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1.
bioRxiv ; 2024 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-38979374

RESUMO

Process Analytical Technologies (PAT) used to monitor and control manufacturing processes are crucial for efficient and automated bioprocessing, which is in congruence with lights-off-manufacturing and Industry 4.0 initiatives. As biomanufacturing seeks to realize more high-throughput and automated operation, an increasing need for multimodal analysis of process metrics becomes essential. Herein, we detail a series of methods for analyzing product yield from a bioreactor and how to conduct cross-method comparisons. We employ a model system of Escherichia coli (E. coli) expression of green fluorescent protein (GFP), which is a simple, cost effective model for students and educators to replicate at different scales. GFP is an ideal analytical marker as it is easy to visualize due to its fluorescence which indicates cellular protein expression, cell localization and physiological changes of the cell population. In this study, samples from a 300 L bioreactor with GFP-expressing E. coli are analyzed to improve product yield and bioprocessing efficiency. Utilizing a fed-batch process for enhanced cell density and product titer, this bioreactor runs on a 24-hour schedule from inoculation to GFP induction and final harvest. To reliably quantify relative GFP expression and E. coli proliferation, we provide simple protocols and example results for comparing three different analytical methods: (1) in-line bioreactor measurements, (2) plate reader assays, and (3) microscopy. The GFP and cell density results follow similar trends based on the various inline and offline analytical methods and show a peak of GFP expression and cell density between 12.5 and 18 hours post inoculation.

2.
bioRxiv ; 2023 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-37961220

RESUMO

Microphysiological systems (MPS) incorporate physiologically relevant microanatomy, mechanics, and cells to mimic tissue function. Reproducible and standardized in vitro models of tissue barriers, such as the blood-tissue interface (BTI), are critical for next-generation MPS applications in research and industry. Many models of the BTI are limited by the need for semipermeable membranes, use of homogenous cell populations, or 2D culture. These factors limit the relevant endothelial-epithelial contact and 3D transport, which would best mimic the BTI. Current models are also difficult to assemble, requiring precise alignment and layering of components. The work reported herein details the engineering of a BTI-on-a-chip (BTI Chip) that addresses current disadvantages by demonstrating a single layer, membrane-free design. Laminar flow profiles, photocurable hydrogel scaffolds, and human cell lines were used to construct a BTI Chip that juxtaposes an endothelium in direct contact with a 3D engineered tissue. A biomaterial composite, gelatin methacryloyl and 8-arm polyethylene glycol thiol, was used for in situ fabrication of a tissue structure within a Y-shaped microfluidic device. To produce the BTI, a laminar flow profile was achieved by flowing a photocurable precursor solution alongside phosphate buffered saline. Immediately after stopping flow, the scaffold underwent polymerization through a rapid exposure to UV light (<300 mJ·cm-2). After scaffold formation, blood vessel endothelial cells were introduced and allowed to adhere directly to the 3D tissue scaffold, without barriers or phase guides. Fabrication of the BTI Chip was demonstrated in both an epithelial tissue model and blood-brain barrier (BBB) model. In the epithelial model, scaffolds were seeded with human dermal fibroblasts. For the BBB models, scaffolds were seeded with the immortalized glial cell line, SVGP12. The BTI Chip microanatomy was analyzed post facto by immunohistochemistry, showing the uniform production of a patent endothelium juxtaposed with a 3D engineered tissue. Fluorescent tracer molecules were used to characterize the permeability of the BTI Chip. The BTI Chips were challenged with an efflux pump inhibitor, cyclosporine A, to assess physiological function and endothelial cell activation. Operation of physiologically relevant BTI Chips and a novel means for high-throughput MPS generation was demonstrated, enabling future development for drug candidate screening and fundamental biological investigations.

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