems for life support on these longer duration missions. A key challenge will likely be supplementing pre-packaged meals with specific nutrients that will be deficient due to problems in long-term storage or low abundance. Vitamin K is one such nutrient that may be important as a supplement. Production of vitamin K for nutrient supplementation during spaceflight will likely require genetic engineering of microorganisms to increase vitamin titers. A microbial bioreactor system that could efficiently convert CO2 to nutritional supplements would be a valuable component for a future advanced life support system. We are exploring biological systems to determine the feasibility of using bioreactors to convert CO2 to higher-value products. We are examining the performance of photosynthetic bacteria engineered to produce sugars, determining rates of production and reliability. We are also engineering microbes to produce higher titers of vitamin K and other potentially important nutrients. The results of this research will offer demonstrations of potential technologies that could be developed further in the future. This work will also provide valuable information for understanding basic science questions about the use of genetically engineered microbes in the microgravity environment.

Bioregenerative life support↗
NASA NTRS1986

ECUT (Energy Conversion and Utilization Technologies Program). Biocatalysis Project

Presented are the FY 1985 accomplishments, activities, and planned research efforts of the Biocatalysis Project of the U.S. Department of Energy, Energy Conversion and Utilization Technologies (ECUT) Program. The Project's technical activities were organized as follows: In the Molecular Modeling and Applied Genetics work element, research focused on (1) modeling and simulation studies to establish the physiological basis of high temperature tolerance in a selected enzyme and the catalytic mechanisms of three species of another enzyme, and (2) determining the degree of plasmid amplification and stability of several DNA bacterial strains. In the Bioprocess Engineering work element, research focused on (1) studies of plasmid propagation and the generation of models, (2) developing methods for preparing immobilized biocatalyst beads, and (3) developing an enzyme encapsulation method. In the Process Design and Analysis work element, research focused on (1) further refinement of a test case simulation of the economics and energy efficiency of alternative biocatalyzed production processes, (2) developing a candidate bioprocess to determine the potential for reduced energy consumption and facility/operating costs, and (3) a techno-economic assessment of potential advancements in microbial ammonia production.

Source record↗
NASA NTRS2012

NASA Tech Briefs, May 2012

Topics covered include: An "Inefficient Fin" Non-Dimensional Parameter to Measure Gas Temperatures Efficiently; On-Wafer Measurement of a Multi-Stage MMIC Amplifier with 10 dB of Gain at 475 GHz; Software to Control and Monitor Gas Streams; Miniaturized Laser Heterodyne Radiometer (LHR) for Measurements of Greenhouse Gases in the Atmospheric Column; Anomaly Detection in Test Equipment via Sliding Mode Observers; Absolute Position of Targets Measured Through a Chamber Window Using Lidar Metrology Systems; Goldstone Solar System Radar Waveform Generator; Fast and Adaptive Lossless Onboard Hyperspectral Data Compression System; Iridium Interfacial Stack - IrIS; Downsampling Photodetector Array with Windowing; Optical Phase Recovery and Locking in a PPM Laser Communication Link; High-Speed Edge-Detecting Line Scan Smart Camera; Optical Communications Channel Combiner; Development of Thermal Infrared Sensor to Supplement Operational Land Imager; Amplitude-Stabilized Oscillator for a Capacitance-Probe Electrometer; Automated Performance Characterization of DSN System Frequency Stability Using Spacecraft Tracking Data; Histogrammatic Method for Determining Relative Abundance of Input Gas Pulse; Predictive Sea State Estimation for Automated Ride Control and Handling - PSSEARCH; LEGION: Lightweight Expandable Group of Independently Operating Nodes; Real-Time Projection to Verify Plan Success During Execution; Automated Performance Characterization of DSN System Frequency Stability Using Spacecraft Tracking Data; Web-Based Customizable Viewer for Mars Network Overflight Opportunities; Fabrication of a Cryogenic Terahertz Emitter for Bolometer Focal Plane Calibrations; Fabrication of an Absorber-Coupled MKID Detector; Graphene Transparent Conductive Electrodes for Next- Generation Microshutter Arrays; Method of Bonding Optical Elements with Near-Zero Displacement; Free-Mass and Interface Configurations of Hammering Mechanisms; Wavefront Compensation Segmented Mirror Sensing and Control; Long-Life, Lightweight, Multi-Roller Traction Drives for Planetary Vehicle Surface Exploration; Reliable Optical Pump Architecture for Highly Coherent Lasers Used in Space Metrology Applications; Electrochemical Ultracapacitors Using Graphitic Nanostacks; Improved Whole-Blood-Staining Device; Monitoring Location and Angular Orientation of a Pill; Molecular Technique to Reduce PCR Bias for Deeper Understanding of Microbial Diversity; Laser Ablation Electrodynamic Ion Funnel for In Situ Mass Spectrometry on Mars; High-Altitude MMIC Sounding Radiometer for the Global Hawk Unmanned Aerial Vehicle; PRTs and Their Bonding for Long-Duration, Extreme-Temperature Environments; Mid- and Long-IR Broadband Quantum Well Photodetector; 3D Display Using Conjugated Multiband Bandpass Filters; Real-Time, Non-Intrusive Detection of Liquid Nitrogen in Liquid Oxygen at High Pressure and High Flow; Method to Enhance the Operation of an Optical Inspection Instrument Using Spatial Light Modulators; Dual-Compartment Inflatable Suitlock; Large-Strain Transparent Magnetoactive Polymer Nanocomposites; Thermodynamic Vent System for an On-Orbit Cryogenic Reaction Control Engine; Time Distribution Using SpaceWire in the SCaN Testbed on ISS; and Techniques for Solution- Assisted Optical Contacting.

Source record↗
NASA NTRS2015

International Internship Report for Asher Williams

For the 2015 NASA I (sup 2) Internship Program, I was selected to work in Dr. John Hogan's laboratory on a Human Nutrient Production in Space (Bio-Nutrients) Project involving Research & Development in advanced microbial strategies for the production of nutrients within crewed spacecraft and habitats. Long-term space missions encounter the hurdle of substantial degradation of certain nutrients in food and supplements with time, potentially resulting in nutrient deficiency and serious health problems. The goal of the Bio-Nutrients Project is to enable rapid, safe, and reliable in situ production of needed nutrients using minimal mass, power, and volume. A platform technology is being developed to employ hydratable single-use packets that contain an edible growth medium and a food microbe engineered to produce target human nutrients. In particular, we examined the production of the carotenoids lutein and zeaxanthin in a spore-forming strain of the yeast Saccharomyces cerevisiae. Carotenoids are important antioxidants required for ocular health, a problematic area for some astronauts on long-duration ISS missions...To meet the first-year milestones for the Bio-Nutrients project, my specific task was to design and run preliminary tests on a disposable bioreactor for in situ production of human nutrients in space.

yeast↗
NASA NTRSDate not supplied

BioNutrients: Microbial on-demand production of short shelf-life micronutrients in space

Long duration deep space missions pose a significant challenge when considering adequate nutrition for human spaceflight. Many micronutrients supplied in pre-packaged foods for low Earth orbit flights have a short shelf life and will degrade over multi-year missions. One strategy to mitigate the risk of micronutrient malnutrition is to grow fresh foods to supplement a pre-packaged diet. The BioNutrients project aims to develop systems for growing edible microorganisms that are genetically engineered to produce short shelf life micronutrients. BioNutrients-1 (BN-1) is a five-year spaceflight project currently underway on the International Space Station (ISS) that tests long-term storage and production of two carotenoids, β-carotene and zeaxanthin, in genetically engineered yeast strains. BN-1 on-orbit and ground control activities indicate that the nutrient production system will be stable for at least two years in ambient storage conditions. BN-1 is also testing the effects of long-term ISS storage on viability and gene expression using “stasis packs” containing a series of microorganisms useful in food and biomanufacturing processes. Stasis pack analysis is distinguishing microorganisms with full viability after two years of storage from other species that lose viability rapidly. In parallel, BioNutrients-2 (BN-2) is being developed as a six-month flight experiment that will expand the range of foods produced on ISS to include yogurt, kefir, and probiotic yeast. BN-2 will improve upon hardware design by transitioning from a hard-shell polycarbonate bioreactor to a flexible fluorinated ethylene propylene (FEP) bag. This change reduces the mass and volume required to store production packs. The long-term goal of BioNutrients is to develop the capability for rapid and safe delivery of multiple micronutrients in single-use production packs. We expect synthetic biology approaches similar to BioNutrients will play an important role for astronaut health in future NASA missions to the Moon and Mars.

Synthetic Biology↗
NASA NTRS2012

Paramyxovirus Infection Mimics In Vivo Cellular Dynamics in Three-Demensional Human Bronchio-Epithelial Tissue-Like Assemblies

Respiratory syncytial virus and parainfluenza virus cause severe respiratory disease, especially in infants, children and the elderly. An in vitro model that accurately mimics infection of the human respiratory epithelium (HRE) would facilitate vaccine development greatly. Monolayer cultures traditionally used to study these viruses do not accurately and precisely differentiate the replication efficiencies of wild type and attenuated viruses. Therefore, we engineered novel three-dimensional (3D) tissue-like assemblies (TLAs) of human broncho-epithelial (HBE) cells to produce a more physiologically relevant in vitro model of the HRE. TLAs resemble HRE structurally and by expression of differentiated epithelial cell markers. Most significantly, wild type viruses exhibited a clear growth advantage over attenuated strains in TLAs unlike monolayer cultures. In addition, the TLAs responded to virus infection by secreting pro-inflammatory mediators similar to the respiratory epithelia of infected children. These characteristics make the TLA model a valuable platform technology to develop and evaluate live, attenuated respiratory virus vaccine candidates for human use. Respiratory virus diseases, the most frequent and least preventable of all infectious diseases, range in severity from the common cold to severe bronchiolitis and pneumonia . Two paramyxoviruses, respiratory syncytial virus (RSV) and parainfluenza virus type 3 (PIV3), are responsible for a majority of the most severe respiratory diseases of infants and young children. RSV causes 70% of all bronchiolitis cases and is a major cause of morbidity and mortality worldwide, especially in infants. PIV3 causes 10-15% of bronchiolitis and pneumonia during infancy, second only to RSV, and 40% of croup in infants To date, licensed vaccines are not available to prevent these respiratory diseases. At present, traditional monkey kidney (Vero and LLC-MK2) and human (HEp-2) tissue culture cells and small animal models (mouse, cotton rat, guinea pig, ferret, and hamster) fail to accurately imitate viral replication and human disease states (8). Lacking an authentic model has impeded the development and evaluation of live, attenuated vaccine candidates. Development of a physiologically relevant in vitro tissue culture model that reproduces characteristics of the HRE, the primary target of RSV and PIV3, would aid in predicting clinical attenuation and safety of vaccine candidates. Successful tissue engineering of a 3D human intestinal model using novel NASA technology inspired the development of a tri-culture 3D model for the HRE. Sequential layering of primary mesenchymal cells (comprised of normal human fibroblasts and endothelial cells) followed by BEAS-2B epithelial cells derived from human bronchi and tracheae were recapitulated on Cultisphere and/or cytodex3 microcarriers in cylindrical vessels that rotate horizontally creating an organized epithelial structure. Horizontal rotation randomizes the gravity vector modeling aspects of microgravity. Mesenchymal and epithelial cells grown under these conditions reproduce the structural organization, multi-cellular complexity, and differentiation state of the HRE. The opportunity to study respiratory viruses in a nasal epithelium model is invaluable because the most promising respiratory virus vaccine candidates are live attenuated viruses for intranasal administration. Here we characterize the interactions of respiratory viruses and epithelial cells grown under modeled microgravity in comparison to gravity-ladened monolayers. 3D HBE TLAs and traditional monolayers (2D) are infected at 35 C, the upper temperature of the upper HRE, to simulate in vivo infection conditions. Growth kinetics of wild type (wt) RSV and PIV3 viruses were compared in 2D and 3D cells to that of strains attenuated in humans or rhesus macaques. This novel 3D HBE model also offers an opportunity to study whether the epithelial cell function, especially in host defenses recapitulated by mimicking the structural organization of the HRE. In vivo, airway epithelial cells play a significant and dynamic role in host defense by blocking paracellular permeability and modulating airway function through cellular interactions or tight junctions. As regulators of the innate immune response, epithelial cells constitutively express cytokines, chemokines, and colony stimulating factors including RANTES, IL-8, IL-6, GM-CSF, and G-CSF for proactive host defense. In response to viral infection, epithelial cells induce potent immuno-modulatory and pro-inflammatory cytokines that recruit phagocytic and inflammatory cells to clear the virus and enhance protection. Although disease pathogenesis is classically attributed to the cytopathic effects of the pathogen, severe disease states associated with RSV and PIV3 are attributed to the inflammatory response, especially in infants. RSV is a potent inducer of cytokines and pro-inflammatory mediators in epithelial cells in vivo. A differentiated human epithelial model independent of the complete functional immune system will help elucidate the role of epithelial cells in respiratory disease. We reported here, virus and host cell interactions in 3D HBE TLAs are similar to that in vivo. Because the epithelial cell organization of the TLAs impacts not only the expression of airway epithelial characteristics, but also cellular communication, the TLAs represent a more physiologically relevant model of the HRE than BEAS-2B or other non-tumour monolayer models of respiratory disease. As a result, wild type respiratory viruses have a clear growth advantage over attenuated viruses in TLAs unlike traditional monolayers. In addition, the TLAs respond to wild type virus infection by secreting pro-inflammatory mediators characteristic of infected HRE. TLAs expressing microbial defense mechanisms provide an excellent model to study the interactions of respiratory pathogens with their host and to identify the innate immunity mediators. Therefore, 3D HBE TLAs offer advantages for the study of respiratory viruses and the development of viral vaccine candidates.

Deatly, Anne M.↗