Session 2: High-throughput and high-content analyses

Standardized in vitro high-throughput and high-content analyses serve efficiently to broadly assess nanomaterials safety and influences of different dispersion and testing protocols
OpenTox Euro 2014 speaker: Roland Grafström
PRESENTING AUTHOR: 

Prof. Roland Grafström

INSTITUTION / COMPANY : 

Health R&D, Knowledge Intensive Products and Services, VTT Technical Research Centre of Finland, Turku, Finland
Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden

AUTHOR(S): 

Roland Grafström and Vesa Hongisto

WHEN: 
Mon, 22. Sep. 2014

 

BIOGRAPHY:
Winner of over 80 international competitive research grants, including from US and European sources. Current grants from the Scientific Research Council, the Cancer and Allergy Fund and the Fund for Research without Animal Experiments in Sweden. Partner/team member 2011-2016 in EU projects: FP7-HEALTH-2010-Alternative-Testing, through “SEURAT/ToxBank” - Integrated data analysis and servicing”, NMP-2012- “NANoREG” “Regulatory testing of nanomaterials”, NMP-2012-NANOSOLUTIONS “Systematic investigations of the mechanisms and effects of engineered nanomaterial interactions with living systems and/or the environment”, and NMP-2013-eNanoMapper “A Database and ontology framework for nanomaterials design and safety assessment”.

Of special relevance to this lecture is the coordination of safety and nanotechnology development studies for a lead group of 12 professors/senior scientists and 400+ persons at diverse VTT sites in Finland. Leading this work has included handling of internal projects and customer offerings according to a "safe by design strategy" as well as the promotion of methods development, research concepts and cross-disciplinary interactions.

CURRENT EMPLOYMENT:
Full Professor in Biochemical Toxicology, Institute of Environmental Medicine, Karolinska Institutet (KI), Stockholm, Sweden, 2000- (60% time effort, research time: 50% of the 60%)
Visiting Professor (part time): VTT Biomedical Technology, Technical Research Centre of Finland, Turku, Finland, 2008- (40% time effort, research time: 50% of the 40 %)

ABSTRACT CONTENT / DETAILS:

There is global concern that known engineered nanomaterials (ENMs) in use and those to be synthesized in coming years will adversely influence environmental and health safety. Future ENM production is expected to be substantial, and the application of high-throughput screening (HTS) technologies would potentially permit for human cell-based synchronous safety evaluations of the ENMs to be generated.

Complicating the application of this technology, diverse nanomaterial dispersion and cell testing protocols generates variables that might influence the safety interpretation of the results. We report now on a standardized protocol for HTS-based safety analysis of ENMs in vitro.

It applies the BEAS-2B human lung epithelial cell line, a common model system for studying effects of ENMs, and the Promega’s CellTiter-Glo assay for cellular ATP content as a surrogate measure for alterations in cell numbers and viability.

Assessed under a 384-well HTS format, established reference ENMs, including oxide forms of iron, titanium, zinc and copper, and so far untested, customer-based ENMs, demonstrated dose-dependent toxicity over a wide range of concentrations, indicating manifold differences in potency among different ENMs.

Many factors, including the ENM dispersion protocol and volume, storage time of the dispersions, the cell density, the exposure time, as well as the length of growth and exposure of cells with or without serum, were found to influence the testing results.

Thousands of experiments together indicated variable sensitivity of the ENMs to the protocol parameters, e.g., a particular dispersion protocol could variably act to increase, decrease or not affect the ENM toxicity effect relative another dispersion protocol. Importantly, it could be demonstrated that the ENMs as such would not influence the toxicity assay readout.

Quantification under the HTS format of cell surface areas by microscopic imaging under time lapse demonstrated the feasibility of high-content screening for morphological toxicity.

Such data can potentially validate effects indicated by the ATP assay. Interestingly, a modified, yet still fully functional, form of an original customer-produced ENM was found to generate significantly lower toxicity relative to the parent ENM. Taken together, we demonstrate standardized, multi-assay HTS protocols for ENM safety testing.

The technology permits rapid sorting of possible influences of testing variables. The results argue for the implementation of HTS methods for proactive ENM safety evaluation, applicable generally to support novel ENM production under a safe-by-design concept.

Our planned continued collection of thousands of HTS results under a standardized ISA-Tab-nano format will ultimately lead to a valuable resource for ENMs ranking.