High-throughput bioprinting of the nasal epithelium using patient-derived nasal epithelial cells.

Progenitor human nasal epithelial cells (hNECs) are an essential cell source for the reconstruction of the respiratory pseudostratified columnar epithelium composed of multiple cell types in the context of infection studies and disease modeling. Hitherto, manual seeding has been the dominant method for creating nasal epithelial tissue models through biofabrication. However, this approach has limitations in terms of achieving the intricate three-dimensional (3D) structure of the natural nasal epithelium. 3D bioprinting has been utilized to reconstruct various epithelial tissue models, such as cutaneous, intestinal, alveolar, and bronchial epithelium, but there has been no attempt to use of 3D bioprinting technologies for reconstruction of the nasal epithelium. In this study, for the first time, we demonstrate the reconstruction of the nasal epithelium with the use of primary hNECs deposited on Transwell inserts via droplet-based bioprinting (DBB), which enabled high-throughput fabrication of the nasal epithelium in Transwell inserts of 24-well plates. DBB of progenitor hNECs ranging from one-tenth to one-half of the cell seeding density employed during the conventional cell seeding approach enabled a high degree of differentiation with the presence of cilia and tight-junctions over a 4 weeks air-liquid interface culture. Single cell RNA sequencing of these cultures identified five major epithelial cells populations, including basal, suprabasal, goblet, club, and ciliated cells. These cultures recapitulated the pseudostratified columnar epithelial architecture present in the native nasal epithelium and were permissive to respiratory virus infection. These results denote the potential of 3D bioprinting for high-throughput fabrication of nasal epithelial tissue models not only for infection studies but also for other purposes, such as disease modeling, immunological studies, and drug screening.

High-Throughput Bioprinting of the Nasal Epithelium using Patient-derived Nasal Epithelial Cells.

Human nasal epithelial cells (hNECs) are an essential cell source for the reconstruction of the respiratory pseudostratified columnar epithelium composed of multiple cell types in the context of infection studies and disease modeling. Hitherto, manual seeding has been the dominant method for creating nasal epithelial tissue models. However, the manual approach is slow, low-throughput and has limitations in terms of achieving the intricate 3D structure of the natural nasal epithelium in a uniform manner. 3D Bioprinting has been utilized to reconstruct various epithelial tissue models, such as cutaneous, intestinal, alveolar, and bronchial epithelium, but there has been no attempt to use of 3D bioprinting technologies for reconstruction of the nasal epithelium. In this study, for the first time, we demonstrate the reconstruction of the nasal epithelium with the use of primary hNECs deposited on Transwell inserts via droplet-based bioprinting (DBB), which enabled high-throughput fabrication of the nasal epithelium in Transwell inserts of 24-well plates. DBB of nasal progenitor cells ranging from one-tenth to one-half of the cell seeding density employed during the conventional cell seeding approach enabled a high degree of differentiation with the presence of cilia and tight-junctions over a 4-week air-liquid interface culture. Single cell RNA sequencing of these cultures identified five major epithelial cells populations, including basal, suprabasal, goblet, club, and ciliated cells. These cultures recapitulated the pseudostratified columnar epithelial architecture present in the native nasal epithelium and were permissive to respiratory virus infection. These results denote the potential of 3D bioprinting for high-throughput fabrication of nasal epithelial tissue models not only for infection studies but also for other purposes such as disease modeling, immunological studies, and drug screening.

Stimulation Effects and Mechanisms of Different Adjuvants on a Norovirus P Particle-Based Active Amyloid-ß Vaccine.

BACKGROUND: Adjuvants are important components of vaccines and effectively enhance the immune response of specific antigens. However, the role of adjuvants or combinations of adjuvants in stimulating immunogenicity of the amyloid-ß (Aß) vaccine, as well as molecular mechanisms underlying such stimulation still remain unclear. A previous study of ours developed a norovirus P particle-based active Aß epitope vaccine, PP-3copy-Aß1-6-loop123, which stimulates a high titer of Aß-specific antibodies in mouse Alzheimer's disease (AD) models. OBJECTIVE: The most effective and safe adjuvant that maximizes the immunogenicity of our protein vaccine was determined. METHODS: We investigated four adjuvants (CpG, AS02, AS03, and MF59), and combinations of those, for capacity to enhance immunogenicity, and performed transcriptome analysis to explore mechanisms underlying the role of these in AD immunotherapy. RESULTS: Addition of the adjuvant, AS02, remarkably improved the immunogenicity of the PP-3copy-Aß1-6-loop123 vaccine without triggering an Aß-specific T-cell response. Combinations of adjuvants, particularly CpG +  AS02 and CpG + AS03, elicited a significantly elevated and prolonged Aß-specific antibody response. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that a combination of two adjuvants was more effective in activating immune-related pathways, thereby enhancing the immunogenicity of PP-3copy-Aß1-6-loop123. CONCLUSION: These findings demonstrated that adjuvants can be used as enhancers in AD protein vaccination, and that a combination of CpG and AS-related adjuvants may be a very effective adjuvant candidate suitable for further clinical trials of the PP-3copy-Aß1-6-loop123 vaccine. Our studies also revealed potential mechanisms underlying the stimulation of immune response of protein vaccines by adjuvants.

An atorvastatin calcium and poly(L-lactide-co-caprolactone) core-shell nanofiber-covered stent to treat aneurysms and promote reendothelialization.

Aneurysmal subarachnoid hemorrhage is a common complication caused by an intracranial aneurysm that can lead to hemorrhagic stroke, brain damage, and death. Knowing this clinical situation, the purpose of this study was to develop a controlled-release stent covered with a core-shell nanofiber mesh, fabricated by emulsion electrospinning, for the treatment of aneurysms. By encapsulating atorvastatin calcium (AtvCa) in the inner of poly (L-lactide-co-caprolactone) (PLCL) nanofibers, the release period of AtvCa was effectively extended. The morphology and inner structure of the core-shell nanofibers were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), respectively. The release of AtvCa from the nanofiber system continued for more than ten weeks without a significant initial burst release. The nanofiber mesh structure degraded gradually but maintained its fiber morphology before neovascularization. The results of this study further elucidated the reendothelialization mechanism of AtvCa by analyzing the nitric oxide (NO) expression from seeded HUVECs. The in vivo studies demonstrated that the PLCL-AtvCa covered stents were capable of separating the aneurysm dome from the blood circulation, leading to the abolishment of the aneurysm. Moreover, the AtvCa controlled release promoted the in vitro proliferation of HUVECs on the nanofiber meshes, and the PLCL-AtvCa covered stents induced in vivo neovascularization. STATEMENT OF SIGNIFICANCE: Intracranial aneurysms are pathological dilatations of blood vessels that have developed an abnormally weak wall structure, thus prone to rupture. Covered stents had been demonstrated to be a method for the treatment of intracranial aneurysm. We prepared a controlled-release stent covered with a core-shell nanofiber mesh, fabricated by emulsion electrospinning, which encapsulated atorvastatin calcium in the inner portion of nanofibers. The results of this study further elucidated the reendothelialization mechanism of AtvCa by analyzing the nitric oxide (NO) expression from seeded HUVECs. The generated AtvCa-load covered stents separated the aneurysm dome from the blood circulation, and keep long-term patency of the parent artery. But also induced neovascularization, thus provide further protection against recurrence of aneurysms after nanofiber meshes degradation.