scholarly journals Co-culture of human alveolar epithelial (A549) and macrophage (THP-1) cells to study the potential toxicity of ambient PM2.5: a comparison of growth under ALI and submerged conditions

2020 ◽  
Vol 9 (5) ◽  
pp. 636-651
Author(s):  
Guanghe Wang ◽  
Xiaofeng Zhang ◽  
Xinyan Liu ◽  
Jing Zheng
Keyword(s):  
High Throughput ◽  
Animal Studies ◽  
Fine Particulate Matter ◽  
Toxicity Testing ◽  
Culture Conditions ◽  
Ambient Atmosphere ◽  
Submerged Condition ◽  
Culture Methods ◽  
Submerged Conditions

Abstract Fine particulate matter (PM2.5) in the ambient atmosphere is strongly associated with detrimental health effects. However, these particles from various sources and regions are unlikely equally toxic. While animal studies are impractical for high-throughput toxicity testing, appropriate in vitro models are urgently needed. Co-culture of A549 and THP-1 macrophages grown at air–liquid interface (ALI) or under submerged conditions was exposed to same concentrations of ambient PM2.5 to provide accurate comparisons between culture methods. Following 24-h incubation with PM2.5 collected in Harbin in China, biological endpoints being investigated include cytotoxicity, reactive oxygen species (ROS) levels and pro-inflammatory mediators. The co-culture grown under submerged condition demonstrated a significant increase in ROS levels and all tested pro-inflammatory indicators [interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor-α] in mRNA expression and released protein levels. Similar but a declining response trend was observed using the same PM2.5 incubation after grown at ALI. We further observed a significant increase of PM2.5-induced phosphorylation of p38 MAPK and activation of NF-κB p65 in a dose-dependent trend for co-cultures grown under submerged condition. These results provide important implications that culture conditions (ALI versus submerged) can induce different extents of biological responses to ambient PM2.5; the co-culture grown at ALI is less likely to produce false-positive results than submerged culture. Hence, culture conditions should be discussed when comparing in vitro methods used for high-throughput PM2.5 toxicity assessment in future.

scholarly journals Development of a Scalable, High-Throughput-Compatible Assay to Detect Tau Aggregates Using iPSC-Derived Cortical Neurons Maintained in a Three-Dimensional Culture Format

2016 ◽  
Vol 21 (8) ◽  
pp. 804-815 ◽  
Author(s):  
X. Medda ◽  
L. Mertens ◽  
S. Versweyveld ◽  
A. Diels ◽  
L. Barnham ◽  
...  
Keyword(s):  
High Throughput ◽  
Cortical Neurons ◽  
High Throughput Screening ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Novel Treatments ◽  
Entry Points ◽  
Induced Pluripotent ◽  
Tau Aggregation

Tau aggregation is the pathological hallmark that best correlates with the progression of Alzheimer’s disease (AD). The presence of neurofibrillary tangles (NFTs), formed of hyperphosphorylated tau, leads to neuronal dysfunction and loss, and is directly associated with the cognitive decline observed in AD patients. The limited success in targeting β-amyloid pathologies has reinforced the hypothesis of blocking tau phosphorylation, aggregation, and/or spreading as alternative therapeutic entry points to treat AD. Identification of novel therapies requires disease-relevant and scalable assays capable of reproducing key features of the pathology in an in vitro setting. Here we use induced pluripotent stem cells (iPSCs) as a virtually unlimited source of human cortical neurons to develop a robust and scalable tau aggregation model compatible with high-throughput screening (HTS). We downscaled cell culture conditions to 384-well plate format and used Matrigel to introduce an extra physical protection against cell detachment that reduces shearing stress and better recapitulates pathological conditions. We complemented the assay with AlphaLISA technology for the detection of tau aggregates in a high-throughput-compatible format. The assay is reproducible across users and works with different commercially available iPSC lines, representing a highly translational tool for the identification of novel treatments against tauopathies, including AD.

scholarly journals Integrating in vitro metabolomics with a 96-well high-throughput screening platform

Metabolomics ◽  
2022 ◽  
Vol 18 (1) ◽  
Author(s):  
Julia M. Malinowska ◽  
Taina Palosaari ◽  
Jukka Sund ◽  
Donatella Carpi ◽  
Mounir Bouhifd ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Toxicity Testing ◽  
Exposure Period ◽  
Solvent System ◽  
Extraction Methods ◽  
Metabolic Phenotype ◽  
Chemical Safety ◽  
Relative Standard

Abstract Introduction High-throughput screening (HTS) is emerging as an approach to support decision-making in chemical safety assessments. In parallel, in vitro metabolomics is a promising approach that can help accelerate the transition from animal models to high-throughput cell-based models in toxicity testing. Objective In this study we establish and evaluate a high-throughput metabolomics workflow that is compatible with a 96-well HTS platform employing 50,000 hepatocytes of HepaRG per well. Methods Low biomass cell samples were extracted for metabolomics analyses using a newly established semi-automated protocol, and the intracellular metabolites were analysed using a high-resolution spectral-stitching nanoelectrospray direct infusion mass spectrometry (nESI-DIMS) method that was modified for low sample biomass. Results The method was assessed with respect to sensitivity and repeatability of the entire workflow from cell culturing and sampling to measurement of the metabolic phenotype, demonstrating sufficient sensitivity (> 3000 features in hepatocyte extracts) and intra- and inter-plate repeatability for polar nESI-DIMS assays (median relative standard deviation < 30%). The assays were employed for a proof-of-principle toxicological study with a model toxicant, cadmium chloride, revealing changes in the metabolome across five sampling times in the 48-h exposure period. To allow the option for lipidomics analyses, the solvent system was extended by establishing separate extraction methods for polar metabolites and lipids. Conclusions Experimental, analytical and informatics workflows reported here met pre-defined criteria in terms of sensitivity, repeatability and ability to detect metabolome changes induced by a toxicant and are ready for application in metabolomics-driven toxicity testing to complement HTS assays.

scholarly journals Significance of incorporating measures of sperm production and function into rat toxicology studies

Reproduction ◽  
2001 ◽  
pp. 207-216 ◽  
Author(s):  
SD Perreault ◽  
AM Cancel
Keyword(s):  
Sperm Quality ◽  
Reproductive Toxicity ◽  
Toxicity Testing ◽  
Culture Conditions ◽  
Test Substance ◽  
Vitro Fertilization ◽  
Sperm Analysis ◽  
Computer Aided ◽  
Motion Characteristics

The rat is the preferred species for reproductive toxicity testing. The inclusion of measures of rat sperm quality, such as motility and morphology, into reproductive test protocols often increases the sensitivity of the test to detect effects, and provides the toxicologist and risk assessor with valuable information about the nature of the reproductive toxicity of the test substance. Technical advances in computer-aided sperm analysis have made it possible to evaluate motion characteristics of rat spermatozoa. This technology can provide an objective means of classifying the motion of rat spermatozoa as progressive or non-progressive, as required in test protocols. More specific tests of rat sperm function are being applied for the purpose of evaluating modes and mechanisms of toxicant action. Computer-aided sperm analysis can be used to evaluate sperm motion during cultures that support sperm capacitation and to identify hyperactivated spermatozoa. Under the same culture conditions, acrosome-specific stains can be used to identify effects of toxicants on the acrosome reaction. These approaches, in combination with in vitro fertilization in rats, can pinpoint sperm functional deficits and thereby assist the toxicologist in addressing hypotheses regarding the cellular-molecular bases of toxicant-induced male infertility.

scholarly journals Impact of cell culture methods on the outcomes of the in vitro inflammatory response in nasal polyps

2011 ◽  
Vol 49 (5) ◽  
pp. 562-569
Author(s):  
L. Fernández-Bertolín ◽  
J. Mullol ◽  
I. Alobid ◽  
J. Roca-Ferrer ◽  
C. Picado ◽  
...  
Keyword(s):  
Cell Viability ◽  
Nasal Mucosa ◽  
Nasal Polyp ◽  
Cytokine Production ◽  
Culture Conditions ◽  
Culture Methods ◽  
Gm Csf ◽  
Research Outcomes ◽  
Cytokine Levels

Background: In vitro culture of nasal polyp cells is frequently used in the investigation of inflammatory mechanisms and effect of treatments in nasal polyposis. Research outcomes may, however, be influenced by the culture methodology used. Methods: Nasal polyp and nasal mucosa in vitro fibroblast cultures were pre-treated with foetal bovine serum (FBS)-free culture medium or medium supplemented with either FBS or charcoal-stripped (cs) FBS. Cells were then stimulated with FBS or csFBS, with or without different doses of dexamethasone for 4 and 24h. IL-6, IL-8, GM-CSF and VEGF release and cell viability were measured. Results: The highest cytokine levels were found in growth-arrested cells stimulated with 10% FBS. csFBS poorly stimulated cytokine release. Nasal polyp released larger IL-8 amounts than nasal mucosa fibroblasts. Dexamethasone decreased cytokine production dose- and time-dependently in both nasal mucosa and nasal polyp fibroblasts. The IC25 of IL-8 inhibition by dexamethasone was higher in nasal polyp than in nasal mucosa fibroblasts. Cell viability did not differ among treatments. Conclusions: Cytokine production by in vitro cultured nasal fibroblasts is affected by the culture conditions used and is inhibited by dexamethasone in both fibroblast types. Our results highlight the importance of culture methodology on nasal polyp research outcomes.

scholarly journals Application of Horticultural and Tissue Culture Methods for Ex Situ Conservation of Endangered Primula farinosa L.

2020 ◽  
Vol 89 (1) ◽  
Author(s):  
Ewa Sitek ◽  
Barbara Nowak ◽  
Michał Fecowicz ◽  
Zbigniew Gajewski ◽  
Piotr Dańda ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Culture Conditions ◽  
Ex Situ ◽  
In Vitro Cultivation ◽  
Culture Methods ◽  
Transient Nature ◽  
Micropropagated Plants ◽  
The Difference

Our study aimed at active conservation of the last location of <em>Primula farinosa</em>, an endangered species in Poland, and assessed reproduction by seeds and plant propagation on sterile media in tissue culture conditions. We identified gibberellic acid (GA<sub data-id="subscript-1">3</sub>) as the key factor stimulating germination of <em>P. farinosa</em> seeds. Growing juvenile plants under controlled temperature of 18/16 °C day/night yielded good quality plant material without mycorrhization. In tissue culture, the most favorable medium for shoot propagation was MS supplemented with the lowest tested concentration of indole-3-butyric acid (IBA; 0.05 mg dm<sup data-id="superscript-1">−3</sup>) and 6-benzyl-aminopurine (BAP; 0.1 mg dm<sup data-id="superscript-2">−3</sup>). The rooting ability of shoots was high and comparable for all auxins used. 2C DNA content of seed-derived and micropropagated plants did not indicate any change in the ploidy level during in vitro cultivation. Plants derived from seeds and tissue cultures were compared in a 2-year study. Of all the characteristics compared, only the number of flowers per inflorescence was lower for micropropagated plants when compared with the seed-origin plants in the first year of observation. The difference was of transient nature and was not observed in the second year of the study. Effective protocols for in vivo and in vitro propagation of <em>P. farinosa</em> were developed, which can be used in practical species protection.

scholarly journals Cell-Type-Specific High Throughput Toxicity Testing in Human Midbrain Organoids

2021 ◽  
Vol 14 ◽  
Author(s):  
Henrik Renner ◽  
Katharina J. Becker ◽  
Theresa E. Kagermeier ◽  
Martha Grabos ◽  
Farsam Eliat ◽  
...  
Keyword(s):  
Human Physiology ◽  
High Throughput ◽  
Toxicity Testing ◽  
Fluorescent Imaging ◽  
Model Systems ◽  
Cell Type ◽  
Human Organ ◽  
Cell Type Specific

Toxicity testing is a crucial step in the development and approval of chemical compounds for human contact and consumption. However, existing model systems often fall short in their prediction of human toxicity in vivo because they may not sufficiently recapitulate human physiology. The complexity of three-dimensional (3D) human organ-like cell culture systems (“organoids”) can generate potentially more relevant models of human physiology and disease, including toxicity predictions. However, so far, the inherent biological heterogeneity and cumbersome generation and analysis of organoids has rendered efficient, unbiased, high throughput evaluation of toxic effects in these systems challenging. Recent advances in both standardization and quantitative fluorescent imaging enabled us to dissect the toxicities of compound exposure to separate cellular subpopulations within human organoids at the single-cell level in a framework that is compatible with high throughput approaches. Screening a library of 84 compounds in standardized human automated midbrain organoids (AMOs) generated from two independent cell lines correctly recognized known nigrostriatal toxicants. This approach further identified the flame retardant 3,3′,5,5′-tetrabromobisphenol A (TBBPA) as a selective toxicant for dopaminergic neurons in the context of human midbrain-like tissues for the first time. Results were verified with high reproducibility in more detailed dose-response experiments. Further, we demonstrate higher sensitivity in 3D AMOs than in 2D cultures to the known neurotoxic effects of the pesticide lindane. Overall, the automated nature of our workflow is freely scalable and demonstrates the feasibility of quantitatively assessing cell-type-specific toxicity in human organoids in vitro.

scholarly journals New substrates for stem cell control

2018 ◽  
Vol 373 (1750) ◽  
pp. 20170223 ◽  
Author(s):  
Sara Schmidt ◽  
Annamaria Lilienkampf ◽  
Mark Bradley
Keyword(s):  
Stem Cells ◽  
Toxicity Testing ◽  
Biological Properties ◽  
Culture Conditions ◽  
Theme Issue ◽  
Serum Free ◽  
Physical Attributes ◽  
Natural Tissue

The capacity to culture stem cells in a controllable, robust and scalable manner is necessary in order to develop successful strategies for the generation of cellular and tissue platforms for drug screening, toxicity testing, tissue engineering and regenerative medicine. Creating substrates that support the expansion, maintenance or directional differentiation of stem cells would greatly aid these efforts. Optimally, the substrates used should be chemically defined and synthetically scalable, allowing growth under defined, serum-free culture conditions. To achieve this, the chemical and physical attributes of the substrates should mimic the natural tissue environment and allow control of their biological properties. Herein, recent advances in the development of materials to study/manipulate stem cells, both in vitro and in vivo, are described with a focus on the novelty of the substrates’ properties, and on application of substrates to direct stem cells. This article is part of the theme issue ‘Designer human tissue: coming to a lab near you’.

scholarly journals The future of toxicity testing: a focus on in vitro methods using a quantitative high-throughput screening platform

2010 ◽  
Vol 15 (23-24) ◽  
pp. 997-1007 ◽  
Author(s):  
Sunita J. Shukla ◽  
Ruili Huang ◽  
Christopher P. Austin ◽  
Menghang Xia
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Toxicity Testing ◽  
In Vitro Methods ◽  
The Future ◽  
Screening Platform

scholarly journals Knockout tissue models: Targeted gene disruption in three dimensions

2014 ◽  
Vol 36 (3) ◽  
pp. 14-18
Author(s):  
Alice Carstairs ◽  
Julia Marshall ◽  
Paul Genever
Keyword(s):  
Gene Disruption ◽  
Animal Studies ◽  
Genetically Modified ◽  
Three Dimensions ◽  
Culture Methods ◽  
Targeted Gene Disruption ◽  
The Uk ◽  
Tissue Models ◽  
Genetically Modified Animals

The use of animal studies in scientific research is increasing, mostly attributed to the rising requirement for genetically modified animals. In 2012, 4.11 million animal procedures were conducted within the UK, the majority of which involved genetically modified animals; a first since records began. Although animal models bring clear scientific insight, they have their limitations and alternative research methods are actively sought. The development of new targeted gene disruption technologies, such as transcription activatorlike effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR), combined with innovative 3D human cell culture methods can provide an attractive in vitro alternative.

scholarly journals Human Pluripotent Stem Cells: A Unique Tool for Toxicity Testing in Pancreatic Progenitor and Endocrine Cells

2021 ◽  
Vol 11 ◽  
Author(s):  
Erin M. MacFarlane ◽  
Jennifer E. Bruin
Keyword(s):  
High Throughput ◽  
Endocrine Cells ◽  
Toxicity Testing ◽  
Diabetes Incidence ◽  
Clear Understanding ◽  
Target Tissue ◽  
Β Cell ◽  
Obesity And Diabetes ◽  
Pancreatic Endocrine Cells

Diabetes prevalence is increasing worldwide, and epidemiological studies report an association between diabetes incidence and environmental pollutant exposure. There are &gt;84,000 chemicals in commerce, many of which are released into the environment without a clear understanding of potential adverse health consequences. While in vivo rodent studies remain an important tool for testing chemical toxicity systemically, we urgently need high-throughput screening platforms in biologically relevant models to efficiently prioritize chemicals for in depth toxicity analysis. Given the increasing global burden of obesity and diabetes, identifying chemicals that disrupt metabolism should be a high priority. Pancreatic endocrine cells are key regulators of systemic metabolism, yet often overlooked as a target tissue in toxicology studies. Immortalized β-cell lines and primary human, porcine, and rodent islets are widely used for studying the endocrine pancreas in vitro, but each have important limitations in terms of scalability, lifespan, and/or biological relevance. Human pluripotent stem cell (hPSC) culture is a powerful tool for in vitro toxicity testing that addresses many of the limitations with other β-cell models. Current in vitro differentiation protocols can efficiently generate glucose-responsive insulin-secreting β-like cells that are not fully mature, but still valuable for high-throughput toxicity screening in vitro. Furthermore, hPSCs can be applied as a model of developing pancreatic endocrine cells to screen for chemicals that influence endocrine cell formation during critical windows of differentiation. Given their versatility, we recommend using hPSCs to identify potential β-cell toxins, which can then be prioritized as chemicals of concern for metabolic disruption.

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