Original article
A method for rapid dose–response screening of environmental chemicals using human embryonic stem cells

https://doi.org/10.1016/j.vascn.2012.07.003Get rights and content

Abstract

Introduction

Human embryonic stem cells (hESC) provide an invaluable model for assessing the effects of environmental chemicals and drugs on human prenatal development. However, hESC are difficult to adapt to 96-well plate screening assays, because they survive best when plated as colonies, which are difficult to count and plate accurately. The purpose of this study is to present an experimental method and analysis procedure to accomplish reliable screening of toxicants using hESC.

Methods

We present a method developed to rapidly and easily determine the number of cells in small colonies of hESC spectrophotometerically and then accurately dispense equivalent numbers of cells in 96-well plates. The MTT assay was used to evaluate plating accuracy, and the method was tested using known toxicants.

Results

The quality of the plate set-up and analysis procedure was evaluated with NIH plate validation and assessment software. All statistical parameters measured by the software were acceptable, and no drift or edge effects were observed. The 96-well plate MTT assay with hESC was tested by performing a dose–response screen of commercial products, which contain a variety of chemicals. The screen was done using single wells/dose, and the reliability of this method was demonstrated in a subsequent screen of the same products repeated three times. The single and triple screens were in good agreement, and NOAELs and IC50s could be determined from the single screen. The effects of vapor from volatile chemicals were studied, and methods to monitor and avoid vapor effects were incorporated into the assay.

Discussion

Our method overcomes the difficulty of using hESC for reliable quantitative 96-well plate assays. It enables rapid dose–response screening using equipment that is commonly available in laboratories that culture hESC. This method could have a broad application in studies of environmental chemicals and drugs using hESC as models of prenatal development.

Introduction

When evaluating the risk of exposure to environmental chemicals or drugs, it is important to consider how chemicals affect embryos and fetuses, which are usually the most sensitive stages of the human life cycle (Grandjean, Bellinger, Bergman, Cordier, et al., 2008). While direct experimentation with human embryos is not feasible, human embryonic stem cells (hESC) provide an invaluable model for evaluating the effects of environmental chemicals on prenatal development (Talbot & Lin, 2011). Several strategies exist for using hESC in toxicological studies (Adler et al., 2008, Krtolica et al., 2009, Talbot and Lin, 2011). Because they are pluripotent, hESC can be differentiated into any type of cell, and the effects of chemicals on differentiation or on differentiated cells can then be studied. As an alternative, hESC themselves can be used directly in toxicological assays, and the effects on various endpoints, including pluripotency, proliferation, apoptosis, survival and morphology, can be accessed (Lin, Fonteno, Weng, & Talbot, 2010). An advantage of the latter strategy is that the time to perform an assay can be greatly reduced to days or even hours, depending on the endpoint (Lin et al., 2009, Lin et al., 2010). A second advantage is that hESC model epiblast cells (Nichols and Smith, 2009, Tesar et al., 2007) which function in germ layer formation. Harm to the epiblast could result in death of the embryo or in major congenital malformations during subsequent development.

It would be highly desirable to be able to perform dose–response screens on environmental chemicals or drugs using hESC. However, hESC have proven difficult to adapt to such screens as they do not survive well when plated as single cells, which are easy to count and plate accurately. To assure survival, hESC are generally plated as small colonies or alternatively they can be plated as single cells with the inclusion of a Rho-associated kinase (ROCK) inhibitor, such as Y-27632, in the culture medium (Watanabe et al., 2007). However, both of these methods have drawbacks. Small colonies are difficult to count and plate accurately making quantitative assays difficult to perform and the inclusion of a ROCK inhibitor can influence the outcome of the assay (Fujimura, Usuki, Kawamura, & Izumo, 2011).

To overcome the above obstacles, we have developed a spectrophotometric method to rapidly measure density of hESC in small colonies, accurately plate equal numbers of cells/well in 96-well plates, and obtain highly reproducible dose–response curves using the MTT assay. The counting, plating, and screening methods that we present are straightforward, rapid, highly reproducible, and can be done in any laboratory with basic equipment and facilities for culturing hESC.

We have applied our counting and plating method to a dose–response screen of 12 electronic cigarette refill products. Electronic cigarette refill solutions (also called e-juice) contain nicotine, flavorings, and a humectant, such as propylene glycol, and are used to replenish fluid in used electronic cigarette cartridges (Trtchounian and Talbot, 2010, Williams and Talbot, 2011). Electronic cigarette refill solutions were chosen for screening as they represent environmental chemical products that have not yet been subjected to cytotoxicity testing; however, we know that some solutions contain chemicals that are toxic to hESC and other cell types (unpublished data).

Section snippets

Cell culture

H9 hESC from WiCell (Madison, WI) were maintained on Matrigel (Fisher Scientific, Bedford, MA) coated 6-well plates (Falcon, Fisher Scientific, Chino, CA) in mTeSR®1 medium (Stem Cell Technologies, Vancouver, BC, Canada) using methods previously described in detail (Lin & Talbot, 2011). Medium was changed daily, and cells were used when 60–80% confluent. To subculture or prepare hESC for experiments, wells were washed with Dulbecco's Phosphate Buffered Saline (DPBS) containing magnesium and

Establishing a rapid method to count and plate hESC in suspensions of small colonies

hESC do not attach and grow well when plated as single cells (Fig. 1A); however, excellent plating efficiencies can be obtained when hESC are plated as small colonies (Fig. 1B). Fig. 1C shows a field of small hESC that was used to generate the standard curve in Fig. 1D, as described in the Methods section. Percent transmittance readings were taken three times for the same sample in a spectrophotometer and produced a coefficient of variation of 1.5%, demonstrating the reproducibility of this

Discussion

A method is presented that allows hESC to be reproducibly plated in 96-well plates with uniform confluency from well to well and from day to day. By spectrophotometerically determining cell concentration in suspensions of hESC colonies and by swirling and resuspending colonies between each pipetting, we were able to obtain cell concentration data, make dilutions, and plate colonies rapidly and accurately. The rapidity with which these steps can be performed enhanced cell survival and plating

Acknowledgments

This work was supported by the Tobacco-Related Disease Research Program of California. V. Bahl was supported by a Dean's Pre-doctoral Fellowship. R. Behar was supported by a Dean's Pre-doctoral Fellowship and a TRDRP Cornelius Hopper Diversity Award. Dr. S. Lin was supported by a TRDRP Postdoctoral Fellowship. We thank Dr. Evan Snyder for providing the mNSC (clone C17.2).

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