Respiratory Burst Evaluation of THP-1 Cell Chip Using a Scanning Electrochemical Microscopy

                      

1. Introduction

  Previously, we electrochemically studied respiratory burst (rapid oxygen consumption) in response to a stimulant of THP-1 (cell line from a patient with myelocytic leukemia) ¹⁾. In this study, we compared the oxygen consumption during ordinary respiration and respiratory burst of THP-1 by fabricating a 3D THP-1 cell-culture chip, using SECM (Scanning Electrochemical Microscopy).

 2.  Experiments

2-1  Fabrication of 3D THP-1 cell chip

  Fig. 1(A) is a schematic diagram of a pyramid well subjected to anisotropic etching on a silicon substrate. 1μL of a cell collagen suspension was inserted into this pyramid well. It was then placed in an incubator at 37°C under a CO₂ concentration of 5 % for 10 minutes to gelate the collagen, forming a THP-1 cell culture chip.  The cell count was set at 4.5x10³ cells /well.  Fig. 1 (B) is an optical micrograph of the 3D THP-1 cell chip.

2-2   Evaluation of respiration of 3D THP-1 cell chip

   Fig. 2 is a schematic diagram of measurement of cell chip respiration by SECM. For the measurements, the working electrode was a φ20 μm platinum electrode, the counter electrode and reference electrode was a silver-silver chloride electrode, and the measurement solutions were phosphate buffered saline (PBS) and 11.4 mM glucose, 10 mL, respectively.With the initial position of the working electrode from the silicon substrate at 20 μm in the Z direction, the potential was held at -0.5 V vs. Ag/AgCl. This electrode was moved back and forth in the Z direction 3 times from the vicinity of the cells up to 500 μm at a speed of 10 μm/s, and the change in the value of the oxygen reduction current was measured.

2-3  Respiratory burst evaluation of 3D THP-1 cell chip  

  In fabricating the cell chip for respiratory burst measurement, phorbol 12-myrystate 13-acetate (PMA) was added to the THP-1 cells to a final concentration of 20 nM as a stimulant, and measurement was performed under the same conditions as those of Experiment 2-2 about 30 minutes later.

3.  Results and discussion

3-1 Respiration activity of 3D THP-1 cell chip, and quantitative evaluation of respiratory burst

Fig. 3 shows the measurement results using a micro electrode moved back and forth in the Z direction 3 times, to determine the difference of oxygen reduction current at a distant position and in the vicinity of the cells by SECM. Fig.3 (A) is during ordinary respiration, and (B) is during respiratory burst.

  The difference Δ I of the oxygen reduction current between the vicinity of the cells and the distant position during ordinary respiration of THP-1 was approx. 80 pA, and ΔI was approx. 160 pA during respiratory burst of THP-1. Based on the above results, Table 1 shows Δ C converted to oxygen concentration and respiration rate F with a bulk dissolved oxygen concentration of 209 μM,²⁾ and an oxygen diffusion coefficient of 2.18 x 10^-5 cm²/s. The respiration rate during ordinary respiration was computed to be 1.42 x 10^-14 mol/s, while the respiration rate of respiratory burst was computed to be 5.82 x 10^-14 mol/s. This shows that during respiratory burst, the oxygen consumption was about 4 times higher than during ordinary respiration. In future, to compute the amount of respiratory burst in cells more precisely, we plan to make measurements in single cells by suitably arranging electrode sizes and measurement wells.

References

1) S. Kasai, T. Numata, H. Shiku, H. Abe, T. Matsue, Y. Niiseki, 215th ECS meeting, (2009).

2) Shigeo Aoyagi, doctoral thesis, Tohoku University (2006)