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The COVID-19 pandemic has led to high global demand for vaccines to safeguard public health. To that end, our institute has developed a recombinant viral vector vaccine utilizing a modified vesicular stomatitis virus (VSV) construct, wherein the G protein of VSV is replaced with the spike protein of SARS-CoV-2 (rVSV-ΔG-spike). Previous studies have demonstrated the production of a VSV-based vaccine in Vero cells adsorbed on Cytodex 1 microcarriers or in suspension. However, the titers were limited by both the carrier surface area and shear forces. Here, we describe the development of a bioprocess for rVSV-ΔG-spike production in serum-free Vero cells using porous Fibra-Cel ® macrocarriers in fixed-bed BioBLU ® 320 5p bioreactors, leading to high-end titers. We identified core factors that significantly improved virus production, such as the kinetics of virus production, the use of macrospargers for oxygen supply, and medium replenishment. Implementing these parameters, among others, in a series of GMP production processes improved the titer yields by at least two orders of magnitude (2e9 PFU/mL) over previously reported values. The developed process was highly effective, repeatable, and robust, creating potent and genetically stable vaccine viruses and introducing new opportunities for application in other viral vaccine platforms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Cohen, Simon, Hazan, Tal, Tzadok, Levin, Girshengorn, Mimran, Natan, Baruhi, David, Rosen, Shmaya, Borni, Cohen, Lupu, Kedmi, Zilberman, Jayson, Monash, Dor, Diamant, Goldvaser, Cohen-Gihon, Israeli, Lazar, Shifman, Beth-Din, Zvi, Oren, Makovitzki, Lerer, Mimran, Toister, Zichel, Adar and Epstein.)

Recent advances in the field of cell therapy have proposed new solutions for tissue repair and regeneration using various cell delivery approaches. Here we studied ex vivo a novel topical delivery system of encapsulated cells in hybrid polyethylene glycol-fibrinogen (PEG-Fb) hydrogel microspheres to respiratory tract models. We investigated basic parameters of cell encapsulation, delivery and release in conditions of inflamed and damaged lungs of bacterial-infected mice. The establishment of each step in the study was essential for the proof of concept. We demonstrated co-encapsulation of alveolar macrophages and epithelial cells that were highly viable and equally distributed inside the microspheres. We found that encapsulated macrophages exposed to bacterial endotoxin lipopolysaccharide preserved high viability and secreted moderate levels of TNFα, whereas non-encapsulated cells exhibited a burst TNFα secretion and reduced viability. LPS-exposed encapsulated macrophages exhibited elongated morphology and out-migration capability from microspheres. Microsphere degradation and cell release in inflamed lung environment was studied ex vivo by the incubation of encapsulated macrophages with lung extracts derived from intranasally infected mice with Yersinia pestis , demonstrating the potential in cell targeting and release in inflamed lungs. Finally, we demonstrated microsphere delivery to a multi-component airways-on-chip platform that mimic human nasal, bronchial and alveolar airways in serially connected compartments. This study demonstrates the feasibility in using hydrogel microspheres as an effective method for topical cell delivery to the lungs in the context of pulmonary damage and the need for tissue repair., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cohen, Vagima, Mouhadeb, Toister, Gutman, Lazar, Jayson, Artzy-Schnirman, Sznitman, Ordentlich, Yitzhaki, Seliktar, Mamroud and Epstein.)