Membrane permeabilization of mammalian cells using bursts of high magnetic field pulses

Abstract

Background: Cell membrane permeabilization by pulsed electromagnetic fields (PEMF) is a novel contactless method which results in effects similar to conventional electroporation. The non-invasiveness of the methodology, independence from the biological object homogeneity and electrical conductance introduce high flexibility and potential applicability of the PEMF in biomedicine, food processing, and biotechnology. The inferior effectiveness of the PEMF permeabilization compared to standard electroporation and the lack of clear description of the induced transmembrane transport are currently of major concern.

Methods: The PEMF permeabilization experiments have been performed using a 5.5 T, 1.2 J pulse generator with a multilayer inductor as an applicator. We investigated the feasibility to increase membrane permeability of Chinese Hamster Ovary (CHO) cells using short microsecond (15 µs) pulse bursts (100 or 200 pulses) at low frequency (1 Hz) and high dB/dt (>10⁶ T/s). The effectiveness of the treatment was evaluated by fluorescence microscopy and flow cytometry using two different fluorescent dyes: propidium iodide (PI) and YO-PRO®-1 (YP). The results were compared to conventional electroporation (single pulse, 1.2 kV/cm, 100 µs), i.e., positive control.

Results: The proposed PEMF protocols (both for 100 and 200 pulses) resulted in increased number of permeable cells (70 ± 11% for PI and 67 ± 9% for YP). Both cell permeabilization assays also showed a significant (8 ± 2% for PI and 35 ± 14% for YP) increase in fluorescence intensity indicating membrane permeabilization. The survival was not affected.

Discussion: The obtained results demonstrate the potential of PEMF as a contactless treatment for achieving reversible permeabilization of biological cells. Similar to electroporation, the PEMF permeabilization efficacy is influenced by pulse parameters in a dose-dependent manner.

Keywords: CHO cells; Electropermeabilization; Electroporation; Magnetopermeabilization; Propidium iodide; YO-PRO-1.

Figure 1. The waveform of the high dB/dt magnetic field pulse.

Each magnetic pulse was 15 µs wide and had a peak of 5.5 T (Acquired using Tektronix DPO4034, post-processed in OriginLab 8.5).

Figure 2. Finite-element method (FEM) model of the pulsed magnetic field inductor.

(A) Spatial distribution of the electric field at highest dB/dt of the magnetic pulse; (B) spatial distribution of the magnetic field. The electric field was the highest at the edges of the polymerase-chain-reaction (PCR) tube and decreased to the zero in the centre of the sample while the magnetic field was approximately homogeneous through the whole sample.

Figure 3. Light and fluorescence microscopy images using PI and YP fluorescent dye assays.

(A, B, G, H) untreated control; (C, D, I, J) cells after the 50 × 4, 5.5 T pulsing protocol; (E, F, K, L) cells after the single 100 µs, 1.2 kV/cm pulsing protocol. Arrows are used to highlight the cells with weak mean fluorescence, which were counted as PI and YP positive during analysis.

Figure 4. The dependence of fluorescence intensity spectra (PI, YP) on the treatment parameters.

After pulsed treatment a shift of fluorescence spectra was detected indicative of membrane permeabilization, where (A) PI assay; (B) YP assay; violet, untreated control; yellow, 50 × 2, 5.5 T PEMF; green, 50 ×4, 5.5 T PEMF; orange, PEF (1 × 100 µs, 1.2 kV/cm).

Figure 5. Membrane permeabilization of mammalian cells using bursts of high magnetic field pulses.

The percentage of permeabilized cells (A), as well as the median fluorescence (B) increased with the treatment intensity using both PI and YP assays. Asterisk (*) highlights statistically significant difference versus control (P < 0.05).

Figure 6. Temperature rise in the cell suspension during PEMF treatment.

The temperature rise during PEMF treatment (5.5 T, 50 pulses) did not exceed 14.5°reaching 36 °C and followed by a rapid decline (due to cooling) when the pulsing stops.