Three-Dimensional (RWV) Experimental Protocol
Three-dimensional neural cells and tissues were cultured by the method described above, except  that the TVEMF RWV was modified to incorporate an electromagnetic coil. The coil was  wrapped around the core of the vessel so it emitted the same electromagnetic field strength as in  the two-dimensional configuration. All other conditions were identical to the two-dimensional
experimental conditions.

Analytical Methods
Cell Metabolic Parameters
The following metabolic parameters were monitored on a daily basis during the culture of the RWV model: pH, dissolved O2, dissolved CO2, glucose concentration, cell viability, and LDH  (lactate dehydrogenase). Dissolved O2, CO2, pH, and glucose are routinely measured in our  laboratories (Goodwin et al., 1992, 1993b; Jessup et al., 1994; Scharz et al., 1992). At 24-hour
intervals, pH, CO2, and O2 were determined on a Corning 238-model clinical blood gas analyzer  (Corning, Corning, New York). The glucose concentration was determined by a Beckman 2  model clinical glucose analyzer (Beckman Instruments, Inc., Fullerton, California). Samples for  determination of LDH levels were collected each 24-hour period. LDH analyses were performed
on a clinical Kodak Biolyzer (Kodak, Inc., New Haven, Connecticut) using dry chemistry  technique.

Light Microscopy
Two-dimensional samples of each experiment were photographed by a Nikon T330 microscope  equipped with advanced Hoffman Modulation optics (Nikon Inc., New York, New York).

Scanning Electron Microscopy
Samples for scanning electron microscopy were harvested every 48 hours of culture and were washed  once in PBS-CMF, fixed in a solution containing 3% glutaraldehyde, 2% paraformaldehyde in 0.1M  cacodylate buffer at pH 7.4, processed by RMC, Inc., and scanned at 10 kV.

Genetic Discovery Array
Samples of human NHNP cells grown in conventional flask culture were trypsinized and placed  into petri dishes in a 55-ml RWV. After 17-21 days of culture on 5 mg/ml cytodex-3 beads, cells were washed once with ice-cold phosphate buffered saline. The cells were lysed and mRNA  selected with biotinylated oligo (dT) then separated with streptavidin paramagnetic particles
(Poly A Ttract System 1000, Promega Madison, WI). 32P-labeled cDNA probes were generated  by reverse transcription with 32P dCTP. The cDNA probes were hybridized to identical gene  discovery array filters (Incyte Genome Systems Inc., St. Louis, Missouri). The gene discovery array filters contain over 12,000 unique human genes from the I.M.A.G.E. Consortium (LLNL)1 cDNA Libraries, which are robotically arrayed on each of a pair of filter membranes. Gene   expression were then detected by phosphor imaging and analyzed using the Gene Discovery  Software (Incyte Genome Systems Inc., St. Louis, Missouri).

Statistical Analysis
All experimental and control groups were analyzed in triplicate or quadruplicate, depending on  the availability of materials. These were averaged for determination of mean and standard error.
Frequencies in phenotypic analysis were compared by chi-square. Proliferation as assessed by  3H-TdR uptake was compared by analysis of variance for multiple groups, using the Student’s test for paired groups (Zar, 1974).

Two-Dimensional Experiments
NHNP pool cells exposed to a TVEMF, whether or not in direct contact with the electrode,
displayed an accelerated growth rate and different morphology as compared to non-waveform  cells (controls) (Tables 1 and 2).


After the application of the TVEMF, the cells preferentially aligned, while cells without  waveform exposure showed a normal random pattern. Cells in direct contact with the electrode  remained stimulated up to 72 hours after the TVEMF was removed (Table 3), while those not in  direct contact with the electrode continued to experience an accelerated and long-term
stimulation in growth pattern even 168 hours after the TVEMF was removed (Table 4). Viability  was also higher in the cells exposed to the waveform (Table 4). In each case, cell growth returned to normal rates without further TVEMF stimulation.
To examine the accelerated growth pattern, called the Corona Effect, after treatment with  TVEMF, the same treatment was applied to the cells without harvesting. Several petri dishes,each from the TVEMF group and non-waveform groups, were chosen randomly. Cells still  attached in sheet form were lifted off of the electrodes carefully and placed in new petri dishes  with medium, and then photograph 24 hours later (Figs. 4-6).



Thomas J. Goodwin, Ph.D.
Lyndon B. Johnson Space Center / 2003

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