Pneumatically Driven Microfluidic Platform and Fully Automated Particle Concentration System for the Capture and Enrichment of Pathogens.

In this study, we developed a pneumatically driven microfluidic platform (PDMFP) operated by a fully automated particle concentration system (FAPCS) for the pretreatment of micro- and nano-sized materials. The proposed PDMFP comprises a 3D network with a curved fluidic chamber and channel, five on/off pneumatic valves for blocking fluid flow, and a sieve valve for sequential trapping of microbeads and target particles. Using this setup, concentrated targets are automatically released into an outlet port. The FAPCS mainly comprises solenoid valves, glass reservoirs, a regulator, pressure sensor, main printed circuit board, and liquid crystal display touch panel. All pneumatic valves in the microfluidic platform as well as the working fluids in the glass reservoirs are controlled using FAPCS. The flow rate of the working fluids is measured to demonstrate the sequential programed operation of the proposed pretreatment process using FAPCS. In our study, we successfully achieved rapid and efficient enrichment using PDMFP-FAPCS with fluorescence-labeled Escherichia coli. With pretreatment-10 min for the microbead concentration and 25 min for target binding-almost all the target bacteria could be captured. A total of 526 Gram-negative bacteria were attached to 82 beads, whereas Gram-positive bacteria were attached to only 2 of the 100 beads. Finally, we evaluated the PDMFP-FAPCS for SARS-CoV-2 receptor-binding domain (RBD)-based outer membrane vesicles (OMVs) (RBD-OMVs). Specific probes involved in PDMFP-FAPCS successfully isolated RBD-OMVs. Thus, PDMFP-FAPCS exhibits excellent enrichment of particles, including microbes and nanovesicles, and is an effective pretreatment platform for disease diagnosis and investigation.

Microfluidic Acoustophoresis for Flowthrough Separation of Gram-Negative Bacteria using Aptamer Affinity Beads.

This article describes the fabrication and operation of microfluidic acoustophoretic chips using a microfluidic acoustophoresis technique and aptamer-modified microbeads that can be used for the fast, efficient isolation of Gram-negative bacteria from a medium. This method enhances the separation efficiency using a mix of long, square microchannels. In this system, the sample and buffer are injected into the inlet port through a flow controller. For bead centering and sample separation, AC power is applied to the piezoelectric transducer via a function generator with a power amplifier to generate acoustic radiation force in the microchannel. There is a bifurcated channel at both the inlet and outlet, enabling simultaneous separation, purification, and concentration. The device has a recovery rate of >98% and purity of 97.8% up to a 10x dose concentration. This study has demonstrated a recovery rate and purity higher than the existing methods for separating bacteria, suggesting that the device can separate bacteria efficiently.

Square microchannel enables to focus and orient ellipsoidal Euglena gracilis cells by two-dimensional acoustic standing wave.

Flow cytometry has become an indispensable tool for counting, analyzing, and sorting large cell populations in biological research and medical practice. Unfortunately, it has limitations in the analysis of non-spherically shaped cells due to the variation of their alignment with respect to the flow direction and, hence, the optical interrogation axis, resulting in unreliable cell analysis. Here, we present a simple on-chip acoustofluidic method to fix the orientation of ellipsoidal cells and focus them into a single, aligned stream. Specifically, by generating acoustic standing waves inside a 100 ⋅ 100 µm square-shaped microchannel, we successfully aligned and focused up to 97.7% of a population of Euglena gracilis (an ellipsoidal shaped microalgal species) cells in the center of the microchannel with high precision at a volume rate of 25 to 200 µL min-1. Uniform positioning of ellipsoidal cells is essential for making flow cytometry applicable to the investigation of a greater variety of cell populations and is expected to be beneficial for ecological studies and aquaculture.

Pneumatically Driven Microfluidic Platform for Micro-Particle Concentration.

The present article introduces a method for fabricating and operating a pneumatic valve to control particle concentration using a microfluidic platform. This platform has a three-dimensional (3D) network with curved fluid channels and three pneumatic valves, which create networks, channels, and spaces through duplex replication with polydimethylsiloxane (PDMS). The device operates based on the transient response of a fluid flow rate controlled by a pneumatic valve in the following order: (1) sample loading, (2) sample blocking, (3) sample concentration, and (4) sample release. The particles are blocked by thin diaphragm layer deformation of the sieve valve (Vs) plate and accumulate in the curved microfluidic channel. The working fluid is discharged by the actuation of two on/off valves. As a result of the operation, all particles of various magnifications were successfully intercepted and disengaged. When this technology is applied, the operating pressure, the time required for concentration, and the concentration rate may vary depending on the device dimensions and particle size magnification.

In vitro study on anti-inflammatory effects of epigallocatechin-3-gallate-loaded nano- and microscale particles.

PURPOSE: This study aimed to develop an anti-inflammation system consisting of epigallo-catechin-3-gallate (EGCG) encapsulated in poly(lactide-co-glycolic acid) (PLGA) particles to promote wound healing. METHODS: Nano- and microscale PLGA particles were fabricated using a water/oil/water emulsion solvent evaporation method. The optimal particle size was determined based on drug delivery efficiency and biocompatibility. The particles were loaded with EGCG. The anti-inflammatory effects of the particles were evaluated in an in vitro cell-based inflammation model. RESULTS: Nano- and microscale PLGA particles were produced. The microscale particles showed better biocompatibility than the nanoscale particles. In addition, the microscale particles released ~60% of the loaded drug, while the nanoscale particles released ~50%, within 48 hours. Thus, microscale particles were selected as the carriers. The optimal EGCG working concentration was determined based on the effects on cell viability and inflammation. A high EGCG dose (100 µM) resulted in poor cell viability; therefore, a lower dose (≤50 µM) was used. Moreover, 50 µM EGCG had a greater anti-inflammatory effect than 10 µM concentration on lipopolysaccharide-induced inflammation. Therefore, 50 µM EGCG was selected as the working dose. EGCG-loaded microparticles inhibited inflammation in human dermal fibroblasts. Interestingly, the inhibitory effects persisted after replacement of the drug-loaded particle suspension solution with fresh medium. CONCLUSION: The EGCG-loaded microscale particles are biocompatible and exert a sustained anti-inflammatory effect.

MG-63 cells proliferation following various types of mechanical stimulation on cells by auxetic hybrid scaffolds.

BACKGROUND: Mechanical properties and cyto-compatibility of a composite scaffold which possessed negative (-) Poisson's ratio (NPR) was investigated for effective load transfer from auxetic scaffold to cell. METHODS: Organic/inorganic composite scaffolds were prepared by mixing hydroxyapatite (HA) to poly(lactide-co-glycolide) (PLGA). To induce NPR in composite scaffold, 3-directional volumetric compression was applied during the scaffold fabrication at adequate temperature(60°C). The pore size of scaffold ranged between 355-400 µm. RESULTS: Poisson's ratios of NPR scaffolds and control scaffolds were -0.07 and 0.16 at 10 % strain. For stable physical stimulating to loaded cells, ceramic/polymer composite scaffold was prepared by incorporating HA in PLGA to increase mechanical strength. Compressive strength of the HA/PLGA composite scaffold (15 wt. % HA to PLGA) was about 21.7 % higher than that of PLGA-only scaffold. The recovery rates of the NPR composite scaffold after applying compression in the dry and wet states were 90 % and 60 %, respectively. Also the composite scaffold was shown to have better hydrophilicity (61.9°) compared to the PLGA-only scaffolds (65.3°). Cell proliferation of osteoblast-like cell line (MG-63) in the composite scaffold was 20 % higher than in PLGA-only scaffold at static compressive stimulation. For dynamic compressive stimulation (15 min cyclic interval), cell proliferation in the composite scaffold was 2 times higher than that of in PLGA-only scaffold. In conclusion, NPR composite (HA/PLGA) scaffold was effective in isotropic compressive load delivery for osteogenic cell proliferation. CONCLUSION: This composite scaffold with stimulation can be used as tissue engineered scaffold and dynamic cell culture system for bone tissue regeneration.

MG-63 osteoblast-like cell proliferation on auxetic PLGA scaffold with mechanical stimulation for bone tissue regeneration.

BACKGROUND: Auxetic scaffolds (experimental) was fabricated by using poly(D, L-lactic-co-glycolic acid), 50:50, (PLGA) for effective bone cell proliferation with mechanical stimulation. METHODS: Negative Poisson's ratio in scaffold, 3-directional volumetric compression was applied during the scaffold fabrication at adequate temperature (60 °C). The pore size of scaffold ranged between 355 and 400 µm. RESULTS: The porous morphology of the prepared auxetic scaffolds had shown partially concave and dent shapes in SEM image as expected. The lowest Poisson's ratios of experimental group was -0.07 at 60 °C/10 min. Compressive strength of experimental group was shown about 3.12 times higher than control group (conventional scaffold) in dry state at 25 °C. The compressive strengths of both groups were tended to be decreased dramatically in wet state compared to in dry state. However, compressive strengths of experimental group were higher 3.08 times and 1.88 times in EtOH/PBS (25 °C) and EtOH/PBS/DMEM (37 °C) than control group in wet state, respectively. Degradation rate of the scaffolds showed about 16 % weight loss in 5 weeks. In cell attachment test, experimental group showed 1.46 times higher cell proliferation than control group at 1-day with compressive stimulation. In 3-day culture, the experimental group showed 1.32 times higher than control group. However, there was no significant difference in cell proliferation in 5-day cultivation. CONCLUSION: Overall, negative Poisson's ratio scaffolds with static mechanical stimulation could affect the cell proliferation at initial cultivation time.