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ACS Applied Bio Materials May 2021Prostate-specific membrane antigen (PSMA) is a viable diagnostic biomarker for the detection and treatment of prostate cancer. Although numerous imaging techniques and...
Prostate-specific membrane antigen (PSMA) is a viable diagnostic biomarker for the detection and treatment of prostate cancer. Although numerous imaging techniques and fluorescent probes have been developed, targeted imaging and intraoperative surgery continue to remain as a proof-of-concept with a severe lack of tools having high affinity and penetrative capacity. three-dimensional cell culture has gained immense interest in cancer research and drug discovery programs as it yields important physiological information and serves an excellent model for bioimaging and penetration analysis studies. Current techniques employed in spheroid formation include liquid overlay and hanging drop methods, both of which are low-yielding and technically demanding. We describe for the first time a simple-to-use platform, μSpherePlatform, an inexpensive, high-throughput method yielding morphologically homogeneous spheroids in bulk for analyzing penetrative capacity and imaging ability of PCa diagnostics. Microwell arrays made of agarose have been fabricated using a commercial hairbrush as a master template. This procedure has been described in detail, and arrays of spheroids (100-120 spheroids/6-well plate) with >95% success rates have been produced from PCa cell lines (LNCaP and DU-145). A PSMA-targeted fluorescent conjugate was synthesized and evaluated in the spheroids developed using μSpherePlatform by multiphoton imaging. A synthetic 3D scaffold strategy is reported herein, which (1) correlates perfectly with the model, (2) is amenable for automated analysis, (3) shows a negligible lot to lot variation, (4) is simplistic, (5) is useful for high-throughput assays, (6) is extremely compatible with imaging techniques, (7) generates PCa spheroids within 48 h, and (8) forms large size-controllable spheroids of diameter 500-1300 μm. The μSpherePlatform thus provides a significant contribution to multimodal analyses of cancer diagnostics and deep-tissue imaging studies.
Topics: Biocompatible Materials; Cell Culture Techniques, Three Dimensional; Cell Line, Tumor; Humans; Male; Materials Testing; Molecular Structure; Particle Size; Prostate-Specific Antigen; Prostatic Neoplasms
PubMed: 35006838
DOI: 10.1021/acsabm.1c00086 -
High-throughput organo-on-pillar (high-TOP) array system for three-dimensional ex vivo drug testing.Biomaterials May 2023The development of organoid culture technologies has triggered industrial interest in ex vivo drug test-guided clinical response prediction for precision cancer therapy....
The development of organoid culture technologies has triggered industrial interest in ex vivo drug test-guided clinical response prediction for precision cancer therapy. The three-dimensional culture encapsulated with basement membrane (BM) components is extremely important in establishing ex vivo organoids and drug sensitivity tests because the BM components confer essential structures resembling tumor histopathology. Although numerous studies have demonstrated three-dimensional culture-based drug screening methods, establishing a large-scale drug-screening platform with matrix-encapsulated tumor cells is challenging because the arrangement of microspots of a matrix-cell droplet onto each well of a microwell plate is inconsistent and difficult to standardize. In addition, relatively low scales and lack of reproducibility discourage the application of three-dimensional organoid-based drug screening data for precision treatment or drug discovery. To overcome these limitations, we manufactured an automated organospotter-integrated high-throughput organo-on-pillar (high-TOP) drug-screening platform. Our system is compatible with various extracellular matrices, including BM extract, Matrigel, collagen, and hydrogel. In addition, it can be readily utilized for high-content analyses by simply exchanging the bottom plates without disrupting the domes. Our system demonstrated considerable robustness, consistency, reproducibility, and biological relevancy in three-dimensional drug sensitivity analyses using Matrigel-encapsulated ovarian cancer cell lines. We also demonstrated proof-of-concept cases representing the clinical feasibility of high-TOP-assisted ex vivo drug tests linked to clinical chemo-response in ovarian cancer patients. In conclusion, our platform provides an automated and standardized method for ex vivo drug-sensitivity-guided clinical response prediction, suggesting effective chemotherapy regimens for patients with cancer.
Topics: Female; Humans; Cell Culture Techniques; Reproducibility of Results; Drug Evaluation, Preclinical; Drug Discovery; Organoids; Ovarian Neoplasms; High-Throughput Screening Assays
PubMed: 36924663
DOI: 10.1016/j.biomaterials.2023.122087 -
Chemosphere Feb 2023Directly measurement of the bioavailable concentration of soil contaminants is essential for their accurate risk assessment. In this study, we successfully modified and...
Directly measurement of the bioavailable concentration of soil contaminants is essential for their accurate risk assessment. In this study, we successfully modified and identified the key genetic elements (pobR1-3) for the bio-detection of p-nitrophenol and synthesized five novel whole-cell biosensors (Escherichia coli BL21/pPNP-mrfp, E. coli BL21/pPNP-CFP, E. coli BL21/pPNP-YFP, E. coli BL21/pPNP-GFP, and E. coli BL21/pPNP-amilCP) to directly detect the concentration of p-nitrophenol in soils. These biosensor methods contained a simple biosensor activation and sample extraction step, a cost-effective detection means, and a fast detection process (5 h) by using a 96-microwell plate with a low background value and high-reliability equation for p-nitrophenol detection. These biosensors had a detection limit of 6.21-25.2 μg/kg and a linear range of 10-10000 μg/kg for p-nitrophenol in four soils. All biosensors showed better detection performance in the detection of p-nitrophenol in soil samples. The biosensors method can help to quickly and directly assess the actual bioavailable fractions of p-nitrophenol in soils, thus facilitating to understand the environmental cycling of p-nitrophenol.
Topics: Soil; Escherichia coli; Reproducibility of Results; Biosensing Techniques
PubMed: 36410515
DOI: 10.1016/j.chemosphere.2022.137306 -
ACS Applied Bio Materials Mar 2022We herein describe a highly versatile platform approach for the in situ and real-time screening of microbial biocatalysts for enhanced production of bioproducts using...
We herein describe a highly versatile platform approach for the in situ and real-time screening of microbial biocatalysts for enhanced production of bioproducts using photonic crystal hydrogels. This approach was demonstrated by preparing optically diffracting films based on polymerized -isopropylacrylamide that contracted in the presence of alcohols and organic acids. The hydrogel films were prepared in a microwell plate format, which allows for high-throughput screening, and characterized optically using a microwell plate reader. While demonstrating the ability to detect a broad range of relevant alcohols and organic acids, we showed that the response of the films correlated strongly with the octanol-water partition coefficient (log ) of the analyte. Differences in the secretion of ethanol and succinic acid from strains of and , respectively, were further detected via optical characterization of the films. These differences, which in some cases were as low as ∼3 g/L, were confirmed by high-performance liquid chromatography, thereby demonstrating the sensitivity of this approach. Our findings highlight the potential utility of this multiplexed approach for the detection of small organic analytes in complex biological media, which overcomes a major challenge in conventional optical sensing methods.
Topics: Acids; Alcohols; Culture Media; Hydrogels; Octanols; Organic Chemicals
PubMed: 35166523
DOI: 10.1021/acsabm.1c01267 -
Journal of AOAC International Jun 2024Galidesivir (GDV) is a promising new antiviral drug for the potent and safe treatment of a broad spectrum of viral diseases, including COVID-19. In the literature, no...
BACKGROUND
Galidesivir (GDV) is a promising new antiviral drug for the potent and safe treatment of a broad spectrum of viral diseases, including COVID-19. In the literature, no analytical method exists for the determination of GDV in bulk and dosage form.
OBJECTIVE
The aim of this study was the development of versatile green and simple microwell spectrophotometric methods (MW-SPMs) for the determination of GDV in its bulk form and capsules.
METHODS
Three MW-SPMs were developed involving the oxidation of GDV by ammonium metavanadate (AMV), chromium trioxide (CTO), and potassium iodate (PIO) in an acid medium. The reactions were carried out in 96-well plates at room temperature and the absorbances of chromogenic reaction products were measured by an absorbance microplate reader at 780, 595, and 475 nm for AMV, CTO, and PIO, respectively. Variables influencing the reactions were carefully investigated and optimized.
RESULTS
Linear relations with excellent correlation coefficients (0.9991-0.9997) were found between the absorbances and GDV concentrations in a range of 25-500 µg/mL. The limits of detection were ≥8.3 µg/mL. The accuracy and precision of the three MW-SPMs were confirmed by recovery and replicate analysis, respectively. The recovery values were 98.6-101.2% and the relative standard deviations were ≤1.02%. The proposed MW-SPMs were successfully applied to the analysis of GDV in bulk drug and capsules with high accuracy and precisions. The greenness of MW-SPMs was confirmed by three comprehensive metric tools.
CONCLUSIONS
The proposed MW-SPMs combined the inherent advantages of microwell-based analysis and the use of common laboratory reagents for the involved reaction. These advantages include high-throughput, readily automation, reduced samples/reagents volume, precise measurements, and versatility. The advantages of the use of common laboratory reagents include availability, consistency, compatibility, safety, and cost-effectiveness.
HIGHLIGHTS
Overall, the proposed MW-SPMs are versatile valuable tools for the quantitation of GDV during its pharmaceutical manufacturing.
PubMed: 38870529
DOI: 10.1093/jaoacint/qsae047 -
Biofabrication Jul 2020Quantification of intratumoral heterogeneity is essential for designing effective therapeutic strategies in the age of personalized medicine. In this study, we used a...
Quantification of intratumoral heterogeneity is essential for designing effective therapeutic strategies in the age of personalized medicine. In this study, we used a piezoelectric inkjet printer to enable analysis of intratumoral heterogeneity in a bladder cancer for the first time. Patient-derived tumor organoids were dissociated into single cell suspension and used as a bioink. The individual cells were precisely allocated into a microwell plate by drop-on-demand inkjet printing without any additive or treatment, followed by culturing into organoids for further analysis. The sizes and morphologies of the organoids were observed, so as the expression of proliferation and apoptotic markers. The tumor organoids also showed heterogeneous responses against chemotherapeutic agent. Further, we quantified mRNA expression levels of representative luminal and basal genes in both type of tumor organoids. These results verify the heterogeneous expression of various genes among individual organoids. This study demonstrates that the fully automated inkjet printing technique can be used as an effective tool to sort cells for evaluating intratumoral heterogeneity.
Topics: Biomarkers, Tumor; Bioprinting; Cell Shape; Cisplatin; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Ink; Neoplasms; Organoids; Single-Cell Analysis
PubMed: 32428886
DOI: 10.1088/1758-5090/ab9491 -
Frontiers in Toxicology 2022The methods outlined here are part of a series of papers designed specifically for genotoxicity assessment of nanomaterials (NM). Common Considerations such as NM...
The methods outlined here are part of a series of papers designed specifically for genotoxicity assessment of nanomaterials (NM). Common Considerations such as NM characterization, sample preparation and dose selection, relevant to all genotoxicity assays, are found in an accompanying paper. The present paper describes methods for evaluation of mutagenicity in the mammalian (mouse) () gene occurring in L5178Y mouse lymphoma (ML) cells and in the designated gene in human lymphoblastoid TK6 cells. Mutations change the functional genotype from TK to TK, detectable as cells surviving on media selective for the lack of thymidine kinase (TK) function. Unlike cells with TK enzyme function, the TK cells are unable to integrate the toxic selection agent, allowing these cells to survive as rare mutant colonies. The ML assay has been shown to detect a broad spectrum of genetic damage, including both small scale (point) mutations and chromosomal alterations. This assay is a widely used mammalian cell gene mutation assay for regulatory purposes and is included in the core battery of genotoxicity tests for regulatory decision-making. The TK6 assay is an assay using a human cell line derived similarly via mutagenic manipulations and optimal selection. Details are provided on the materials required, cell culture methods, selection of test chemical concentrations, cytotoxicity, treatment time, mutation expression, cloning, and data calculation and interpretation. The methods describe the microwell plate version of the assays without metabolic activation.
PubMed: 35757197
DOI: 10.3389/ftox.2022.864753 -
PloS One 2020Fluorescent reporters are an important tool for monitoring dynamics of bacterial populations at the single cell and community level. While there are a large range of...
Fluorescent reporters are an important tool for monitoring dynamics of bacterial populations at the single cell and community level. While there are a large range of reporter constructs available-particularly for common model organisms like E. coli-fewer options exist for other species, including E. faecalis, a gram-positive opportunistic pathogen. To expand the potential toolkit available for E. faecalis, we exchanged the original fluorescent reporter in a previously developed plasmid (pBSU101) with one of eight fluorescent reporters and confirmed that all constructs exhibited detectable fluorescence in single E. faecalis cells and mixed biofilm communities. To identify promising constructs for bulk-level experiments, we then measured the fluorescence spectra from E. faecalis populations in microwell plate (liquid) cultures during different phases of aerobic growth. Cultures showed density- and reporter-specific variations in fluorescent signal, though spectral signatures of all reporters become clear in late-exponential and stationary-phase populations. Based on these results, we identified six pairs of reporters that can be combined with simple spectral unmixing to accurately estimate population composition in 2-strain mixtures at or near stationary phase. This approach offers a simple and scalable method for selection and competition experiments in simple two-species populations under aerobic growth conditions. Finally, we incorporated codon-optimized variants of blue (BFP) and red (RFP) reporters and show that they lead to increased fluorescence in exponentially growing cells. As a whole, the results inform the scope of application of different reporters and identify both single reporters and reporter pairs that are promising for fluorescence-based assays at bulk and single-cell levels in E. faecalis.
Topics: Biofilms; Enterococcus faecalis; Fluorescence; Luminescent Proteins; Plasmids; Single-Cell Analysis
PubMed: 32369497
DOI: 10.1371/journal.pone.0232539 -
Advanced Healthcare Materials Sep 2022For high-throughput anti-cancer drug screening, microwell arrays may serve as an effective tool to generate uniform and scalable tumor spheroids. However, microwell...
For high-throughput anti-cancer drug screening, microwell arrays may serve as an effective tool to generate uniform and scalable tumor spheroids. However, microwell arrays are commonly anchored in non-oxygen-permeable culture plates, leading to limited oxygen supply for avascular spheroids. Herein, a polydimethylsiloxane (PDMS)-based oxygen-permeable microwell device is introduced for generating highly viable and functional hepatocellular carcinoma (HCC) spheroids. The PDMS sheets at the bottom of the microwell device provide a high flux of oxygen like in vivo neighboring hepatic sinusoids. Owing to the better oxygen supply, the generated HepG2 spheroids are larger in size and exhibit higher viability and proliferation with less cell apoptosis and necrosis. These spheroids also exhibit lower levels of anaerobic cellular respiration and express higher levels of liver-related functions. In anti-cancer drug testing, spheroids cultured in PDMS plates show a significantly stronger resistance against doxorubicin because of the stronger stem-cell and multidrug resistance phenotype. Moreover, higher expression of vascular endothelial growth factor-A produces a stronger angiogenesis capability of the spheroids. Overall, compared to the spheroids cultured in conventional non-oxygen-permeable plates, these spheroids can be used as a more favorable model for early-stage HCCs and be applied in high-throughput anti-cancer drug screening.
Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Culture Techniques; Dimethylpolysiloxanes; Doxorubicin; Humans; Liver Neoplasms; Oxygen; Spheroids, Cellular; Vascular Endothelial Growth Factor A
PubMed: 35841538
DOI: 10.1002/adhm.202200863 -
Molecules (Basel, Switzerland) May 2023This study discusses the development and validation of a universal microwell spectrophotometric assay for TKIs, regardless of the diversity in their chemical structures....
Development of Green and High Throughput Microplate Reader-Assisted Universal Microwell Spectrophotometric Assay for Direct Determination of Tyrosine Kinase Inhibitors in Their Pharmaceutical Formulations Irrespective the Diversity of Their Chemical Structures.
This study discusses the development and validation of a universal microwell spectrophotometric assay for TKIs, regardless of the diversity in their chemical structures. The assay depends on directly measuring the native ultraviolet light (UV) absorption of TKIs. The assay was carried out using UV-transparent 96-microwell plates and the absorbance signals were measured by a microplate reader at 230 nm, at which all TKIs had light absorption. Beer's law correlating the absorbances of TKIs with their corresponding concentrations was obeyed in the range of 2-160 µg mL with excellent correlation coefficients (0.9991-0.9997). The limits of detection and limits quantitation were in the ranges of 0.56-5.21 and 1.69-15.78 µg mL, respectively. The proposed assay showed high precision as the values of the relative standard deviations for the intra- and inter-assay precisions did not exceed 2.03 and 2.14%, respectively. The accuracy of the assay was proven as the recovery values were in the range of 97.8-102.9% (±0.8-2.4%). The proposed assay was successfully applied to the quantitation of all TKIs in their pharmaceutical formulations (tablets) with reliable results in terms of high accuracy and precision. The assay greenness was evaluated, and the results proved that the assay fulfils the requirements of green analytical approach. The proposed assay is the first assay that can analyse all TKIs on a single assay system without chemical derivatization or modifications in the detection wavelength. In addition, the simple and simultaneous handling of a large number of samples as a batch using micro-volumes of samples gave the assay the advantage of high throughput analysis, which is a serious demand in the pharmaceutical industry.
Topics: Tyrosine Kinase Inhibitors; Drug Compounding; Spectrophotometry; Tablets; High-Throughput Screening Assays
PubMed: 37241790
DOI: 10.3390/molecules28104049