(BEING CONTINUED FROM 19/02/14 )
Materials and Methods
Normal human neuronal progenitor cells (NHNP) were pooled from three donors to diminish
donor-to-donor variations in response. As controls, NHNP were grown in conventional tissue
culture following standard cell culturing procedures in tissue culture flasks obtained through
Clonetics Corporation, San Diego, California.
Cell Culture Protocols
For two-dimensional culture, GTSF-2 medium with 10% FBS, Ciprofloxacin and Fungizone was
used to culture the cells (Goodwin et al., 1993a). 1 X PBS, Collagenase, DNase and trypsin were
purchased from Clonetics San Diego, California, and used Corning T-75 flasks (Corning Inc.,
Corning, New York) for initial cell culture to obtain the appropriate number of cells for each
experiment. Briefly, cells to be cultured were enzymatically dissociated with the referenced
reagents from T-flasks, washed once with PBS-CMF and assayed for viability by trypan dye
exclusion (GIBCO, Grand Island, New York). Cells were grown on 100-mm petri dishes (tissue
culture treated to prevent adherence) or grown on the actual electrodes inside the petri dishes.
Electrodes were made of platinum and stainless steel. Cell cultures were maintained in a
humidified Forma CO2 incubator (Forma, Inc.) at 37°C at a CO2 concentration of 6%.
For three-dimensional culture, NHNP cells were prepared as described above and an RWV was
sequentially inoculated with 5 mg/ml Cytodex-3 type I collagen-coated microcarriers
(Pharmacia) and freshly digested NHNP cells, yielding a cell density of 2.5.x 105 cells/ml in a
55-ml vessel. Tissues were cultured for 17 to 21 days or until 3- to 5-mm diameter tissue masses
A waveform (TVEMF) generator of original design and capability was developed and used to
generate the waveform in a strength of 1-6 mA (AC) square wave, 10 Hz variable duty cycle,
which was pulse-width modulated (Goodwin et al. patent cases MSC 22633-1; US Patent,
6,485,963, B1 and MSC 22633-2 Notice to Issue). NHNP cells were subjected to these
extremely low-level magnetic fields (ELF waves) (~10 – 200 mGauss), which are far less than
the field strength of the Earth. Figure 1 illustrates the experimental arrangements for twodimensional
FIGURE 1. Notice the silver-faced box in the left of photo. The generator is
attached to the experiment in the incubator by a ribbon cable.
Two-Dimensional Experimental Protocols
Initially, a metal electrode was placed inside a petri dish and centered. NHNP were seeded at
2.5 × 105 cells in 0.7 ml of media and carefully dropped on the electrode in a concentrated
bubble (Figure 2).
FIGURE 2. The NHNP cells produced a whitish fingerprint on each of the
electrodes under TVEMF.
Cells were incubated for 2 days. The second day after cell inoculation is considered day 0 of the
experiment protocol. At day 0, each dish was given 15 ml of media and waveform was applied
to the electrodes. Cells were fed with 15 ml of media at day 3 and with 13 ml every three days
thereafter at day 6, 9, and 12. At days 14 and 17, the cells were fed again with 15 ml of media.
At days 17 to 21, the cells were incubated for 10 minutes in a Collagenase/DNase cocktail, then
trypsin was directly applied to the cocktail and the cells were further incubated for 3 more
minutes. Before the complete media was added to deactivate the trypsin, the cocktail mix was
pipetted up and down several times. The cells were washed twice with 1 X PBS, reapplied with
the media, and placed on ice. The cells were observed under a dissecting microscope, counted,
and assessed for viability.
An identical protocol was followed in similar experiments with the exception that, instead of the
electrode being placed within the petri dishes, in media, it was attached to the underside of the
TVEMF treated dishes, so that the cells had no direct contact with the metal surface.
(TO BE CONTINUED)
Thomas J. Goodwin, Ph.D.
Lyndon B. Johnson Space Center / 2003