(BEING CONTINUED FROM 8/8/15 )
Three-Dimensional Experiments The ability to influence and, to some degree, control, the growth patterns of NHNP cells in the two-dimensional aspect is a great investigational tool. The question that emanated from this result was, did we have the ability to construct a three-dimensional model and have it emulate the same genetic influence of the TVEMF as shown in Figures 4 and 5? To answer this question, we turned to a new technology called RWV.
Rotating- Wall Vessel Background The RWV was created at the Johnson Space Center in 1985 to address the problems associated with attempting to simulate microgravity conditions in an Earth-bound lab. It was designed to maximize the conditions of free fall while allowing continuous cell culturing over prolonged periods of time. This instrument uses a cylindrical tube with suspended particles that is rotated inline with the horizontal axis. The particles move in conjunction with the fluid and the wall of the vessel, the effects of gravity are randomized, modeling some aspects of microgravity, while reducing to a minimum shear and turbulence (Dedolph and Dipert, 1971; Cogoli and Gmiinder, 1991; Schwarz et al., 1992; Prewett et al., 1993, Goodwin et al., 1993). Several fluid dynamic operating principles define the RWV. First, it is composed of a solid body rotation about a horizontal axis characterized by (a) co-location of particles of different sedimentation rates, (b) extremely low fluid shear stress and turbulence, and (c) threedimensional spatial freedom. Second, oxygenation is by active or passive diffusion so that only dissolved gasses are present in the reactor chamber. There are no gas bubbles and no gadfluid interface (Wolf and Schwarz, 1991; Schwarz et al., 1992; Goodwin et al., 1993). Rotating- Wall Vessel Experimental Data We modified an RWV by adding an electromagnetic field coil to allow observation of TVEMF influence on NHNP cells in three-dimensional aspect. Using the RWV, we replicated our preliminary results from the two-dimensional studies several times, and have analyzed the gene expression using gene arrays. We monitored cell proliferation, orientation, morphology, glucose metabolism, and pH (Figs. 7-1 1). Our results show a stable, reliable model to study the control of high-level cellular processes by application of low-amplitude, time-varying micromagnetic fields. Figures 7 to 11 show that metabolic parameters evidenced little or no differences between TVEMF and control three-dimensional culture. This would indicate that, although cellular growth was enhanced and more directed, additional cellular energy was not required to accomplish this effect. The RWV-grown tissues also emulated the same basic genetic response as the two-dimensional tissues at similar fields strengths. This study focused on the use of NHNP cells because of their importance in human nervous system development and maintenance. However, we have developed two-dimensional and now three-dimensional bioreactors that can potentially accommodate other cell lines. Initial results with the NHNP cells were quite startling, using extremely low-level magnetic fields (-10 – 200 mGauss), below the magnetic field strength of the Earth itself (approximately 500 mGauss). We found the low-amplitude, rapidly time-varying magnetic fields exerted a very potent effect on the proliferation, morphology, and gene expression of the cells in culture, both in standard 2-dimensional culture plates (Fig 12) as well as cells organized into 3-dimensional tissue clusters (Fig 8) in the RWV.
(TO BE CONTINUED)
Thomas J. Goodwin, Ph.D.
Lyndon B. Johnson Space Center / 2003