Final Report: Developmental Neurotox Assay Using Scalable Neurons and Astrocytes in High-Content Imaging

EPA Contract Number: EPD13018
Title: Developmental Neurotox Assay Using Scalable Neurons and Astrocytes in High-Content Imaging
Investigators: Majumder, Anirban
Small Business: ArunA Biomedical, Inc.
EPA Contact: Manager, SBIR Program
Phase: I
Project Period: May 15, 2013 through November 14, 2013
Project Amount: $79,993
RFA: Small Business Innovation Research (SBIR) - Phase I (2013) RFA Text |  Recipients Lists
Research Category: Small Business Innovation Research (SBIR) , SBIR - Innovation in Manufacturing


There is a stated need both within the computational toxicology programs at EPA and regulatory agencies worldwide for rapid, cost effective in vitro methods to identify chemicals that pose a hazard to the developing human brain. As an alternative to costly animal studies, a number of in vitro assays utilizing primary cell lines have been tested in an effort to find and devise effective models to prioritize chemicals for further in depth toxicity studies. However, isolation of primary neural tissue is cost- and labor-intensive, and primary cells cannot be produced in the numbers required for high throughput screening (HTS). To address this critical cell sourcing limitation, ArunA Biomedical has developed HTS-scalable, human embryonic stem cell (hESC) derived cellular model systems, hNP1™ Human Neural Progenitor Cells and hN2™ Differentiated Human Neuronal Cells, which are commercially available to advance developmental neurotoxicity assays. It has been increasingly appreciated that multi-cellular systems provide a more physiologically relevant in vitro system to assess the effect of drug candidates on complex tissue systems. In this project, the company addressed this major gap by developing a multi-cellular neural system [neurons (hN2™) and astrocytes, referred to as AstroPro™], that more faithfully recapitulates early neural development. Moreover, the use of pluripotent progenitor cells enables scalable, high content screening (HCS)-amenable formats for toxicant screening and adverse outcome pathway computational analyses. In this study, ArunA Biomedical assessed the feasibility of using a neuron-astrocyte co-culture system for high throughput neurotoxicity screening, and reinforced the need for a more representative system by demonstrating that known neurotoxins do indeed evoke different neuronal responses in a multi-cellular assay environment.

Summary/Accomplishments (Outputs/Outcomes):

During Phase I, ArunA Biomedical optimized the production of assay-ready neurons and astrocytes, developed protocols for cryopreservation and plating of co-culture systems, and carried out experiments to test feasibility of co-culturing astrocytes and neurons at assay-optimized ratios. The company also performed experiments to document the toxic effects of two known neurotoxins on neurite outgrowth, a neuro-developmentally relevant endpoint, in comparison to the single cell type neuron-only assay to determine differences in response to neurotoxins.
ArunA Biomedical successfully met or exceeded all of its proposed milestone goals. For Objective 1, the company was able to manufacture populations consisting of greater than 95 percent progenitor cell-derived astrocytes, without the need for cell purification, which expressed several astrocyte-specific markers, and most importantly, displayed functional characteristics of primary astrocytes, namely inducible expression of ApoE. ArunA Biomedical established protocols to produce cryopreserved differentiated astrocytes at > 70 percent post-thaw viabilities, and was able to co-culture thawed differentiated astrocytes and neurons for at least seven days, which is a sufficient length of time for toxicity testing. In Objective 2, the company determined that a 3:1 neuron: astrocyte ratio at a density of 20,000 cells per well (96-well format) was the optimal plating scheme to enable quantification of a sufficient number of traceable neurons to assess the response of neurites to potential neurotoxins. Indeed, ArunA Biomedical observed that the presence of astrocytes influenced the response of neurons to two known neurotoxins, Bis-1 and lead acetate. These data demonstrated the importance of developing culture systems that more closely recapitulate the in vivo environment.


The primary innovation was a screening assay for neurotoxins that incorporated the two common cell types present in the brain, making the assay more representative of human neural development. Additionally, utilizing easily scalable human source cells ensured assay scalability to meet virtually any throughput demand. Potential applications/products include high content screening (HCS) assays such as neurite outgrowth assays for use in screening neurotoxins, drug toxicity drug development-related HCS, and neuro-developmental research. More generally, ArunA Biomedical's co-culture system will provide a valuable and unique in vitro model to examine interactions between astrocytes and neurons.

Supplemental Keywords:

astrocytes, neurons, neurotoxicology, stem cells, neurotoxicity assay

Relevant Websites:

SBIR Phase II:

Developmental Neurotox Assay using Scalable Neurons and Astrocytes in High Content Imaging