Optimization of cytotoxicity assay by real-time, impedance-based cell analysis

Abstract

This paper presents an optimized procedure for assessing an immune-mediated cytotoxicity, produced after the addition of human and baboon serum to transgenic porcine fibroblasts. This procedure is performed with the xCELLigence Real-Time Cell Analyzer (RTCA). The xCELLigence system measures the impedance variations in the culture media of a 96-well microelectronic plate, and shows the changes in cell number and morphology in a real-time plot. However, different factors need to be optimized before developing an RTCA assay. Thus, we studied the influence of several variables, such as the number of cells seeded, the time the cells were allowed to grow before the tests, the serum concentration and the addition of rabbit complement. The findings were confirmed by the WST-1 classical cytotoxicity test. The results showed that 7.5 × 10(3) cells seeded per well produced the adequate CI in 10 h. The area under the curve and the CImin versus concentration values showed a very high correlation index (r(2) = 0.966 and r(2) = 0.92 for the first 50 h after challenge, respectively), proving that CI variations are directly proportional to the quantity of serum added. The addition of complement resulted in lower CImin values. Therefore, both the cytolysis level with and without exogenous complement addition had to be assessed. There was a high correlation between the relative cytotoxicity assessed by WST-1 and the CI obtained by RTCA when exogenous complement was not added (r(2) = 0.827; p < 0.001). The correlation was average when rabbit complement was added (r(2) = 0.523; p = 0.046). In conclusion, culture conditions have an important influence on RTCA cytotoxicity assays.

Real-time cell analysis of the inhibitory effect of vitamin K2 on adhesion and proliferation of breast cancer cells

“…The RTCA xCELLigence platform used in this study is highly advantageous in comparison to these assays as it facilitates label-free, continuous monitoring of cellular response to compounds [31,32]. It has been shown to correlate very well with the conventional adhesion, viability, migration, and invasion assays and proven to be a highly accurate platform to monitor cell behavior [27,[33][34][35][36][37][38].…”

PD-1 blockade enhances the vaccination-induced immune response in glioma

“…After overnight tumor culture to allow adherence of the cells to the well bottom, T cells were added to tumor cell cultures at a 10:1 T cell/tumor ratio. Cell index values (relative cell impedance) were collected over 15 hours and normalized to the maximal cell index value immediately prior to effector cell plating using RTCA Software 2.0 (Acea Biotechnology) as described previously (53). PD-1 mAb was added to the T cell cultures prior to coculture at 10 μg/ml where necessary.…”

Impedimetric Toxicity Assay in Microfluidics Using Free and Liposome-Encapsulated Anticancer Drugs

“…40 EIS-based detection has been widely used to monitor cell cultures under static conditions. 21,23,27,35,41 However, systems that combine the advantages of perfusion cell culture with label-free electrochemical and optical detection for multiparameter analysis are an emerging field of research. 32, 33,42−45 The here-presented compact microfluidic cell culture and electrochemical analysis platform with in-built fluid handling and detection 46,47 represents an improvement to the existing μTAS devices where the down-scaling of the liquid handling and electrochemical detection is often overlooked.…”