Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Summary

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

Abstract

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations – most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT_2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Introduction

Neuronal cell death plays a critical role in the pathophysiology of many brain-related disorders¹. The availability of reliable and high-throughput in vitro toxicity assays is critical to gain better insight into the mechanisms of neurotoxicity and to help select neuroprotective molecules as therapeutic candidates in drug development². However, there are many limitations to most widely used in vitro neurotoxicity assays.They assess neurotoxicity/neuroprotection at a single time-point not allowing kinetic resolution; often use label or probe which can interfere with the signaling pathways and limit additional studies in the same ....

Protocol

1. Preparation

1. Place the real-time cell analyzer’s station in a tissue culture incubator set at 37 °C and with 5% CO₂. Carry out all cell culture handling and pharmacological treatments in a tissue culture hood under sterile conditions.
2. NOTE: Neuronal cell lines requiring different temperature settings compared to standard conditions for culture, should be adjusted accordingly. For weakly adherent cell lines, use coating agents to facilitate the interaction of cells with the gold microelectrodes in the bottom of the wells of the E-Plate 96.
3. Prepare 1,000X stock solutions of the pharmacological compounds for cell cu....

Results

Serum deprivation leads to decrease in Cell Index values, which can be monitored continuously with the real-time cell analyzer

Neurotoxic stimuli to the cells lead to a decrease in Cell Index values, which can be monitored in real-time with the presented technique and the dynamics of which is dependent on the specific neurotoxic stimulus and the cell type studied. Figure 2 demonstrates the increase in Cell Index when SK-N-SH cells are grown in proliferation me.......

Discussion

The current protocol presents the utility of a real-time cell analyzer to assess continuously and under label-free conditions the neuroprotective/neurotoxic effects of compounds in neuronal cell lines and to gain insight into the second messenger pathways involved in the effect.

Even though the real-time cell analyzer’s utility to study cytotoxicity and effects of drugs on cell proliferation is generally recognized, only a few studies have used it in neuronal related cell types. We have .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
xCELLigence  RTCA SP system bundle	ACEA Biosciences	No: 00380601030	Consists of RTCA Analyzer, RTCA SP Station and RTCA Control Unit
E-plate 96	ACEA Biosciences	No: 05232368001	For culturing the cells, inserted in the RTCA SP Station
SK-N-SH cells	ATCC (in partnership with LGC Standards)	HTB-11	Can be replaced by another adherent neuronal cell line of interest
DMEM/F12	Sigma-Aldrich	D8437	Cell culture medium
Fetal bovine serum	Life Technologies	16140-063	Supplements proliferation, but not serum deprivation medium
Tissue culture flask T175	Sarstedt	83.1812.302	For culturing cells, which will be later plated on the E-Plate 96
0.05% Trypsin-EDTA	Life Technologies	25300-054	For trypsinization of cells cultured in tissue culture flasks
Scepter 2.0 cell counter	Merck Millipore	PHCC20060	Automated cell counter
Phosphate-buffered saline	Life Technologies	10010-015	For washing the cells
(±)-DOI hydrochloride	Sigma-Aldrich	D101	5-HT2A agonist for cell culture treatment
LY-294002 hydrochloride	Sigma-Aldrich	L9908	PI3-K inhibitor for cell culture treatment
Lisuride maleate	Tocris Bioscience	4052	Compound with 5-HT2A agonistic activity for cell culture treatment
Dimethyl sulfoxide	Sigma-Aldrich	D4540	For dissolving LY-294002 and lisuride maleate