Optimized alamarBlue assay protocol for drug dose-response determination of 3D tumor spheroids

Eilenberger, Christoph; Kratz, Sebastian Rudi Adam; Rothbauer, Mario; Ehmoser, Eva-Kathrin; Ertl, Peter; Küpcü, Seta

doi:10.1016/j.mex.2018.07.011

Cited by 48 publications

(30 citation statements)

References 6 publications

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“…Fluorescence intensity of reduced form of resazurin was measured spectrophotometrically at 570 nm excitation and 580–600 nm as emission wavelength (Wu et al, 2011 ; Osterman et al, 2016 ; Tang et al, 2020 ) by using Thermo Scientific Varioskan Flash multi-plate reader. The percentage of cell proliferation was reported relative to untreated cells (100% viability) according the following equation using relative fluorescence units (RFU) (Eilenberger et al, 2018 ):…”

Section: Methodsmentioning

confidence: 99%

Stimuli-Responsive, Plasmonic Nanogel for Dual Delivery of Curcumin and Photothermal Therapy for Cancer Treatment

et al. 2021

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Nanogels (Ng) are crosslinked polymer-based hydrogel nanoparticles considered to be next-generation drug delivery systems due to their superior properties, including high drug loading capacity, low toxicity, and stimuli responsiveness. In this study, dually thermo-pH-responsive plasmonic nanogel (AuNP@Ng) was synthesized by grafting poly (N-isopropyl acrylamide) (PNIPAM) to chitosan (CS) in the presence of a chemical crosslinker to serve as a drug carrier system. The nanogel was further incorporated with gold nanoparticles (AuNP) to provide simultaneous drug delivery and photothermal therapy (PTT). Curcumin's (Cur) low water solubility and low bioavailability are the biggest obstacles to effective use of curcumin for anticancer therapy, and these obstacles can be overcome by utilizing an efficient delivery system. Therefore, curcumin was chosen as a model drug to be loaded into the nanogel for enhancing the anticancer efficiency, and further, its therapeutic efficiency was enhanced by PTT of the formulated AuNP@Ng. Thorough characterization of Ng based on CS and PNIPAM was conducted to confirm successful synthesis. Furthermore, photothermal properties and swelling ratio of fabricated nanoparticles were evaluated. Morphology and size measurements of nanogel were determined by transmission electron microscopy (TEM), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). Nanogel was found to have a hydrodynamic size of ~167 nm and exhibited sustained release of curcumin up to 72 h with dual thermo-pH responsive drug release behavior, as examined under different temperature and pH conditions. Cytocompatibility of plasmonic nanogel was evaluated on MDA-MB-231 human breast cancer and non-tumorigenic MCF 10A cell lines, and the findings indicated the nanogel formulation to be cytocompatible. Nanoparticle uptake studies showed high internalization of nanoparticles in cancer cells when compared with non-tumorigenic cells and confocal microscopy further demonstrated that AuNP@Ng were internalized into the MDA-MB-231 cancer cells via endosomal route. In vitro cytotoxicity studies revealed dose-dependent and time-dependent drug delivery of curcumin loaded AuNP@Ng/Cur. Furthermore, the developed nanoparticles showed an improved chemotherapy efficacy when irradiated with near-infrared (NIR) laser (808 nm) in vitro. This work revealed that synthesized plasmonic nanogel loaded with curcumin (AuNP@Ng/Cur) can act as stimuli-responsive nanocarriers, having potential for dual therapy i.e., delivery of hydrophobic drug and photothermal therapy.

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Section: Methodsmentioning

confidence: 99%

Stimuli-Responsive, Plasmonic Nanogel for Dual Delivery of Curcumin and Photothermal Therapy for Cancer Treatment

et al. 2021

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“…In summary, optimization is crucial for data reliability regardless of the model type (e.g. 2D monolayers, 3D spheroids or in vivo models) used to evaluate drug sensitivity 45 . We identified new critical experimental parameters (e.g.…”

Section: Discussionmentioning

confidence: 99%

Optimization of cell viability assays to improve replicability and reproducibility of cancer drug sensitivity screens

Larsson

Engqvist

Biermann

et al. 2020

Sci Rep

140

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cancer drug development has been riddled with high attrition rates, in part, due to poor reproducibility of preclinical models for drug discovery. Poor experimental design and lack of scientific transparency may cause experimental biases that in turn affect data quality, robustness and reproducibility. Here, we pinpoint sources of experimental variability in conventional 2D cell-based cancer drug screens to determine the effect of confounders on cell viability for MCF7 and HCC38 breast cancer cell lines treated with platinum agents (cisplatin and carboplatin) and a proteasome inhibitor (bortezomib). Variance component analysis demonstrated that variations in cell viability were primarily associated with the choice of pharmaceutical drug and cell line, and less likely to be due to the type of growth medium or assay incubation time. Furthermore, careful consideration should be given to different methods of storing diluted pharmaceutical drugs and use of DMSO controls due to the potential risk of evaporation and the subsequent effect on dose-response curves. Optimization of experimental parameters not only improved data quality substantially but also resulted in reproducible results for bortezomib-and cisplatin-treated HCC38, MCF7, MCF-10A, and MDA-MB-436 cells. Taken together, these findings indicate that replicability (the same analyst re-performs the same experiment multiple times) and reproducibility (different analysts perform the same experiment using different experimental conditions) for cell-based drug screens can be improved by identifying potential confounders and subsequent optimization of experimental parameters for each cell line. Cancer drug candidates currently have the lowest overall success rates and are 23% less likely to succeed in phase III clinical trials compared with other therapeutic areas 1-3. At a cost of approximately $3 billion per approved drug, over a decade may have passed from target discovery to drug approval (long development time) and tens of thousands of drug candidates would have likely been dropped due to drug safety and/or efficacy issues 4. Cell-based pharmacogenomics screens are commonly used during the preclinical drug screening process to identify druggable targets by characterizing the biological effects associated with drug response and toxicity. However, low interlaboratory reproducibility of cell-based pharmacogenomics screens, lack of robust disease models that recapitulate the natural progression of human cancers, and drug-associated toxicity issues contribute to the high drug attrition rates in oncology 5-13. Biomedical researchers and the pharmaceutical industry are, therefore, developing strategies to improve early-phase drug screening, e.g. novel preclinical models of disease and drug repurposing 9,14. Drug-dose response assays (e.g. MTT assay) performed in two-dimensional (2D) cell culture are typically used to evaluate drug efficacy and potency in cells exposed to a drug for up to 72 hours 15-17. However, it has been challenging to develop robust drug sensitivi...

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“…The development of 3D models such as spheroids made it possible to engineer several cancer-like microenvironments in vitro . Many papers claim to have developed their protocols to build tumors such as glioblastomas, colorectal, breast, liver, lungs, among others ( Kelm et al, 2003 ; Hirschhaeuser et al, 2010 ; Chimenti et al, 2017 ; Eilenberger et al, 2018 ; Froehlich et al, 2018 ; Oraiopoulou et al, 2019 ; Foglietta et al, 2020 ; Lee et al, 2020 ).…”

Section: D Models Recapitulating the Tumorigenesis In Vitromentioning

confidence: 99%

Recapitulating Tumorigenesis in vitro: Opportunities and Challenges of 3D Bioprinting

Kronemberger

Miranda

Tavares

et al. 2021

Front. Bioeng. Biotechnol.

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Cancer is considered one of the most predominant diseases in the world and one of the principal causes of mortality per year. The cellular and molecular mechanisms involved in the development and establishment of solid tumors can be defined as tumorigenesis. Recent technological advances in the 3D cell culture field have enabled the recapitulation of tumorigenesis in vitro, including the complexity of stromal microenvironment. The establishment of these 3D solid tumor models has a crucial role in personalized medicine and drug discovery. Recently, spheroids and organoids are being largely explored as 3D solid tumor models for recreating tumorigenesis in vitro. In spheroids, the solid tumor can be recreated from cancer cells, cancer stem cells, stromal and immune cell lineages. Organoids must be derived from tumor biopsies, including cancer and cancer stem cells. Both models are considered as a suitable model for drug assessment and high-throughput screening. The main advantages of 3D bioprinting are its ability to engineer complex and controllable 3D tissue models in a higher resolution. Although 3D bioprinting represents a promising technology, main challenges need to be addressed to improve the results in cancer research. The aim of this review is to explore (1) the principal cell components and extracellular matrix composition of solid tumor microenvironment; (2) the recapitulation of tumorigenesis in vitro using spheroids and organoids as 3D culture models; and (3) the opportunities, challenges, and applications of 3D bioprinting in this area.

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Optimized alamarBlue assay protocol for drug dose-response determination of 3D tumor spheroids

Abstract: Graphical abstract

Cited by 48 publications

References 6 publications

Stimuli-Responsive, Plasmonic Nanogel for Dual Delivery of Curcumin and Photothermal Therapy for Cancer Treatment

Stimuli-Responsive, Plasmonic Nanogel for Dual Delivery of Curcumin and Photothermal Therapy for Cancer Treatment

Optimization of cell viability assays to improve replicability and reproducibility of cancer drug sensitivity screens

Recapitulating Tumorigenesis in vitro: Opportunities and Challenges of 3D Bioprinting

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