Engineered human broncho-epithelial tissue-like assemblies
Inventors
Assignees
National Aeronautics and Space Administration NASA
Publication Number
US-8338114-B1
Publication Date
2012-12-25
Expiration Date
2027-04-19
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Abstract
Three-dimensional human broncho-epithelial tissue-like assemblies (TLAs) are produced in a rotating wall vessel (RWV) with microcarriers by coculturing mesenchymal bronchial-tracheal cells (BTC) and bronchial epithelium cells (BEC). These TLAs display structural characteristics and express markers of in vivo respiratory epithelia. TLAs are useful for screening compounds active in lung tissues such as antiviral compounds, cystic fibrosis treatments, allergens, and cytotoxic compounds.
Core Innovation
The invention relates to the engineering of three-dimensional human broncho-epithelial tissue-like assemblies (TLAs) generated in a rotating wall vessel (RWV) by coculturing mesenchymal bronchial-tracheal cells (BTC) and bronchial epithelium cells (BEC) on microcarriers. These TLAs exhibit structural features and express molecular markers characteristic of in vivo respiratory epithelia, including polarization, tight junctions, desmosomes, microvilli, and differentiation markers such as villin, keratins, and lung-specific proteins.
The problem addressed by the invention arises from limitations in existing in vitro models of lung epithelium. Two-dimensional monolayer cultures and traditional three-dimensional air-liquid interface cultures fail to replicate the complex cellular interactions, architecture, and longevity of native respiratory epithelia. Furthermore, traditional cultures are limited in scale and require complex culture media, complicating long-term growth and large-scale production. Prior 3D aggregates derived from cell lines lack functional and structural fidelity to in vivo airway epithelia. Therefore, there is a need for a scalable, physiologically relevant human respiratory epithelial model that maintains differentiation markers and functional characteristics over extended periods.
The invention overcomes these limitations by using RWV technology to coculture primary human mesenchymal BTCs and immortalized human bronchial epithelial cells on collagen-coated microcarriers, producing large-scale, well-differentiated TLAs that mimic in vivo human respiratory epithelia structurally and functionally. This model supports culture for more than 40 days without loss of cell markers, sustains viral infections, and is amenable to extensive analysis and manipulation. The TLAs provide a platform for studying cell-cell interactions, host-pathogen responses, and potential therapeutic assays with simplified culture media requirements and scalable production.
Claims Coverage
The patent contains two independent claims focused on methods of assaying test compounds using three-dimensional human broncho-epithelial tissue-like assemblies generated in rotating wall vessels. The claims cover inventive features related to TLA production conditions, assay procedures, and viral infection methods.
Method for producing tissue-like assemblies in a rotating wall vessel
Inoculating a rotating wall vessel containing microcarriers and culture media (with controlled density differences from 90% to 95% or 105% to 110%) with mesenchymal bronchial-tracheal cells followed by a growth period of 24 to 240 hours, then inoculating with bronchial epithelium cells to generate a coculture, and growing the coculture for 24 to 960 hours to produce TLAs expressing markers and structural characteristics of in vivo respiratory epithelia.
Assaying test compounds using tissue-like assemblies in infection models
Dividing cocultures into experimental and control TLAs, infecting experimental TLAs with respiratory viruses (including RSV, parainfluenza, HIV, and other specified viruses), allowing viral proliferation up to about 504 hours, contacting experimental TLAs with antiviral test compounds, and measuring phenotypic, genetic, or quantitative traits to determine therapeutic, allergenic, or cytotoxic activity.
Features of the rotating wall vessel culture apparatus and conditions
Utilization of a rotating wall vessel culture chamber rotatable about a horizontal longitudinal axis with means for controlled rotation, oxygenation, and waste removal, operating under conditions achieving collocation of culture media and cells, minimal relative motion with chamber boundaries, and freedom for three-dimensional spatial orientation to support TLA growth.
Infection and viral proliferation protocol in TLAs
A defined infection protocol involving virus absorption for about one hour at room temperature (20° to 25° C), washing infected TLAs three times with phosphate buffered saline, feeding with culture media, removing air bubbles to minimize shear, and incubating in a humidified environment at about 5% CO2 and 35° C, with periodic culture media replacement (approximately 65% every 48 hours).
Immunohistochemical measurement of marker expression
Measuring markers such as cytokeratin 8 and 18, Factor VIII, tubulin, PECAM-1, and others by immunohistochemistry in TLAs, with specific quantitative staining patterns (e.g., 99% staining for cytokeratin 18 and tubulin, 50-75% for cytokeratin 8 and Factor VIII), confirming differentiation and functional similarity to in vivo respiratory epithelia.
The independent claims cover inventive aspects of producing physiologically relevant 3D human broncho-epithelial TLAs in RWVs with specific density parameters, methods for assaying therapeutic and cytotoxic compounds using these assemblies infected with respiratory viruses, detailed infection and culture conditions, and specific marker-based assessments confirming in vivo-like differentiation.
Stated Advantages
The method enables production of large-scale, functionally accurate three-dimensional human airway tissue models that maintain differentiated epithelial and mesenchymal cell markers for prolonged culture periods exceeding 40 days.
The TLAs require less complex culture media due to specific cell-cell interactions and production of essential growth and differentiation factors, facilitating longer-term and scalable tissue culture.
The model supports productive infection with respiratory viruses such as human respiratory syncytial virus and parainfluenza virus, allowing study of host-virus interactions independent of the immune system.
The RWV culture system provides ease of manipulation, consistent culture conditions, and the ability to sustain viral production and cellular repair during infections.
Documented Applications
Screening of compounds for antiviral activity against respiratory viruses using TLA cultures infected with viruses such as RSV and parainfluenza virus.
Identification and development of cystic fibrosis treatments by creating TLAs with cystic fibrosis epithelial cells to assay changes in mucous production, surfactants, and other CF-related traits.
Quantitative and qualitative assessment of allergenic activity by exposing TLAs to known and unknown allergens or irritants and measuring inflammatory and apoptotic markers.
Determination of cytotoxicity of test compounds by observing apoptotic responses and other markers in TLAs.
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