Low-energy electromagnetic frequencies induce
conspicuous, reproducible, and lasting effects in human
and animal cells

METHODS

The frequencies were transmitted using two F-SCAN 2 oscillators, one was a prototype and the other a series instrument. The frequency generator was connected to a potentiometer with 12 steps that reduced the voltage of the frequencies from 10V (maximum output voltage) to 0.8V (step 1). The potentiometer was connected to a spider box with 6 parallel pairs of wires with a length of 1m each. This enabled parallel treatment of six cell cultures. The EMF were transmitted to the medium with the cells using sterilized wires of 24 carat gold with a diameter of 0.4mm. The gold electrodes were attached to glass platelets of 10x26x1mm, which were made from microscope slides. Two flattened gold wires were wound around the ends of the glass platelets and glued with Araldite so that the length of the free end of the gold wires was 4cm long. The distance between the two wires on the glass platelet was 1.8cm. Each gold wire was connected at a distance of 2 to 3cm from the glass platelet to an insulated tin wire (inner diameter, ID 0.4mm) with a length of 30cm. The junctions were isolated from the surroundings by a 2cm long glass capillary each whose end was sealed with Araldite. This design should allow EMF transmission to the medium exclusively via the gold wires. The free ends of the insulated wires were pulled through two holes next to the membrane of the lid of the culture bottle. These free ends were then connected to one pair of cables from the spider box for the EMF treatment. The cells were treated in 25cm² Falcon cell culture bottles made of plastic in a non-sterile environment at room temperature. The control cultures without frequency treatment were also kept at room temperature for the same length of time, which was up to one hour. The controls did not have electrodes. After EMF treatment, the wires to the spider box were disconnected, and the culture bottles with the electrodes and tin wires were incubated again at 37°C. During the EMF treatment, care was taken to ensure that the glass platelets lay flat on the bottom and in the middle of the culture bottle. They were completely covered by the nutrient solution; the length of the gold wire above the medium was at least 0.5cm. During the two-month treatment period, the cells grew around and also on the electrodes.

The frequency treatments of the cell cultures were carried out in a medium with 1/4 of the usual serum concentration to keep the cell growth rate low. As soon as the cultures were confluent, the cells were partially resuspended with EDTA solution. About 10% of the cells were transferred to a new culture bottle, which was then fitted with a new sterile electrode. The cell groups were not completely dissociated in this step. All work with the cells was carried out in a sterile bench. There were cases of contamination, but they were rare. After completion of the frequency treatments, the cells were cultivated for 10 passages under standard conditions at 37°C to re-accustom them to the standard medium. Microscopic examinations were already possible during these ten passages.

Each experiment comprised a total of 30 to 35 EMF treatments. The first 18 treatments were performed every second day and the followings every day. Before each frequency treatment, a sweep was carried out for 2 minutes over the entire frequency range of the subsequent EMF. The sweep excited every 1000th frequency in the target oscillation range from 1.0 to 6x10⁶ Hertz three times for a fraction of a second. The excitation of the EM field of the cells is supposed to increase their sensitivity to the subsequently applied EMF. If the time between the sweep and the EMF treatment was more than 1 hour, the sweep was repeated.

Each EM treatment of the cells comprised 7 or 8 frequencies: frequency group A with or without final frequency C and frequency group B, without a final frequency. Frequency group B was originally intended as a control EMF treatment. After completion of the overall treatment with A, frequency C was applied on its own on three consecutive days after the sweep and without EMF A. Each frequency was applied for 3 minutes. Standard group A comprised the following frequencies: 5,555,555.4Hz; 5,555,554.3Hz; 555,555.6Hz; 555,555.1Hz; 555.7Hz; 111.1Hz; 112.2Hz, and final frequency C with 122.2Hz. Frequency group B: 5,555,556.9Hz; 5,555,556.2Hz; 555,554.0Hz; 555,553.2Hz; 556.7Hz; 109.2Hz and 110.0Hz. The actual treatment time for the sweep plus EMF A was 23 minutes.
Three established cell lines and one primary cell line were used: 1) THP-1, human monocytes; this cell culture was obtained from a patient with acute monocytic leukemia, acquired from ATCC; #TIB-202. 2) 3T3-L1 mouse fibroblast cells; pre-adipocyte cells (ATCC #CRL-173). 3) B16-F1 mouse melanoma cells (ATCC #CRL-6323). 4) Rafi; primary cell culture of rat fibroblasts. These were established from a one day-old laboratory rat. In contrast to the Rafi cells, THP-1, 3T3-L1, and B16-F1 are mutated to cancerous cells. The rat fibroblasts were cultivated after EMF treatments at low oxygen concentrations to minimize the likelihood of genetic changes (Lit.2). The cell lines were assigned new designations after completion of the EMF treatment. After treatment with A frequencies, the designations of the cell lines changed to THP-J, 3T3-J, B16-J, and Rafi-J. After treatment with B frequencies, the designations changed to 3T3-K and Rafi-K (see Table 3).

The EMF treatments of the cells were carried out with several culture flasks (n = 2 to 4) in parallel, and the identically treated cultures were pooled after completion of the treatment. Exceptions were 3T3-K1 and 3T3-K2, the Rafi and most of the B16-J/K cells. For the EMF treatments, the number of cells used to start a culture in 25 cm² Falcon culture flask was 40 cells for B16-F1, 400 for THP-1, and 500 for 3T3-L1. The treatments were carried out in 2005 and 2006, with exception of the B16-J cells for the investigations on melanin synthesis which were carried out in 2007. Rafi cells were treated in 2007 with 400 cells per culture flask. For Rafi and the second experiment with B16, the batch was extended from 2–4 to 6 culture flasks with parallel treatment. They were not pooled at the end of the EMF treatment.
Culture conditions: The cell cultures were kept at 37°C in standard incubators for mammalian cells with 5% CO₂. Where stated, the cells were cultivated in nitrogen with 1 to 3% oxygen and 5% CO2. THP-1 cells were cultivated in RPMI medium (Gibco 1640) with 25mM Hepes, 50 units/ml penicillin/streptomycin, 7.5% NBS (without thermal inactivation), and 1mM Glutamax; 3T3-L1 cells were cultivated in DMEM high glucose medium (Gibco) with 25mM Hepes, 50 units/ml penicillin/streptomycin, 10% NBS (without thermal inactivation), and 1mM Glutamax; B16-F1 cells were cultivated in MEM-Earle medium (Biochroma) with 25mM Hepes, 50 units/ml penicillin/streptomycin, 8% NBS (without thermal inactivation), 1mM Glutamax, and 1% vitamin solution (MEM non-essential amino acids) of a 100x stock; Rafi were cultivated in MEM high glucose medium (Gibco) with 25mM Hepes, 50 units/ml penicillin/streptomycin, 5% NBS (without thermal inactivation), and 1mM Glutamax. The EDTA solution consisted of 10mM EDTA in a phosphate-buffered isotonic solution containing 0.1% BSA. Serum-free cell cultivation was carried out in Ultraculture (12–725F) medium from Cambrex containing 1mM Glutamax and 50 units/ml penicillin/streptomycin. The PMA stock solution was dissolved in chloroform at a concentration of 0.1mg/ml and then diluted to 10ng per ml medium for treatment of the cells.

Various counting methods were used for cell growth analyses: The most sensitive method was cell count with the counter from Beckmann (Z2 Cell Coulter). At least 1000 cells in three independent samples were determined. Great care was taken with all cell lines to ensure that all cell groups were completely dissociated into individual cells using EDTA solution. Loosening of the adherent cells by EDTA solution from the flask and from attachment to each other required up to about one hour incubation at 37°C, before dissociating the cells by aspirating gently with a pipette. The cell number of the resuspended cells remained stable in the EDTA solution containing BSA at 4°C for 24 hours.

To simplify the growth assay, microtiter plates were used with 96 wells instead of 12 or 24 wells. Growth was determined indirectly by measuring the color intensity after staining the fixed cells with methylene blue. Although a growth assay in microtiter plates and measurement with a plate reader is much less labor-intensive, providing a greater throughput for triplicate determinations, this method was significantly less sensitive: a difference of 100% in the direct determination of the cell count decreased to 30% or less in the methylene blue assay. Nevertheless, the reproducibility of the methylene blue assay was good. Many growth tests with 3T3, B16, and all of the experiments with Rafi cells were carried out with the methylene blue assay in spite of the lower sensitivity.

Confidence intervals in the statistical evaluations were calculated with a P value of 95%.
Fluorescence staining of the DNA of giant cells was carried out with DAPI (4',6-diamidino-2-phenylindole dihydrochloride).
Determination of the survival rate: The cells were resuspended and stored in EDTA solution at 4°C at identical cell density. The fraction of viable cells was determined microscopally once a day by staining with methylene blue.
The seeding efficiency SE was determined as follows: cells of a culture with about 80% confluency were resuspended to individual cells and kept on ice. 50,000 cells were distributed into 4 wells each of a 6-well plate in MEM medium containing 4% NBS and 1% FBS. The wells were pretreated with 5ml PBS containing 0.1% BSA solution, kept overnight at 37°C, then rinsed once with MEM medium. 4ml medium was added to each well for the assay and the plate equilibrated to 37°C. After 30 minutes, 50,000 cells in 1ml MEM medium were added to each well and the cell cultures incubated at 37°C for 2 hours. Cells that had not adhered were aspirated along with the incubation medium. The adherent cells were washed once and then counted: 100,000 adherent cells gave an SE of 50% per experiment.

The growth assays of the Rafi cell lines were carried out as follows: 6000 cells with an SE of 35% were added to each well in the first row of a 96-well plate and diluted 1:2 in each of the following rows. If the SE value was less than 35%, correspondingly more cells were used, and for a higher SE value correspondingly fewer cells.