A practical note on the use of cytotoxicity assays

Weyermann, Jörg; Lochmann, Dirk; Zimmer, Andreas

doi:10.1016/j.ijpharm.2004.09.018

Cited by 416 publications

(283 citation statements)

References 19 publications

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“…Different assays are used to measure cell viability such as MTT assays, LDH assays and apoptosis/necrosis assays among others. MTT [3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazoliumbromide] assays, used to demonstrate the viability of cells through the reduction of the pale yellow MTT dye to a dark blue formazan product by the activity of succinate dehydrogenase present in the mithocondria of living cells, LDH (lactate dehydrogenase) assays, that consists in measuring the release of this LDH stable cytoplasmic enzyme that is rapidly released into the cell-culture supernantant upon damage of the plasma membrane and apoptosis/necrosis assays detects changes in cell membrane permeability (Weyermann et al, 2005).…”

Section: Cytotoxicity Biodistribution and Clearancementioning

confidence: 99%

Silver Nanoparticles Interactions with the Immune System: Implications for Health and Disease

Klippstein¹,

Fernández-Montesinos²,

Castillo³

et al. 2010

Silver Nanoparticles

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Section: Cytotoxicity Biodistribution and Clearancementioning

confidence: 99%

Silver Nanoparticles Interactions with the Immune System: Implications for Health and Disease

Klippstein¹,

Fernández-Montesinos²,

Castillo³

et al. 2010

Silver Nanoparticles

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“…It is considered that the samples in the supernatant would interact with LDH as previously reported for SDS. 27) On the other hand, LDH activity in the basolateral chamber was dependent on the applied concentration of SDS or DOX (Fig. 4).…”

Section: Resultsmentioning

confidence: 92%

Safety Evaluation of Dry Powder Formulations by Direct Dispersion onto Air–Liquid Interface Cultured Cell Layer

Asai

Okuda

Yamauchi

et al. 2016

Biological & Pharmaceutical Bulletin

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Most safety evaluations of dry powder inhalers (DPIs) using cultured cells have been performed with dry powder formulations dissolved in a medium. However, this method is not considered to be suitable to evaluate the safety of inhaled dry powder formulations correctly since it cannot reflect the actual phenomenon on the respiratory epithelial surface. In this study, we established a novel in-vitro safety evaluation system suitable for DPIs by combining an air-liquid interface cultured cell layer and a device for dispersing dry powders, and evaluated the safety of candidate excipients of dry powders for inhalation. The safety of excipients (sugars, amino acids, cyclodextrins, and positive controls) in solutions was compared using submerged cell culture systems with a conventional 96-well plate and Transwell ® . The sensitivity of the cells grown in Transwell ® was lower than that of those grown in the 96-well plate. Dry powders were prepared by spray-drying and we evaluated their safety with a novel in-vitro safety evaluation system using an air-liquid interface cultured cell layer. Dry powders decreased the cell viability with doses more than solutions. On the other hand, dissolving the dry powders attenuated their cytotoxicity. This suggested that the novel in-vitro safety evaluation system would be suitable to evaluate the safety of DPIs with high sensitivity.Key words dry powder; safety evaluation; air-liquid interfaced culture; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; A549 cell Pulmonary drug delivery has been investigated as not only a local treatment for respiratory diseases such as asthma or chronic obstructive pulmonary disease, but also as an alternative route for the systemic delivery of macromolecule drugs such as insulin, calcitonin, and heparin. 1-4)Among the three major systems for pulmonary deliverynebulizers, pressurized metered-dose inhalers (pMDIs), and dry powder inhalers (DPIs)-DPIs have several advantages, including favorable portability, a low cost, and the lack of a need for propellants. Furthermore, the handling of DPIs is easier than that of pMDIs because of breath-actuated passive aerosolization. 5)For effective pulmonary deposition after inhalation, in general, the optimal aerodynamic diameter of drug particles is less than 6 µm.6,7) However, micronized drug particles tend to be highly cohesive and poorly flowable, leading to low-level performance. To solve these problems, it is usual for an excipient, such as coarse lactose monohydrate, to be formulated to physically attach to fine active ingredients. 8,9) It has been recently reported that the addition of amino acids or cyclodextrins (CyDs) as excipients improves pulmonary delivery. 10,11) As for fundamental research on DPIs, evaluation of the physical properties such as particle size and pulmonary deposition is mainstream, whereas biological evaluation such as of the efficacy and safety is essential for clinical application. 12)These biological evaluations of dry powder formulations are often conduct...

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“…For comparison purposes, staurosporine (1 μM) and Triton-X (0.01%) were used as positive inducers of apoptosis (Zhang et al, 1998) and necrosis (Weyermann et al, 2005), respectively. Cells exposed to media only were used as controls.…”

Section: Transmission Electron Microscopy (Tem)mentioning

confidence: 99%

Characterization of cell death caused by diplodiatoxin and dipmatol, toxic metabolites of Stenocarpella maydis

Masango

Ellis

Botha

2015

Toxicon

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Highlights• Diplodiatoxin and dipmatol induced necrosis in Neuro-2a, CHO-K1 and MDBK cells.• Diplodiatoxin and dipmatol induced caspase-dependent apoptosis in vitro.• Mitochondrial damage, cytoplasmic vacuoles and nuclear fragmentation were observed. AbstractDiplodiosis, a neuromycotoxicosis of cattle and sheep grazing on mouldy cobs infected by Stenocarpella maydis, is considered the last major veterinary mycotoxicosis for which the causative mycotoxin is still unknown. The current study was aimed at characterizing the cell death observed in mouse neuroblastoma (Neuro-2a), Chinese hamster ovary (CHO-K1) and Madin-Darby bovine kidney (MDBK) cell lines exposed to the S. maydis metabolites (i.e. diplodiatoxin and dipmatol) by investigating the roles of necrosis and apoptosis. Necrosis was investigated using the lactate dehydrogenase (LDH) leakage and propidium iodide (PI) flow cytometry assays and apoptosis was evaluated using the caspase-3/7 and Annexin V flow cytometry assays. In addition, transmission electron microscopy (TEM) was used to correlate the cell death pathways observed in this study with their typical morphologies. Both diplodiatoxin and dipmatol (750 μM) induced necrosis and caspase-dependent apoptosis in Neuro-2a, CHO-K1 and MDBK cells. Ultrastructurally, the two mycotoxins induced mitochondrial damage, cytoplasmic vacuolation and nuclear fragmentation in the three cell lines. These findings have laid a foundation for future studies aimed at elucidating in detail the mechanism of action of the S. maydis metabolites.

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A practical note on the use of cytotoxicity assays

Cited by 416 publications

References 19 publications

Silver Nanoparticles Interactions with the Immune System: Implications for Health and Disease

Silver Nanoparticles Interactions with the Immune System: Implications for Health and Disease

Safety Evaluation of Dry Powder Formulations by Direct Dispersion onto Air–Liquid Interface Cultured Cell Layer

Characterization of cell death caused by diplodiatoxin and dipmatol, toxic metabolites of Stenocarpella maydis

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