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

Abstract

Nonexcitable cell types, fibroblasts of heart muscle or astrocytes, are well known for their spontaneous Ca(2+) 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: ECIS; blebbing; connexin 43; lamellipodia; murine fibroblast; synchronization.

Figure 1

(A) Impedance measurement graph of cells grown on ECIS wells from 0 to 78 hrs. Inset shows the optical microscopic images of cells on circular detecting microelectrodes of ECIS wells, in which the images were taken (a) once after the cell addition, (b) after 10 hrs, and (c) after 15 hrs. (B) Impedance measurement of L929 cells with connexin hemichannel inhibitor (FFA). Abbreviations: ECIS, electric cell-substrate impedance-sensing system; FFA, flufenamic acid.

Figure 2

Inverse microscopic images of cells grown on Petri dishes taken at different time periods. A–I are images taken at 30, 30.5, 31, 55, 55.5, 56, 70, 70.5, and 71 hrs, respectively. Notes: White arrows showing examples of lamellipodium extension and contraction, black arrows indicate retraction of lamellipodium to form approximately round shape, and block arrows show some examples of blebbing activity of L929 cells.

Figure 3

(A) Power spectrum analysis of L929 cellular oscillation compared to vero cells at different time periods from (a) 30 to 40 hrs and (b) 55 to 70 hrs. (B) Time-frequency plots for cellular oscillation. (a) L929 cells (b) vero cells. Note: Black circles show oscillation frequencies. Abbreviations: ATP, adenosine triphosphate; ECIS, electrical cell-substrate impedance-sensing system.

Figure 4

L929 cellular oscillatory mechanism and relationship with cellular blebbing motility. Note: Small black arrows along the plasma membrane indicate cellular oscillatory movement and blue arrow indicates blebbing motility. Abbreviations: ATP, adenosine triphosphate; ECIS, electric cell-substrate impedance-sensing system.

Figure 5

Fluorescent imaging of calcium flux from L929 cells without drug (FFA) and with drug at different time periods (0 hrs and 40 hrs). Abbreviation: FFA, flufenamic acid.

Figure 6

Quantitative analysis of ATP release from L929 cells without drug (FFA) and with drug at different time period (A) at 0 hrs (B) at 40 hrs. Abbreviations: ATP, adenosine triphosphate; FFA, flufenamic acid.