Development of a partition-controlled dosing system for cell assays

Area of Science:

• Environmental Toxicology
• Cell Biology
• Analytical Chemistry

Background:

• Dosing hydrophobic and volatile chemicals in cell assays is challenging due to solubility issues and concentration loss over time.
• Conventional methods often require cosolvents and struggle with chemical degradation, evaporation, or binding to assay components.

Purpose of the Study:

• To develop a partition-controlled dosing system for maintaining stable concentrations of hydrophobic chemicals in cell-based assays.
• To evaluate the system's efficacy using benzo(a)pyrene and dichlorobenzenes in ethoxyresorufin-O-deethylase (EROD) and cytotoxicity assays.

Main Methods:

• Utilized polydimethylsiloxane (PDMS) sheets loaded with test chemicals as a reservoir for controlled release into cell culture medium.
• Employed rainbow trout (Oncorhynchus mykiss) cell lines (RTL-W1 and RTgill-W1) in a 24-well plate format with cells grown on inserts.
• Measured chemical concentrations and determined median effect concentrations (EC50) under controlled exposure conditions.

Main Results:

• The PDMS system achieved equilibrium within 24 hours, even for highly hydrophobic benzo(a)pyrene.
• The system maintained constant chemical concentrations in the medium for over 72 hours, compensating for >95% loss.
• Apparent bioassay sensitivity increased, with EC50 values 1.3-7.0 times lower compared to conventional dimethyl sulfoxide (DMSO) spiking methods.

Conclusions:

• The partition-controlled dosing system provides stable and reliable exposure conditions for hydrophobic chemicals in cell assays.
• This method enhances bioassay sensitivity and accuracy by overcoming challenges associated with chemical instability and non-specific binding.
• Significant binding of test chemicals to serum constituents was observed, highlighting the importance of accounting for free concentrations.