USOO6485963B1

(12) United States Patent (10) Patent No.: US 6,485,963 B1

Wolf et al. (45) Date of Patent: Nov. 26, 2002

(54) GROWTH STIMULATION OF BIOLOGICAL 4,703,010 A 10/1987 Yunker et al. .............. 435/173
CELLS AND TISSUE BY 4,762,795. A 8/1988 Masson ...................... 435/287
ELECTROMAGNETIC FIELDS AND USES E. A
2-a-s- 2
2. pathwski et al. .......
aSIllS. . . . . . . . . . . . . . . . . . . . . . . .
i
THEREOF 5,270,205 A 12/1993 Rogalsky .................... 435/285
(75) Inventors: David A. Wolf, Houston; Thomas J. SE
2- - -2
A 3.1994 Minuth ....................... 435/285
/1994 Clarke et al. ................. 623/11
Goodwin, Friendswood, both of TX 6,022,733 A 2/2000 Tam et al. ............... 435/287.1
(US) 6,066.495 A * 5/2000 Fofonoff et al. ......... 435/289.1
(73) Assignee: The United States of America as * cited by examiner
represented by the Administrator of Primary Examiner David A. Redding
the National Aeronautics and Space (74) Attorney, Agent, or Firm-James M. Cate
Administration, Washington, DC (US) (57) ABSTRACT
(*) Notice: Subject to any disclaimer, the term of this The present invention provides Systems- 0for growing two or
patent is extended or adjusted under 35
U.S.C. 154(b) by 0 days. three dimensional mammalian cells within a culture medium
facilitated by an electromagnetic field, and preferably, a time
varying electromagnetic field. The cells and culture medium
(21) Appl. No.: 09/587,028 are contained within a fixed or rotating culture vessel, and
(22) Filed: Jun. 2, 2000 the electromagnetic field is emitted from at least one elec
7 trode. In one embodiment, the electrode is Spaced from the
(51) Int. Cl.' ................................................. C12M 1/10 vessel. The invention further provides methods to promote
(52) U.S. Cl. .................................. 435/298.2; 435/299.1 neural tissue regeneration by means of culturing the neural
(58) Field of Search ........................... 435/173.1, 1738, cells in the claimed System. In one embodiment, neuronal
435/298.2, 299.1 cells are grown within longitudinally extending tissue
Strands extending axially along and within electrodes com
(56) References Cited prising electrically conductive channels or guides through
U.S. PATENT DOCUMENTS which a time varying electrical current is conducted, the
conductive channels being positioned within a culture
2.996,429 A 8/1961 Toulmin, Jr. ................. 167/78 medium.
3,133,003 A 5/1964 Schaefer et al. .............. 195/81
4,377,639 A 3/1983 Lee ............................ 435/285 18 Claims, 11 Drawing Sheets
U.S. Patent Nov. 26, 2002 Sheet 1 of 11 US 6,485,963 B1

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U.S. Patent Nov. 26, 2002 Sheet 2 of 11 US 6,485,963 B1

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U.S. Patent Nov. 26, 2002 Sheet 3 of 11 US 6,485,963 B1
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FGURE 4
U.S. Patent Nov. 26, 2002 Sheet S of 11 US 6,485,963 B1

FIGURE 5
U.S. Patent Nov. 26, 2002 Sheet 6 of 11 US 6,485,963 B1

FIGURE 6
U.S. Patent Nov. 26, 2002 Sheet 7 of 11 US 6,485,963 B1
U.S. Patent Nov. 26, 2002 Sheet 8 of 11 US 6,485,963 B1

FIGURE 8
U.S. Patent Nov. 26, 2002 Sheet 9 of 11 US 6,485,963 B1

FIGURE 9
U.S. Patent Nov. 26, 2002 Sheet 10 of 11 US 6,485,963 B1

14A

YP

FIGURE 10
U.S. Patent Nov. 26, 2002 Sheet 11 of 11 US 6,485,963 B1

FIG 11B

FIG. 1 1A
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1 2
GROWTH STIMULATION OF BIOLOGICAL This three dimensional Spatial arrangement has not been
CELLS AND TISSUE BY reproduced by conventional in vitro culture. Investigators,
ELECTROMAGNETIC FIELDS AND USES Borgens RB et al, and others, have utilized Static electric
THEREOF fields in an attempt to enhance nerve growth in culture.
ORIGIN OF THE INVENTION
(Valentini et al) with Some Success to either alter embryonic
development or achieve isolated nerve axon directional
The invention described herein was made by an employee growth. However, actual potentiation of growth or genetic
of the United States Government and may be manufactured activity causing Such, have not been achieved. Mechanical
and used by or for the Government of the United States of devices intended to help grow and orient three dimensional
America for governmental purposes without the payment of mammalian neuronal tissue are currently available. Fukuda
any royalties thereon or therefor. et al. (7) used Zones formed between stainless steel shaving
blades to orient neuronal cells or axons. Additionally, elec
BACKGROUND OF THE INVENTION trodes charged with electrical potential were employed to
1. Field of the Invention enhance axon response. Aebischer (8) described an
15 electrically-charged, implantable tubular membrane for use
The present invention relates generally to the fields of in regenerating Severed nerves within the human body.
biophysics, tissue regeneration, tissue culture, and neurobi However, none of these devices utilize channels of cell
ology. More specifically, the present invention relates to the attractive material, neither do they apply a time varying
use of an electromagnetic field, and preferably, a time electromagnetic field, or a Static electrical or magnetic field.
varying electromagnetic field, for potentiation of or control Additionally, no use is made of Simulated or actual micro
ling the growth of biological cells and tissue, Such as gravity techniques for pure neuronal, or mixed, neuronal and
mammalian tissue. More specifically, the present invention feeder cell cultures. The prior art is deficient in its lack of
relates to the use of an electromagnetic field for controlling effective means for growing three dimensional mammalian
the growth of neural cells and tissues. The preferred embodi neuronal tissue in the proximity of, or directly upon the
ment utilizes two-dimensional conducting plate electrodes 25 Surface, of a current carrying electrode (which may be
and may be applied to conventional, two dimensional tissue bioattractive and directly adherent to the cells). Furthermore,
cultures or to three-dimensional cultures. Three dimensional
cultures may be achieved in actual microgravity or by the use of a time varying current to induce a corresponding
time varying electromagnetic field, in the vicinity of the
rotating wall vessel technology which simulates the physical growing culture, to potentiate or Spatially direct cell growth
conditions of microgravity, and in other, conventional three is not part of the prior art.
dimensional matrix based cultures. The electromagnetic
field, preferably a time varying electromagnetic field, is SUMMARY OF THE INVENTION
achieved in the vicinity of the electrode by passing, through The present invention relates to a system and method for
the electrode, a time varying current. culturing biological cells, Such as mammalian cells, within
2. Description of the Related Art 35 a culture medium. The cells are exposed to an electromag
Growth of a variety of both normal and neoplastic mam netic field, which, in the preferred embodiment, is a time
malian tissues in both mono-culture and co-culture has been varying electromagnetic field. In the preferred embodiment,
established in both batch-fed and perfused rotating wall this field is generated by a conductive electrode, adjacently
vessels (1-2), and in conventional plate or flask based Spaced from the incubating cells, carrying a time varying
culture Systems. In Some applications, growth of three 40 electrical current. The electrode, in one case, is in direct
dimensional Structure, e.g., tissues, in these culture Systems galvanic contact with the culture media and cells, and in
has been facilitated by Support of a solid matrix in the form another case, it is placed external to the culture apparatus in
of biocompatible polymers and microcarriers. In the case of a galvanically isolated condition. Preferably, a 10 hertz
Spheroidal growth, three-dimensional Structure has been Square wave of 1-6 milliamperes, and with nearly Zero time
achieved without matrix support (3–6). NASA rotating wall 45 average, is passed through the electrode, Suitably from
tissue culture technologies have extended this three dimen corner to opposite comer of a Square metallic conductive
Sional capacity for a number of tissues and has allowed the plate. The cells, Such as neurons in this case, were, in one
tissue to express different genes and biomolecules. Neuronal embodiment, grown directly on the electrode Surface, com
tissue has been largely refractory, in terms of controlled posed of a biocompatable material. In another embodiment,
growth induction and three dimensional organization, under 50 the cells were grown within a container under the influence
conventional culture conditions. Actual microgravity, and to of a time varying electromagnetic field from an electrode
a lesser extent, rotationally simulated microgravity, have external of and adjacent to the container, galvanically iso
permitted Some enhanced nerve growth (Lelkes et al). lated from the media and culture within the container.
Attempts to electrically Stimulate growth have utilized Static The growing cells may actually be attracted and trophi
electric fields, Static magnetic fields, and the direct passage 55 cally Supported by more Supportive electrode material or
of current through the culture medium, though not the coatings. Furthermore, channels may be incorporated in the
induction of a time varying electromagnetic field in the culture vessel and lined with growth substrate which may be
culture region. electrically conductive. In one embodiment, a time varying
Neuronal tissue comprises elongated nerve cells com electromagnetic field is induced in the region of the channel
posed of elongated axons, dendrites, and nuclear areas. 60 by passing the time varying current through a conductor
AXons and dendrites are chiefly responsible for transmission placed along the channel. This arrangement will further
of neural Signals over distance and longitudinal cell orien direct growth by the combined effect of the field and trophic
tation is critical for proper tissue formation and function. materials.
The nucleus plays the typical role of directing nucleic acid In the preferred embodiment, the presence of the time
synthesis for the control of cellular metabolic function, 65 varying electromagnetic field potentiates the growth of
including growth. In Vivo, the neuronal tissue is invariably nerve and other tissue. The time varying field may be
Spatially associated with a System of feeder, or glial, cells. induced by either: 1) a time varying current within a
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conductor, or 2) a time varying Voltage between fixed nected to the ends of the channels, and an external power
conductors. In one embodiment, for example, the culture is Source is provided for applying the time varying electrical
placed nearby a conductor through which a time varying current defining the waveform through the channels. In
current is passed, or between parallel plates upon which a another embodiment, the channel consists of a pair of
time varying Voltage is applied. In both cases, a time varying parallel, mutually spaced conductors acroSS which a time
electromagnetic field results within the area of interest, i.e., varying Voltage is applied. This also achieves the time
in the region of the cell culture. varying electromagnetic field but restricts it to the region
The System and proceSS are utilized in combination with between the parallel electrodes, which is advantageous for
known tissue culture processes to produce enhanced cell directing localized growth according to a desired physical
growth, directed cell growth, and tissue formation and pattern. The present invention also relates to a System and
organization. method for culturing primarily two dimensional mammalian
As will be understood from the description to follow, the cells facilitated by a time varying electromagnetic field. The
System is operable to up or down regulate the activity of electrodes may either be in direct galvanic contact or gal
Specific genes. In general, growth promoting genes are up Vanically isolated from the target cells. The present inven
regulated and growth inhibitory genes are down regulated. 15 tion provides a Strategy to re-engineer nerve tissue and
The effect is shown to persist for some period after termi myoneural junctions and can be used medically for axonal
nation of the applied time varying field. This persistent, regeneration.
growth promoting effect Subsides after a period of Some In one embodiment of the present invention, there is
days, and the cells return to a growth State characterized by provided a System for growing three dimensional mamma
controls, having never been exposed to the fields. This is lian cells, comprising a rotating wall vessel containing a
beneficial in certain applications, in that medical applica cell-rich medium and a formed cell growth Substrate. A time
tions for clinical medical care, i.e. nerve regeneration, are varying electromagnetic field is applied to enhance tissue
therefore safer than if the “pseudo transformed” state per growth which may occur on a shaped Substrate. The elec
Sisted. The Set of gene transformations, associated with the tromagnetic field may be generated by means Such as by
time varying electromagnetic field, also promote the ability 25 directing the current waveform directly through a conduc
of the growing tissue to adhere and thrive on Substrates by tive Substrate (or Substrate layer) or by projecting the field
the induction of genes leading to the Secretion of extracel from an external antenna, or electrode adjacent to and
lular materials favorable to the tissue microenvironment. Spaced from the medium, the Spacing being Sufficiently
Several methods of producing the time varying electro Small relative to the Strength of the electromagnetic field to
magnetic field in the vicinity of the living tissue culture are induce effectual levels of electromagnetic field within the
encompassed. In one embodiment, an array of conductive medium, in accordance with the particular application. A
current carrying elements (or Voltaic electrodes) are time varying electromagnetic field may be emitted from a
arranged so as to intensify or focus the time varying elec nearby plate or other Suitable “antennae, or a time varying
tromagnetic (EM) field onto the culture. Each embodiment voltage may be applied across Suitable electrodes (such as
is characterized by a method for application of the time 35 plates) to produce the time varying electromagnetic field.
varying field to the target tissue, Such as neuronal, for The field generation System may either be rotating with the
Stimulation of growth, or repair or induction of changes in vessel or fixed, and Spaced from, the rotating vessel. The
gene activity patterns. The term "field generator” is used rotating wall vessel can be a rotating wall perfused vessel or
herein to represent these various embodiments for generat a rotating wall batch-fed vessel.
ing the time varying electromagnetic fields. In its simplest 40 The time varying electromagnetic field is advantageously
form, it is a conductive electrode, placed near the target produced by a varying electrical potential in the form of a
cells, through which current is directed from a controlled Square wave having a frequency of approximately ten cycles
waveform current Source. per Second. In one embodiment, a current of about ten
AS Suggested above, in one embodiment, the field gen milliamps, conducted between opposite comers of a metallic
erator is in the form of a conductive channel mounted on or 45 conductor, produces a Stimulatory time varying electromag
embedded in a disc of biocompatable material. (FIG. 11) netic field extending Several centimeters from the plate
One or more of these discS may be then placed within a Surface. In practice, the range of frequency and oscillating
rotational bioreactor So as to obtain the beneficial culture electromagnetic field Strength is a parameter which may be
conditions associated therewith. The combination of the Selected to for achieving the desired Stimulation of particular
Stimulatory electromagnetic field with the rotational 50 tissues, cells, or genes, and for providing the appropriate
environment, known to permit morphological expression amount of up/down regulation of these genes.
beyond conventional culture, is particularly effective. This is In one embodiment of the present invention, the cell
because the induced pattern of growth enhancing genes is growth Susbtrates or carriers are spherical disks containing
permitted to be ultimately expressed, as cell growth and multiple parallel channels (FIG. 10) which are coated with
tissue formation, without mechanical inhibition from the 55 a bioattractive material. The bioattractive material has a
culture apparatus. Also the inherent growth advantages well longitudinal axis acroSS which the time varying electrical
known in the rotational Systems is Synergistic with the potential is applied and through which a time varying
growth Stimulation derived from the time varying electro current is conducted. The mammalian cells adhere to the
magnetic field. The conditions may be further optimized by bioattractive material and are free to orient, as they grow.
utilizing actual microgravity, in Space. In this application, 60 Representative bioattractive materials include titanium, Zir
mechanical rotation of the cell culture vessel is not required conium and platinum.
but may be utilized to achieve mixing and Sufficient mass The class of mammalian cells preferably is Selected from
transfer to Sustain a healthy culture. Other forms of mixing the group consisting of neuronal cells, normal human neu
may be introduced as necessary to achieve adequate mass ronal progenitor cells (NHNP), and a cell responding to the
transfer for each embodiment. 65 time varying electromagnetic field. It will be understood by
In one embodiment, illustrated in FIGS. 10 and 11, those of ordinary skill in the art that the teachings of the
Slip-ring contacts or their equivalents are electrically con present invention apply to other cell types.
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In another embodiment of the present invention, there is FIG.2 shows normal human neuronal progenitor (NHNP)
provided a method of culturing mammalian cells in the cells grown in conventional tissue culture procedures.
claimed System, comprising the Steps of inoculating the cells FIG. 3 shows the perimeter of non-waveform influenced
into the vessel containing a culture medium, rotating the normal human neuronal progenitor cells 24 hours after the
vessel to enhance the proliferation of the cells and, in one experiment.
embodiment, to initiate the attachment of the cells to micro FIG. 4 shows neural tube formation within normal human
carrier spheres or beads Suspended within the culture neuronal progenitor cells under the influence of waveform.
medium, applying a time varying electromagnetic field to
the culture medium, cells, and cell carriers, and measuring FIG. 5 shows neural tube generation within normal
the growth of the cells. Preferably, the vessel is rotated at a human neuronal progenitor cells under the influence of
waveform.
speed from about 2 RPM to 30 RPM, and the time varying
electromagnetic field is generated by a time varying current FIG. 6 shows the composition of waveform-influenced
passed through a conductor with RMS value of about 1 to neural tissue 24 hours after the exposure.
1,000 ma. In one embodiment, a range of about 1 mA to 6 FIG. 7 shows the waveform-influenced normal human
mA is used. 15 neuronal progenitor cells 24 hours after the exposure.
In still another embodiment of the present invention, there FIG. 8 shows a close-up of waveform-influenced normal
is provided a System for growing two-dimensional neural human neuronal progenitor cells.
cells, comprising a petri dish containing a cell culture FIG. 9 shows waveform-influenced normal human neu
medium and an electrode placed in the center of the petri ronal progenitor cells 24 hours after the exposure.
dish. In this embodiment, the electrode serves as the field FIG. 10 shows prototype of the system 1, consisting of
generator. Preferably, the neural cells are applied directly on Silicon plateS2, fluid coupling 3, Slip rings 4, a rotating wall
the electrode. As a result, the neutral cells exhibit acceler pressure vessel (RWPV) 5, electrical conductor 6 (i.e., an
ated growth. electrical conductive bioattractive inlay strip), a perfusion
In yet another embodiment of the present invention, there 25 inlet 7, perfusion outlet 8, a stand for the rotating wall
is provided a System for growing two-dimensional neural preSSure vessel 9 and a Source of time varying electrical
cells further comprising a slide placed on the electrode. current 10.
Preferably, the neural cells are applied, e.g., bubbled, on the FIG. 11A is a plan view of one of the disc-shaped silicon
Slide instead of directly contacting the electrode, and plates 2, showing a central opening 11 about which rotation
preferably, the current producing the waveform is applied at occurs, the electrical conductorS 6, and electrical contacts 12
a strength range of from about 1 mA to about 100 mA, and, connected to the strips at opposite ends thereof. FIG. 11B is
in one embodiment, suitably from about 1 mA to 6 m.A. a side view of the Silicon plate 2, demonstrating the elec
In still another embodiment of the present invention, there trical contacts 12 and the electrical conductive strips, in the
is provided a method of treating an individual having form of bioattractive inlays 6.
diseased neuronal cells, comprising the Steps of growing 35
neuronal cells in the claimed two- or three-dimensional DETAILED DESCRIPTION OF THE
Systems and transplanting the neuronal cells into the indi INVENTION
vidual. Such diseases include Parkinson's disease, diseases AS used herein, the term “bioattractive material” shall
of neuromuscular junction and Alzheimer's Disease. Neural refer to materials to which a cellular material will attach.
trauma can also be treated in Same methodology.
In yet another embodiment of the present invention, the
40 As used herein, the term “longitudinally orient” shall refer
time varying electromagnetic field (or electrical potential) to orientation in an elongated cordlike fashion.
induces cellular response including cellular control of As used herein, the term “parallel channels' shall refer to
growth and differentiation at gene level. Preferably, the electric channels which are designed to provide constant
cellular control of growth and differentiation is to SuppreSS output to all the electrodes Simultaneously.
45
or enhance growth regulatory functions at gene level. Still AS used herein, the term "cell carriers' shall refer to
preferably, the gene is associated with increased tissue and microcarrier beads, Scaffolds and matrices which Support the
cell proliferation. growth and/or attachment of cellular materials.
Other and further aspects, features, and advantages of the As used herein, the term “rotating wall batch-fed vessel.”
present invention will be apparent from the following 50 shall refer to slow turning lateral vessel (STLV) and high
description of the presently preferred embodiments of the aspect rotating vessel (HARV).
invention given for the purpose of disclosure. AS used herein, the term “Corona Effec?t” shall refer to
the accelerated growth pattern of neuronal cells electrically
BRIEF DESCRIPTION OF THE DRAWINGS potentiated by waveform.
So that the matter in which the above-recited features, 55 In one preferred embodiment, the present invention is
advantages and objects of the invention, as well as others directed to the growth of three dimensional mammalian
which will become clear, are attained and can be understood neuronal tissue using an electrically conductive Strip in the
in detail, more particular descriptions of the invention form of a channel or mold coated with a bioattractive or
briefly Summarized above may be had by reference to biocompatable material to which an electrical potential is
certain embodiments thereof which are illustrated in the 60 applied to longitudinally orient the neural cells or axons as
appended drawings. These drawings form a part of the they adhere to the bioattractive material which is Suspended
Specification. It is to be noted, however, that the appended in an axon rich medium.
drawings illustrate preferred embodiments of the invention A Specifically, in the present embodiment, the apparatus
and therefore are not to be considered limiting in their Scope. includes a bioreactor chamber vessel employing electrically
FIG. 1 shows a petri dish with cells in a concentrated 65 insulative, biocompatable spherical disks of a material Such
bubble placed on the metal electrode in the center of the as Silicon. These disks rotate inside the pressure vessel. Each
dish. disk has multiple parallel channels cut into its Surface. The
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channels have a Semicircular cross-section and contain an into the individual. Such diseases include Parkinson's
electrically conductive inlay in the form of a channel-shaped disease, diseases of neuromuscular junction and Alzheimer's
conductive Strip of a bioattractive material Such as Disease. Neural trauma can also be treated with this same
Zirconium, titanium and platinum. Each channel Strip 6 has methodology.
an electrical contact on each longitudinal end that is used to In yet another embodiment of the present invention, the
create and control an electrical potential along the length of waveform (or electrical potential) induces a cellular
the strip. The vessel is filled with a medium and the disks are response including cellular control of growth and differen
rotated within a medium containing axons. The cells adhere tiation at gene level. Preferably, the cellular control of
to the electrically conductive bioattractive inlay material. growth and differentiation is to Suppress or enhance growth
The desired longitudinal cell orientation and therefore the regulatory functions at gene level. Still preferably, the gene
Structure of the resulting tissue is affected and/or controlled is associated with embryogenesis.
by the electrical Stimulus. The following examples are given for the purpose of
The present invention is also directed to the growth of two illustrating various embodiments of the invention and are
dimensional mammalian neuronal tissue using electrodes. not meant to limit the present invention in any fashion.
The electrodes are either in direct contact or not in contact 15
with the target cells. EXAMPLE 1.
Cells
In one embodiment of the present invention, there is Normal human neuronal progenitor cells (NHNP) were
provided a System for growing three dimensional mamma
lian cells, comprising a rotating wall vessel containing a pooled from three donors. AS controls, normal human neu
cell-rich medium, cell carriers placed within the vessel and ronal progenitor cells were grown in conventional tissue
an electrical potential applied to the cell carrier. Preferably, culture following Standard cell culturing procedures.
the rotating wall vessel can be a rotating wall perfused vessel EXAMPLE 2
or a rotating wall batch-fed vessel. Materials
In one embodiment of the present invention, the cell 25
GTSF-2 medium with 10% FBS, Ciprofloxacin and Fun
carriers are spherical disks containing multiple parallel giZone was used to culture the cells. 1XPBS, Collagenase,
channels, which are coated with a bioattractive material. DNase and Trypsin were purchased from Clonetics. The
More preferably, the bioattractive material has a longitudinal cells were grown on 12-100 mm Petri dishes (tissue culture
axis acroSS which the electrical potential is applied. The coated or not coated). Electrodes were made of platinum and
mammalian cells adhere to the bioattractive material and are Stainless Steel. A waveform generator was used to generate
therefore oriented longitudinally upon the electrical Stimu the waveform in a strength of 1-6 mA (AC) square wave, 10
lus. Representative bioattractive materials include titanium, HZ variable duty cycle.
Zirconium and platinum. EXAMPLE 3
In the methods of the present invention, the mammalian Electrically Potentiating Cell Growth. When Electrode is in
cell is Selected from the group consisting of a neuronal cell, 35 Direct Contact with the Target Cells
a normal human neuronal progenitor cell (NHNP) and a cell Initially, a metal electrode was placed inside a petri dish
responding to waveform. A perSon having ordinary skill in and centered.
this art will be able to apply the teachings of the present Normal human neuronal progenitor cells were Seeded at
invention to other cell types. 2x10 cells in 0.7 ml of media and carefully dropped on the
In another embodiment of the present invention, there is 40 electrode in a concentrated bubble (FIG. 1). Cells were
provided a method of culturing mammalian cells in the incubated for 2 dayS. Second day after Seeding is considered
claimed System, comprising the Steps of inoculating the cells day 0 of the experiment. At day 0, each dish was given 15
into the vessel, rotating the vessel to initiate the attachment ml of media and waveform was applied to Seven electrodes.
of the cell to the cell carriers, applying an electrical potential Cells were observed under a dissecting microscope and fed
to the cell carriers and measuring the growth of the cells. 45 with 15 ml of media at day 3, and 13 ml every three days at
Preferably, the vessel is rotated at a speed from about 10 day 6, 9 and 12. At day 14, the cells were fed again with 13
RPM to 30 RPM, and the electrical potential is applied at a ml of media. At day 17, the cells were incubated for 10
Strength range of from about 1 mA to about 6 mA. minutes in a Collagenase/DNase cocktail, then trypsin was
In still another embodiment of the present invention, there directly applied to the cocktail and the cells were further
is provided a System for growing two-dimensional neural 50 incubated for 3 more minutes. Before the media was added
cells, comprising a petri dish containing a cell culture to deactivate trypsin, the cocktail mix was pipetted up and
medium and an electrode placed in the center of the petri down several times. The cells were washed twice with
dish. The electrode is charged with a waveform. Preferably, 1xPBS, reapplied with the media and placed on ice. The
the neural cells are bubbled directly on the electrode. As a cells were counted, assessed for viability.
result, the neutral cells exhibit accelerated growth. 55 To examine the accelerated growth of cells 48 and 72
In yet another embodiment of the present invention, there hours after waveform was discontinued, cells were treated
is provided a System for growing two-dimensional neural the same as above, except that after day 14 treatment, instead
cells further comprising a slide placed on the electrode. of harvesting, two dishes from the non-waveform group
Preferably, the neural cells are bubbled on the slide instead (control) and two dishes from the waveform group were
of directly contacting the electrode. Preferably, the wave 60 randomly chosen and re-seeded at 9x10 cells in two new
form is applied at a strength range of from about 1 mA to petri dishes each, with a total of four dishes. Cells from one
about 6 mA. set (#11 waveform and control #6) were counted and pho
In Still yet another embodiment of the present invention, tographed 48 hours after Seeding, and cells from the Second
there is provided a method of treating an individual having set (#12 waveform and control #7) were counted and pho
diseased neuronal cells, comprising the Steps of growing 65 tographed 72 hours after Seeding.
neuronal cells in the two or three dimensional Systems To examine accelerated growth pattern “Corona Effect”
disclosed herein and transplanting the grown neuronal cells after the electrical potentiation, the same treatment was
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applied to the cells without harvesting. A dish each from
waveform group and non-waveform group were chosen TABLE 1-continued
randomly. Cells still attached in sheet from were lifted off of
the electrodes carefully and placed in new petri dishes with Cell Count and Viability at Harvest (day 17
medium, and then photographed 24 hours later. NHNP-POOL CELL COUNT VIABILITY HARVEST

EXAMPLE 4 Waveform 10 1,000,000 98% 17 days

Control 1 500,000 98% 17 days
Electrically Potentiatiny Cell Growth. When Electrode is not Control 2 400,000 98% 17 days
in Direct Contact with the Target Cells Control 3 3OOOOO 98% 17 days
Initially, a metal electrode was placed inside a petri dish Control 4
Control 5
500,000
400,000
98%
98%
17 days
17 days
and centered. A Slide was carefully placed on the electrode
under Sterile conditions. Normal human neuronal progenitor *Cells were in direct contact with the electrode.
cells were seeded at 2x10 cells in 0.7 ml of media and
bubbled on the slide. Cells were incubated for 2 days. 25 ml
of media were applied and two 1000 ul pipetman blue tips 15 TABLE 2
were placed in the dish to anchor the slide to bottom of the Cell Count and Viability at Harvest (day 18
dish. The Second day after Seeding was considered day 0 of
the experiment. At day 0, each dish was given 25 ml of NHNP-POOL CELL COUNT VIABILITY HARVEST
media and waveform was applied to Six of the twelve Waveform 1 1,000,000 100% 18 days
electrodes.
Waveform 5 1,100,000 100% 18 days
Cells were observed under a dissecting microscope and Control 1 800,000 100% 18 days
fed with 25 ml of media every three days at day 3, 6, 9 and Control 5 800,000 100% 18 days
12. At day 14, the cells were fed again with 25 ml of media. *Cells were not in direct contact with the electrode.
At day 18, the cells were incubated for 10 minutes in a
Collagenase/DNase cocktail, then typsin was directly 25
applied to the cocktail and the cells were further incubated TABLE 3
for 3 more minutes. Before the media was added to deac
tivate trypsin, the cocktail mix was pipetted up and down *Cell Count at Various Times after Removal of Waveform
several times. The cells were washed twice with 1xPBS, CELLS COUNT HOURS OFFELECTRODE
reapplied with the media and placed on ice. The cells were
counted, assessed for viability and then replated at 100,000 Waveform 11 520,000 Counted and re-seeded at 96,000/plate
per plate. The remaining waveform and non waveform Slides Control 6
Waveform 11
112,000
274,000
Counted and re-seeded at 96,000/plate
48 hours off electrode
were fixed and refrigerated for staining at a later date. Control 6 48,000 48 hours off electrode
Waveform 12 576,000 Counted and re-seeded at 96,000/plate
EXAMPLE 5 35 Control 7 96,000 Counted and re-seeded at 96,000/plate
Electrically Potentiated Growth of Cells Waveform 12 228,000 72 hours off electrode
Control 7 120,000 72 hours off electrode
Normal human neuronal progenitor-pool cells exposed to
a time varying electromagnetic field (waveform), either in *Cells were in direct contact with the electrode.
direct contact or not in direct contact with the electrode, 40
displayed an accelerated growth rate and different morphol TABLE 4
ogy as compared to non waveform cells (control), i.e., cells
not Subject to the time varying electromagnetic field (see Cell Count and Viability at Various Times after Harvest
Table 1 and Table 2, FIGS. 2-9). After the application of the
time varying electromagnetic field or waveform, the cells 45
TIME AFTER
HARVEST
preferentially aligned, while cells without waveform expo NHNP-POOL CELL COUNT VIABILITY (Hours)
Sure showed random pattern. Cells in direct contact with the
electrode remained Stimulated up to at least 72 hours after Waveform 1 56,000 85% 24
Waveform 5 40,000 85% 24
waveforn was removed (Table 3); while those not in direct Control 1 36,000 65% 24
contact with the electrode once removed from waveform Control 5 28,000 65% 24
50
continued to experience accelerated and long term Stimula Waveform 1 188,000 98% 48
tion growth pattern even after 168 hours (Table 4). Viability Waveform 5 212,000 98% 48
was also higher in the cells exposed to the waveform (Table Control 1 74,000 87% 48
Control 5 162,000 90% 48
4). Cells were suspended easily with the Collagenase/DNase Waveform 1 3,400,000 100% 12O
then trypsin Sequence. 55
Waveform 5 3,400,000 100% 12O
Control 1 900,000 99% 12O
TABLE 1. Control 5 900,000 99% 12O
Waveform 1 4,000,000 100% 168
Waveform 5 3,800,000 100% 168
Cell Count and Viability at Harvest (day 17 Control 1 980,000 97% 168
NHNP-POOL CELL COUNT VIABILITY HARVEST Control 5 900,000 95% 168
60
Waveform 1 860,000 98% 17 days *Cells were not in direct contact with the electrode.
Waveform 2 1,000,000 98% 17 days
Waveform 3 1,000,000 98% 17 days EXAMPLE 6
Waveform 4 1,300,000 98% 17 days
Waveform 7 1,000,000 98% 17 days Waveform Gene Array Display (GAD) Results
Waveform 8 940,000 98% 17 days 65 Normal Human Neural Progenitor cells or human adult
Waveform 9 700,000 98% 17 days astrocytes were exposed to waveform and non-waveform
growth conditions for 17 days. Upon completion of the
 US 6,485,963 B1
11 12
exposure period cells were harvested via trypsinization and 31. Adenylosuccinate lyase Incyte PD: 1653326}
poly-RNA was prepared from the respective groups of cells. 32. EST Incyte PD: 1798393}
RNA samples were quick frozen and shipped to Synteni 33. Homo Sapiens HP protein (HP) mRNA, complete cds
Corporation for GAD analysis. Below are the results of a {Incyte PD:30841223}
Survey of the response of over 10,000 human genes. The 34. ESTs, Moderately similar to T10C6C.elegans Incyte
results were divided into two categories (Table 5 and Table PD: 1923186}
6). Those genes down regulated or Suppressed by the wave 35. Chromosome condensation 1 {Incyte PD:3180854}
form and those up regulated or enhanced in activity by the 36. Calmodulin 1 (phosphorylase kinase, delta) {Incyte
waveform.
An analysis of the data indicates a Significant down PD:2803306}
regulation of maturation and regulatory genes. These matu 37. Centromere protein A (17kD) Incyte PD:2444942
ration and regulatory genes are normally associated with the 38. V-jun avian sarcoma virus 17 oncogene homolog{Incyte
differentiated or non-growth profile of normal cells. PD: 1920177)
However, there is a Significant up regulation of Some 150 39. Human glutathione-S-transferase homolog mRNA, com
genes which are mainly associated with growth and cellular plete cds {Incyte PD: 1862232
proliferation. Neither two nor three dimensional growth of
15 40. Homo Sapiens gene for protein involved in Sexual
neural cells has been achieved prior to this event with the
development, complete cds {Incyte PD:3033934}
positive outcome of enhanced growth and apparent gene 41. EST Incyte PD:2630992}
regulatory control. 42. Human low-Mr GTP-binding protein (RAB32) mRNA,
partial cds {Incyte PD: 1662688}
TABLE 5 43. Annexin III (lipocortin Ill) {Incyte PD: 1920650}
Down Regulated Genes in Descending Order (Highest to 44. Hydroxymethylbilane synthase Incyte PD: 1509204}
lowest) 45. Synteni: HK4 (HK 4.2000.Y}
1. Homo Sapiens (clone Zap2) mRNA fragment Incyte 46. Ribosomal protein L7a Incyte PD:2579602}
PD: 1661837} 47. Human mRNA for myosin regulatory light chain Incyte
2. CDC28 protein kinase 2 Incyte PD: 1384823} 25 PD:78783}
3. Synteni: YCFR 22 YC 22.2000.W} 48. Ferredoxin reductase Incyte PD: 1819763}
4. ESTs, Moderately similar to cell growth regulating nucle 49. Human copper transport protein HAH1 (HAH1) mRNA,
olar protein LYAR M.musculus Incyte PD:2233551} complete cds {Incyte PD:2313349
5. KERATIN, TYPE II CYTOSKELETAL 7 (Incyte 50. Human G protein gamma-11 subunit mRNA, complete
PD: 1649959} cds {Incyte PD: 1988.432}
6. MITOTTC KINESIN-LIKE PROTEIN-1 (Incyte 51. Synteni: HK4 (HK 4.2000.W}
PD:2640427 52. Human XIST, coding sequence a mRNA (locus
7. EST Incyte PD:674714} DXS399E) {Incyte PD: 1514318}
8. Synteni: YCFR 22 {YC 22.2000.X} 53. Ribosomal protein, large, P0 Incyte PD:3511355
9. Synteni: YCFR 26 {YC 26.0062.N} 35 54. Homo Sapiens clone 23714 mRNA sequence Incyte
10. Synteni: YCFR 22 {YC 22.2000.Z} PD: 1728368}
11. Transcription factor 6-like 1 (mitochondrial transcription 55. Human mRNA for Apoll Human (MER5(Aop1
factor 1-like) {Incyte PD:3371995} Mouse)like protein), complete cds Incyte PD:2527879
12. Interferon-inducible 56-KDa protein Incyte 56. Synteni: HK4 (HK 4.2000.Z}
PD: 1215596) 40 57. Proteasome (proSome, macropain) Subunit, beta type, 5
13. EST Incyte PD: 1794375} {Incyte PD:2503119}
14. Homo Sapiens mitotic feedback control protein Madp2 58. Human PINCH protein mRNA, complete cds {Incyte
homolog mRNA, complete cds {Incyte PD:2414624} PD: 126888}
15. EST Incyte PD: 151026} 59. Homo Sapiens peroxisome assembly protein PEX10
16. Homo Sapiens Pig3 (PIG3) mRNA, complete cds 45 mRNA, complete cds {Incyte PD:998279
{Incyte PD:2395269} 60. Homo Sapiens short chain L-3-hydroxyacyl-CoA dehy
17. General transcription factor IIIA Incyte PD: 1527070} drogenase (SCHAD) mRNA, complete cds {Incyte
18. Cellular retinoic acid-binding protein human, Skin, PD: 1638850}
mRNA, 735 nt) Incyte PD:585432} 61. Neuroblastoma RAS viral (v-ras) oncogene homolog
19. EST Incyte PD: 1755159} 50 {Incyte PD:2816984}
20. Homo Sapiens mRNA for KIAA0285 gene, complete cds 62. H.Sapiens mRNA for b4 integrin interactor Incyte
{Incyte PD: 1738053} PD: 1932850}
21. ESTs, Weakly similar to F25H5.h C. elegans Incyte 63. Human forkhead protein FREAC-1 mRNA, complete
PD: 1923567) cds {Incyte PD: 1449920}
22. Homo Sapiens mRNA expressed in Osteoblast, complete 55 64. Human mRNA for protein D123, complete cds {Incyte
cds {Incyte PD:2537863} PD: 1920522}
23. EST Incyte PD:3204745} 65. H. Sapiens mRNA for A-kinase anchoring protein
24. Homo Sapiens mRNA for serine/threonine protein kinase AKAP95 Incyte PD: 1628787}
SAK Incyte PD:2732630} 66. Carbonyl reductase Incyte PD: 1633249}
25. Homo Sapiens serum-inducible kinase mRNA, complete 60 67. EST Incyte PD:2060973}
cds {Incyte PD: 1255087 68. ESTs, Highly similar to GUANINE NUCLEOTIDE
26. Carbonic anhydrase II Incyte PD:2474163} BINDING PROTEING(I)/G(S)/G(O) GAMMA-7 SUB
27. EST Incyte PD:660376} UNIT Rattus norvegicus Incyte PD: 1640161}
28. GRANCALCIN (Incyte PD: 1671852} 69. Homo Sapiens Na+/Ca+exchanger mRNA sequence
29. N-CHIMAERIN Incyte PD:1852659} 65 {Incyte PD:2880435}
30. Homo Sapiens Pig10 (PIG 10) M3RNA, complete cds 7O. STRESS-ACTIVATED PROTEIN KINASE UNK1
{Incyte PD:1731.061} {Incyte PD:3331719)
 US 6,485,963 B1
13 14
71. Homo Sapiens leupaxin mRNA, complete cds {Incyte 109. Human mRNA for proteasome subunit HsC10-II, com
PD: 1595756} plete cds {Incyte PD: 1737833}
72. CLEAVAGE SIGNAL-1 PROTEIN Incyte 110. Homo Sapiens mRNA for ST1 C2, complete cds
PD:2054053} {Incyte PD:3993007)
73. EST Incyte PD: 1798965} 111. Human dual specificity phosphatase tyrosine?serine
74. Human DNA from overlapping chromosome 19 cosmids mRNA, complete cds {Incyte PD: 1514573}
R31396, F2545. 1, and R31076 containing COX6B and 112. Human stimulator of TAR RNA binding (SRB) mRNA,
UPKA, genomic sequence {Incyte PD: 1320685} complete cds {Incyte PD:2057162}
75. INTERFERON-INDUCED 17 KD PROTEIN Incyte 113. EST Incyte PD:2507206}
PD:2862971} 114. H.Sapiens mRNA for Ndr protein kinase Incyte
76. Human homolog of yeast IPP isomerase Incyte PD:3318571}
PD: 1526240} 115. ESTs, Weakly similar to Grb2-related adaptor protein
77. Translation elongation factor 1 gamma Incyte H.sapiens Incyte PD: 1857259
PD:3138196} 116. ESTs, Highly similar to Tbc1 M. musculus Incyte
78. Tropomyosin alpha chain (skeletal muscle) {Incyte 15 PD: 1889147}
PD: 1572555} 117. GTPase-activating protein ras p21 (RASA) Incyte
79. Aplysia ras-related homolog 9 {Incyte PD:2733928} PD: 147344}
80. ATP SYNTHASE ALPHA CHAIN, MITOCHON 118. Human mRNA for KIAA0123 gene, partial cds {Incyte
DRIAL PRECURSOR Incyte PD:3206210} PD: 1752436}
81. Homo Sapiens androgen receptor associated protein 24 119. Synteni: YCFR 22 YC 22.2000.Y}
(ARA24) mRNA, complete cds {Incyte PD:552654} 120. Human non-histone chromosomal protein (NHC)
82. Glucagon Incyte PD: 1333075} mRNA, complete cds {Incyte PD: 1748670}
83. Human enhancer of rudimentary homolog mRNA, com 121. Thioredoxin Incyte PD:2606240
plete cds {Incyte PD: 1704472} 122. FATTY ACID-BINDING PROTEIN, EPIDERMAL
84. TRANSCRIPTIONAL ENHANCER FACTOR TEF-1 25 {Incyte PD:2537805}
{Incyte PD:2957175} 123. Proteasome component C2 Incyte PD:2195309
85. Ubiquitin-like protein Incyte PD: 1754454} 124. Homo Sapiens heat shock protein hsp40 homolog
86. Human RGP4 mRNA, complete cds {Incyte mRNA, complete cds {Incyte PD:2844989}
PD:617517) 125. Human amyloid precursor protein-binding protein 1
87. Cellular retinol-binding protein Incyte PD: 1612969} mRNA, complete cds {Incyte PD: 1663083}
88. Ornithine decarboxylase 1 Incyte PD: 1930235} 126. Homo Sapiens DNA binding protein homolog (DRIL1)
89. EST Incyte PD:3605632} mRNA, complete cds {Incyte PD:2538333}
90. EST Incyte PD:2057260} 127. Human Has2 mRNA, complete cds {Incyte
91. ESTs, Weakly similar to CAMP-DEPENDENT PRO PD:3602403}
TEIN KINASE TYPE 2 Saccharomyces cerevisiae) 35 128. EST Incyte PD: 1749678}
{Incyte PD:2055611} 129. Homo Sapiens golgi SNARE (GS27) mRNA, complete
92. Human p37NB mRNA, complete cds {Incyte cds {Incyte PD:3279439}
PD: 1407110} 130. ESTs, Weakly similar to UBIOUITIN-ACTIVATING
93. Human mRNA for suppressor for yeast mutant, complete ENZYME E1 HOMOLOG H. sapiens Incyte
cds {Incyte PD:288.8814} 40 PD: 1710472}
94. EST Incyte PD:3142705} 131. Synteni: YCFR 22 {YC 22.2000N}
95. ESTs, Weakly similar to KO1H12.1 Celegans Incyte 132. Voltage-dependent anion channel 2 Incyte
PD:56197} PD:2189062}
96. Cell division cycle 2, G1 to S and G2 to M Incyte 133. Human rap2 mRNA for ras-related protein Incyte
PD: 1525795} 45 PD:3334979}
97. EST Incyte PD: 1794.175} 134. Acid phosphatase 1, soluble Incyte PD:620871}
98. EST Incyte PD: 1489557) 135. Human clone 23840 mRNA, partial cds {Incyte
99. ESTs, Weakly similar to PROTEIN PHOSPHATASE PD: 1830083}
PP2A, 72 KD REGULATORY SUBUNIT H.sapiens 136. Human mRNA for KIAAO008 gene, complete cds
{Incyte PD:2379045} 50 {Incyte PD: 1970111}
10O. CAMP-DEPENDENT PROTEIN KINASE TYPE 137. H. Sapiens mRNA for protein-tyrosine-phosphatase
II-ALPH A REGULATORY CHAIN Incyte (tissue type: foreskin) Incyte PD:444957
PD: 1649731} 138. Human B-cell receptor associated protein (hBAP)
101. ESTs, Weakly similar to transcription factor mRNA, partial cds {Incyte PD:2545562}
H.Sapiens Incyte PD: 1637517 55 139. ESTs, Highly similar to ring finger protein H.Sapiens
102. ATP synthase, H+transporting, mitochondrial F1 {Incyte PD:2860918
complex, O Subunit (oligomycin Sensitivity conferring 140. H.sapiens mRNA for CLPP (Incyte PD:2675481}
protein) Incyte PD:2193246} 141. APOPTOSIS REGULATOR BCL-X (Incyte
103. RAS-LIKE PROTEIN TC21{Incyte PD:2505425} PD: 1855683}
104. Small nuclear ribonucleoprotein polypeptides B and B1 60 142. PROTEASOME COMPONENT C13 PRECURSOR
{Incyte PD:2071473} {Incyte PD:2668334}
105. EST Incyte PD: 1922084} 143. Sorting nexin 1 {Incyte PD: 1508407
106. Proliferating cell nuclear antigen Incyte PD:2781405 144. Human Voltage dependent anion channel form 3
107. ESTs, Highly similar to HIGH MOBILITY GROUP mRNA, complete cds {Incyte PD:2051154}
LIKE NUCLEAR PROTEIN 2Saccharomyces 65 145. H.Sapiens mRNA for translin Incyte PD: 986855}
cerevisiae) {Incyte PD:2669174} 146. Human DEAD-box protein p72 (P72) mRNA, com
108. EST Incyte PD: 1844150} plete cds {Incyte PD: 1750553}
 US 6,485,963 B1
15 16
147. Ras homolog gene family, member G (rho G) {Incyte 8. ESTs, Weakly similar to metaxin H.Sapiens Incyte
PD: 1342744} PD: 1754461}
148. EST Incyte PD: 1377794} 9. Plasminogen Incyte PD:2515873}
149. Human FEZ2 mRNA, partial cds {Incyte PD:2623268} 10. Human mRNA for CC chemokine LARC precursor,
150. Human homolog of Drosophila discs large protein, complete cds {Incyte PD:2220923}
isoform 2 (hdlg-2) mRNA, complete cds {Incyte 11. Synteni: YCFR 21 {YC 21.0062.N)
PD:2203554} 12. Homo Sapiens Amplified in Breast Cancer (AIB1)
151. ALCOHOL DEHYDRO GENASE { Incyte mRNA, complete cds Incyte PD:2634.478
PD: 1634342} 13. Homo Sapiens clone 24640 mRNA sequence Incyte
152. 3-hydroxymethyl-3-methylglutaryl-Coenzyme A lyase
(hydroxymethylglutaricaciduria) {Incyte PD: 1695917 PD: 1560143}
153. ENOYL-COA HYDRATASE, MITOCHONDRIAL 14. Synteni: YCFR 21 {YC 21.2000.N}
PRECURSOR (Incyte PD:2235870} 15. EST Incyte PD: 143912}
154. Proteasome (proSome, macropain) Subunit, beta type, 6 16. Human transcription factor SIM2long form mRNA,
{Incyte PD:2989852} complete cds {Incyte PD:996.104}
155. INTERFERON GAMMA UP-REGULATED I-5111
15 17. EST Incyte PD:284.1478}
PROTEIN PRECURSOR (Incyte PD:2211625} 18. PUTATIVE DNA BINDING PROTEIN A20 Incyte
156. Epimorphin Incyte PD:3438987 PD: 1878791}
157. H.Sapiens RY-1 mRNA for putative nucleic acid bind 19. Protein tyrosine phosphatase, receptor type, mu polypep
ing protein Incyte PD: 1805712) tide {Incyte PD:987736}
158. EST Incyte PD: 1905120} 20. Human clone A9A2BRB5(CAC)n/(GTG)n repeat
159. KD HOUSEKEEPING PROTEIN Incyte containing mRNA Incyte PD: 1987975}
PD: 1819287) 21. Endothelin converting enzyme 1 Incyte PD: 1963819.
160. Cytochrome c oxidase subunit VIIb Incyte 22. BB1 (Incyte PD: 1966148}
23. Pleiotrophin (heparin binding growth factor 8, neurite
PD:2060789} 25 growth-promoting factor 1) {Incyte PD:2989411}
161. EST Incyte PD:661516} 24. Argininosuccinate synthetase Incyte PD: 1981145}
162. Homo Sapiens nuclear VCP-like protein NVLp.2 25. Human breast epithelial antigen BA46mRNA, complete
(NVL.2) mRNA, complete cds {Incyte PD: 1445507 cds {Incyte PD: 1319020}
163. EST Incyte PD: 1251588} 26. Human clone 46690 brain expressed mRNA from chro
164. EST Incyte PD:1665871} mosome X Incyte PD: 1669780
165. Homo Sapiens inositol polyphosphate 4-phosphatase 27. Human plectin (PLEC1) mRNA, complete cds {Incyte
type 11-alpha mRNA, complete cds {Incyte PD: 1907232}
PD:3032739} 28. Homo Sapiens mRNA for calmegin, complete cols
166. Homo Sapiens arsenite translocating ATPase (ASNA1) {Incyte PD:2498216}
mRNA, complete cds {Incyte PD: 1666094 35 29. EST Incyte PD:769182}
167. Human SnRNP core protein Sm D3 mRNA, complete 30. Amyloid beta (A4) precursor-like protein 2 Incyte
cds {Incyte PD: 1624865} PD:3876715}
168. Homo Sapiens clone 23777 putative taansmembrane 31. Polymerase (RNA) II (DNA directed) polypeptide A
GTPase mRNA, partial cds {Incyte PD:2554541} (220 kD) {Incyte PD: 1382059}
169. Homo Sapiens regulator of G protein signaling RGS12 40 32. GLUCOSE TRANSPORTERTYPE 3, BRAIN (Incyte
(RGS) mRNA, complete cds {Incyte PD:3618382} PD:2745082}
170. Human Ki nuclear autoantigen mRNA, complete cds 33. Homo Sapiens sarco-/endoplasmic reticulum Ca-ATPase
{Incyte PD: 1308112} 3 (ATP2A3) mRNA, alternatively spliced, partial cds
171. Homo Sapiens peroxisomal phytanoyl-CoA alpha {Incyte PD:688411}
hydroxylase (PAHX) mRNA, complete cds {Incyte 45 34. Human c-jun proto oncogene (JUN), complete cds, clone
PD:4073867) hCJ-1 (Incyte PD: 1969563
172. PLACENTAL CALCIUM-BINDING PROTEIN
{Incyte PD: 1222317) 35. Microtubule-associated protein 1A Incyte PD:702684}
173. PRE-MRNA SPLICING FACTOR SF2, P32 SUB 36. Clusterin (complement lysis inhibitor, testosterone
repressed prostate message 2, apolipoprotein J) {Incyte
UNIT PRECURSOR (Incyte PD: 1552335} 50 PD:2966620}
174. Human clone C4E 1.63 (CAC)n/(GTG)n repeat 37. NADH-CYTOCHROME B5 REDUCTASE {Incyte
containing mRNA Incyte PD: 1928789} PD: 1901142)
175. Human glioma pathogenesis-related protein (GliPR) 38. Protein-tyrosine kinase 7 Incyte PD:996229}
mRNA, complete cds {Incyte PD:477045} 39. Alpha-1 type XVI collagen Incyte PD: 1963.529
176. Homeo box A9 (Incyte PD:459651} 55 40. EST Incyte PD:2839121}
TABLE 6 41. Homo Sapiens mRNA for DEC 1, complete cds {Incyte
Waveform Up Regulated Genes in Ascending Order (Lowest PD: 1732479}
to Highest) 42. Human endogenous retroviral protease mRNA, com
1. NEUROMEDINB PRECURSOR (Incyte PD:2754315} plete cds {Incyte PD: 1347636}
2. Synteni: YCFR 21 {YC 21.0031.N} 60 43. ATPase, Na+/K+transporting, alpha 1 polypeptide
3. ATRIAL NATRTC PEPTIDE CLEARANCE RECEP {Incyte PD: 1730609}
TOR PRECURSOR (Incyte PD: 1353606} 44. Laminin,alpha 4 Incyte PD:1851696
4. Synteni: YCFR 85 {YC 85.2000.Y} 45. Hexabrachion (tenascin C, cytotactin) Incyte
5. Homo Sapiens CHD3mRNA, complete cds {Incyte PD: 1453450}
PD: 1965248} 65 46. Human mRNA for KIAA0325 gene, partial cds {Incyte
6. EST Incyte PD:565872} PD: 1995315}
7. Synteni: YCFR 46 Cy3 {YC 46.2000. Z} 47. Integrin beta-5 subunit {Incyte PD:418731}
 US 6,485,963 B1
17 18
48. Microfibrillar-associated protein 4{Incyte PD: 1659231} 87. Synteni: YCFR 45 (YC 45.2000.X}
49. Fibulin 1 (Incyte PD: 1320658 88. Synde can 4 (amphiglycan, ryudocan) Incyte
50. Protein serine/threonine kinase stk2 Incyte PD:3214670}
PD: 1518981} 89. Synteni: YCFR 21 {YC 21.0500. N}
51. ESTs, Weakly similar to HYPOTHETICAL 16.1 KD 90. Human pre-B cell enhancing factor (PBEF) mRNA,
PROTEIN IN SEC 17-QCR1 INTERGENIC REGION complete cds {Incyte PD: 1641590}
Saccharomyces cerevisiae) {Incyte PD: 1923722} 91. Cytochrome P450, subfamily IIC (mephenytoin
52. Homo Sapiens carbonic anhydrase precursor (CA 12) 4-hydroxylase) Incyte PD: 168865}
mRNA, complete cds {Incyte PD:3766382} 92. Latent transforming growth factor beta binding protein
53. H. Sapiens mRNA for SIX1 protein Incyte 1{Incyte PD: 1313183}
PD:3208486) 93. Lysyl hydroxylase Incyte PD: 1759127
54. Plasminogen activator inhibitor, type I {Incyte 94. Human mRNA for KIAA0230 gene, partial cds {Incyte
PD: 1445767} PD: 1449824}
55. Human mRNA for SHPS-1, complete cds {Incyte 95. Human mRNA for dihydropyrimidinase related protein
PD:2180684} 15 2, complete cds {Incyte PD:2784546}
56. Collagen, type V, alpha 1 Incyte PD: 1672442 96. H.Sapiens garp gene mRNA, complete CDS Incyte
57. Homo Sapiens monocarboxylate transporter (MCT3) PD:3572014}
mRNA, complete cds {Incyte PD: 1343253} 97. EST Incyte PD:724880}
58. Human follistatin gene Incyte PD: 1577614} 98. ESTs, Weakly similar to TRANSMEMBRANE PRO
59. Human putative RNA binding protein (RBP56) mRNA, TEIN SEX PRECURSOR H. sapiens Incyte
complete cds {Incyte PD: 1907369 PD: 1511346}
60. Homo Sapiens mRNA for PRP8 protein, complete cds 99. Human contactin associated protein (Caspr) mRNA,
{Incyte PD:3616229} complete cds {Incyte PD:2309047
61. Homo Sapiens CAGH13 mRNA, complete cds {Incyte 100. Human cysteine-rich fibroblast growth factor receptor
PD: 1432042} 25 (CFR-1) mRNA, complete cds {Incyte PD:2204871}
62. EST Incyte PD:2953888} 101. EST Incyte PD:2580841}
63. Intercellular adhesion molecule 1 (CD54), human rhi 102. Collagen, type V, alpha Incyte PD: 1887959}
novirus receptor Incyte PD: 1556061 103. H.Sapiens RNA for type VI collagen alpha3 chain
64. Human p120E4F transcription factor mRNA, complete {Incyte PD: 1314882}
cds {Incyte PD: 1940164} 104. Protein kinase C substrate 80K-H (Incyte
65. Collagen, type VI, alpha 1 Incyte PD:2672056} PD: 1723971}
66. Human mRNA for pM5 protein Incyte PD: 1578951} 105. Fibrillin 1 (Marfan syndrome) {Incyte PD: 1448051
67. ALZHEIMERS DISEASE AMYLOID A4 PROTEIN 106. Collagen, type XI, alpha 1 Incyte PD:3598222}
PRECURSOR (Incyte PD: 126370} 107. H.Sapiens mRNA for extracellular matrix protein col
68. Human mRNA for KIAA0062 gene, partial cds {Incyte 35 lagen type)aV, C-terminus Incyte PD:2208990}
PD:3138128} 108. Collagen, type II, alpha 1 (primary osteoarthritis,
69. Human clone HSH1 HMG CoA synthase mRNA, partial spondyloepiphyseal dysplasia, congenital) {Incyte
cds {Incyte PD: 1807407} PD:2518178}
70. Filamin 1 (actin-binding protein-280) {Incyte 109. ESTs, Weakly similar to unknown S.cerevisiae
PD: 1708528} 40 {Incyte PD:2171401}
71. Synteni: YCFR 85 {YC 85.2000.X} 110. EST Incyte PD: 1923572}
72. Synteni: YCFR 46 Cy3 {YC 46.2000.W} 111. Human insulin-like growth factor binding protein 5
73. Homologue of mouse tumor rejection antigen gp96 (IGFBP5) mRNA (Incyte PD: 1686585}
{Incyte PD:2679349} 112. Human mRNA for KIAA0242 gene, partial cds {Incyte
74. Tissue inhibitor of metalloproteinase 3 (Sorsby fundus 45 PD: 1940994}
dystrophy, pseudoinflammatory) Incyte PD:418041 113. Complement component 1, S Subcomponent Incyte
75. Human XMP mRNA, complete cds {Incyte PD: 1904751}
PD: 1887661} 114. Human chromosome 17 unknown product mRNA,
76. Cytochrome P450, Subfamily XIA (cholesterol side complete cds {Incyte PD:2849603}
chain cleavage) Incyte PD:2368282} 50 115. Homo Sapiens lySOSomal pepstatin insensitive protease
77. Granulin Incyte PD:812141 (CLN2) mRNA, complete cds {Incyte PD:3500996}
78. Human extracellular matrix protein 1 (ECM1) mRNA, 116. Collagen, type IV, alpha 2 Incyte PD: 1906574}
complete cds {Incyte PD: 1965806} 117. ESTs, Moderately similar to ZINC FINGER PROTEIN
79.78 KID GLUCOSE REGULATED PROTEIN PRECUR HF.12 Homo sapiens Incyte PD:3729155}
SOR (Incyte PD:2884613} 55 118. Homo Sapiens stanniocalcin precursor (STC) mRNA,
80. Synteni: YCFR 21 {YC 21.2000.X} complete cds {Incyte PD:2222921
81. Homo Sapiens mRNA for serin protease with IGF 119. P55-C-FOS PROTO-ONCOGENE PROTEIN Incyte
binding motif, complete cds {Incyte PD: 1958902} PD:341491}
82. Inhibitor of DNA binding 1, dominant negative helix 120. EST Incyte PD:2424631}
loop-helix protein Incyte PD: 1687060} 60 121. EST Incyte PD: 1940710}
83. Solute carrier family 6 (neurotransmitter transporter, 122. Thrombospondin 1 {Incyte PD:2055534}
taurine), member 6 Incyte PD: 1516886. 123. Complement component C1r Incyte PD: 1664320
84. Hormone receptor (growth factor-inducible nuclear pro 124. REGULATOR OF G-PROTEIN SIGNALLING
tein N10) {Incyte PD: 1958,560} 2{Incyte PD: 1218114}
85. Fibulin 2 Incyte PD: 1901.095} 65 125. INTEGRAL MEMBRANE PROTEIN E16 Incyte
86. Kinase insert domain receptor (a type III receptor PD: 1911012}
tyrosine kinase) Incyte PD:2220338} 126. Collagen, type I, alpha 1 Incyte PD:782235}
 US 6,485,963 B1
19 20
127. H.Sapiens mRNA for adipophilin Incyte PD: 1985104} about 1,000 mA, in the present embodiment. The time
128. EST Incyte PD: 1979450} varying current is Suitably an alternating current, as indi
129. EST Incyte PD:690994} cated above, although in other embodiments it is a pulsating
130. Cathepsin D (lysosomal aspartyl protease) {Incyte DC current. The current is conducted from the Source 10
PD:3940755} along first and Second conductorS 13A and 13B to Slip rings
131. Matrix metalloproteinase 2 (gelatinase A, 72 kD 4. The slip rings are non-rotatable relative to the vessel 5,
gelatinase, 72 kD type IV collagenase) {Incyte and therefore rotate with the vessel during operation. Cur
PD: 1558081} rent received through conductors 13A and 13B is conducted
132. Cyclin D2 Incyte PD: 1618422} through the associated Slip rings to first and Second Sets of
133. EST Incyte PD:2636514} conductors, represented by first and Second conductors 14A
134. COMPLEMENT C3 PRECURSOR Incyte and 14B, which are preferably insulated with an insulative
PD: 1513989} material, not shown, compatible with the fluids and products
135. Homo Sapiens secreted frizzled related protein mRNA, within the bioreactor chamber. Each conductor is mechani
complete cds {Incyte PD:428236 cally connected to a respective peripheral portion of each of
136. INSULIN-LIKE GROWTH FACTOR BINDING 15
the respective discs, and electrically connected with an end
PROTEIN 3 PRECURSOR (Incyte PD: 1447903} portion of one of the conductive strips 6 (FIGS. 1A and 1B).
137. Fibronectin 1 {Incyte PD:3553729} As viewed in FIG. 10, conductor 14A and the associated slip
138. Early growth response protein 1 Incyte PD: 1705208} ring are indicated to be of positive (+) polarity, and conduc
139. Human autoantigen DFS70 mRNA, partial cds {Incyte tor 14 B and its associated Slip ring are indicated to be of
PD:42920} relatively negative (-) polarity. AS Suggested above, upon
140. Prostaglandin-endoperoxide Synffiase 2 (prostaglandin the current changing in polarity, conductor 14A will momen
G/HSynthase and cyclooxygenase) Incyte PD:3139163} tarily have a negative potential relative to conductor 14B,
141. Synteni: YCFR 43 {YC 43.2000.W} thereby permitting a time varying current, which in this
142. Synteni: YCFR 43 {YC 43.2000.Z} embodiment is an alternating current, to flow through con
143. Synteni: YCFR 23 {YC 23.0062.N} 25 ductor 6 which. In other embodiments, the time-varying
144. Synteni: YCFR 43 {YC 43.2000.Y} current may be in the form of a pulsating DC current,
145. Homo Sapiens phosphomevalonate kinase mRNA, Suitably a Square wave or other waveform, rather than an
complete cds {Incyte PD: 1497123} alternating current, in which case the conductorS 14A and
146. Synteni: YCFR 43 {YC 43.2000.X} 14B and their associated slip rings remain of the same
147. Synteni: YCFR 23 {YC 23.0031.N} polarity but of differing potentials.
148. CARTILAGE GLYCOPROTEIN-39 PRECURSOR
{Incyte PD: 157510} After inoculation, the rotating wall vessel 5 is rotated at
149. Synteni: YCFR 23 {YC 23.0125.N} an appropriate Speed and Single cellular material begins to
150. Synteni: YCFR 23 {YC 23.0250.N} attach onto the Surface of the biocompatable material 6.
151. Synteni: YCFR 23 {YC 23.4000.N} After initial growth of one 24- or 48-hour period, electrical
152. Human germline oligomeric matrix protein (COMP)
35 Stimulation, i.e., potentiation, begins via continuous low
mRNA, complete cds {Incyte PD:2636634} level or pulsatile electrical flow through each disk in Series.
Discussion
EXAMPLE 7 Use of the methods of the present invention to control the
Prototype of the System proliferative rate of normal human adult astrocytes and
FIGS. 10 and 11 are partially diagrammatic representa 40 normal human neural progenitor cells (NHNP) has been
tions of one embodiment of the system 1. Spherical disks 2 demonstrated. The procedure is applicable to, but not limited
of biocompatable material are arranged along the horizontal to, the control of normal human neural cells in both two
Spin filter or horizontal oxygenator of a Standard rotating dimensional and threeimensional culture. AS presented in
wall perfused vessel or rotating wall batch-fed vessel 5. the molecular genetic data shown in Table 5 and Table 6,
The electrically conductive, bioattractive Strips 6 are each 45 many of the genetic responses in both up regulated and down
suitably embedded in or affixed to each of the disks such that regulated genes are maturation and growth regulatory in
each disk has a positive and negative pole 12, associated nature. An inspection reveals these genes are also primarily
with positive and negative terminals 12 connected to respec involved in the eipbryogenic process. Therefore it is rea
tive endportions of the strips, as shown in FIG. 11A. As will Sonable to conclude that control over the embryogenic
be more fully described, in one preferred embodiment in 50 development process can be achieved via the presently
which an alternating current is applied to the Strips, the demonstrated methodology.
polarity of the Strips changes cyclically in correspondence AS Shown in Table 6, Specific genes Such as human
with the change in polarity of the applied current. Each germline oligomeric matrix protein, proStaglandin endoper
biocompatable disk is preferably two-sided, allowing oxide Synthase 2, early growth response protein 1, and
growth of tissue on both the left and right portions of the 55 insulin like growth factor binding protein 3 precursor are
spherical disk. Sterilization of the reactor core is effected by highly up regulated, while Keratin Type II cytoskellatal 7,
one of multiple Sterilization procedure, either ethylene oxide mytotic kinesin like protein 1, transcription factor 6 like 1,
Sterilization, autoclave Sterilization if the polymer perrnits, mytotic feedback control protein, and cellular retinoic acid
or in the case of the batch-fed vessel, sterilization with binding protein are down regulated (Table 5). Each of these
hydrogen peroxide. After Sterilization and Sufficient detoxi 60 two sets or classes of genes are only examples from the Sum
fication procedures, cellular material is Seeded into the of approximately 320 genes changes expressed as a conse
reactor at a level to be determined according to the cell line quence of exposure to electrical potentiation.
of interest. AS is clearly demonstrated in the human body, the
A Source of time varying current 10, Suitably a laboratory bioelectric, biochemical process of electrical nerve Stimula
current Source with adjustable wave-forn output connected 65 tion is a documented reality. The present invention demon
to a remote power Source, not shown, is operable to provide Strates that the same phenomena can be potentiated in a
a time varying current, Suitably of a value of about 1 mA to Synthetic atmosphere, i.e., in rotating wall cell culture ves
 US 6,485,963 B1
21 22
Sels. AS may be understood from the forgoing discussion, wherein Said electrical current flows through Said channels
this electrical potentiation can be used for a number of along Said longitudinal axes.
purposes. 5. The system of claim 1, wherein said bioattractive
The following references were cited herein. material is Selected from the group consisting of titanium,
1. Schwarz et al., U.S. Pat. No. 4,988,623, (1991). Zirconium and platinum.
2. Schwarz et al., U.S. Pat. No. 5,026,650, (1991). 6. The system of claim 1, wherein said biological cells
3. Goodwin, et al., In Vitro Cell Dev. Biol., 28A:47–60 comprise mammalian cells Selected from the group consist
(1992). ing of neuronal cells, normal human neuronal progenitor
4. Goodwin, et al., Proc. Soc. Exp. Biol. Med., 202:181-192 cells and cells responding to waveform.
(1993). 7. The system of claim 1, wherein said rotating wall vessel
5. Goodwin, et al., J. Cell Biochem., 51:301–311 (1993). is Selected from the group consisting of a rotating wall
6. Goodwin, et al., In Vitro Cell Dev. Biol. Anim., perfused vessel and a rotating wall batch-fed vessel.
33:366-374 (1997). 8. The system of claim 1, wherein said electrical current
7. Fukuda et al., U.S. Pat. No. 5,328,843
8. Aebischer, U.S. Pat. No. 5,030,225 15 induces a cellular response at the gene level.
Any patents or publications mentioned in this specifica 9. The system of claim 8, wherein said cellular response
tion are indicative of the levels of those skilled in the art to is cellular control of growth and differentiation at the gene
which the invention pertains. These patents and publications level.
are herein incorporated by reference to the same extent as if 10. The system of claim 1, wherein said electrical current
each individual publication was specifically and individually induces cellular control of growth and differentiation, for
indicated to be incorporated by reference. Suppressing or enhancing growth regulatory functions at
One skilled in the art will readily appreciate that the gene level.
present invention is well adapted to carry out the objects and 11. The System of claim 10, wherein Said gene is associ
obtain the ends and advantages mentioned, as well as those ated with embryogenesis.
inherent therein. The present examples along with the 25 12. The system of claim 1, wherein said electrical current
methods, procedures, treatments, molecules, and Specific is associated with a time varying potential.
compounds described herein are presently representative of 13. The system of claim 12, wherein said time varying
preferred embodiments, are exemplary, and are not intended electrical potential is in the form of a Square wave.
as limitations on the Scope of the invention. Changes therein 14. A method of culturing biological cells in the System of
and other uses will occur to those skilled in the art which are
claim 1, comprising the Steps of
encompassed within the Spirit of the invention as defined by
the Scope of the claims. Inoculating Said cells into Said vessel;
What is claimed is: rotating said vessel to initiate the attachment of Said cells
1. A System for growing three-dimensional biological to cell carriers,
cells, comprising: 35 applying an electrical potential to Said cell carriers, and
a rotating wall vessel containing a cell-rich medium; measuring the growth of Said cells.
cell carriers placed within Said vessel; and 15. The method of claim 14, wherein said vessel is rotated
an electrical current passed through said cell carriers for at a speed of from about 10 RPM to about 30 RPM.
producing an electromagnetic field within Said cell-rich 40 16. The method of claim 15, wherein said electrical
medium. potential is associated with a current applied at a strength
2. The System of claim 1, wherein Said cell carriers are range of from about 1 mA to about 6 mA.
Selected from the group consisting of Spherical disks and 17. The method of claim 14, wherein said electrical
bioScaffold matrices containing multiple parallel channels. potential is a time varying potential.
3. The System of claim 2, wherein Said channels are 45 18. The method of claim 17, wherein said time varying
coated with a bioattractive material. potential is in a Square wave.
4. The System of claim 3, wherein Said channels, coated
with Said bioattractive material, have longitudinal axes, and k k k k k
 UNITED STATES PATENT AND TRADEMARK OFFICE
CERTIFICATE OF CORRECTION

PATENT NO. : 6,485,963 B1 Page 1 of 1

APPLICATIONNO. : 09/587028
DATED : November 26, 2002
INVENTOR(S) : Wolfetal.

It is certified that error appears in the above-identified patent and that said Letters Patent is
hereby corrected as shown below:

On the Title page, Item (75) should read,

David A. Wolf, Houston, Texas
Thomas J. Goodwin, Houston, Texas
Robert G. Dennis, Chapel Hill, North Carolina

Signed and Sealed this

Twenty-fourth Day of June, 2008

WDJ
JON. W. DUDAS
Director of the United States Patent and Trademark Office