A study of electrochemical biosensor for analysis of three-dimensional (3D) cell culture

https://doi.org/10.1016/j.bios.2012.02.039Get rights and content

Abstract

Cell culture has a fundamental role not only in regenerative medicine but also in biotechnology, pharmacology, impacting both drug discovery and manufacturing. Although cell culture has been generally developed for only two-dimensional (2D) culture systems, three-dimensional (3D) culture is being spotlighted as the means to mimic in vivo cellular conditions. In this study, a method for cytotoxicity assay using an electrochemical biosensor applying 3D cell culture is presented. In order to strengthen the advantage of a 3D cell culture, the experimental condition of gelation between several types of sol–gels (alginate, collagen, matrigel) and cancer cells can be optimized to make a 3D cell structure on the electrode, which will show the reproducibility of electrical measurement for long-term monitoring. Moreover, cytotoxicity test results applying this method showed IC50 value of A549 lung cancer cells to erlotinib. Thus, this study evaluates the feasibility of application of the electrochemical biosensor for 3D cell culture to cytotoxicity assay for investigation of 3D cell response to drug compounds.

Highlights

▸ Characterization of an electrochemical biosensor for analysis of 3D cell culture. ▸ Optimization for enhancement of electrical signal effectiveness in 3D cell culture. ▸ Electrochemical measurement of cell viability according to cell seeding density. ▸ Drug dose response on the electrochemical biosensor applying 3D cell culture. ▸ We confirmed the feasibility of application of the electrochemical biosensor for analysis of 3D cell culture to cytotoxicity assay.

Introduction

Generally, a cell-based biosensor has been developed for only two-dimensional (2D) culture systems. Although some drug compounds are effective in vitro, they might not be effective in vivo because of the difference between in vitro and in vivo conditions. Since a 2D culture forming mono layer has contact inhibition among cells and might change the original characteristic of cell morphology and functionality, unlike three-dimensional (3D) cultures forming multi layers (Mueller-Klieser, 1997, Petersen et al., 1992). 3D cell models, however, can mimic in vivo cellular conditions because they have a 3D scaffold that supports cell growth and cell functions including morphogenesis, cell metabolism and cell-to-cell interactions (Lee et al., 2008a, Yang et al., 2008). For these reasons, optimization of the experimental condition in the scaffold matrix of 3D cell cultures is needed to develop a biosensor for cytotoxicity assay on 3D cell culture.

Currently, optical detectors have been used as a commercial analytical bio-device to observe cell responses, although they remain expensive due to their high-cost modules and fluorescent materials. They also have experimental limitations because they, like most optical devices, are not portable size. On the other hand, electrical detection systems have unique advantages such as simultaneous real-time analysis, high-cost efficiency, versatile fabrication, label-free biosensors, and portable-size devices and simply operated analytical instruments (Albers et al., 2003, Mehrvar and Abdi, 2004, Jeong et al., 2009). Based on the advantages, electrochemical techniques are a powerful tool that can be applied when studying the cell immobilization, adhesion, proliferation and apoptosis (Ding et al., 2007). Moreover, electrochemical detection systems provide precise and real-time information of cell status by monitoring of metabolic compounds in cell cultures, such as glucose, lactate and oxygen to deeply understand the mechanisms of various types of cells (Boero et al., 2011, Zhang et al., 2011, Rodrigues et al., 2008, Kaya et al., 2003). Recently, by combining the electrochemical detection systems with nanoscale materials, nanostructured biosensors have been developed in order to overcome detection limit and improve sensitivity (Boero et al., 2011). Furthermore, multi-channel measurement systems are capable of real-time monitoring which can be effectively used in drug discovery as a high-throughput screening method (Yue et al., 2008, Andreescu and Sadik, 2005).

In this study, our current work addresses the development of a novel electrochemical biosensor for cytotoxicity assay on 3D cell culture that combines the advantages mentioned above. Although the common electrochemical biosensor has a unique advantage of being able to measure cell signals in real time unlike optical biosensors, its process of measuring accurate electrical signals from a 3D cell population is complex. Thus, to make 3D cell structure on an electrode, we optimized and evaluated the gelatin condition between several types of sol–gels and A549 cancer cells and confirmed the reproducibility of electrical measurements for long-term monitoring. In addition, cytotoxicity test results showed that our 3D-cell-based biosensor monitoring can be an alternative method for high-throughput drug discovery screening.

Section snippets

Cell cultures

A549 (human lung adenocarcinoma epithelial cell line) cells were cultured for continuous logarithmic-phase growth in a medium provided by the Roswell Park Memorial Institute (RPMI 1640, GIBCO, US) and 10% fetal bovine serum (FBS, GIBCO, US) in T-150 tissue culture flasks. Cells were incubated at 37 °C in 5% CO2, 95% air humidified atmosphere. Culture medium was maintained fresh by changing every other day as needed.

Reagents and preparation of the three-dimensional cell chips

Alginic acid sodium salt (low viscosity, Sigma, US) was dissolved in sterile

Characterization of the electrochemical biosensor applying 3D cell culture

Electrochemical biosensors having an advantage of label-free have been used to electronically monitor cell activity in a culture. Interestingly, although most investigations have been done with 2D cultures, we could effectively measure cell signals in a 3D culture. Cells mixed with sol–gel were spotted on the working electrode, and gel–cells mixture was gelated on the gold electrode, as illustrated in Fig. 1. Faradaic impedance investigation can be effective in analyzing cell activity by

Conclusion

We optimized the condition of gelation between several types of sol–gel and A549 cancer cells to enhance signal effectiveness and verified the reproducibility of electrical measurement for long-term monitoring by an electrochemical biosensor applying 3D cell culture. In addition, we confirmed the feasibility of application of the electrochemical biosensor for analysis of 3D cell culture to cytotoxicity assay by investigating the drug efficacy of erlotinib with the biosensor. Thus, our work

Acknowledgments

I am thankful to all my colleagues including SH Yi in AMD lab, HS Lee, KH Kim in SMC who supported me in any respect during the completion of the project.

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