Microplates with Integrated Oxygen Sensors for Kinetic Cell Respiration Measurement and Cytotoxicity Testing in Primary and Secondary Cell Lines

Abstract

This paper presents a cytotoxicity and cell respiration assay that is nondestructive and kinetic. It makes use of 96-well microplates integrated with oxygen sensors. The oxygen signal monitored on-line gives an indication of the cell viability. We show its application for suspension cell lines (Chinese hamster ovary and HL60 cells) as well as adherent (Caco2 cells) and primary (rat hepatocytes) cells using well-known cytotoxic compounds (sodium azide, diclofenac, clozapine, sodium dodecyl sulfate, 2-thiouracil, tamoxifen, and tranylcypromine). The 50% lethality concentration (LC50) obtained from the assay is compared with the standard 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl-2H-tetrazolium bromide end-point assay. The cells can be grown directly in the plates, and the assay requires no further reagents or processing. The cells can be harvested for further analysis, if required. The on-line dynamic measurement allows the calculation of LC50 as a function of exposure time. LC50 was shown to decrease with time in HL60 cells. The dynamics of this process was considerably different for the three compounds sodium dodecyl sulfate, tamoxifen, and diclofenac, indicating a large potential of application of this method for cell death studies. The assay system can be applied to almost any cell-based systems with little adaptation. The assay is robust, flexible, and applicable for medium- to high-throughput systems requiring only minimal handling and no additional agent.

Quercetin treatment changes fluxes in the primary metabolism and increases culture longevity and recombinant α1-antitrypsin production in human AGE1.HN cells

“…Toxicity screening was performed using respiration measurements in 96-well OxoPlates (PreSens GmbH, Regensburg, Germany (Deshpande et al 2005;John et al 2003)) applying the equipment and procedures as described in recent publications Noor et al 2009). A1AT concentration was measured as described recently (Niklas et al 2011a) using an activity assay based on the inhibition of trypsin by A1AT.…”

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME

“…intermittant periods of perifusion with drug cocktails for repetitively determining biotransformation capacity. Even here, the HepaChip ® could be equipped with additional sensors and analytical tools working online and allowing time-dependent measurements, for example, integrated oxygen sensors measuring the cell respiration allow the calculation of LC 50 s in cytotoxicity testing and require only minimal handling and no additional agents (Deshpande et al 2005). …”

High throughput screening of monoamine oxidase (MAO-N-D5) substrate selectivity and rapid kinetic model generation

“…The initial value for the Michaelis constant of O 2 (K O2 ) was taken from previous studies of MAO-N-D5 that reported a value of 0.7 mM [17]. Using air as the O 2 source, the maximum concentration that could be reached in the microwell reactions at 37 • C was 0.21 mM [35], which is approximately four times lower than the literature K O2 value. The Michaelis-Menten profiles (Fig.…”