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•Cell cluster removal device based on spiral inertial microfluidics.•Cell cluster removal with high volumetric throughput, removal efficiency, and biocompatibility.•The first ...demonstration of the selective removal of large CHO cell clusters from bioreactors using microfluidics.•The first report on the large-particle trapping effect in spiral inertial microfluidic devices.
Suspension cultures of Chinese hamster ovary (CHO) cells are extensively employed in biopharmaceutical production. During cultivation, CHO cell clusters often form due to cell death and other irregularities, substantially reducing cell growth and productivity. In this study, we introduce a novel method for the selective removal of large CHO cell clusters from bioreactors, utilizing the large-particle trapping effect in spiral inertial microfluidics. A single spiral inertial microfluidic device effectively removed large clusters from CHO cell culture, demonstrating high volumetric throughput, removal efficiency, and biocompatibility. This straightforward, agent-free, and continuous-flow approach can be applied to remove large mammalian cell clusters across various scales and modes of operation (discontinuous or continuous) during cell line development, bioprocess optimization, and biomanufacturing.
Process analytical technology (PAT) is critical for the manufacture of high-quality biologics as it enables continuous, real-time and on-line/at-line monitoring during biomanufacturing processes. The ...conventional analytical tools currently used have many restrictions to realizing the PAT of current and future biomanufacturing. Here we describe a nanofluidic device for the continuous monitoring of biologics' purity and bioactivity with high sensitivity, resolution and speed. Periodic and angled nanofilter arrays served as the molecular sieve structures to conduct a continuous size-based analysis of biologics. A multiparameter quality monitoring of three separate commercial biologic samples within 50 minutes has been demonstrated, with 20 µl of sample consumption, inclusive of dead volume in the reservoirs. Additionally, a proof-of-concept prototype system, which integrates an on-line sample-preparation system and the nanofluidic device, was demonstrated for at-line monitoring. Thus, the system is ideal for on-site monitoring, and the real-time quality assurance of biologics throughout the biomanufacturing processes.
Online monitoring of monoclonal antibody product titers throughout biologics process development and production enables rapid bioprocess decision-making and process optimization. Conventional ...analytical methods, including high-performance liquid chromatography and turbidimetry, typically require interfacing with an automated sampling system capable of online sampling and fractionation, which suffers from increased cost, a higher risk of failure, and a higher mechanical complexity of the system. In this study, a novel nanofluidic system for continuous direct (no sample preparation) IgG titer measurements was investigated. Tumor necrosis factor α (TNF-α), conjugated with fluorophores, was utilized as a selective binder for adalimumab in the unprocessed cell culture supernatant. The nanofluidic device can separate the bound complex from unbound TNF-α and selectively concentrate the bound complex for high-sensitivity detection. Based on the fluorescence intensity from the concentrated bound complex, a fluorescence intensity versus titer curve can be generated, which was used to determine the titer of samples from filtered, unpurified Chinese hamster ovary cell cultures continuously. The system performed direct monitoring of IgG titers with nanomolar resolution and showed a good correlation with the biolayer interferometry assays. Furthermore, by variation of the concentration of the indicator (TNF-α), the dynamic range of the system can be tuned and further expanded.
Removing nonviable cells from a cell suspension is crucial in biotechnology and biomanufacturing. Label-free microfluidic cell separation devices based on dielectrophoresis, acoustophoresis, and ...deterministic lateral displacement are used to remove nonviable cells. However, their volumetric throughputs and test cell concentrations are generally too low to be useful in typical bioreactors in biomanufacturing. In this study, we demonstrate the efficient removal of small (<10 μm) nonviable cells from bioreactors while maintaining viable cells using inertial microfluidic cell sorting devices and characterize their performance. Despite the size overlap between viable and nonviable cell populations, the devices demonstrated 3.5-28.0% dead cell removal efficiency with 88.3-83.6% removal purity as well as 97.8-99.8% live cell retention efficiency at 4 million cells per mL with 80% viability. Cascaded and parallel configurations increased the cell concentration capacity (10 million cells per mL) and volumetric throughput (6-8 mL min-1). The system can be used for the removal of small nonviable cells from a cell suspension during continuous perfusion cell culture operations.
Medium perfusion is critical in maintaining high cell concentration in cultures. The conventional membrane filtration method for medium exchange has been challenged by the fouling and clogging of the ...membrane filters in long‐term cultures. In this study, we present a miniature auto‐perfusion system that can be operated inside a common‐size laboratory incubator. The system is equipped with a spiral microfluidic chip for cell retention to replace conventional membrane filters, which fundamentally overcomes the clogging and fouling problem. We showed that the system supported continuous perfusion culture of Chinese hamster ovary (CHO) cells in suspension up to 14 days without cell retention chip replacement. Compared to daily manual medium change, 25% higher CHO cell concentration can be maintained at an average auto‐perfusion rate of 196 ml/day in spinner flask at 70 ml working volume (2.8 VVD). The auto‐perfusion system also resulted in better cell quality at high concentrations, in terms of higher viability, more uniform and regular morphology, and fewer aggregates. We also demonstrated the potential application of the system for culturing mesenchymal stem cells on microcarriers. This miniature auto‐perfusion system provides an excellent solution to maintain cell‐favorable conditions and high cell concentration in small‐scale cultures for research and clinical uses.
A miniature auto‐perfusion bioreactor system with a spiral microfluidic cell retention device was developed in this study. The clogging‐free cell retention technology, utilizing inertial microfluidics principles, was applicable to suspension cells and cell‐laden microcarriers. By continuous culture media perfusion, the system could maintain cell‐favourable conditions and high cell concentration in small‐scale long‐term cultures, which is suitable for research and clinical applications. α.
Although droplet microfludics is a promising technology for handling a number of liquids of a single type of analyte, it has limitations in handling thousands of different types of analytes for ...multiplex assay. Here, we present a novel "liquid-capped encoded microcapsule", which is applicable to various liquid format assays. Various liquid drops can be graphically encoded and arrayed without repeated dispensing processes, evaporation, and the risk of cross-contamination. Millions of nanoliter-scale liquids are encapsulated within encoded microcapsules and self-assembled in microwells in a single dispensing process. The graphical code on the microcapsule enables identification of randomly assembled microcapsules in each microwell. We conducted various liquid phase assays including enzyme inhibitor screening, virus transduction, and drug-induced apoptosis tests. The results showed that our liquid handling technology can be utilized widely for various solution phase assays.
Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource to evaluate a variety of pulmonary diseases, ...both for research and clinical purposes. However, due to the heterogeneous and viscous nature of patient mucus, there is currently no reliable dissociation method that does not damage the host immune cells in the patient airway secretion. In this research, we introduce a sample preparation method that uses inertial microfluidics for the patient's immune assessment. Regardless of the heterogeneous fluidic properties of the clinical samples, the proposed method recovers more than 95% of neutrophils from airway secretion samples that are diluted 1,000-fold with milliliters of clean saline. By recirculating the concentrated output stream to the initial sample reservoir, a high concentration, recovery, and purity of the immune cells are provided; recirculation is considered a trade-off to the single-run syringe-based operation of inertial microfluidics. The closed-loop operation of spiral microfluidics provides leukocytes without physical or chemical disturbance, as demonstrated by the phorbol 12-myristate 13-acetate (PMA)-induced elastase release of sorted neutrophils.
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