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Journal of Colloid and Interface Science Oct 2022Microscopic self-propelled motors (SPMs) are an area of active research, but very little investigation has been conducted on millimetre-scale or macroscopic SPMs and...
HYPOTHESIS
Microscopic self-propelled motors (SPMs) are an area of active research, but very little investigation has been conducted on millimetre-scale or macroscopic SPMs and exploring their potential in biomedical research. In this study, we tested if 3D reactive inkjet (RIJ) printing could be used for precise fabrication of millimetre-scale self-propelled motors (SPMs) with well-defined shapes from regenerated silk fibroin (RSF) by converting water soluble RSF (silk I) to insoluble silk fibroin (silk II). Secondly, we compared the different propulsion behaviour of the SPMs to put forward the best geometry and propulsion mechanism for potential applications in enhancing the sensitivity of diffusion-rate limited biomedical assays by inducing fluid flow.
EXPERIMENTS
SPMs with four different geometric shapes and propelled by two different mechanisms (catalysis and surface tension gradient) were fabricated by 3D RIJ printing and compared. For bubble propulsion, the structures were selectively doped in specific regions with the enzyme catalase in order to produce motion via bubble generation and detachment in hydrogen peroxide solutions. For surface tension propulsion, PEG-doped structures were propelled through surface tension gradients caused by leaching of PEG surfactant in deionized water.
FINDINGS
The results demonstrated the ability of 3D inkjet printing to fabricate SPMs with desired propulsion mechanism and fine-tune the propulsion by precisely fabricating the different geometric shapes. The resulting 3D structures were capable of generating motion without external actuation, thereby enabling applications in biomedicine such as micro-stirring small fluid volumes to enhance biological assay sensitivity. The surface tension gradient caused by the leaching of surfactant led to faster propulsion velocities with smooth deceleration, whereas, in comparison, catalysis-induced bubble propulsion tended to be jerky and uneven in deceleration, and therefore less suitable for aforementioned applications. Computational fluid dynamic simulations were used to compare the various experimental SPMs ability to enhance mixing when deployed within 96-well plate microwells, to reveal the effect of both SPM shape and motion character on performance, and show viability for small scale mixing applications.
Topics: Fibroins; Printing, Three-Dimensional; Silk; Surface-Active Agents; Water
PubMed: 35576653
DOI: 10.1016/j.jcis.2022.05.011 -
Frontiers in Bioengineering and... 2022Islet encapsulation devices serve to deliver pancreatic beta cells to type 1 diabetic patients without the need for chronic immunosuppression. However, clinical...
Islet encapsulation devices serve to deliver pancreatic beta cells to type 1 diabetic patients without the need for chronic immunosuppression. However, clinical translation is hampered by mass transport limitations causing graft hypoxia. This is exacerbated in devices relying only on passive diffusion for oxygenation. Here, we describe the application of a cylindrical perfusion system to study oxygen effects on islet-like clusters immobilized in alginate hydrogel. Mouse insulinoma 6 islet-like clusters were generated using microwell plates and characterized with respect to size distribution, viability, and oxygen consumption rate to determine an appropriate seeding density for perfusion studies. Immobilized clusters were perfused through a central channel at different oxygen tensions. Analysis of histological staining indicated the distribution of viable clusters was severely limited to near the perfusion channel at low oxygen tensions, while the distribution was broadest at normoxia. The results agreed with a 3D computational model designed to simulate the oxygen distribution within the perfusion device. Further simulations were generated to predict device performance with human islets under and conditions. The combination of experimental and computational findings suggest that a multichannel perfusion strategy could support viability and function of a therapeutic islet dose.
PubMed: 35519615
DOI: 10.3389/fbioe.2022.884071 -
Archives of Microbiology May 2022The depletion of dissolved oxygen in a defined synthetic medium can be measured in real time, using a micro-well plate format, associated with a fluorescent plate...
Microtiter plate with built-in oxygen sensors: a novel approach to investigate the dynamics of Pseudomonas aeruginosa growth suppression in the presence of divalent cations and antibiotics.
The depletion of dissolved oxygen in a defined synthetic medium can be measured in real time, using a micro-well plate format, associated with a fluorescent plate reader. This technology is appropriate for investigating the effect of antibiotics on cell kinetics because there is a direct correlation between the latter and the amount of dissolved oxygen in the medium of an assay. In this study, the metabolic activity of the opportunistic human pathogen Pseudomonas aeruginosa PA01 was investigated using the OxoPlate OP96U optical sensor technology. The response of P. aeruginosa to aminoglycoside antibiotics when Caand Mg ions are present in the Evans defined synthetic medium was measured. The results revealed that the effect of antibiotics on P. aeruginosa is influenced by the concentration of divalent cations present in the test medium, although the efficiency of Ca in supressing antibiotic activity was found to be greater than that of Mg. By comparison to tobramycin, the effect of amikacin is largely inhibited by the Caand Mgconcentrations. The study results underscore that the reliability of the observation of growth inhibitors is enhanced by the oxygen consumption measurements. Thus, the OxoPlate OP96U system is proven to be an accurate method to test the effectiveness of antibiotic treatments against P. aeruginosa.
Topics: Anti-Bacterial Agents; Cations, Divalent; Humans; Microbial Sensitivity Tests; Oxygen; Pseudomonas aeruginosa; Reproducibility of Results; Tobramycin
PubMed: 35508818
DOI: 10.1007/s00203-022-02877-y -
Acta Naturae 2022The aim of this work is to develop a 3D cell culture model based on cell spheroids for predicting the functional activity of various compounds in vivo. Agarose gel molds...
The aim of this work is to develop a 3D cell culture model based on cell spheroids for predicting the functional activity of various compounds in vivo. Agarose gel molds were made using 3D printing. The solidified agarose gel is a matrix consisting of nine low-adhesive U-shaped microwells of 2.3 × 3.3 mm for 3D cell spheroid formation and growth. This matrix is placed into a single well of a 12-well plate. The effectiveness of the cell culture method was demonstrated using human ovarian carcinoma SKOVip-kat cells stably expressing the red fluorescent protein Katushka in the cytoplasm and overexpressing the membrane-associated tumor marker HER2. The SKOVip-kat cell spheroids were visualized by fluorescence microscopy. The cell concentration required for the formation of same-shape and same-size spheroids with tight intercellular contacts was optimized. To verify the developed model, the cytotoxicity of the targeted immunotoxin anti-HER2 consisting of the anti-HER2 scaffold DARP 9_29 and a fragment of the Pseudomonas aeroginosa exotoxin, DARP-LoPE, was studied in 2D and 3D SKOVip-kat cell cultures. The existence of a difference in the cytotoxic properties of DARP-LoPE between the 2D and 3D cultures has been demonstrated: the IC value in the 3D culture is an order of magnitude higher than that in the monolayer culture. The present work describes a universal tool for 3D cultivation of mammalian cells based on reusable agarose gel molds that allows for reproducible formation of multicellular spheroids with tight contacts for molecular and cell biology studies.
PubMed: 35441052
DOI: 10.32607/actanaturae.11603 -
Applied and Environmental Microbiology May 2022The capacity to defluorinate polyfluorinated organic compounds is a rare phenotype in microbes but is increasingly considered important for maintaining the environment....
The capacity to defluorinate polyfluorinated organic compounds is a rare phenotype in microbes but is increasingly considered important for maintaining the environment. New discoveries will be greatly facilitated by the ability to screen many natural and engineered microbes in a combinatorial manner against large numbers of fluorinated compounds simultaneously. Here, we describe a low-volume, high-throughput screening method to determine defluorination capacity of microbes and their enzymes. The method is based on selective binding of fluoride to a lanthanum chelate complex that gives a purple-colored product. It was miniaturized to determine biodefluorination in 96-well microtiter plates by visual inspection or robotic handling and spectrophotometry. Chemicals commonly used in microbiological studies were examined to define usable buffers and reagents. Base-catalyzed, purified enzyme and whole-cell defluorination reactions were demonstrated with fluoroatrazine and showed correspondence between the microtiter assay and a fluoride electrode. For discovering new defluorination reactions and mechanisms, a chemical library of 63 fluorinated compounds was screened with Pseudomonas putida F1 in microtiter well plates. These data were also calibrated against a fluoride electrode. Our new method revealed 21 new compounds undergoing defluorination. A compound with four fluorine substituents, 4-fluorobenzotrifluoride, was shown to undergo defluorination to the greatest extent. The mechanism of its defluorination was studied to reveal a latent microbial propensity to defluorinate trifluoromethylphenyl groups, a moiety that is commonly incorporated into numerous pharmaceutical and agricultural chemicals. Thousands of organofluorine chemicals are known, and a number are considered to be persistent and toxic environmental pollutants. Environmental bioremediation methods are avidly being sought, but few bacteria biodegrade fluorinated chemicals. To find new organofluoride biodegradation, a rapid screening method was developed. The method is versatile, monitoring chemical, enzymatic, and whole-cell biodegradation. Biodegradation of organofluorine compounds invariably releases fluoride anions, which was sensitively detected. Our method uncovered 21 new microbial defluorination reactions. A general mechanism was delineated for the biodegradation of trifluoromethylphenyl groups that are increasingly being used in drugs and pesticides.
Topics: Biodegradation, Environmental; Fluorides; Fluorine; Pseudomonas putida
PubMed: 35435713
DOI: 10.1128/aem.00288-22 -
Bioactive Materials Sep 2022While most studies of mechanical stimulation of cells are focused on two-dimensional (2D) and three-dimensional (3D) systems, it is rare to study the effects of cyclic...
While most studies of mechanical stimulation of cells are focused on two-dimensional (2D) and three-dimensional (3D) systems, it is rare to study the effects of cyclic stretching on cells under a quasi-3D microenvironment as a linkage between 2D and 3D. Herein, we report a new method to prepare an elastic membrane with topographic microstructures and integrate the membrane into a microfluidic chip. The fabrication difficulty lay not only in the preparation of microstructures but also in the alignment and bonding of the patterned membrane to other layers. To resolve the problem, we designed and assembled a fast aligner that is cost-effective and convenient to operate. To enable quasi-3D microenvironment of cells, we fabricated polydimethylsiloxane (PDMS) microwell arrays (formed by micropillars of a few microns in diameter) with the microwell diameters close to the cell sizes. An appropriate plasma treatment was found to afford a coating-free approach to enable cell adhesion on PDMS. We examined three types of cells in 2D, quasi-3D, and 3D microenvironments; the cell adhesion results showed that quasi-3D cells behaved between 2D and 3D cells. We also constructed transgenic human mesenchymal stem cells (hMSCs); under cyclic stretching, the visualizable live hMSCs in microwells were found to orientate differently from in a 3D Matrigel matrix and migrate differently from on a 2D flat plate. This study not only provides valuable tools for microfabrication of a microfluidic device for cell studies, but also inspires further studies of the topological effects of biomaterials on cells.
PubMed: 35356817
DOI: 10.1016/j.bioactmat.2021.12.010 -
Scientific Reports Mar 2022Pluripotent stem-cell derived cells can be used for type I diabetes treatment, but we require at least 10-10 islet-like clusters per patient. Although thousands of...
Pluripotent stem-cell derived cells can be used for type I diabetes treatment, but we require at least 10-10 islet-like clusters per patient. Although thousands of uniform cell clusters can be produced using a conventional microwell plate, numerous obstacles need to be overcome for its clinical use. In this study, we aimed to develop a novel bag culture method for the production of uniform cell clusters on a large scale (10-10 clusters). We prepared small-scale culture bags (< 10 clusters) with microwells at the bottom and optimized the conditions for producing uniform-sized clusters in the bag using undifferentiated induced pluripotent stem cells (iPSCs). Subsequently, we verified the suitability of the bag culture method using iPSC-derived pancreatic islet cells (iPICs) and successfully demonstrate the production of 6.5 × 10 uniform iPIC clusters using a large-scale bag. In addition, we simplified the pre- and post-process of the culture-a degassing process before cell seeding and a cluster harvesting process. In conclusion, compared with conventional methods, the cluster production method using bags exhibits improved scalability, sterility, and operability for both clinical and research use.
Topics: Cell Differentiation; Diabetes Mellitus, Type 1; Humans; Induced Pluripotent Stem Cells; Pluripotent Stem Cells
PubMed: 35338209
DOI: 10.1038/s41598-022-09124-w -
Frontiers in Bioengineering and... 2021Microbial resource mining of electroactive microorganism (EAM) is currently methodically hampered due to unavailable electrochemical screening tools. Here, we introduce...
Microbial resource mining of electroactive microorganism (EAM) is currently methodically hampered due to unavailable electrochemical screening tools. Here, we introduce an electrochemical microwell plate (ec-MP) composed of a 96 electrochemical deepwell plate and a recently developed 96-channel multipotentiostat. Using the ec-MP we investigated the electrochemical and metabolic properties of the EAM models and with acetate and lactate as electron donor combined with an individual genetic analysis of each well. Electrochemical cultivation of pure cultures achieved maximum current densities ( ) and coulombic efficiencies () that were well in line with literature data. The co-cultivation of and led to an increased current density of of 88.57 ± 14.04 µA cm (lactate) and of 99.36 ± 19.12 µA cm (lactate and acetate). Further, a decreased time period of reaching and biphasic current production was revealed and the microbial electrochemical performance could be linked to the shift in the relative abundance.
PubMed: 35242754
DOI: 10.3389/fbioe.2021.821734 -
Activity of Free and Liposome-Encapsulated Essential Oil from against Persister-Derived Biofilm of .Antibiotics (Basel, Switzerland) Dec 2021The high virulence of , a pathogen fungus considered as a global threat for public health, is due to its peculiar traits such as its intrinsic resistance to conventional...
The high virulence of , a pathogen fungus considered as a global threat for public health, is due to its peculiar traits such as its intrinsic resistance to conventional antifungals. Its biofilm lifestyle certainly promotes the prolonged survival of after disinfection or antifungal treatments. In this work, for the first time, we detected persister cells in a biofilm of in a microwell plate model, following caspofungin treatment. Furthermore, we showed how persisters can progressively develop a new biofilm in situ, mimicking the re-colonization of a surface which may be responsible for recalcitrant infections. Plant-derived compounds, such as essential oils, may represent a valid alternative to combat fungal infections. Here, essential oil, as free or encapsulated in liposomes, was used to eradicate primary and persister-derived biofilms of , confirming the great potential of alternative compounds against emergent fungal pathogens. As in other species, the action of essential oils against involves ROS production and affects the expression of some biofilm-related genes.
PubMed: 35052903
DOI: 10.3390/antibiotics11010026 -
Zhejiang Da Xue Xue Bao. Yi Xue Ban =... Oct 2021To investigate the active compounds from on the heart and brain of mice at simulated high altitude.Fifty healthy male adult BALB/c mice were randomly divided into normal...
To investigate the active compounds from on the heart and brain of mice at simulated high altitude.Fifty healthy male adult BALB/c mice were randomly divided into normal control group, hypoxic model group, acetazolamide group, petroleum ether extract of (PESI) group and octacosan group with 10 mice in each group. Acetazolamide group, PESI group and octacosan group were treated with acetazolamide PESI (200 mg/kg) or octacosan by single tail vein injection, respectively. Except normal control group, the mice were exposed to a simulated high altitude of for in an animal decompression chamber. After the mice were sacrificed by cervical dislocation, the heart and brain were histologically observed by HE staining; superoxide dismutase (SOD) activity, total anti-oxidant capacity (T-AOC) and the content of malondialdehyde (MDA) in plasma, heart and brain tissues were detected by WST-1 method, ABTS method and TBA method, respectively; lactic acid and lactate dehydrogenase (LDH) activity in plasma, heart and brain tissues were detected by colorimetric method and microwell plate method, respectively; ATP content and ATPase activity in heart and brain tissues were detected by colorimetric method. PESI and octacosane significantly attenuated the pathological damages of heart and brain tissue at simulated high altitude; increased SOD activity, T-AOC and LDH activity, and decreased the contents of MDA and lactic acid in plasma, heart and brain tissues; increased the content of ATP in heart and brain tissues; increased the activities of Na-K ATPase, Mg ATPase, Ca ATPase and Ca-Mg ATPase in myocardial tissue; and increased the activities of Mg ATPase, Ca-Mg ATPase in brain tissue. PESI and octacosan exert anti-hypoxic activity by improving the antioxidant capacity, reducing the free radical levels, promoting the anaerobic fermentation, and alleviating the energy deficiency and metabolic disorders caused by hypoxia in mice.
Topics: Altitude; Animals; Brain; Heart; Male; Malondialdehyde; Mice; Mice, Inbred BALB C; Superoxide Dismutase
PubMed: 34986540
DOI: 10.3724/zdxbyxb-2021-0344