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Biotechnology and Bioengineering Apr 2024Recent advancements in bioengineering have introduced potential alternatives to liver transplantation via the development of self-assembled liver organoids, derived from...
A highly efficient cell culture method using oxygen-permeable PDMS-based honeycomb microwells produces functional liver organoids from human induced pluripotent stem cell-derived carboxypeptidase M liver progenitor cells.
Recent advancements in bioengineering have introduced potential alternatives to liver transplantation via the development of self-assembled liver organoids, derived from human-induced pluripotent stem cells (hiPSCs). However, the limited maturity of the tissue makes it challenging to implement this technology on a large scale in clinical settings. In this study, we developed a highly efficient method for generating functional liver organoids from hiPSC-derived carboxypeptidase M liver progenitor cells (CPM+ LPCs), using a microwell structure, and enhanced maturation through direct oxygenation in oxygen-permeable culture plates. We compared the morphology, gene expression profile, and function of the liver organoid with those of cells cultured under conventional conditions using either monolayer or spheroid culture systems. Our results revealed that liver organoids generated using polydimethylsiloxane-based honeycomb microwells significantly exhibited enhanced albumin secretion, hepatic marker expression, and cytochrome P450-mediated metabolism. Additionally, the oxygenated organoids consisted of both hepatocytes and cholangiocytes, which showed increased expression of bile transporter-related genes as well as enhanced bile transport function. Oxygen-permeable polydimethylsiloxane membranes may offer an efficient approach to generating highly mature liver organoids consisting of diverse cell populations.
Topics: Humans; Induced Pluripotent Stem Cells; Oxygen; Cell Differentiation; Liver; Cell Culture Techniques; Organoids; Dimethylpolysiloxanes; Metalloendopeptidases; GPI-Linked Proteins
PubMed: 38184815
DOI: 10.1002/bit.28640 -
Biotechnology Journal Jan 2024The ApxII toxin and the outer membrane lipoprotein (Oml) of Actinobacillus pleuropneumoniae are important vaccine antigens against porcine contagious pleuropneumonia...
The ApxII toxin and the outer membrane lipoprotein (Oml) of Actinobacillus pleuropneumoniae are important vaccine antigens against porcine contagious pleuropneumonia (PCP), a prevalent infectious disease affecting the swine industry worldwide. Previous studies have reported the recombinant expression of ApxII and Oml in Escherichia coli; however, their yields were not satisfactory. Here, we aimed to enhance the production of ApxII and Oml by constructing a bicistronic expression system based on the widely used T7 promoter. To create efficient T7 bicistronic expression cassettes, 16 different fore-cistron sequences were introduced downstream of the T7 promoter. The expression of three vaccine antigens Oml1, Oml7, and ApxII in the four strongest bicistronic vectors were enhanced compared to the monocistronic control. Further optimization of the fermentation conditions in micro-well plates (MWP) led to improved production. Finally, the production yields reached unprecedented levels of 2.43 g L of Oml1, 2.59 g L of Oml7, and 1.21 g L of ApxII, in a 5 L bioreactor. These three antigens also demonstrated well-protective immunity against A. pleuropneumoniae infection. In conclusion, this study establishes an efficient bicistronic T7 expression system that can be used to express recombinant proteins in E. coli and achieves the hyper-production of PCP vaccine proteins.
Topics: Swine; Animals; Bacterial Proteins; Escherichia coli; Pleuropneumonia, Contagious; Recombinant Proteins; Actinobacillus Infections; Vaccines, Subunit
PubMed: 38178735
DOI: 10.1002/biot.202300187 -
Surgical Infections Feb 2024Chronic prosthetic joint infections (PJI) are associated with substantial morbidity because conventional antibiotic agents lack activity to bacteria in biofilms that...
Chronic prosthetic joint infections (PJI) are associated with substantial morbidity because conventional antibiotic agents lack activity to bacteria in biofilms that necessitates prosthetic removal to attempt definitive cure. However, these are complex infections that go beyond biofilms and bacteria can be present in various other different states such as synovial fluid aggregates. Consequently, the purpose of this study was to assess the propensity of historically preserved PJI clinical isolates to form synovial fluid aggregates and if aggregation occurred then what is proclivity to be tolerant to high doses of antibiotic agents. Historically preserved chronic PJI clinical isolates from 2021 were evaluated for their ability to form synovial fluid aggregates under static and dynamic conditions in 24-microwell plates. Tolerance to vancomycin, gentamicin, or amphotericin was conducted by adding high concentrations of these antibiotic agents to synovial fluid microbial aggregates. All clinical isolates formed synovial fluid aggregates under dynamic conditions, which with the use of scanning electron microscopy showed dense collections of bacteria with synovial fluid polymers. However, under static conditions only formed aggregates. Importantly, all the microbes in these aggregates were tolerant to high concentrations of antibiotic agents. This study demonstrates that synovial fluid aggregation occurred with all bacterial and fungal species assessed. Therefore, the findings here have important clinical ramifications given the extent that this phenomenon occurs across microbial species and the propensity for the microbes in these aggregates to be tolerant to antibiotic agents.
Topics: Humans; Synovial Fluid; Anti-Bacterial Agents; Biofilms; Arthritis, Infectious; Vancomycin; Bacteria; Prosthesis-Related Infections
PubMed: 38150525
DOI: 10.1089/sur.2023.242 -
Analytical Methods : Advancing Methods... Dec 2023This study presents the development of a polyester microplate for detecting the S-protein of the SARS-CoV-2 virus in saliva and nasopharyngeal swab samples using direct...
This study presents the development of a polyester microplate for detecting the S-protein of the SARS-CoV-2 virus in saliva and nasopharyngeal swab samples using direct enzyme-linked immunosorbent assay (ELISA) technology. The polyester microplate was designed to contain 96 zones with a 3 mm diameter each, and a volume of 2-3 μL. The experimental conditions including reagent concentration and reaction time were optimized. The microplate image was digitized and analyzed using graphical software. The linear range obtained between protein S concentrations and pixel intensity was 0-10 μg mL, with a correlation coefficient of 0.99 and a limit of detection of 0.44 μg mL. The developed methodology showed satisfactory intraplate and interplate repeatability with RSD values lower than 7.8%. The results achieved through immunoassay performed on polyester microplates were consistent with those of the RT-PCR method and showed a sensitivity of 100% and 90% and specificity of 85.71% and 100% for saliva and nasopharyngeal samples, respectively. The proposed direct immunoassay on polyester microplates emerges as an alternative to conventional immunoassays performed on commercial polystyrene plates, given the low cost of the device, low consumption of samples and reagents, lower waste generation, and shorter analysis time. Moreover, the immunoassay has shown great potential for diagnosing COVID-19 with precision and accuracy.
Topics: Humans; Saliva; Spike Glycoprotein, Coronavirus; Colorimetry; COVID-19; Immunoassay
PubMed: 38073521
DOI: 10.1039/d3ay01755a -
Molecules (Basel, Switzerland) Nov 2023Ruxolitinib (RUX) is a potent drug that has been approved by the Food and Drug Administration for the treatment of myelofibrosis, polycythemia vera, and...
Spectrophotometric Study of Charge-Transfer Complexes of Ruxolitinib with Chloranilic Acid and 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone: An Application to the Development of a Green and High-Throughput Microwell Method for Quantification of Ruxolitinib in Its Pharmaceutical Formulations.
Ruxolitinib (RUX) is a potent drug that has been approved by the Food and Drug Administration for the treatment of myelofibrosis, polycythemia vera, and graft-versus-host disease. This study describes the formation of colored charge-transfer complexes (CTCs) of RUX, an electron donor, with chloranilic acid (CLA) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), the π-electron acceptors. The CTCs were characterized using UV-visible spectrophotometry. The formation of CTCs in methanol was confirmed via formation of new absorption bands with maximum absorption at 530 and 470 nm for CTCs with CLA and DDQ, respectively. The molar absorptivity and other physicochemical and electronic properties of CTCs were determined. The molar ratio was found to be 1:1 for both CTCs with CLA and CTCs with DDQ. The site of interaction on RUX molecules was assigned and the mechanisms of the reactions were postulated. The reactions were employed as basis for the development of a novel green and one-step microwell spectrophotometric method (MW-SPM) for high-throughput quantitation of RUX. Reactions of RUX with CLA and DDQ were carried out in 96-well transparent plates, and the absorbances of the colored CTCs were measured by an absorbance microplate reader. The MW-SPM was validated according to the ICH guidelines. The limits of quantitation were 7.5 and 12.6 µg/mL for the methods involving reactions with CLA and DDQ, respectively. The method was applied with great reliability to the quantitation of RUX content in Jakavi tablets and Opzelura cream. The greenness of the MW-SPM was assessed by three different metric tools, and the results proved that the method fulfills the requirements of green analytical approaches. In addition, the one-step reactions and simultaneous handling of a large number of samples with micro-volumes using the proposed method enables the high-throughput analysis. In conclusion, this study describes the first MW-SPM, a valuable analytical tool for the quality control of pharmaceutical formulations of RUX.
Topics: Drug Compounding; Reproducibility of Results; Benzoquinones; Spectrophotometry; Tablets
PubMed: 38067605
DOI: 10.3390/molecules28237877 -
ACS Biomaterials Science & Engineering Jan 2024Cell distribution is one of the primary factors that can affect cell morphology and behaviors, as it determines cell-cell interactions. Despite the importance of cell...
Cell distribution is one of the primary factors that can affect cell morphology and behaviors, as it determines cell-cell interactions. Despite the importance of cell distribution, the seeding process of in vitro cell culture still highly relies on the traditional method using manual pipetting. Because manual pipetting cannot ensure a uniform cell distribution and has the possibility of compromising experimental reproducibility, an accurate and systemic seeding method that enables uniform cell seeding over versatile culture substrates is required. Here, we developed a perforated plate-based cell seeding device called the CellShower, which enabled uniform cell seeding over a large area of cell culture substrates. The working principles of the CellShower are based on the laminar filling flow and capillary force in microfluidics, and the design of the CellShower was optimized with numerical simulations. The versatility of the CellShower in view of uniform cell seeding was demonstrated by applying it to various types of culture substrates from a conventional culture dish to culture substrates having nanotopography, porous structures, and 3D concave structures. The CellShower and its operating principles are expected to contribute to enhancing the accuracy and reproducibility of biological experiments.
Topics: Reproducibility of Results; Cell Culture Techniques; Porosity
PubMed: 38048415
DOI: 10.1021/acsbiomaterials.3c01203 -
Communications Biology Nov 2023The ability to perform sophisticated, high-throughput optogenetic experiments has been greatly enhanced by recent open-source illumination devices that allow independent...
The ability to perform sophisticated, high-throughput optogenetic experiments has been greatly enhanced by recent open-source illumination devices that allow independent programming of light patterns in single wells of microwell plates. However, there is currently a lack of instrumentation to monitor such experiments in real time, necessitating repeated transfers of the samples to stand-alone analytical instruments, thus limiting the types of experiments that could be performed. Here we address this gap with the development of the optoPlateReader (oPR), an open-source, solid-state, compact device that allows automated optogenetic stimulation and spectroscopy in each well of a 96-well plate. The oPR integrates an optoPlate illumination module with a module called the optoReader, an array of 96 photodiodes and LEDs that allows 96 parallel light measurements. The oPR was optimized for stimulation with blue light and for measurements of optical density and fluorescence. After calibration of all device components, we used the oPR to measure growth and to induce and measure fluorescent protein expression in E. coli. We further demonstrated how the optical read/write capabilities of the oPR permit computer-in-the-loop feedback control, where the current state of the sample can be used to adjust the optical stimulation parameters of the sample according to pre-defined feedback algorithms. The oPR will thus help realize an untapped potential for optogenetic experiments by enabling automated reading, writing, and feedback in microwell plates through open-source hardware that is accessible, customizable, and inexpensive.
Topics: Optogenetics; Feedback; Escherichia coli; Algorithms; Spectrum Analysis
PubMed: 38001175
DOI: 10.1038/s42003-023-05532-4 -
Biosensors & Bioelectronics Feb 2024Culture plating is worldwide accepted as the gold standard for quantifying viable foodborne pathogens. However, it is time-consuming (1-2 days) and requires specialized...
Culture plating is worldwide accepted as the gold standard for quantifying viable foodborne pathogens. However, it is time-consuming (1-2 days) and requires specialized laboratory and personnel. This study reported a deep learning enhanced digital microfluidic platform for multiplex detection of viable foodborne pathogens. The new method used a Time-Lapse images driven EfficientNet-Transformer Network (TLENTNet) to type and quantify the bacteria through spatiotemporal features of bacterial growth and digital enumeration of bacterial culture. First, the bacterial sample was prepared with LB medium and injected into a pre-vacuumed microfluidic chip with an array of 800 microwells to encapsulate at most one bacterium in each well. Then, a programmed sliding microscopic platform was used to scan all microwells every 15 min, capturing time-lapse images of bacterial growth within each microwell. Finally, the TLENTNet was used to facilitate bacterial typing and quantification. Under optimal conditions, this platform was able to detect four bacterial species (S.typhimurium, E. coli O157:H7, S. aureus and B. cereus) with an average accuracy of 97.72% and a detection limit of 63 CFU/mL in 7 h.
Topics: Food Microbiology; Microfluidics; Staphylococcus aureus; Deep Learning; Biosensing Techniques; Escherichia coli O157; Bacteria
PubMed: 38000308
DOI: 10.1016/j.bios.2023.115837 -
Nanoscale Dec 2023Thanks to their unique nanoscale properties, nanomedicines can overcome some of the shortcomings of conventional therapies. For better predictive screening, it is...
Thanks to their unique nanoscale properties, nanomedicines can overcome some of the shortcomings of conventional therapies. For better predictive screening, it is important to assess their performance in three-dimensional (3D) multicellular tumour spheroids (MCTS) that can recapitulate the physiological barriers found in real tumours. Today, the evaluation of drug delivery nanosystems in MCTS is mainly explored by means of microscopy techniques that are invasive and require fluorescent labels which modify the composition and fate of the carriers. In recent years, a new quantitative microscopy technique based on Brillouin light scattering (BLS) has been proposed that uses the interaction of laser light with picosecond timescale density fluctuations in the sample. Because it is label-free, all-optical and non-destructive, BLS has gained interest in the pharmaceutical and biomedical fields. In this work, we implemented a fast BLS spectrometer and used the Brillouin frequency shift at the center of the MCTS as a quantitative readout for drug efficacy. We first investigated the ability of this setup to quantify drug efficacy in MCTS grown in classical multiwell plates and concluded that the low number of samples available in the multiwells limits the statistical significance of the results. To improve the throughput, we then combined the microscope with agarose microwells designed to fabricate a large number of MCTS and test 50 MCTS in less than a minute. Using this platform, we assessed the efficacy of polymeric nanoparticles (NPs) loaded with a platinum derivative anticancer drug (dichloro(1,2-diaminocyclohexane)platinum(II)) in reducing the growth of colorectal cancer cells (HCT-116) in MCTS. We observe a time- and dose-dependent decrease in the frequency shift, revealing the progressive loss of mechanical integrity in the MCTS. These results demonstrate that BLS probing of MCTS grown in agarose microwells is a promising tool for high-throughput screening of nanocarriers in 3D models.
Topics: Cell Line, Tumor; Microscopy; Sepharose; Antineoplastic Agents; Spheroids, Cellular
PubMed: 37990811
DOI: 10.1039/d3nr03502f -
Molecules (Basel, Switzerland) Oct 2023This study describes the development of two highly sensitive immunosensor platforms for the trace determination of copper ions, Cu(II), in drinking water. These...
Development and Comparative Evaluation of Two Highly Sensitive Immunosensor Platforms for Trace Determination of Copper Ions in Drinking Water Using a Monoclonal Antibody Specific to Copper-EDTA Complex.
This study describes the development of two highly sensitive immunosensor platforms for the trace determination of copper ions, Cu(II), in drinking water. These platforms were a microwell-based enzyme-linked immunosorbent assay (ELISA) and a kinetic exclusion assay (KinExA) with a KinExA 3200 immunosensor. Both ELISA and KinExA were developed utilizing the same antibody and coating reagent. The antibody was a mouse monoclonal antibody, designated as 8D66, that specifically recognized Cu(II)-ethylenediamine tetraacetic acid complex (Cu(II)-EDTA) but did not recognize Cu(II)-free EDTA. The 8D66 monoclonal antibody was generated by the fusion of spleen cells of an immunized BALB/c mouse with SP2/0-Ag14 myeloma cells. The immunogen was a protein conjugate of Cu(II)-EDTA with keyhole limpet hemocyanin protein. The coating reagent was Cu(II)-EDTA covalently linked to bovine serum albumin protein (Cu(II)-EDTA-BSA). Both assays involved the competitive binding reaction between Cu(II)-EDTA complexes, formed in the sample solution, and Cu(II)-EDTA-BSA conjugate which has been immobilized onto ELISA plates (in ELISA) or polymethylmethacrylate beads (in KinExA) for a limited quantity of binding sites of the 8D66 antibody. In ELISA, color signals were generated by a peroxidase-labeled secondary antibody and 3,3',5,5'-tetramethylbenzidine substrate. In KinExA, a fluorescein isothiocyanate-labeled secondary antibody was used to generate KinExAgram (trend-line fluorescence responses vs. time). The conditions of both ELISA and KinExA were investigated, and the optimum procedures were established. Both ELISA and KinExA were validated, and all validation parameters were acceptable. Many different metal ions that are commonly encountered in drinking water did not interfere with the Cu(II) analysis by both ELISA and KinExA. Both assays were applied to the determination of Cu(II) in drinking water with satisfactory accuracy and precision. Both assays were compared favorably with inductively coupled plasma atomic emission spectroscopy in terms of their abilities to accurately and precisely determine Cu(II) in drinking water samples. A comparative evaluation of ELISA and KinExA revealed that KinExA had a higher sensitivity and better precision than ELISA, whereas both assays had comparable accuracy. Both ELISA and KinExA were superior to the existing atomic spectrometric methods for Cu(II) in terms of sensitivity, convenience, and analysis throughputs. The proposed ELISA and KinExA are anticipated to effectively contribute to assessing Cu(II) concentrations and control the exposure of humans to its potential toxicities.
Topics: Humans; Animals; Mice; Copper; Antibodies, Monoclonal; Edetic Acid; Drinking Water; Biosensing Techniques; Immunoassay; Antigens; Indicators and Reagents
PubMed: 37894495
DOI: 10.3390/molecules28207017