Real-time electrical measurement of L929 cellular spontaneous and synchronous oscillation

Abstract

Mohana Marimuthu1, Cheolsoo Park2, Sanghyo Kim1*, Cheol Soo Choi3*1College of Bionanotechnology, Kyungwon University, Seongnam-si, Gyeonggi-do, Republic of Korea; 2Department of Electrical and Electronic Engineering, Imperial College London, London, UK; 3Korea Mouse Metabolic Phenotyping Centre, Lee Gil Ya Cancer and Diabetes Institute, Gachon University of Medicine and Science, Yeonsu-gu, Incheon, Republic of Korea *These authors contributed equally to this work.Abstract: Nonexcitable cell types, fibroblasts of heart muscle or astrocytes, are well known for their spontaneous Ca2+ oscillations. On the other hand, murine fibroblast (L929) cells are known to be deficient in cell–cell adhesive proteins and therefore lack gap junctions for cellular communication. However, these cells exhibit a unique property of collectively synchronized and spontaneous oscillation, as revealed by real-time monitoring of cells cultured on a 250-µm diameter microelectrode for more than 3 days using an electrical cell-substrate impedance-sensing system (ECIS). Live-cell imaging is a widely used technique for oscillation detection, but it has limitations relating to cellular physiological environment maintenance for microscopic analysis and for prolonged periods of study. The present research emphasizes an electrical-sensing technique (ECIS) capable of overcoming the most important issues inherent in live-cell imaging systems for the detection of L929 cellular spontaneous and synchronized oscillation in real-time for longer periods. Possible mechanisms involved in L929 oscillation were elucidated to be periodic extension/contraction of lamellipodia continued as blebbing, which is produced by signals from the actomyosin complex initiated by connexin hemichannel opening and adenosine triphosphate (ATP) release. By applying the connexin hemichannel inhibitor, flufenamic acid, the hindrance of ATP release and calcium transients were analyzed to elucidate this hypothesis.Keywords: murine fibroblast, synchronization, ECIS, connexin 43, blebbing, lamellipodia