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Advanced Healthcare Materials Jan 2016During the last decades, the engineering of well-defined 3D tissues has attracted great attention because it provides in vivo mimicking environment and can be a building... (Review)
Review
During the last decades, the engineering of well-defined 3D tissues has attracted great attention because it provides in vivo mimicking environment and can be a building block for the engineering of bioartificial organs. In this Review, diverse engineering methods of 3D tissues using microscale devices are introduced. Recent progress of microtechnologies has enabled the development of microplatforms for bottom-up assembly of diverse shaped 3D tissues consisting of various cells. Micro hanging-drop plates, microfluidic chips, and arrayed microwells are the typical examples. The encapsulation of cells in hydrogel microspheres and microfibers allows the engineering of 3D microtissues with diverse shapes. Applications of 3D microtissues in biomedical fields are described, and the future direction of microplatform-based engineering of 3D micro-tissues is discussed.
Topics: Biomedical Technology; Humans; Microfluidics; Microtechnology; Spheroids, Cellular; Tissue Engineering; Tissue Scaffolds
PubMed: 25880830
DOI: 10.1002/adhm.201500107 -
Carbohydrate Research Jan 2019PNGases are crucial targets and valuable tools in analyzing asparagine-linked carbohydrate moieties (N-glycans) of glycoproteins. Activity tests of PNGases have been...
PNGases are crucial targets and valuable tools in analyzing asparagine-linked carbohydrate moieties (N-glycans) of glycoproteins. Activity tests of PNGases have been little improved since their discovery four decades ago, and still rely on observing deglycosylation patterns of glycoproteins or glycopeptides using SDS-PAGE or HPLC analysis. These techniques cannot be easily adapted for automated sampling and high-throughput procedures. Herein, we describe a PNGase activity assay which relies on the conversion of WST-1, a yellowish, water-soluble tetrazolium dye (sodium 2-(4-Iodophenyl)-3-(4-nitro-phenyl)-5-(2,4-disulfophenyl)-2H-tetrazolate), into a blue formazan dye. In this work, we showed that WST-1 could be reduced by N-glycans, which were enzymatically released from glycoprotein substrates. After optimization of the assay conditions, the robustness of the method was challenged by quantifying the activity of various PNGase isoforms at different purification stages using a microwell plate reader. Furthermore, the assay could be used to obtain steady-state kinetics of PNGase H wild-type and mutant variants, which showed significant differences in their enzymatic reaction rates. The simplicity and robustness of this method might be of benefit for the detection of PNGase activity in routine applications of large amounts of samples.
Topics: Bacteria; Bacterial Proteins; Biological Assay; Colorimetry; Glycosylation; Kinetics; Mutation; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Polysaccharides; Substrate Specificity; Tetrazolium Salts
PubMed: 30476755
DOI: 10.1016/j.carres.2018.11.007 -
Microorganisms Nov 2022induces heavy mortality in aquaculture. The detection of is often time consuming and uncertain, making it difficult to manage the disease. We validated a previously...
induces heavy mortality in aquaculture. The detection of is often time consuming and uncertain, making it difficult to manage the disease. We validated a previously published real-time quantitative PCR (qPCR) assay to confirm the presence of in fish and in water using environmental DNA (eDNA) quantification. Analytical sensitivity and specificity of the assay was assessed in silico, in vitro and the qPCR assay was compared with microbiological cultivation methods to detect and quantify in water samples from a controlled fish exposure experiment and from fish farms. Furthermore, we compared the use of an agar cultivation method and the qPCR assay to detect directly from mucus samples taken from the fish surface. The analytical sensitivity and specificity of the qPCR assay were high. The qPCR assay detected 100% of -positive water samples. In a field study, the qPCR assay and a microwell plate (MWP) enumeration method correlated significantly. Furthermore, the qPCR assay could be used to confirm the presence of in skin mucus. Thus, the qPCR assay could complement diagnostic methods in specifically detecting saprolegniosis in fish and used as a surveillance method for pathogen in aquaculture environments.
PubMed: 36363778
DOI: 10.3390/microorganisms10112186 -
PloS One 2021Three-dimensional spheroid cultures have been shown to better physiologically mimic the cell-cell and cell-matrix interactions that occur in solid tumors more than...
Three-dimensional spheroid cultures have been shown to better physiologically mimic the cell-cell and cell-matrix interactions that occur in solid tumors more than traditional 2D cell cultures. One challenge in spheroid production is forming and maintaining spheroids of uniform size. Here, we developed uniform, high-throughput, multicellular spheroids that self-assemble using microwell plates. DU145 and PC3 cells were cultured as 2D monolayers and 3D spheroids to compare sensitization of TRAIL-resistance cancer cells to TRAIL mediated apoptosis via chemotherapy based on dimensionality. Monocultured monolayers and spheroids were treated with soluble TRAIL alone (24 hr), DTX or CBZ alone (24 hr), or a combination of taxane and TRAIL (24 + 24 hr) to determine the effectiveness of taxanes as TRAIL sensitizers. Upon treatment with soluble TRAIL or taxanes solely, monolayer cells and spheroids exhibited no significant reduction in cell viability compared to the control, indicating that both cell lines are resistant to TRAIL and taxane alone in 2D and 3D. Pretreatment with CBZ or DTX followed by TRAIL synergistically amplified apoptosis in 2D and 3D DU145 cell cultures. PC3 spheroids were more resistant to the combination therapy, displaying a more additive effect in the DTX + TRAIL group compared to 2D. There was a downregulation of DR4/5 expression in spheroid form compared to monolayers in each cell line. Additionally, normal fibroblasts (NFs) and cancer-associated fibroblasts (CAFs) were cocultured with both PCa cell lines as spheroids to determine if CAFs confer additional resistance to chemotherapy. We determined that co-cultured spheroids show similar drug resistance to monocultured spheroids when treated with taxane plus TRAIL treatment. Collectively, these findings suggest how the third dimension and cocultures of different cell types effect the sensitization of androgen-independent prostate cancer cells to TRAIL, suggesting therapeutic targets that could overcome TRAIL-resistance in metastatic castration-resistant prostate cancer (mCRPC).
Topics: Apoptosis; Bridged-Ring Compounds; Cell Line, Tumor; Drug Resistance, Neoplasm; Humans; Male; Prostatic Neoplasms; Spheroids, Cellular; TNF-Related Apoptosis-Inducing Ligand; Taxoids
PubMed: 33661931
DOI: 10.1371/journal.pone.0246733 -
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 -
Scientific Reports Jul 2021Recent statistics report that more than 3.7 million new cases of cancer occur in Europe yearly, and the disease accounts for approximately 20% of all deaths....
Recent statistics report that more than 3.7 million new cases of cancer occur in Europe yearly, and the disease accounts for approximately 20% of all deaths. High-throughput screening of cancer cell cultures has dominated the search for novel, effective anticancer therapies in the past decades. Recently, functional assays with patient-derived ex vivo 3D cell culture have gained importance for drug discovery and precision medicine. We recently evaluated the major advancements and needs for the 3D cell culture screening, and concluded that strictly standardized and robust sample preparation is the most desired development. Here we propose an artificial intelligence-guided low-cost 3D cell culture delivery system. It consists of a light microscope, a micromanipulator, a syringe pump, and a controller computer. The system performs morphology-based feature analysis on spheroids and can select uniform sized or shaped spheroids to transfer them between various sample holders. It can select the samples from standard sample holders, including Petri dishes and microwell plates, and then transfer them to a variety of holders up to 384 well plates. The device performs reliable semi- and fully automated spheroid transfer. This results in highly controlled experimental conditions and eliminates non-trivial side effects of sample variability that is a key aspect towards next-generation precision medicine.
Topics: Artificial Intelligence; Cell Culture Techniques; Cell Line, Tumor; Deep Learning; Drug Screening Assays, Antitumor; High-Throughput Screening Assays; Humans; Neoplasms; Precision Medicine; Spheroids, Cellular
PubMed: 34285291
DOI: 10.1038/s41598-021-94217-1 -
Journal of Personalized Medicine Apr 2021Altered miRNA expression and DNA methylation have highly active and diverse roles in carcinogenesis. Simultaneous detection of the molecular aberrations may have a...
Altered miRNA expression and DNA methylation have highly active and diverse roles in carcinogenesis. Simultaneous detection of the molecular aberrations may have a synergistic effect on the diagnosis of malignancies. Herein, we develop a high-throughput assay for detecting multiple miRNAs and DNA methylation using droplet digital PCR (ddPCR) coupled with a 96-microwell plate. The microplate-based ddPCR could absolutely and reproducibly quantify 15 miRNAs and 14 DNA methylation sites with a high sensitivity (one copy/µL and 0.1%, respectively). Analyzing sputum and plasma of 40 lung cancer patients and 36 cancer-free smokers by this approach identified an integrated biomarker panel consisting of two sputum miRNAs (miRs-31-5p and 210-3p), one sputum DNA methylation (RASSF1A), and two plasma miRNAs (miR-21-5p and 126) for the diagnosis of lung cancer with higher sensitivity and specificity compared with a single type of biomarker. The diagnostic value of the integrated biomarker panel for the early detection of lung cancer was confirmed in a different cohort of 36 lung cancer patients and 39 cancer-free smokers. The high-throughput assay for quantification of multiple molecular aberrations across sputum and plasma could improve the early detection of lung cancer.
PubMed: 33946992
DOI: 10.3390/jpm11050359 -
Scientific Reports Aug 2018Microwell platforms show great promise in single-cell studies and protein measurements because of their low volume sampling, rapid analysis and high throughput screening...
Microwell platforms show great promise in single-cell studies and protein measurements because of their low volume sampling, rapid analysis and high throughput screening ability. However, the existing actuation mechanisms to manipulate the target samples and fabrication procedures involved in the microwell-based microfluidic devices are complex, resource-intensive and require an external power source. In this work, we present proof of concept of a simple, power-free and low-cost closed magnet digital microfluidics device for isolating biological entities in femtoliter-sized microwells. The target biological entities were encapsulated in magnetic liquid marbles and shuttled back and forth between micropatterned top and bottom plates in the microdevice to obtain high loading efficiency and short processing time. The microdevice performance was studied through fluorescent detection of three different entities: microbeads, bovine serum albumin (BSA) and Escherichia coli, captured in the microwell array. Almost 80% of the microwells were loaded with single microbeads in five shuttling cycles, in less than a minute. Further, a low volume of BSA was compartmentalized in the microwell array over a two order range of concentration. The microdevice exhibits two unique features: lotus leaf stamps were used to fabricate micropatterns (microwells and micropillars) on top and bottom plates to impart functionality and cost-effectiveness, and the target samples were actuated by a permanent magnet to make the microdevice power-free and simple in operation. The developed biomimetic microdevice is therefore capable of capturing a multitude of biological entities in low-resource settings.
Topics: Animals; Biomimetic Materials; Biomimetics; Cattle; Escherichia coli; Fluorescein-5-isothiocyanate; Fluorescence; Hydrophobic and Hydrophilic Interactions; Magnets; Microfluidics; Nanoparticles; Serum Albumin, Bovine; Signal Processing, Computer-Assisted; Wettability
PubMed: 30143719
DOI: 10.1038/s41598-018-31274-z -
Life (Basel, Switzerland) Jun 2021Research in fields studying cellular response to surface tension and mechanical forces necessitate cell culture tools with tunability of substrate stiffness. We created...
Research in fields studying cellular response to surface tension and mechanical forces necessitate cell culture tools with tunability of substrate stiffness. We created a scalable hydrogel dish design to facilitate scaffold-free formation of multiple spheroids in a single dish. Our novel design features inner and outer walls, allowing efficient media changes and downstream experiments. The design is easily scalable, accommodating varying numbers of microwells per plate. We report that non-adherent hydrogel stiffness affects spheroid morphology and compaction. We found that spheroid morphology and viability in our hydrogel dishes were comparable to commercially available Aggrewell™800 plates, with improved tunability of surface stiffness and imaging area. Device function was demonstrated with a migration assay using two investigational inhibitors against EMT. We successfully maintained primary-derived spheroids from murine and porcine lungs in the hydrogel dish. These features increase the ability to produce highly consistent cell aggregates for biological research.
PubMed: 34204955
DOI: 10.3390/life11060517 -
Molecules (Basel, Switzerland) Sep 2023Olaparib (OLA) is a PARP inhibitor drug which has been recently approved by the Food and Drug Administration (FDA) for the treatment of ovarian and breast cancer. A...
Development and Comparative Evaluation of Two Different Label-Free and Sensitive Fluorescence Platforms for Analysis of Olaparib: A Recently FDA-Approved Drug for the Treatment of Ovarian and Breast Cancer.
Olaparib (OLA) is a PARP inhibitor drug which has been recently approved by the Food and Drug Administration (FDA) for the treatment of ovarian and breast cancer. A convenient analytical tool for the quantitation of OLA in its dosage form and plasma samples was urgently needed. This study describes, for the first time, the development of two different label-free and sensitive fluorescence-based platforms for the pharmaceutical and bioanalysis of OLA. These platforms were microwell-assisted with a fluorescence microplate reader (MW-FLR) and high-performance liquid chromatography with fluorescence detection (HPLC-FD). Both MW-FLR and HPLC-FD employed the native fluorescence of OLA as an analytical signal. The MW-FLR involved measuring the fluorescence signals in 96-well white-opaque plates. The HPLC-FD involved chromatographic separation of OLA and duvelisib (DUV), as an internal standard on a Nucleosil-CN HPLC column (250 mm length × 4.6 mm i.d., 5 µm particle diameter) with a mobile phase composed of acetonitrile: water (25:75, ) pumped at a flow rate of 1.7 mL/min. Elution of OLA and DUV was detected using a fluorescence detector. The optimal conditions of both MW-FLR and HPLC-FD were established, and they were validated according to the guidelines of the International Council for Harmonization for the validation of analytical procedures. The linear ranges of MW-FLR and HPLC-FD were 25-1000 and 5-200 ng/mL, respectively, with limits of detection of 15 and 1.7 ng/mL, respectively. The accuracy and precision of both platforms were confirmed as the recovery values were ≥98.2% and the relative standard deviations (RSD) were ≤2.89%. Both methodologies were satisfactorily applied to the quantitation of OLA in its commercial dosage form (Lynparza tablets) and plasma samples with high accuracy and precision. The greenness of both MW-FLR and HPLC-FD was assessed using two different multiple parameter-based metric tools, and the results proved their greenness and adherence to the requirements of green analytical approaches. Both platforms have simple procedures and acceptable levels of analytical throughput. In conclusion, the proposed MW-FLR and HPLC-FD are valuable tools for routine use in quality control and clinical laboratories for the quantitation of OLA for the purposes of pharmaceutical quality control, pharmacokinetic studies, and bioequivalence testing.
Topics: Humans; Female; Breast Neoplasms; Chromatography, High Pressure Liquid; Phthalazines; Tablets
PubMed: 37764300
DOI: 10.3390/molecules28186524