Proliferation Monitoring of Immunocytes Using Low Frequency Impedance Measurement

 

1.  Introduction

    We previously performed a study of the fine behaviors in the differentiation of THP-1 (a human myeloid leukemia cell line) into macrophages¹⁾. In this study, using a combination of an incubator and an impedance analyzer, we first drew a Nyquist plot of the natural proliferation process non-invasively. We then plotted the impedance at the low frequency of 25 mHz temporally, and compared it with the proliferation curve (profile) of THP-1.

2.  Experiments

2-1. Measurement system

 Fig. 1 shows a schematic diagram of the immunocyte differentiation monitoring system used in this study. The system consists of an impedance analyzer, potentiostats (1255, 1287, Solartron), and an incubator (4020, Asahi Life Science). For the measurement cell in the incubator, we installed a pair of platinum electrodes (5mm x 5 mm x 0.05 mm; inter-electrode distance 8 mm) in a petri dish (φ8 mm).

2-2. Proliferation curve of THP-1

  To determine the number of THP-1 cells, starting with 2.7x10⁵cells/mL, we cultured the cells in a RPMI1640 culture medium in an incubator, counted the number of cells each day, and plotted a proliferation curve.

2-3  Impedance measurement relative to proliferation of THP-1

  The measurement solution was 200mL of a solution obtained by adding 11.4 mM glucose to phosphate buffer.  10 μL of THP-1 cultured in Section 2-2 was introduced into this solution, and the impedance was measured at an applied voltage of 10 mV_rms and frequency of 25 mHz - 5 kHz each day.

3.  Results and discussion

      Fig. 2 shows the proliferation curve of THP-1. It was found that Day 1 was the lag phase from the day of initiation of culture, Day 2 to Day 4 was the log phase, and Day 4 to Day 6 was the stationary phase.

      Fig. 3 shows the Nyquist plot of the impedance in the phase of the proliferation curve of THP-1. In the lag phase on Day 1 at 25 mHz, the impedance was less than 5 kΩ, but in the log phase, it increased to approximately 15 kΩ, while in the stationary phase, the impedance slightly decreased, but still exceeded 12 kΩ.  In the death phase, the impedance decreased rapidly, and fell below 8 kΩ.

     Table 1 summarizes the absolute values (lag phase, stationary phase) of impedance at 25 mHz, and the changes (log phase, death phase) per day in the absolute values of impedance. The impedance in each period mostly coincided with the proliferation curve.

   This suggests that it is possible to estimate the proliferation of floating cells from the impedance in the low-frequency region. In future, we aim to draw up a calibration curve, and study the proportion of living cell counts and dead cell counts together with morphological changes.

Reference

[1] S.Kasai, K.Shoji, M.Tada, H.Kiriu, R.Ishii, H.Kodama, 222^ndECS Meeting, (2012)

Table 1 Magnitude |Z| of impedance at 25mHz in each phase of the THP-1 proliferation curve

 

	Period	|Z|
Lag phase	Day   1-2	2.2   x103 Ω, effectively constant
Log phase	Day   2-4	4.6   x103 Ω/day
Stationary   phase	Day   4-6	2.3   x104 Ω, slight decrease
Death phase	Day   6-9	2.7   x103 Ω/day