U.S. patent application number 13/859206 was filed with the patent office on 2013-10-10 for methods for culturing cells in an alternating ionic magnetic resonance (aimr) multiple-chambered culture apparatus.
The applicant listed for this patent is Thomas J. Goodwin, Moses J. Kushman. Invention is credited to Thomas J. Goodwin, Moses J. Kushman.
Application Number | 20130267003 13/859206 |
Document ID | / |
Family ID | 49292593 |
Filed Date | 2013-10-10 |
United States Patent
Application |
20130267003 |
Kind Code |
A1 |
Goodwin; Thomas J. ; et
al. |
October 10, 2013 |
Methods for Culturing Cells in an Alternating Ionic Magnetic
Resonance (AIMR) Multiple-Chambered Culture Apparatus
Abstract
Provided herein are methods for culturing cells, tissues or
organoid bodies in the presence of a pulsating alternating ionic
magnetic resonance field and models comprising a tissue-like
assembly of the cells so cultured. The cells, tissues or organoid
bodies are introduced into a culture unit comprising growth and
nutrient modules in which the gravity vector of the growth unit is
continually randomized and cultured in the presence of the
alternating ionic magnetic resonance field.
Inventors: |
Goodwin; Thomas J.; (Kemah,
TX) ; Kushman; Moses J.; (Porter Ranch, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Goodwin; Thomas J.
Kushman; Moses J. |
Kemah
Porter Ranch |
TX
CA |
US
US |
|
|
Family ID: |
49292593 |
Appl. No.: |
13/859206 |
Filed: |
April 9, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61686690 |
Apr 9, 2012 |
|
|
|
Current U.S.
Class: |
435/173.8 ;
435/173.1; 435/394 |
Current CPC
Class: |
C12N 2513/00 20130101;
C12M 35/04 20130101; C12M 23/24 20130101; C12N 5/0062 20130101;
C12N 2529/00 20130101; C12M 35/02 20130101; C12M 27/10 20130101;
C12M 27/20 20130101; C12M 29/04 20130101; C12M 23/28 20130101; C12M
35/06 20130101; C12N 13/00 20130101 |
Class at
Publication: |
435/173.8 ;
435/173.1; 435/394 |
International
Class: |
C12N 13/00 20060101
C12N013/00 |
Claims
1. A method for culturing cells, comprising the steps of:
introducing cells into a culture unit having a growth module and a
nutrient module; randomizing continually the gravity vector of the
growth module; and applying a pulsating alternating ionic magnetic
resonance field to the growth module during culturing of the
cells.
2. The method of claim 1, wherein the introducing step comprises:
placing the cells into the growth module; filling the growth module
with growth media; sealing the growth module to remove observable
gases; attaching the growth module to the nutrient module; and
adding fresh growth medium to the nutrient module.
3. The method of claim 1, wherein the randomizing step comprises:
positioning the nutrient module comprising the culture unit into a
randomizing adapter; and randomizing the gravity vector via a
randomizing mechanism comprising the randomizing adapter.
4. The method of claim 1, wherein the applying step comprises:
positioning the growth module comprising the culture unit into an
electromagnetic chamber; and generating the pulsating alternating
ionic magnetic resonance field in the electromagnetic chamber.
5. The method of claim 1, further comprising the step of:
modulating the alternating ionic magnetic resonance field to
produce overlapping or fluctuating alternating ionic magnetic
resonance frequencies at one or more modal intervals spanning about
6.5 Hz and ranging from about 7.8 Hz to about 59.9 Hz.
6. The culture system of claim 5, wherein the overlapping or
fluctuating alternating ionic magnetic resonance frequencies
produced are about 10, 14, 15, 16, or 32 Hz.
7. The method of claim 1, wherein the nutrient module comprises: an
open-ended body having a proximal end with a diameter of a length
to receive the growth module therein; a first gas-permeable
membrane with a gas port disposed thereon comprising the proximal
end; and a distal end comprising a first sealable opening.
8. The method of claim 7, wherein the growth module comprises: a
body having a proximal end comprising a second gas-permeable
membrane with a plurality of inlet/outlet ports disposed thereon;
and a distal end comprising a baffling system and a semi-permeable
membrane in fluid contact with the first gas-permeable
membrane.
9. The method of claim 1, wherein the growth module and the
nutrient module are disposable.
10. The method of claim 1, wherein the alternating ionic magnetic
resonance field produces one or more of a modification, growth,
differentiation, dedifferentiation, altered gene expression,
altered transcription events or cellular regeneration via
controlling the ionic transport or transcription mechanism in the
cells.
11. The method of claim 1, wherein the alternating ionic magnetic
resonance field changes the mammalian cellular ionic transport
thereby enabling infection and an expression of mammalian viral
epitopes.
12. The method of claim 1, wherein the cells comprise a tissue, an
organoid body, a virally-infected cell, or a bacterially-infected
cell.
13. A culture method for growing mammalian cells or tissues,
comprising the steps of: introducing mammalian cells or tissues
into an alternating ionic magnetic resonance culture apparatus
comprising: a nutrient module, having a proximal end and a sealable
distal end, that contains a nutrient media; a growth module, having
a proximal end and a distal end, that contains the cells or tissue
and that is filled with nutrient media and sealed to remove
observable gases, said distal end of the growth module fluidly
sealed to the proximal end of the nutrient module; a randomizing
adapter electrically connected to a randomizing mechanism and
containing the nutrient module therein; and an electromagnetic
chamber comprising a conductive wire to produce a pulsating
alternating ionic magnetic resonance field; randomizing,
continually, the gravity vector of the growth module with the
randomzing adapter; and generating the pulsating alternating ionic
magnetic resonance field around the growth module during
culturing.
14. The method of claim 13, further comprising the step of:
modulating the alternating ionic magnetic resonance field to
produce overlapping or fluctuating alternating ionic magnetic
resonance frequencies at one or more modal intervals spanning about
6.5 Hz and ranging from about 7.8 Hz to about 59.9 Hz.
15. The method of claim 14, wherein the overlapping or fluctuating
alternating ionic magnetic resonance frequencies produced are about
10, 14, 15, 16, or 32 Hz.
16. The method of claim 13, wherein the nutrient module comprises:
a first gas-permeable membrane with a gas port disposed thereon
comprising the proximal end.
17. The method of claim 16, wherein the growth module comprises: a
second gas-permeable membrane with a plurality of inlet/outlet
ports disposed thereon comprising the proximal end; and a baffling
system and a semi-permeable membrane comprising the distal end in
fluid contact with the first gas-permeable membrane.
18. The method of claim 13, wherein the growth module and the
nutrient module are disposable.
19. The method of claim 13, wherein materials comprising the
alternating ionic magnetic resonance culture apparatus are
sterilizable.
20. The method of claim 13, wherein culturing in the alternating
ionic magnetic resonance field produces one or more of a
modification, growth, differentiation, dedifferentiation, altered
gene expression, altered transcription events or cellular
regeneration via controlling the ionic transport or transcription
mechanism in the cells or tissues.
21. The method of claim 13, wherein culturing in the alternating
ionic magnetic resonance field produces a change in the mammalian
cellular ionic transport thereby enabling infection and an
expression of mammalian viral epitopes.
22. The method of claim 13, wherein the mammalian cells or tissues
are virally or bacterially infected.
23. A model comprising a tissue-like assembly of the cultured
mammalian cells or tissue produced by the method of claim 13.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This non-provisional application claims benefit of priority
under 35 U.S.C. .sctn.119(e) of provisional application U.S. Ser.
No. 61/686,690, filed Apr. 9, 2012, now abandoned, the entirety of
which is hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to the fields of
biophysics, bioelectromechanics, bioengineering, tissue engineering
and cellular regeneration. Specifically, the present invention
relates to an alternating ionic magnetic resonance
multiple-chambered culture apparatus for potentiating or
controlling the growth of biological cells and tissues, such as
mammalian tissue.
[0004] 2. Description of the Related Art
[0005] Prior to the development of bioreactors, cell culture was
limited to systems subjected to the forces of gravity, with most
laboratory cultures producing flat two-dimensional (2D), one cell
thick specimens unlike the natural three-dimensional (3D)
environment of a complex, multi-cellular organism. Most laboratory
experiments therefore had inherent limitations and a strictly
one-dimensional view of understanding how cells grew and interacted
with one another in their natural environment.
[0006] With the development of bioreactors, most of these devices
were "stirred tank" bioreactors that used a vertical aspect
configuration with a stirring device at the bottom of the growth
chamber to mix the cells and fluid medium suspension. Horizontally
rotating bioreactors offered a way to minimize or neutralize the
sedimentation and shear effects caused by gravity by using the
"clinostat principal" in which a fluid medium was rotated about a
horizontal axis thus minimizing the wall effects and impeller
impacts of internal stirring devices and lowering the overall
Reynolds and Coriolis force effect on cells.
[0007] Cells grown in rotating bioreactors were suspended in a
fluid medium and were continually rotated away from the surfaces of
the vessel which enabled cells to adhere to one another and to
grow. This type of suspended cell culture resembled growth
mechanics in a naturally occurring tissue and in a multidimensional
form and thereby promoted more realistic, three-dimensional
cell-to-cell contact signaling. These 3D cells were induced to
regulate and to produce cellular components as if grown within a
complex organism and to produce complex matrices comprising
extracellular matrix molecules, proteins, fibers, and other
cellular components. These aforementioned processes lead to
autoregulation and the ability to self-order in the human mammalian
physiology. Inside a complex organism, these components often
informed a cell of the neighboring environment and triggered a
specific set of responses to that external environment. The cell
grew or it became static, which in turn, determined how the cell
responded with the production of secondary regulators.
[0008] A typical rotating bioreactor had an outer tubular enclosure
with transverse end walls and end caps in the end walls. The outer
tubular enclosure was supported on input and output shaft members
and rotationally driven by an independent drive mechanism.
Coaxially disposed within the outer tubular enclosure was a central
tubular filter member that was rotationally supported on the input
shaft and coupled to the output shaft. The annular space between
the inner and outer tubular members defined a cell culture
chamber.
[0009] Two blade members were positioned about the horizontal axis
and extended lengthwise along the cell culture chamber. The blade
members had radial arms at one end that were rotationally supported
on the output shaft and radial arms at the other end that were
coupled to the input shaft. The input shaft was rotationally driven
by an independent drive means that normally drove the inner and
outer tubular members and the blade members at the same angular
rate and direction so that no relative motion occurred between
these members. Thus, clinostat motion could be achieved for the
particles in the fluid within the cell culture chamber.
[0010] Existing bioreactors, however, are overly complicated
systems and costly to operate with respect to expenditure of
preparation time, upkeep, disposal of non-reusable components. For
example, in existing bioreactors the cell samples and any other
required initial ingredients must be assembled into the culture
system in preparation for an experiment. At the end of the
experiment, it is necessary to disassemble significant portions of
the culture system in order to extract the cells and/or tissue
culture that grew during the experiment. Moreover, because the
culture system mechanisms include rotating fluid couplings, leaks
may develop over time in the seals of these couplings. Finally, the
motors that provide the rotation in existing bioreactors are
integral to the system, contributing to the complexity of the
system and making it difficult to maintain and operate the system.
Accordingly, what is needed is a culture system and method that
mitigates or overcomes some or all of the shortcomings of existing
bioreactors.
[0011] U.S. Pat. Nos. 6,485,963 and 6,673,597 disclose the use of a
time-varying electromagnetic force (TVEMF) in a manner that
stimulates the proliferation of cells grown in culture. In U.S.
Pat. No. 7,179,217, Goodwin et al. disclose the use of a TVEMF
sleeve for treatment of an animal limb. Commercial utilization of
this technology has provided two approaches to culture system
design. The first approach is the use of baffles or plates within
the culture system with a time-varying electromagnetic current
applied across the plates to induce a time-varying electromagnetic
force within the culture chamber. The second approach is to use a
coil wrapped around the rotating culture system chamber and affixed
thereto with a time-varying electromagnetic current applied to the
coil to create a time-varying electromagnetic force within the
culture chamber.
[0012] There are several limitations with existing culture systems
designs that utilize TVEMF in the context of a rotating culture
system chamber. First, the existing TVEMF culture systems have the
electromagnetic device permanently affixed to the culture chamber
unit, which does not allow for the use of disposable modules nor
does it accommodate the self-feeding capability of the current
invention. Instead, existing systems require periodic and frequent
manual exchange of growth media during the culture cycle.
Additionally, since the goal of proliferation of cell cultures is
in many instances the utilization of the cells and tissues for
reintroduction into the human body for tissue regeneration or
treatment of human maladies, the culture system chamber must meet
the rigid standards of the Food and Drug Administration (FDA). If
the EMF inducing device is incorporated into the culture chamber,
it significantly complicates the manufacture and sterilization
process, and would require routine disposal of the EMF inducing
device along with the used culture system chamber. This would
significantly add to the cost of the equipment and culturing
process for FDA approved purposes.
[0013] Another limitation of existing culture systems is that they
utilize TVEMF, which does not effectuate the same stimulatory or
physiological effect on cultured cells as compared with alternating
ionic magnetic resonance. TVEMF fails to stimulate specific ionic
species and membrane channel systems that play a major role in the
regulation of proliferation, differentiation, tissue repair, and
related cellular mechanisms that are inherent to growth,
development and maintenance of a mammalian organism. A further
limitation is that existing culture systems rely on a batch fed or
media perfusion systems to transfer media into and out of the
growth chamber. Each of these methodologies fails to provide
physiological and homeostatic parameters similar to those of a
naturally occurring physiological system.
[0014] Thus, there is a recognized need in the art for culture
systems and methods that utilize an alternating ionic magnetic
resonance field during the three-dimensional culture of cells,
including tissues and/or organoid bodies. Particularly, the prior
art is deficient in methods for culturing cells, tissues and/or
organoid bodies in an alternating ionic magnetic resonance system
comprising a culturing apparatus that utilizes pre-sterilized and
disposable modules and a removable alternating ionic magnetic
resonance chamber. The present invention fulfills this
long-standing need and desire in the art.
SUMMARY OF THE INVENTION
[0015] The present invention is directed to a method for culturing
cells. The method comprises introducing cells into a culture unit
having a growth module and a nutrient module, randomizing
continually the gravity vector of the growth module and applying a
pulsating alternating ionic magnetic resonance field to the growth
module during culturing of the cells. The present invention is
directed to a related method for culturing cells further comprising
the step of modulating the alternating ionic magnetic resonance
field to produce overlapping or fluctuating alternating ionic
magnetic resonance frequencies at one or more modal intervals
spanning about 6.5 Hz and ranging from about 7.8 Hz to about 59.9
Hz.
[0016] The present invention also is directed to a culture method
for growing mammalian cells or tissues. The method comprises
introducing mammalian cells or tissues into an alternating ionic
magnetic resonance culture apparatus. The alternating ionic
magnetic resonance culture apparatus comprises a nutrient module,
having a proximal end and a sealable distal end, that contains a
nutrient media, a growth module, having a proximal end and a distal
end, that contains the cells or tissue and that is filled with
nutrient media and sealed to remove observable gases, said distal
end of the growth module fluidly sealed to the proximal end of the
nutrient module, a randomizing adapter electrically connected to a
randomizing mechanism and containing the nutrient module therein,
and an electromagnetic chamber comprising a conductive wire to
produce a pulsating alternating ionic magnetic resonance field. The
gravity vector of the growth module is randomized continually with
the randomzing adapter and the pulsating alternating ionic magnetic
resonance field is generated around the growth module during
culturing. The present invention is directed to a related method
for culturing cells further comprising the step of modulating the
alternating ionic magnetic resonance field to produce overlapping
or fluctuating alternating ionic magnetic resonance frequencies at
one or more modal intervals spanning about 6.5 Hz and ranging from
about 7.8 Hz to about 59.9 Hz.
[0017] The present invention is directed further to a model
comprising a tissue-like assembly of the cultured cells or tissue
produced by the methods described herein.
[0018] Other and further aspects, features and advantages of the
present invention will be apparent from the following description
of the presently preferred embodiments of the invention given for
the purpose of disclosure
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] So that the matter in which the above-recited features,
advantages and objects of the invention, as well as others which
will become clear, are attained and can be understood in detail,
more particular descriptions and certain embodiments of the
invention briefly summarized above are illustrated in the appended
drawings. These drawings form a part of the specification. It is to
be noted, however, that the appended drawings illustrate preferred
embodiments of the invention and therefore are not to be considered
limiting in their scope.
[0020] FIGS. 1A-1C is an overview of the unassembled (FIG. 1A),
partially assembled (FIG. 1B) and assembled (FIG. 1C) primary
components of the culture apparatus comprising a culture unit, a
randomizing adaptor and a removable adjustable alternating ionic
magnetic resonance module.
[0021] FIGS. 2A-2B are front (FIG. 2A) and back (FIG. 2B) views of
the growth module.
[0022] FIG. 3 illustrates the assembly of the growth module with
the nutrient module to form the culture unit.
[0023] FIG. 4 illustrate culture cell viability parameters of HBTC
cells grown with alternating ionic magnetic resonance. Growth
module samples were taken prior to weekly changing of the media in
the nutrient module and measurements of glucose utilization and
culture pH v. time were made.
[0024] FIG. 5 is a cell growth and tissue assembly curve for HBTC
cells with and without exposure to alternating ionic magnetic
resonance over a twenty day growth period.
[0025] FIGS. 6A-6D are calcium and potassium ion transport
micrographs of HBTC cells grown in alternating ionic magnetic
resonance culture apparatus. FIG. 6A depicts calcium ion staining
of HBTC cells grown alone exposed (left) and unexposed (right) to
alternating ionic magnetic resonance. FIG. 6B depicts calcium ion
staining of HBTC cells grown on cultisphere microcarriers exposed
(left), unexposed (right) to alternating ionic magnetic resonance
and microcarrier control treated with Fura 2AM. FIG. 6C depicts
potassium ion staining of HBTC cells grown on cultisphere
microcarriers exposed (left) and unexposed (right) to alternating
ionic magnetic resonance. FIG. 6D depicts potassium ion staining of
HBTC cells exposed to alternating ionic magnetic resonance (top),
grown without an electric field (middle) and control microcarriers
alone (bottom).
DETAILED DESCRIPTION OF THE INVENTION
[0026] As used herein, the term "a" or "an", when used in
conjunction with the term "comprising" in the claims and/or the
specification, may refer to "one", but it is also consistent with
the meaning of "one or more", "at least one", and "one or more than
one". Some embodiments of the invention may consist of or consist
essentially of one or more elements, method steps, and/or methods
of the invention. It is contemplated that any device or method
described herein can be implemented with respect to any other
device or method described herein.
[0027] As used herein, the term "or" in the claims refers to
"and/or" unless explicitly indicated to refer to alternatives only
or the alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or".
[0028] As used herein, the term "about" refers to a numeric value,
including, for example, whole numbers, fractions, and percentages,
whether or not explicitly indicated. The term "about" generally
refers to a range of numerical values (e.g., +/-5-10% of the
recited value) that one of ordinary skill in the art would consider
equivalent to the recited value (e.g., having the same function or
result). In some instances, the term "about" may include numerical
values that are rounded to the nearest significant figure.
[0029] As used herein, the terms "proximal" and "distal" refer to
components or parts thereof or fields that are nearer or farther
from the growth module, respectively. With respect to the growth
module per se, proximal refers to the side comprising the gas
membrane and distal refers to the side comprising the baffling that
engages with the nutrient module.
[0030] As used herein, the term "animal" refers to a mammal,
preferably a human.
[0031] In one embodiment of the present invention there is provided
a method for culturing cells, comprising the steps of introducing
cells into a culture unit having a growth module and a nutrient
module; randomizing continually the gravity vector of the growth
module; and applying a pulsating alternating ionic magnetic
resonance field to the growth module during culturing of the cells.
Further to this embodiment the method comprises modulating the
alternating ionic magnetic resonance field to produce overlapping
or fluctuating alternating ionic magnetic resonance frequencies at
one or more modal intervals spanning about 6.5 Hz and ranging from
about 7.8 Hz to about 59.9 Hz. Particularly the overlapping or
fluctuating alternating ionic magnetic resonance frequencies
produced are about 10, 14, 15, 16, or 32 Hz.
[0032] In one aspect of both embodiments the introducing step may
comprise placing the cells into the growth module; filling the
growth module with growth media; sealing the growth module to
remove observable gases; attaching the growth module to the
nutrient module; and adding fresh growth medium to the nutrient
module. In another aspect of both embodiments the randomizing step
may comprise positioning the nutrient module comprising the culture
unit into a randomizing adapter; and randomizing the gravity vector
via a randomizing mechanism comprising the randomizing adapter. In
yet another aspect the applying step may comprise positioning the
growth module comprising the culture unit into an electromagnetic
chamber and generating the pulsating alternating ionic magnetic
resonance field in the electromagnetic chamber.
[0033] In all embodiments and aspects thereof the nutrient module
may comprise an open-ended body having a proximal end with a
diameter of a length to receive the growth module therein; a first
gas-permeable membrane with a gas port disposed thereon comprising
the proximal end; and a distal end comprising a first sealable
opening. Also the growth module may comprise a body having a
proximal end comprising a second gas-permeable membrane with a
plurality of inlet/outlet ports disposed thereon; and a distal end
comprising a baffling system and a semi-permeable membrane in fluid
contact with the first gas-permeable membrane. Particularly, the
growth module and the nutrient module are disposable. In addition
the pulsating alternating ionic magnetic resonance field may
produce one or more of a modification, growth, differentiation,
dedifferentiation, altered gene expression, altered transcription
events or cellular regeneration via controlling the ionic transport
or transcription mechanism in the cells. Furthermore the
alternating ionic magnetic resonance field may change the mammalian
cellular ionic transport thereby enabling infection and an
expression of mammalian viral epitopes. Further still the cells may
comprise a tissue, an organoid body, a virally-infected cell, or a
bacterially-infected cell.
[0034] In another embodiment of the present invention there is
provided a culture method for growing mammalian cells or tissues,
comprising the steps of introducing mammalian cells or tissues into
an alternating ionic magnetic resonance culture apparatus
comprising a nutrient module, having a proximal end and a sealable
distal end, that contains a nutrient media; a growth module, having
a proximal end and a distal end, that contains the cells or tissue
and that is filled with nutrient media and sealed to remove
observable gases, the distal end of the growth module fluidly
sealed to the proximal end of the nutrient module; a randomizing
adapter electrically connected to a randomizing mechanism and
containing the nutrient module therein; and an electromagnetic
chamber comprising a conductive wire to produce a pulsating
alternating ionic magnetic resonance field; randomizing,
continually, the gravity vector of the growth module with the
randomzing adapter; and generating the pulsating alternating ionic
magnetic resonance field around the growth module during culturing.
Further to this embodiment the method comprises modulating the
alternating ionic magnetic resonance field, as described supra.
[0035] In both embodiments the nutrient module may comprise a first
gas-permeable membrane with a gas port disposed thereon comprising
the proximal end. Also the growth module may comprise a second
gas-permeable membrane with a plurality of inlet/outlet ports
disposed thereon comprising the proximal end and a baffling system
and a semi-permeable membrane comprising the distal end in fluid
contact with the first gas-permeable membrane. Particularly, the
growth and/or the nutrient modules may be disposable and the
materials comprising the alternating ionic magnetic resonance
culture apparatus may be sterilizable. In addition wherein
culturing in the pulsating alternating ionic magnetic resonance
field produces one or more of a modification, growth,
differentiation, dedifferentiation, altered gene expression,
altered transcription events or cellular regeneration via
controlling the ionic transport or transcription mechanism in the
cells or tissues. Furthermore, culturing in the alternating ionic
magnetic resonance field may change the mammalian cellular ionic
transport thereby enabling infection and an expression of mammalian
viral epitopes. Particularly, the mammalian cells or tissues are
virally or bacterially infected.
[0036] In yet another embodiment of the present invention there is
provided a model comprising a tissue-like assembly of the cultured
mammalian cells or tissue produced by the methods as described
supra.
[0037] The present invention provides methods for the short and
long-term proliferation, growth, enrichment, conditioning,
modification, and/or aggregation of mammalian cells, tissues or
organoid structures in adaptable culture systems in alternating
ionic magnetic resonance fields. alternating ionic magnetic
resonance fields comprise specific bio-electromagnetically relevant
frequencies that mimic the global diurnal cycle believed to
influence cellular behavior and genetic evolution. Particularly, an
alternating ionic magnetic resonance field, such as produced in the
alternating ionic magnetic resonance chamber presented herein,
mimics in part the natural environment that mammalian cells are
exposed to in the normal earth-based living system.
[0038] Thus, the present invention provides methods for
three-dimensional growth of a culture material, such as, but not
limited to, animal, preferably mammalian, cells, tissues, organoid
bodies, etc. The culture material is introduced into the growth
module and grown in the nutrient-rich media provided by the
nutrient module in the presence of an alternating ionic magnetic
resonance field. During growth the gravity vector of the culture
unit is randomized to favor three-dimensional growth. This
maximizes the efficiency of metabolic exchange within the system
while simultaneously providing for the accumulation of valuable
biomolecules in the growth module and the nutrient module. A more
controlled cell growth culture system is thus enabled that can be
manipulated to provide for increased rate of cell growth, faster
differentiation, increased cell fidelity, and the induction or
suppression of selective physiological genes involved in directing
cellular differentiation.
[0039] Specific culture material may be selected and conditions set
to regulate, for example, gene expression and protein activity
within the cultured material. The alternating ionic magnetic
resonance field stimulates the expression and regulation of various
genes, including transcription factors, and alters the activity of
the genome. This results in a modified output of existing cellular
proteins, such as cell transport proteins involved in regulating
ionic concentration, membrane transport and other crucial pathways
in the regulation of growth, development, and differentiation,
dedifferentiation, cell maintenance and aging-related mechanisms in
animals and plants. Regulating cell
differentiation/dedifferentiation via the methods and processes
provided herein may facilitate the development of faster healing
and lifespan extension compositions and applications.
[0040] In systems where it is desired to have the cells adhere to
substrates for the sole purpose of proliferation without promoting
three-dimensional tissue growth, the cells can be grown directly on
a flat, two-dimensional electrode surface composed of a
biocompatible material. In these situations, some cultured cells
may actually be attracted to the supportive electrode material,
coatings, or electrically conductive channels that can be
incorporated into the culture unit to facilitate cell attachment.
For example, the alternating ionic magnetic resonance field is
induced in the region of the channel by passing the alternating
ionic magnetic resonance protocol through a conductor placed along
the channel.
[0041] In some systems microcarrier spheres or beads are included
and suspended within the culture medium to induce adherence of the
cells to the beads. For cell proliferation in these systems, the
culture system growth module is preferably exposed to the
randomization at a range of about 2 to 60 rpm, and the alternating
ionic magnetic resonance is generated by a time-varying current
passed through a conductor with an RMS value of about 0.001 to
10,000 Gauss with a preferred range of about 0.01 to 3000
Gauss.
[0042] Particularly, the present invention provides methods for
up-regulating or increasing viral replication and proliferation
genes and gene products in a culture material, such as, cells,
tissue, etc. Culture material infected with a virus of interest,
grown in the alternating ionic magnetic resonance culture system
induces the up-regulation of genes associated with the virus,
particularly, those associated with replication and proliferation.
In non-limiting examples, such models and systems would be useful
for producing large numbers of virions for vaccine production,
identification of viral genomic adaptation products, tracking of
viral genomic shift during a long term culture, development of
antivirals or antibacterials targeted at blocking replication, and
harvest of human cell-produced proteins that only result from
virally or bacterially infected cells. Correspondingly, the methods
provided herein are applicable to culture materials infected with
or grown with a bacteria of interest to up-regulate
bacterial-associated genes. Methods of identifying proteins or
other products from the media comprising the culture system are
well-known in the art as are methods for development of antivirals
and an antibacterials based on specific compounds, proteins,
nucleic acids, etc.
[0043] Moreover, the present invention provides models of
3-dimensional tissue-like assemblies (TLAs) of cells. The
tissue-like assemblies are stable for at least 3 months, preferably
6 months or longer and share features with the corresponding
2-dimensional tissues/cells or with tissue/cells obtained in vivo.
The cells may be grown with or without an alternating ionic
magnetic resonance field. Alternatively, the model may comprise
tissue-like assemblies of cells infected with a virus or a
bacteria, particularly a pathogen. These tissue-like assemblies
also remain stable for at least three months. Moreover the viral or
bacterial genome remains stable throughout the infection period.
Such models are useful for, but not limited to, the up-regulation
of viral or bacterial associated or induced genes and/or gene
products and/or the study of viral or bacterial adaptive
mechanisms.
[0044] Generally, the culture system comprises a gravity
randomizing, multiphasic culture system having a disposable
self-feeding growth module, a nutrient and growth module that
comprises a culture unit which, optionally, is disposable, and a
removable electromagnetic chamber or unit which, when applied to
the outside of a culture unit, is suitable for delivering
alternating ionic magnetic resonance fields to the contents of the
culture unit. Existing culture systems using PEMF and TVEMF have
been shown to increase the rate of cell growth of the cells
cultured in the system. The alternating ionic magnetic resonance
culture system is a significant improvement on these systems and
incorporates instead the use of an alternating ionic magnetic
resonance device which induces cell regeneration, increases cell
fidelity, modulates cellular transcription and induces the
selective regulation of key physiological genes useful in directing
the differentiation and dedifferentiation process of particular
cells.
[0045] The generated alternating ionic magnetic resonance field, as
described herein, produces a series of controlled resonating
waveforms that mimic the Schumann Resonances, which are global
electromagnetic frequencies that are excited by lightning
discharges, with more precision than are created naturally. The
alternating ionic magnetic resonance-generated resonating waveforms
can be modified or accelerated to specifically regulate or induce a
physiological response in a particular cell system, i.e., a pulsed
emission to preferentially effect the oscillation of specific ion
species in the living cell. Each cell type has the potential to
respond to a given resonating pattern differently than another cell
type based on total ion content and ion species. Moreover, each
physiological response may involve the induction of different
cellular control mechanisms, such as, but not limited to,
stimulated or decreased genomic, proteomic, transcriptomics, and
metabolomic expressions, altered ion flow through the membrane, and
altered gene replication.
[0046] Thus, the alternating ionic magnetic resonance culture
system and apparatus and methods for use can stimulate the
expression and regulation of various genes, including transcription
factors, and to alter the activity of the genome to result in
modified output of existing cellular proteins, such as cell
transport proteins involved in regulating ionic concentration,
membrane transport and other crucial pathways in the regulation of
growth, development, and differentiation, dedifferentiation, cell
maintenance, inflammation, and aging-related mechanisms in animals
and plants. Use of the alternating ionic magnetic resonance culture
system, apparatus and the methods to stimulate gene expression and
regulation, is relevant to both practical commercial applications
as well as applications relating to investigations that focus upon
re-creating initial conditions in the context of evolutionary
biological processes at the cellular and physiological level.
[0047] The alternating ionic magnetic resonance culture system and
apparatus also offers the ease and convenience of using disposable
components for ready compliance with rigid FDA requirements
addressing cleanliness and the avoidance of cross-contamination of
cell species. The use of a disposable culture unit facilitates the
manufacture and use of a system that can easily meet the strict
requirements of the FDA. Components can be manufactured and
packaged in sterile packs for ready use by one of ordinary skill in
the art, much the same as other disposable medical devices are
used. The alternating ionic magnetic resonance chamber of the
current invention facilitates selective reuse of the ionic magnetic
resonance (IMR) device, which contributes to minimizing the costs
associated with culturing cells and tissues for medical
purposes.
[0048] The alternating ionic magnetic resonance culture system
comprises a culture unit, which has a pre-sterilized, disposable,
self-feeding growth module and a pre-sterilized disposable nutrient
module, a removable and interchangeable alternating ionic magnetic
resonance electromagnetic chamber, and a means for continually
randomizing the gravity vector of the growth module and nutrient
module, such as a randomizing adapter. The pre-sterilized and
disposable components minimize cumbersome handling, costs and
difficulties associated with the improper delivery of the IMR
fields in known culture systems and EMF and TVEMF designs.
Alternatively, the growth and nutrient modules may comprise
reusable materials and the alternating ionic magnetic resonance
chamber may be disposable.
[0049] The randomizing adapter holds the culture system in a
horizontal position, whereby a basically cylindrical culture system
can rotate or move clockwise and/or counter clockwise horizontally
about its central radial axis to minimize adherence of the cells to
the reactor walls. The randomizing adapter comprises a randomizing
device or mechanism for continually randomizing the gravity vector
in the growth module or culture system alone within a stationary
nutrient module and a stationary electromagnetic device, in unison
with an electromagnetic device located inside a stationary nutrient
module or together with the nutrient module and electromagnetic
device.
[0050] A continuously randomized culture system provides a
three-dimensional growth environment effectuated by continual
gravity randomization, steady but consistent disruption, such as
oscillation. Minimal turbulence randomization discourages adherence
of eukaryotic cells to the walls of the culture system while
encouraging self-adherence of the cells to one another. The
randomizing adapter accommodates rotations or oscillations of at
least the growth module sufficient to minimize adherence of the
cells to the walls of the chamber. Different cell types have
different adherence factors, so depending on the type of cells to
be cultured, the optimal rate of rotation will fluctuate. The
adherence factor will also become more important as the cells
proliferate within the chamber and become more concentrated,
whereby there is more interaction with the wall of the chamber. As
such, the rate of rotation or oscillation of the chamber often
increases as the density of cells increases in longer runs in the
culture system. Consequently, the continually randomized gravity
vector device optimally comprises a variable setting that can
accommodate growth chamber rotation speeds in the range of 0.01 to
60 rpm, with a preferred range of 2 to 40 rpm. In systems using an
oscillating type of device, periodic oscillations may range in
frequency from being continuous to oscillating every 30 seconds or
even every half hour.
[0051] The randomizing adapter may be a simple system of external
rollers on which the culture system sits, similar to typical tissue
culture roller bottle mechanisms. Alternatively, rotation can be
effectuated by an external electric motor using a system of
fan-belt like connection mechanisms or a direct drive. The
randomizing mechanism may systematically rotate the entire culture
system or the culture unit or the growth module alone. The type of
rotation device will dictate the type of adapter necessary on the
component parts, such as a pulley-like wheel that would be firmly
attached to a spindle incorporated into to the affixed growth
module cap and extends through a sterile liquid tight adapter in
the nutrient module cap.
[0052] The growth module contains a small volume of culture media,
as well as the cells and/or tissue and, optionally, a matrix
material to be cultured, that completely fills the module with no
noticeable air space. It has an integral semi-permeable molecular
membrane incorporated into one of its walls to facilitate the
diffusion of gases, nutrients and wastes between the cell culture
chamber and the extra nutrient-rich media in the surrounding
nutrient module. The molecular membrane of the growth module
contains a diffusible osmotic membrane capable of exclusion
thresholds from 100-500,000 MW with a preferable cutoff range of
2000-12500 MW. The osmotic semi-permeable membrane is generally
composed of a hydrophilic composition, but may comprise a more
structural composite coated with a hydrophilic composition (e.g.
nitrocellulose, polysulphone, polyacetate, or other similar
composite). Unlike a perfused system, the semi-permeable membrane
system facilitates the transport of nutrients and wastes without
the loss of valuable biomolecules from the growth module. The
retention of these biomolecules increases the accuracy and fidelity
of the mammalian organoid recapitulation. Additionally, the
specific membrane exclusion cut off provides a means to enhance the
production of valuable cellular proteomics. This enhancement saves
time, effort and purification costs. The growth module also
comprises means for securement
[0053] The nutrient module may be disposable and serves as a media
reservoir that attaches to or surrounds the growth module. The
nutrient module has at least one sealable opening at one end, which
is sealable with an appropriate cap, e.g., screw-top, snap-top,
crown-top, crimped-top, slide-top, and designed to be large enough
to insert an appropriately sized growth module therein. The cap may
have an adapter assembly for connecting an external movement device
capable of delivering a continual randomized movement to the growth
module or culture system, for example, oscillating or rotating in a
mono- or bidirectional manner. Preferably, the entire culture
system is attached via the wall of the nutrient module to a
bidirectional motor device that slowly randomizes the gravity
vector of the entire system.
[0054] The nutrient module supplies a continually diffusible supply
of fresh material to the cultured cells and is adapted with a gas
port or gas exchange vent fitted with a semi- or gas-permeable
membrane to provide for the exchange of waste gases. Carbon dioxide
and ammonia generated by the tissues in the growth module diffuse
out of nutrient module and atmospheric, i.e., 159 mm Hg, oxygen
diffuses into the nutrient module through the gas-permeable
membrane of the gas port. The gas permeable membrane may be a
dialysis membrane, a thin gas-permeable silicone membrane or a
similar material. The gas port may be incorporated into the
nutrient module cap for convenience or may be located in the wall
of the nutrient module as a separate opening to the outside
environment. The nutrient module includes a mixing device located
externally to the gas permeable membrane.
[0055] These processes maintain homeostatic physiological
conditions in the culture much as see in the human or mammalian
body. The nutrient module is large enough to accommodate the full
volume of the growth module in addition to a sufficient volume of
media to effectuate efficient exchange of nutrients and oxygen from
the fresh media to the growth module and waste products and gases
away from the growth module for removal from the system.
[0056] The growth module may be disposable and/or an internal
module and is typically a cylindrical container, although it may be
any shape, such as, but not limited to, a sphere or bag. The growth
module has a sealable opening at one end that is fitted with and
sealed with an appropriate sterile, liquid-tight cap, for example,
screw-top, snap-top, crown-top, crimped-top, slide-top, that may
have one or more ports for easy assembly, injection, inoculation
and harvest. The sterile liquid-tight cap provides also for the
growth module cap to fit into a liquid-tight randomizing adapter
that allows for rotation of the growth module. The growth module
may be adapted with inlet and outlet ports for the periodic or
continual exchange of media through the chamber and may be equipped
with a baffling system that efficiently directs a slow continual
flow of fresh media and nutrients across the osmotic membrane to
allow more control over nutrient transport between the modules to
aid in maintaining a more controlled, homeostatic environment. Such
a baffling system streamlines the use of fresh media and has the
potential of decreasing the overall amount of media needed during
the course of a culture experiment.
[0057] One wall of the growth module at least partially comprises a
semi-permeable, hydrophilic dialysis membrane that contains the
cells and/or tissue within the confines of the growth module while
allowing the free diffusion of gases, nutrients and metabolic
wastes with the fresh media in the nutrient media-hold compartment.
A second wall in the growth module comprises a gas permeable
membrane that is hydrophobic and controls the resident dissolved
gas coefficient in the growth module.
[0058] The dialysis membrane may be any material with pores large
enough for the transfer of small molecules, but small enough to
retain intact cells within the growth module. It may comprise a
gas-permeable silicone composition or a polyethylene type material
that provides for efficient transport of carbon dioxide, dissolved
in the culture medium, both as a gas and as a solute in the form of
sodium bicarbonate from the growth module to the nutrient module
and for the transport of oxygen into the bioreactor. The dialysis
membrane may be covered with an additional support or membrane
stabilizer that protects the dialysis membrane from mechanical
damage during handling, setup and harvest, as well as during the
culture stage to prevent damage from moving or swirling media in
the modules.
[0059] In addition to permitting transport of carbon dioxide and
oxygen, the dialysis membrane is selected to have a pore size
sufficient to permit the diffusion of other solubilized nutrients,
such as sugars, amino acids, vitamins, ions, etc., from the fresh
media in the nutrient module to the growth module, as well as the
transfer of metabolic byproducts, such as acidic compounds, for
example, lactic acid, toxic gases, e.g. carbon dioxide, toxic
solutes, e.g. ammonium ions, and other low molecular mass products
from the growth module to the nutrient module. The pore size must
be small enough, however, to prevent the transfer of cells and high
molecular weight cell products, such as, secreted proteins,
antibodies, glycoproteins, large nucleic acids, etc., into the
nutrient module.
[0060] The growth module may be disposed inside a larger nutrient
module. The growth module may be filled with cells/tissue and
media, and sealed prior to insertion into the nutrient module, or
may be inserted empty and assembled inside the nutrient module, and
later filled and sealed while inside the nutrient module. The
ability to remove the growth module and move it to another nutrient
module facilitates subsequent processing of the cultured
cells/tissues with minimum hazard of contamination and loss of time
and efficiency.
[0061] In one non-limiting example of a culture unit, a 35 ml or 50
ml capacity growth module is fitted to a 450 ml nutrient module by
a snapping or other connection mechanism or means. The outer
nutrient module holds sufficient media to provide support of cell
growth inside the smaller growth module for a period of several
days or more. In another non-limiting example of a culture unit,
the nutrient module is significantly and substantially larger than
the growth module whereby the volume of the media-hold compartment
now exceeds the volume of the growth module by as much as 100,000
fold. For instance, one or multiple small 1-5 cc growth module(s)
may be completely submersed in a 100-liter nutrient module (similar
to a 25-30 gallon bacterial fermentation tank), or one or more
rod(s) comprising multiple tandem units of smaller 1-5 cc growth
modules may be submersed in an elongated cylindrical nutrient
module. This is more conducive for periodic manual exchanges of
media.
[0062] Preferably, a larger nutrient module having a volume 2 to 50
times that of the growth module is used. With larger nutrient
modules, manual exchange of the media at periodic time intervals
without having to continually feed fresh media into the module is
possible. As such, culture units having larger nutrient modules
need not have inlet and outlet ports for media exchange, but,
optionally, may have one or more sets of ports for convenience in
handling the media. Culture units requiring periodic manual
exchange of the media would preferably have nutrient module volumes
greater than 10 times that of the growth module.
[0063] The growth and nutrient modules may be made of disposable
biocompatible polycarbonate based materials that can be autoclaved
under controlled conditions for reuse if necessary, or they may be
made of more durable components such as glass or stainless steel or
polycarbonates/plastics. The growth module comprising the dialysis
membrane is more adapted to irradiation type sterilization and
better for prepackaged blister-like manufacture and sterilizing.
The nutrient module can be reused and may be made of polycarbonate
or a more stable material such as glass or stainless steel.
[0064] The alternating ionic magnetic resonance chamber comprises
an electromagnetic modulating device configured to deliver a
pulsating alternating ionic magnetic resonance field to cultured
cells/tissue, organoid bodies, etc. within the growth module. The
electromagnetic device may comprise an electrode or set of
electrodes or a removable chamber that is easily interchangeable
depending upon the needs of the system. Preferably the alternating
ionic magnetic resonance chamber is an easily removable chamber
that encompasses or fits around or receives therein the entire
culture system or only the growth chamber. In the form of a
removable chamber, the chamber is a slip-on chamber that holds a
coil designed to be larger than the diameter of the culture unit.
It is made of a relatively rigid electrical conductive material,
e.g., a wire, wound in a cylindrical or rectangular shape that when
connected to a pulsating electromagnetic current creates a
electromagnetic force in the range of about 0.001 to about 10,000
Gauss within the internal portion of the chamber and the
encompassed culture device.
[0065] Preferably, the conductive wire of the alternating ionic
magnetic resonance chamber is made of a conductive ferromagnetic
material coiled about an electromagnetic permeable polymer at about
ten coils per inch. The coil can be encased in a thin flexible
encasement made of a smooth conductive material that provides for
easy handling during assembly and disassembly of the culture system
and convenient cleaning before and after use. Also, the alternating
ionic magnetic resonance field may be generated by a device
producing a pulsating time-varying current passed through a
conductor with an RMS value of about 0.01 to about 10000 mA, with a
preferred range of about 1 to about 5000 mA for some cell
systems.
[0066] The alternating ionic magnetic resonance protocol/signal can
be generated by many commercially available devices that are
commonly referred to as random/arbitrary waveform or waveform
generators, such as units produced by Tektronics, e.g., models
AFG3021B, AFG3022B, AFG3101, AFG3102, AFG3251, AFG3252; and
Agilent, e.g., models 33220A, 33250A, and 33220A-HO1, among
numerous other suppliers. The waveform generator is programmed to
produce the desired series of pulses at the desired frequencies
over a specific time interval. This signal is then connected to the
output or transmission device either directly or though an
amplifier to strengthen/regulate or increase the intensity of the
field if desired. Alternatively, the "signal or waveform protocol"
is programmed onto a custom designed computer chip and the series
of desired signals are emitted from the chip to the transmission
device after it is energized via a power supply that will produce
the desired field strength in the transmission device.
[0067] The alternating ionic magnetic resonance field is a
multivariant field and may be induced by either a multi varying
current within a conductor or by a multi varying voltage between
fixed conductors. For example, the culture is placed near a
conductor through which a time-varying current is passed.
Alternatively, the culture is placed between parallel plates upon
which a time-varying voltage is applied. In both cases, an
alternating ionic magnetic resonance results within the region of
the cell culture.
[0068] Several methods can be used to produce an alternating ionic
magnetic resonance signal, such as delta or square wave, Fourier
curve or a combination of signals within a given time domain. For
example, an array of conductive current carrying (voltaic)
electrodes can be arranged to focus the electromagnetic (EM) field
in the specific chamber holding a culture. An alternating ionic
magnetic resonance can also be applied to enhance tissue growth
that may occur on a shaped or custom designed substrate within the
chamber. The electromagnetic field may be generated by various
means, such as, by directing the current waveform directly through
a conductive substrate or substrate layer or by projecting the
field from an external electrode, for example, a plate, an antenna,
a coil, or a chamber, or from a set of electrodes adjacent to and
spaced apart from, but in the immediate vicinity of, the medium, so
that the relative strength of the electromagnetic field is
effective within the growth chamber. For example, a current of
about 100 milliamps, conducted between opposite corners of a
metallic conductor, produces a stimulatory alternating ionic
magnetic resonance extending several centimeters from the plate
surface.
[0069] Particularly, when the alternating ionic magnetic resonance
field is generated through conductive antennae, external or in
direct contact with the media, e.g., wire, electrode, coil or
similar transmission device, the field is adjacently spaced apart
from the cultured cells and media and carries an alternating ionic
magnetic resonance signal advantageously produced by a varying
electrical potential in the form of a delta or square wave having
the preferred fundamental frequencies of approximately 10-300
cycles per second (Hz). Particularly, one or more overlapping or
fluctuating alternating ionic magnetic resonance frequencies at
fundamental intervals of 10, 14, 15, 16, or 32 Hz, and, optionally,
resonances that fluctuate between about 8 and 14 Hz (rounded
values) can be produced and passed through the antennae or
transmission device. The fundamental intervals include the
respective harmonic intervals extending to 256 Hz, and
incorporating all harmonics of the aforementioned fundamental
frequencies to infinity in the form of a square wave of 0.01-10000
mA with a nearly zero time average
[0070] Preferably, a two-dimensional or a three-dimensional
directional antennae may be utilized and may be applied to
conventional two-dimensional or to three-dimensional tissue
cultures. Three-dimensional cultures may be achieved in actual
microgravity or by continually randomized gravity vector vessel
technology that simulates some of the physical conditions of
microgravity, and/or in other, conventional three-dimensional
matrix based cultures. The electromagnetic field, preferably an
alternating ionic magnetic resonance field, is achieved in the
vicinity of the antennae or coil by passing, through the
directional device, a pulsating electromagnetic field of the
correct frequency, duration, and field strength, for the proper
duration.
[0071] During use of the culture system the range of frequency and
oscillating electromagnetic field strength is a parameter that may
be selected to achieve the desired stimulation of the cultured
material, such as tissues, cells or genes, etc. of interest. The
final field produced can be in the range of 0.001 to 10000 Gauss,
but the preferred range inside the central region of the chamber
cylinder and the growth module of the culture system is in the
range of about 0.01 to about 5000 Gauss.
[0072] A preferred embodiment of the alternating ionic magnetic
resonance culture system is depicted in the figures and described
below. However, such reference is not meant to limit the present
invention in any fashion. The embodiments and variations described
in detail herein are to be interpreted by the appended claims and
equivalents thereof.
[0073] As shown in an unassembled view in FIG. 1A, the alternating
ionic magnetic resonance culture apparatus 100 comprises a
randomizing adapter 110, a culture unit 120, and an AIMR chamber
150. The randomizing adapter has an open, circular proximal end 112
with a diameter sufficient to accommodate the culture unit therein
and a distal end 114 in electrical communication with a randomizing
mechanism 116. The culture unit comprises a growth module 130 at
the proximal end and a nutrient module 140 at the distal end of the
culture unit into which the growth module is fitted and secured at
least via a securing or fastening means 138a,b to a lip, rim or
edge 141a comprising the proximal end 141 of the nutrient module.
The growth and nutrient modules are shown here comprising
substantially cylindrical bodies, however the modules may have
other shapes as long as the nutrient module can securely and
functionally accommodate the growth module and contain nutrient
media therein and the growth module can securely and functionally
contain a culture material for growth therein and receive and
exchange nutrient media and gases.
[0074] The growth module 130 has a front or proximal wall 131
comprising a gas membrane 132 and inlet and outlet ports 133a,b,c
disposed through the gas membrane (see FIG. 2A). The back or distal
wall of the growth module comprises a baffling means or system 136
which when affixed to the proximal end 141 of nutrient module is in
fluid communication therewith (see FIG. 2B). The proximal end 141
of the nutrient module comprises a gas port or gas exchange vent
145 fitted with a semi- or gas-permeable membrane 146 (see FIG. 3).
The distal end 142 of the nutrient module comprises a cap 143
covering an opening 144 into the nutrient module that is adaptable
to engage with the randomizing mechanism. Optionally, the nutrient
module may comprise a means for indicating media volume 147 etched
or disposed on the module surface. The AIMR chamber 150 has
circular proximal 151 and distal 153 ends with a diameter
sufficient to slide or fit over the culture unit and comprises an
electromagnetic device 155, in this instance a coil, disposed
around the exterior thereof and means or device 157 for generating
a pulsating, time-varying electromagnetic current (PTVEC) in
electrical communication with the electromagnetic device.
[0075] FIG. 1B illustrates how the culture unit is accommodated
within the randomizing adapter. The distal end 142 of the nutrient
module 140 comprising the culture unit 120 is disposed within the
proximal end 112 of the randomizing unit 110 and is electrically
engaged with the randomizing mechanism 116 (not shown). This leaves
the growth unit 130 uncovered and available to receive an
alternating ionic magnetic resonance field. Assembled, as shown in
FIG. 1C, the proximal end 151 of the AIMR chamber 150 is disposed
around the proximal end 141 of the nutrient module, particularly
such that at least the growth module 130 is disposed within the
alternating ionic magnetic resonance chamber to receive the
alternating ionic magnetic resonance field generated by the
electromagnetic device 155. A pulse sensor 159 is disposed on the
electromagnetic device.
[0076] With continued reference to FIGS. 1A-1C, FIG. 2A is a front
view of the growth module 130. The front or proximal side 131 of
the growth module comprises a gas membrane 132 disposed across the
surface thereof. The gas membrane comprises a plurality of
protusions, generally represented by 134a,b,c,d,e,f, radially
disposed across the surface of the membrane to increase the surface
area and has a plurality of inlet/outlet ports represented as
133a,b,c disposed through the membrane and in fluid communication
with nutrient media contained within the growth module.
[0077] FIG. 2B is a back view of the growth module 130. The back or
distal side 135 comprises a baffling system 136 disposed therein
and a semi-permeable dialysis membrane 137 comprising at least part
of the distal side. The gas-permeable membranes 132 and 146,
including the inlet/outlet ports 133a,b,c and the gas port 145 are
in fluid contact with the nutrient media in both the growth module
and the nutrient module. The outer edge of the growth module
comprises a plurality of a first securing or fastening means or
components, represented by 138a,b,c,d, extending therefrom that
secure the growth module to the nutrient module at the lip, rim or
edge 141a comprising the proximal end 141 of the nutrient module.
The outer edge of the growth module also comprises a plurality of a
second means, generally represented by 139a,b,c,d, for securing or
fastening the growth module to the nutrient module, such as snaps,
clips or claimp, that are disposed between the primary securing
means. The combination of the first and second securing means forms
a watertight seal between the modules.
[0078] With continued reference to FIGS. 2A-2B, FIG. 3 illustrates
how the growth module 130 is fastened or secured to the nutrient
module 140. The gas port or vent 145 and its disposition in
relation to the gas-permeable membrane 146 is depicted. One can see
that upon fastening the modules together, the baffling system 136
and semi-permeable dialysis membrane 137 in the growth module are
in fluid contact with the gas port 145 and gas-permeable membrane
146 in the nutrient module. The fastening means 138a,b,c,d comprise
raised beveled edges 139a,b,c,d which can slide or snap over the
rim 141a in the nutrient module along 160a,b to secure the growth
module therein.
[0079] The following example(s) are given for the purpose of
illustrating various embodiments of the invention and are not meant
to limit the present invention in any fashion.
Example 1
Preparation of Alternating Ionic Magnetic Resonance Culture
System
Preparation of Cells for Culture/Medium
[0080] Under sterile conditions, a seeder culture is started with
about 35 to 50 ml of a human or mammalian cell suspension
containing approximately 1.times.10.sup.5-5.times.10.sup.6 cells/ml
in a 50 ml cell culture flask.
[0081] The conditions of growing cells in a high density
environment requires the use of a high quality media having a
minimal concentration of glucose at 4 g/l, and a sufficient buffer
component such as NaHCO.sub.3 as found in standard media (3.7 g/l)
which generally provides buffering capacity for a period of up to
2-10 weeks under standard culture conditions. Because the culture
system enables high-density growth, the buffer is generally changed
1 to 2 times per week. The medium in the nutrient module should be
replaced as soon as the color starts to change from salmon-pink to
a yellowish-pink. Due to the high-density growth inside the growth
module, the media will tend to maintain a yellowish color once a
critical mass is achieved.
[0082] Serum concentration is often critical when eukaryotic cells
are grown at high density and should be minimally maintained at
levels normally used in stationary culture, approaching 0-80%
depending on the cell type. When cells are cultured for the
production of secretory molecules, e.g. antibodies, serum
concentrations should be 5-30% inside the growth module. Serum
concentrations in the nutrient module can often be reduced but
should be tested with each individual cell type. Because the use of
serum can create foaming problems in the culture system
environment, an antifoam agent may be used. Cell types that can be
grown without serum should be adapted for such growth prior to
growth in the bioreactor.
[0083] Adherent cells, such as CHO, HEK 293, BHK, will generally
grow first in suspension and then as aggregates. Some adherent cell
types will produce secreted products more effectively if grown in
the presence of a microcarrier type bead to minimize large
aggregates of cells and to optimize cell surface (secretory)
area.
Preparation of the Culture System
[0084] The culture system is assembled under sterile conditions,
preferably in a sterile hood, by attaching to or slipping a
pre-sterilized disposable growth module made of polycarbonate
inside a reusable nutrient module about ten times larger than the
growth module. The growth module is supplied as a pre-sterilized
disposable unit pre-packaged in a sterile blister pack and is
pre-fitted with a sterile cap. It is inserted into the sterile
nutrient module fitted with internal guides to hold the growth
module whereby the growth module snaps tightly into place with a
liquid tight seal in the specially designed opening. The nutrient
module has a separate media opening for periodic exchange of media
in the nutrient module which is vented during assembly to vent
displaced air. The reusable/disposable nutrient module is comprised
of polycarbonate, and sterilized by autoclaving to a maximum of
121.degree. C. for 30 min inside an autoclave bag before assembly
or may be gamma sterilized.
Filling the Culture System
[0085] The filling steps are done with all of the equipment and
solutions equilibrated at the culture temperature to minimize
condensation and gas expansion or contraction in the system after
assembly. The seeder culture/cell suspension is introduced into the
35 ml growth module with a syringe or pipette through a fill port
in the growth module cap taking care to allow for venting of
displaced air. This growth module cap has two ports with snap caps
comprising rubberized septum caps for syringe inoculations and air
removal. One port has a Luer Lock adapter that permits easy filling
by a syringe, while the other port is opened to allow air to escape
during filling. The growth module is filled completely with the
seeder culture and an appropriate media, removing all air from the
growth module. The ports are sealed and last traces of air are
removed by insertion of an empty syringe with a needle into the
rubberized snap caps and withdrawing all air.
[0086] The nutrient module is filled to almost full capacity with
about 450-500 ml nutrient medium through the inlet port while
removing air pressure through the gas-permeable silicone membrane
in the nutrient module. A small air space is maintained to provide
exchange of gases through the gas port. The inlet port is tightly
sealed.
Applying the Electromagnetic Chamber
[0087] Once the modules are assembled, filled and sealed, the
removable electromagnetic chamber, fitted to the diameter of the
nutrient module is slipped over the entire unit from the end distal
FIG. 1C to the end with the filling caps and ports. A flexible
swivel cord adapter on the chamber enables the entire unit to
rotate without interference from the electrical cord that supplies
the current to generate the pulsating electromagnetic field. The
chamber imparts a time-varying electromagnetic force (square/delta
wave, Fourier curve) to the culture system growth chamber and its
contents.
Applying the Gravity Vector Randomization Device
[0088] The assembled culture system is placed on a gravity vector
randomization device inside an incubator chamber set at the
temperature adapted for the specific cell culture, which in this
case is 35-37.degree. C. and set to rotate the human hybridoma
cells at about 5 rpm. The culture system is monitored for leaks and
other problems and incubated while continually randomizing the
gravity vector until the first sample is taken. Different mammalian
cell types require different randomization rates. For example,
murine hybridoma cells are generally grown at 5 to 20 rpm, whereas
human and transfected cells do well with slightly faster rotation
rates of about 10 to 100 rpm. These cell lines are not intended to
be limiting to the current invention as the current culture system
is intended to be adaptable for the growth of any cell type or
tissues that can be adapted to traditional cell culture
methods.
Taking Samples and Harvesting
[0089] Samples are periodically taken from the culture system in
order to assess the growth and development of the cultured
material. Before taking samples, the culture system is taken from
the incubator, removed from the continually randomized gravity
vector device and the electromagnetic chamber is removed. All steps
are done quickly to minimize settling of the cells. The culture
system is then wiped down to minimize contamination and transferred
to a sterile hood. Inside the hood, built up pressure is released
by slowly opening the media port in the nutrient module. The growth
module can then be sampled by opening the fill port with the Luer
lock adapter. The volume removed is replaced with an equal volume
of fresh media and the chamber is resealed, reassembled with the
electromagnetic chamber and reset on the continually randomized
gravity vector device in the incubator chamber.
Changing the Medium in the Nutrient Module
[0090] Replacing the spent medium with fresh medium should be done
about 1-2 times per week and requires dismantling of the culture
system in the same manner as if taking a sample, but the growth
module is left unopened. Instead, the nutrient module fill cap is
removed and the used medium is emptied by carefully pouring out the
contents in a sterile hood. About 350 to 400 ml of fresh media
(37.degree. C.) is poured into the nutrient module and reassembled
as before. Care is taken to minimize any contamination of the
modules or media.
Cell Culture Density
[0091] Growth of high-density cells, such as hybridoma cells,
requires more oxygen, more nutrients and more frequent removal of
waste products and is therefore better accommodated with a
continual flow design nutrient module. For example, the oxygen
requirement of hybridoma cells at about 10.sup.7 cells/ml in a
35-50 ml growth module is about 1.75 mg/hr. Some cell lines do not
grow to high densities (less than 2.times.107 cells/ml) but may be
cultivated for a longer period of time in the culture system for
production and harvest of secreted products with regular changes of
medium or a continual flow nutrient module.
Production of Secreted Cell Products
[0092] Cell products such as monoclonal antibodies, cytokines,
pro-inflammatory molecules, biomolecular markers and all other
soluble biochemical products can be produced in a culture system
once the cells have been cultured to a critical cell density which
depends on the individual properties of the cells cultured.
Hybridoma cells typically produce between 4.times.10.sup.7 and
7.times.10.sup.8 antibody molecules per cell in a 24-hour
period.
Genomic Analysis of Tissue-Like Assemblies (TLAs)
[0093] RNA from tissue-like assemblies of cells grown in GTSF-2,
RPMI 1640, Hams F10, MEM Alpha, L-15, Dulbecco's Modified Eagles
Medium (DMEM), Hams F12, Earls MEM, DMEM/F12, or other media
appropriate to the cell type with out without alternating ionic
magnetic resonance was harvested by removing it from the 3D device
and placing in a 50 ml tube. Media was removed and the tissue-like
assemblies were washed 3.times. with sterile PBS. After washing the
tissue-like assemblies were frozen at -80 C. and stored for
transfer to Asuragen Inc. Samples were sent to Asuragen for
digestion of the RNA and gene array chip analyses on Affymetrix
U133 2.0 plus human genome chips. Digital Chip data was sent to the
laboratory and processed by analyses in Genspring software.
3D TLA Growth Kinetics and Glucose Consumption
[0094] Metabolic parameters of tissue-like assemblies were measured
every 24-48 h over the course of the experiments to monitor a
cellular development profile and to monitor the metabolic status of
the tissues. Glucose consumption was determined using the iStat
clinical blood gas analyzer using an EC8.sup.+ cartridge (Abbott
Laboratories, Abbott Park, Ill.) according to the manufacturer's
instructions (1).
Example 2
Gene Induction in HBTC Cells Grown in the Alternating Ionic
Magnetic Resonance Culture System
HBTC TLA 3D Cell Culture
[0095] A culture of cells comprising fibroblasts, mesenchymal and
secretory cells (HBTC) were cultured in the alternating ionic
magnetic resonance culture system. A mixture of human bronchi and
tracheae primary cells (HBTC; fibroblasts and mesenchymal cells)
were obtained from the lung mucosa of multiple tissue donors
through Cambrex Biosciences (Walkersville, Md.) and were shown to
be free of viral contamination by a survey of a panel of standard
adventitious viruses (e.g. HIV, hepatitis, herpes) conducted by the
supplier (Cambrex). The cells were initially grown as monolayers in
human fibronectin coated flasks (BD Biosciences, San Jose, Calif.)
and propagated in GTSF-2 media supplemented with 10% fetal bovine
serum (FBS). GTSF-2 media, initially described in U.S. Pat. No.
5,846,807, is a tri-sugar-based growth medium containing glucose,
galactose and fructose. U.S. Pat. No. 5,846,807 is herein
incorporated by reference in its entirety.
[0096] The monolayers were grown in a Form a humidified CO.sub.2
incubator with 95% air and 5% CO.sub.2 at constant atmosphere and
at 37.degree. C. The HBTC cells were passaged using enzymatic
dissociation with a solution of 0.1% trypsin and 0.1% EDTA for 15
minutes at 37.degree. C. After incubation with the appropriate
enzymes, the cells were transferred to 50 ml Corning conical
centrifuge tubes and centrifuged at 800 g for 10 minutes. The
pelleted cells were suspended in fresh GTSF-2 medium and diluted
into T-75 flasks using 30 ml of fresh growth medium.
[0097] The culture assembly was inoculated with HBTC (mesenchymal)
cells and grown for several weeks. HBTC cells were first removed
from the T flasks by enzymatic digestion, washed once with calcium-
and magnesium-free phosphate-buffered saline (CMF-PBS), and assayed
for viability by trypan blue dye exclusion (Gibco). Cells were held
on ice in fresh growth medium prior to inoculation of the culture
assembly. The primary inoculum for the culture experiment included
2.times.10.sup.5 cells/ml HBTC cells, which were added to fresh
GTSF-2 media in a 35-ml growth module with 5 mg/ml of Cytodex-3
(Type I, collagen-coated cyclodextran) microcarriers having a
diameter of 120 mm (Pharmacia, Piscataway, N.J., USA). The 450 ml
nutrient module was filled with fresh GTSF-2 media, the culture
assembly was sealed as described above.
[0098] Briefly, the alternating ionic magnetic resonance is
supplied to the culture unit by encompassing the culture assembly
with the removable and adjustable alternating ionic magnetic
resonance coil. At increasing frequencies cultured cells and media
are exposed to an alternating ionic magnetic resonance signal at
fundamental intervals of 10, 14, 15, 16 and 32 Hz including the
harmonic intervals of each of these extending to 256 Hz and
incorporating all harmonics of the aforementioned fundamental
frequencies to infinity in the form of a square wave of 0.01-5000
mA. The alternating ionic magnetic resonance chamber providing the
electromagnetic protocol was placed around the culture device and a
series of stepped resonance pulses at approximately 500 msec
intervals was applied to the outside of the culture assembly. The
culture assembly and the unit was connected to a continuously
randomized gravity device and grown in a Form a humidified CO.sub.2
incubator with 94.5% air and 5.5% CO.sub.2 providing constant
atmosphere at 35.0.degree. C. to mimic that of the nasopharyngeal
epithelium. The HBTC cultures were allowed to grow for a minimum of
24 hours before the medium was changed. Thereafter, fresh medium
was replenished by replacing 65-100% of the spent medium within the
nutrient module once every 96-168 hour period.
[0099] At this point, the media for the culture experiments
comprised GTSF-2 supplemented with 10% fetal bovine serum. As the
cells proliferated, metabolic requirements increased, and the fresh
medium was routinely supplemented with an additional 100 mg/dl of
glucose.
[0100] The culture was sampled periodically over the course of the
experiment, generally at 24-48 hour time points, in order to
establish a cellular development profile. The parameters of glucose
utilization (FIG. 5A) and pH (FIG. 5B) were surveyed via iStat.TM.
clinical blood gas analyzer to determine the relative progress and
health of the cultures and the rate of cellular growth and
viability.
[0101] The cells were monitored as shown in FIGS. 6A to 6D. FIG. 6A
shows photos of the HBTC cells grown in T-flasks (passaged as
necessary to maintain growth over a 20-day period) that have been
infused with the calcium binding fluorescent dye, Fura-2AM. The
cells on the left were exposed to multi-variant electromagnetic
frequency field for the entirety of the 20-day growth period. Cells
in the right panel were not exposed to an electromagnetic field.
Exposure to alternating ionic magnetic resonance altered the
cellular distribution of calcium ions. FIG. 6B shows HBTC cells
grown on cultisphere or Cytodex-3 microcarriers in the alternating
ionic magnetic resonance bioreactor for 21 days. The upper left
panel shows cells exposed to alternating ionic magnetic resonance
for the entire growth period; the cells on the right were not
exposed to an electromagnetic field. Tissue-like assemblies of
cells and microcarrier beads (tissue-like assemblies) were treated
with Fura-2AM. Although the microcarriers shown retain background
levels of fluorescence as evidenced by the photo in the bottom
control panel showing the microcarrier alone, the cells exposed to
alternating ionic magnetic resonance emit a significant signal
above background levels. Arrows indicate clusters of cells attached
to, but not atop a microcarrier bead. FIG. 6C is similar to cells
shown in FIG. 6A, with HBTC cells grown with (left) or without
(right) alternating ionic magnetic resonance, but infused with the
potassium binding fluorescent dye, PBFI-2 AM. FIG. 6D illustrates
potassium ion staining of cells grown in the presence of
microcarrier beads either with (top) or without (middle)
alternating ionic magnetic resonance compared to a microcarrier
alone control treated with PBFI-AM (bottom).
Gene Induction in the Alternating Ionic Magnetic Resonance-Grown
HBTC TLAs
[0102] HBTC cells grown in the alternating ionic magnetic resonance
culture system demonstrate up-regulation of genes within specific
gene families, including levels of expression for various transport
and regenerative genes. Table 1 lists genes up-regulated in an
alternating ionic magnetic resonance field and provides the fold
increase relative to level of gene expression in cells grown
without a magnetic resonance field.
TABLE-US-00001 TABLE 1 Alternating Ionic Magnetic Resonance
Initiated Gene Up-Regulation GENE FAMILY FOLD GENE SYMBOL GENE NAME
INCREASE TRANSPORT FAMILY GENES (1) Calcium Ion Transport KCNMB1
Potassium large conductance +62 Calcium-activated channel,
subfamily M, beta member 1 CABP1 calcium binding protein 1 +52
(calbrain) CACNG1 calcium channel, voltage- +46 dependent, gamma
subunit 1 CABP2 calcium binding protein 2 +36 SLC24A3 solute
carrier family 24 +12 (sodium/potassium/calcium exchanger), member
3 CACNA1C calcium channel, voltage- +3.6 dependent, L type, alpha
1C subunit TACSTD tumor-associated calcium signal +2.8 transducer 2
CACNB1 calcium channel, voltage- +7.3 dependent, beta 1 subunit
CALB3 calbindin 3, vitamin D-dependent +4.2 calcium binding protein
KCNMA1 potassium large conductance +3.0 calcium-activated channel,
subfamily M, alpha member 1 CACL2 chloride channel, calcium +2.5
activated, family member 2 CAMK2A calcium/calmodulin-dependent +2.5
protein kinase (CaM kinase) II alpha S100A5 S100 calcium binding
protein A5 +2.3 (2) Potassium IonTransport KCNMB1 Potassium large
conductance +61 Calcium-activated channel, subfamily M, beta member
1 KCND3 potassium voltage-gated +21 channel, ShaI-related
subfamily, member 3 SLC24A3 solute carrier family 24 +8.6
(sodium/potassium/calcium exchanger), member 3 KCNK15 potassium
channel, subfamily K, +3.7 member 15 KCNK3 potassium channel,
subfamily K, +2.1 member 3 KCNJ12 potassium inwardly-rectifying
+7.6 channel, subfamily J, member 12 KCNQ1 potassium voltage-gated
+3.3 channel, KQT-like subfamily, member 1 KCNAB1 potassium
voltage-gated +3.1 channel, shaker-related subfamily, beta member 1
KCND3 potassium voltage-gated +3.0 channel, ShaI-related subfamily,
member 3 KCND2 potassium voltage-gated +2.8 channel, ShaI-related
subfamily, member 2 SLC12A5 solute carrier family 12, +2.7
potassium-chloride transporter member 5 KCNJ5 potassium
inwardly-rectifying +2.5 channel, subfamily J, member 5 SLC24A1
solute carrier family 24 +2.2 (sodium/potassium/calcium exchanger,
member 1 KCNE1L potassium voltage-gated +2.2 channel, Isk-related
family, member 1-like KCNK7 potassium channel, subfamily K, +2.0
member 7 KCNK4 potassium channel, subfamily K, +1.9 member 4 (3)
ATPase Transport ATP6V0A4 ATPase, H+ transporting, +32 lysosomal V0
subunit a isoform 4 ATP2A3 ATPase, Ca++ transporting, +14
ubiquitous ATP1A2 ATPase, Na+/K+ transporting, +6.5 alpha 2 (+)
polypeptide SERCA3 ATPase, Ca++ transporting, +6 ubiquitous ATP2A1
ATPase, Ca++ transporting, +13 cardiac muscle, fast twitch 1)
ATP6V0A2 ATPase, H+ transporting, +7.7 lysosomal V0 subunit a
isoform 2 ATPase mRNA sequence +7.2 ATP11A ATPase, Class VI, type
11A +5.6 ATP-binding Cassette, sub-family A (ABC1), +4.7 member 6
TAP2 transporter 2, ATP-binding +4.5 cassette, sub-family B
(MDR/TAP) ABCC9 ATP-binding cassette, sub-family +2.7 C (CFTR/MRP),
member 9 ATP4B ATPase, H+/K+ exchanging, beta +2.5 polypeptide
ATP8A1 ATPase, aminophospholipid +2.5 transporter (APLT), Class I,
type 8A, member 1 ABCA8 ATP-binding cassette, sub-family +2.4 A
(ABC1), member 8 ATP6V1B1 ATPase, H+ transporting, +2.4 lysosomal
56/58 kDa, V1 subunit B, isoform 1 ATP5G2 ATP , H+ transporting,
mitochondrial +2.0 synthase F0 complex, subunit c (subunit 9),
isoform 2 ATP1B4 ATPase, (Na+)/K+ transporting, +1.9 beta 4
polypeptide REGENERATION GENES (1) WNTs Family WNT2 wingless-type
MMTV integration +21038 site family member 2 WNT16 wingless-type
MMTV integration +180.8 site family member 16 WNT3 inducible
signaling pathway +45.8 protein 3 WNT4 +78.8 WNT8B +3.3 WNT1 +1.7
(2) Bone Morphogenetic Protein Family (BMP2) Bone Morphogenetic
Protein 2 +166.6 (BMP5) Bone Morphogenetic Protein 5 +88.0 BMP-2
inducible BMP inducible kinase +5.5 kinase BMPY Bone Morphogenetic
Protein Y +2.4 (BMP6) Bone Morphogenetic Protein 6 +1.6 (3) Catenin
Family Catenin cadherin-associated protein, +38.3 delta 2 (neural
plakophilin-related arm-repeat protein) Catenin +2.1 (4) Forkhead
Box Family FOXI1 Forkhead Box I1 +60.4 FOXA2 Forkhead Box A2 +9.0
FOXD1 Forkhead Box D1 +3.4 FOXA1 Forkhead Box A1 +2.0 FOXM1
Forkhead Box M1 +1.6 (5) SOX (SRY (Sex Determining Region Y)-BOX2)
Family SOX2 +2.2 SOX3 +1.9 SOX29 +1.8 SOX17 +1.7 (6) Transforming
Growth Factor (TGF) Family TGFBR2 transforming growth factor, beta
+1.9 receptor II TGFA transforming growth factor, alpha +1.9 TGIF2
TGFB-induced factor 2 (TALE +1.7 family homeobox) (7) Parathyroid
Hormone (PTH) Family PTH parathyroid hormone +180 PTHLH parathyroid
hormone-like +1.8 hormone PTHR2 parathyroid hormone receptor 2
+1.6
Example 3
Gene Induction in NHNP and HBE Cells Grown in the Alternating Ionic
Magnetic Resonance Culture System
NHNP TLA 3D cell culture
[0103] NHNP cells were obtained from Lonza (Walkersville, Md., USA)
and propagated in GTSF-2, a unique media containing glucose,
galactose and fructose supplemented with 10% fetal bovine serum
(FBS), at 37.degree. C. under a 5% CO.sub.2 atmosphere (2-4). NHNP
cells were initially grown as monolayers in human
fibronectin-coated flasks (BD Biosciences, San Jose, Calif.) and
pooled from at least five donors, as described previously (5). NHNP
cell cultures were expanded, tested for viral contaminants as
pre-certified by the manufacturer's production criteria (Lonza),
and cryopreserved in liquid nitrogen. Three-dimensional (3D) NHNP
TLAs were generated by seeding 3.times.10.sup.5 NHNP cells/ml onto
3 mg/ml Cultispher beads (Sigma-Aldrich, St. Louis, Mo.) into a 55
ml rotating wall vessel bioreactor (RWV; Synthecon, Houston, Tex.)
or into the culture unit of the alternating ionic magnetic
resonance culture apparatus and grown at 37.degree. C. under a 5%
CO.sub.2. Cells were allowed to attach to the beads for 48 h in the
bioreactor before re-feeding with GTSF-2 containing 10% FBS. To
maintain the TLA cultures within normal human physiological blood
chemistry parameters (pH 7.2 and a glucose concentration of 80-120
mg/dL), 20-90% of the media was replaced as required with fresh
GTSF2 media every 48 h, facilitating efficient tissue-like assembly
tissue growth and maturation prior to VZV infection. All metabolic
determinations were made using an iStat hand held blood gas
analyzer (Abbott Laboratories, Abbott Park, Ill.). Flow cytometry
analysis confirmed that after 180 days in culture, NHNP TLAs
expressed neuronal progenitor markers CXCR4, CD133, CD105-Endoglin,
CD 90-Thy-1 and CD49f-.alpha.6 Integrin at levels comparable to a
parental NHNP (2D) cell population.
HBE TLA 3D Cell Culture
[0104] Mesenchymal cells (HBTC) from human bronchi and tracheae
were obtained from three donors through Cambrex Biosciences
(Walkersville, Md.). LLC-MK2 and BEAS-2B epithelial cells (6) were
obtained from ATCC (Manassas, Va.). BEAS-2B cells were used instead
of primary cells to provide consistency from batch to batch.
BEAS-2B and HBTC cells were maintained in GTSF-2 medium with 7%
fetal bovine serum (7) in human fibronectin coated flasks (BD
Biosciences, San Jose, Calif.). Vero, HEp-2, and LLC-MK2 cells were
grown at 37.degree. C. in Eagle's modified minimum essential medium
supplemented with 2 mM non-essential amino acids, 100 units
penicillin, 100 .mu.g/ml streptomycin, 0.25 .mu.g/ml amphotericin
B, 10% fetal bovine serum, 2 mM L-glutamine, and 25 mM HEPES buffer
(Gibco-BRL, Gaithersburg, Md.).
[0105] To construct 3D HBE tissue-like assembly cultures, HBTC
cells from a monolayer culture were seeded at 2.times.105 cells/mL
into a 55-mL rotating wall vessel (RWV) (Synthecon, Houston, Tex.)
or into the culture unit of the alternating ionic magnetic
resonance culture apparatus with 4-5 mg/mL of Cytodex-3
microcarriers, type I collagen-coated cyclodextran microcarriers
(Pharmacia, Piscataway, N.J.) at 35.degree. C. Cultures were
allowed to grow for a minimum of 48 hours before the medium was
changed. BEAS-2B cells were seeded at 2.times.10.sup.5 cells/mL 4
to 6 days after HBTCCytodex 3 microcarrier aggregates were formed.
Thereafter, approximately 65% of the media was replaced every 20 to
24 hours. As metabolic requirements increased, the glucose
concentration in GTSF-2 medium was increased to 200 mg/dL.
Tissue-like assembly cultures were grown in RWV to 1 to 2 mm in
diameter using the rotary cell culture system (Synthecon, Houston,
Tex.) or into the culture unit of the alternating ionic magnetic
resonance culture apparatus at 35.degree. C. with appropriate
rotation rate for aggregate suspension. Cell numbers were
determined after treating the tissue-like assemblies with 2000 U/mL
type I collagenase (Invitrogen, Carlsbad, Calif.) at 37.degree. C.
for 10 minutes. Expression levels of epithelial markers in TLAs are
very similar to the levels in normal human lung than in 2D BEAS-2B
and HBTC cells.
Alternating Ionic Magnetic Resonance-Exposed HBE and NHNP TLAs
[0106] A set of human bronchial epithelial (HBE) tissue-like
assembly samples and a set of normal human neural progenitor (NHNP)
tissue-like assembly samples, each set contained in at least 3
rotating wall vessels (RWVs) were exposed to an alternating ionic
magnetic resonance stimulation field of predetermined profile. The
profile substantially comprises a biphasic, square wave with a
frequency of about .about.10 Hz, a wavelength of about 500 ms, a
rising slew rate between about 0.1 T/s (1.0 kG/s) to about 0.50 T/s
(5.0 kG/s), a falling slew rate between about 0.50 T/s (5.0 kG/s)
and about 2.0 T/s (20.0 kG/s), a dwell time of about 10% after each
burst, a duty cycle of about 80% on and about 20% off, and a
resultant B-Field magnitude of about 100 .mu.T (1.0 G). The
experiment was conducted at .about.10 Hz. For reference purposes,
the frequency of Earth's geomagnetic field is 7.83 Hz, thus the
experiment satisfies the criteria of being appreciably different
from the background magnetic field. Exposure was continuous for the
duration of a period of about 15-90 days or about 360-2160 hours. A
gene fold change analysis, as describe in Example 1, was conducted
for the HBE and NHNP TLA samples exposed to the alternating ionic
magnetic resonance stimulation field.
Gene Induction in Alternating Ionic Magnetic Resonance-Grown HBE
and NHNP TLAs
[0107] Although the HBE and NHNP tissue-like assemblies responded
differently when exposed to an alternating ionic magnetic resonance
field under substantially identical conditions, unexpectedly, in
both sets virally-associated and viral oncogenes were activated and
their expression levels were upregulated. Most of the
differentially regulated genes are related to the ability of
viruses to be absorbed or introduced into the human cell to enable
replication and proliferation. Table 2 lists the up-regulated viral
genes initiated by alternating ionic magnetic resonance.
TABLE-US-00002 TABLE 2 Alternating Ionic Magnetic Resonance
Initiated Viral Gene Up-Regulation GENE SYMBOL GENE NAME FOLD
INCREASE HBE TLA GENES RAB6A RAB6A virus associated 2.7991202 RAB5B
RAB5B virus associated 1.5721819 IVNS1ABP influenza virus NS1A
binding protein 1.5685624 JUN jun oncogene 1.6782385 JUNB jun B
proto-oncogene 1.5822183 HTATSF1 HIV-1 Tat specific factor 1
1.8373269 FOSB FBJ murine osteosarcoma viral oncogene homolog B
1.5298892 JUND jun D proto-oncogene 3.2985632 CXADR coxsackie virus
and adenovirus receptor 1.8511372 EVI2A ecotropic viral integration
site 2A 1.922923 EBI2 Epstein-Barr virus induced gene 2
(lymphocyte- 1.7887306 specific G protein-coupled receptor)
IVNS1ABP influenza virus NS1A binding protein 1.7672802 AKT1 v-akt
murine thymoma viral oncogene homolog 1 1.5862192 FOS v-fos FBJ
murine osteosarcoma viral oncogene 4.55682 homolog UBE3A ubiquitin
protein ligase E3A (human papilloma virus 1.5529021 E6-associated
protein EVI2B ecotropic viral integration site 2B 1.566628 RRAS
related RAS viral (r-ras) oncogene homolog 1.6267682 HRB HIV-1 Rev
binding protein 1.6248319 JUND jun D proto-oncogene 2.2347317 FYN
FYN oncogene related to SRC 1.8570358 ITPR1 inositol 1 1.5927685
RAB23 RAB23 1.7071296 AKT3 v-akt murine thymoma viral oncogene
homolog 3 1.8035016 (protein kinase B BIRC6 baculoviral IAP
repeat-containing 6 (apollon) 1.5996437 THRB thyroid hormone
receptor 1.7461618 TPR translocated promoter region (to activated
MET 1.7921942 oncogene) NHNP TLA GENES ETS2 v-ets erythroblastosis
virus E26 oncogene homolog 1.5718486 2 (avian) JUNB jun B
proto-oncogene 2.720869 EGFR epidermal growth factor receptor
(erythroblastic 2.6352847 leukemia viral (v-erb-b) oncogene homolog
BIRC5 baculoviral IAP repeat-containing 5 (survivin) 1.6022671 FOSB
FBJ murine osteosarcoma viral oncogene homolog B 2.0152645 PVRL2
poliovirus receptor-related 2 (herpesvirus entry 1.6020694 mediator
B) SKIV2L superkiller viralicidic activity 2-like (S. cerevisiae)
1.520639 FOS v-fos FBJ murine osteosarcoma viral oncogene 5.8558683
homolog TNFRSF14 tumor necrosis factor receptor superfamily
1.8052368 member 14 (herpesvirus entry mediator) PVR poliovirus
receptor 2.9330401 THRA thyroid hormone receptor (Alpha
(erythroblastic 1.5989169 leukemia viral (v-erb-a) oncogene homolog
MOV10 Mov10 Maloney Leukemia Virus 10 1.8294989 PVRL2 poliovirus
receptor-related 2 (herpesvirus entry 1.5439448 mediator B) ISY1
/// ISY1 splicing factor homolog (S. cerevisiae) /// 1.802375 RAB43
RAB43 MRVI1 murine retrovirus integration site 1 homolog 2.84193
MAFK v-maf musculoaponeurotic fibrosarcoma oncogene 1.6252148
homolog K (avian) RAB4B RAS oncogene family 1.5139388 MRVI1 murine
retrovirus integration site 1 homolog 2.0572836 RAB7B RAS oncogene
family 1.5806108 LOC401233 similar to HIV TAT specific factor 1;
cofactor 2.2209923 required for Tat activation of HIV-1
transcription
Example 4
3-Dimensional TLA Models of Viral Infection
v63G/70R Infection of NHNP TLAs
[0108] Varicella zoster virus (VZV) was propagated in human
melanoma cells (MeWo, American Type Culture Collection, ATCC,
Manassas, Va.) in Dulbecco's minimal essential medium supplemented
with 10% fetal bovine serum, 100 U/ml penicillin and 0.1 mg/ml
streptomycin (Sigma-Aldrich, St. Louis, Mo.) at 37.degree. C. under
a 5% CO.sub.2 (8). Wild-type and recombinant viruses were passaged
on MeWo cells by co-cultivation of infected with uninfected cells
at a ratio of 1/5 (9). MeWo cells for the infection of NHNP
cultures were adapted to GTSF2 medium over two passages prior to
harvest of VZV.
[0109] Cell free VZV was used for the NHNP TLA infections to avoid
transfer of any infected MeWo cells to the TLA culture (10).
Briefly, infected cells were harvested at 96 h post-infection
(p.i.) and resuspended in reticulocyte standard buffer (10 mM NaCl,
1.5 mM MgCl.sub.2, 10 mM Tris-HCl, pH 7.4). Cells were disrupted by
Dounce (type A) homogenization (Cole-Parmer, Vernon Hills, Ill.)
and clarified by centrifugation at 900.times.g for 15 min.
[0110] NHNP tissue-like assemblies were infected in the RWV with
cell-free VZV at a multiplicity of infection (MOI) of 0.1 by
absorption at room temp for 30 min in 20 ml GTSF-2. Then, the RWVs
were filled with fresh GTSF-2/10% FBS and transferred to a
humidified incubator with under 5% CO.sub.2 atmosphere at
37.degree. C. Every 24 h p.i., 55-65% of the culture media was
replaced with fresh GTSF-2 containing 10% FBS. Samples were
collected approximately every other day for .about.70 days to
determine viral genome copies utilizing a dually tagged v63G/70R
recombinant.
[0111] Dually tagged v63G/70R was able to efficiently infect NHNP
tissue-like assemblies, as evidenced by an approximate 50-fold
increase in VZV genome copies from 0 to 18 days post-infection
(dpi). After 18 dpi, VZV genome copy numbers remained constant,
indicating that the virus had established equilibrium between de
novo virus DNA replication and degradation. Glucose utilization was
used to monitor the metabolic activity of infected and uninfected
3D NHNP tissue-like assemblies and MeWo cultures. Each culture was
initially maintained for 39 days to establish a baseline glucose
consumption rate before and after infection with v63G/70R. Upon
infection, glucose utilization rapidly declined in MeWo cells, as
cell death, likely as a consequence of lytic VZV replication, was
evident microscopically (data not shown). In contrast, glucose
uptake in NHNP tissue-like assemblies was not altered as a response
to VZV infection, suggesting that limited, if any, lytic VZV
infection occurred. Confocal analysis of v63G infected NHNP
tissue-like assemblies at 27 dpi revealed that the progenitor
neuronal marker Nestin and the mature neuronal marker .beta.
Tubulin-III colocalized with GFP, indicating that VZV
preferentially established a persistent infection in these
cells.
[0112] GFP/RFP ratios in v63G/70R infected NHNP tissue-like
assemblies remained unaltered for at least 69 days in culture,
suggesting that the VZV genome is stably maintained in NHNP
tissue-like assemblies. In addition, confocal microscopy confirmed
that both ORF63-eGFP and ORF70-mRFP are expressed in NHNP
tissue-like assembly cultures infected with v63G/70R. A stable
viral genome is preserved for an extended period in NHNP
tissue-like assemblies.
Paramyxovirus Infection of HBE TLAs
[0113] Tissue-like assemblies were inoculated at a MOI of 0.1 with
both wtPIV3 and attenuated PIV3 viruses to achieve an effective MOI
of 1 for the cells on the outer surface. After virus absorption at
room temperature for 1 hour, HBE TLA cultures were washed three
times with DPBS (Invitrogen, Carlsbad, Calif.) and fed with media.
All air bubbles were removed from the RWV before rotation to
eliminate shearing of the cells (7). Approximately 65% of the
culture media was replaced every 48 hours. For virus titration,
samples were collected on days 0, 2, 4, 6, 8, and 10. In HBE
tissue-like assemblies, replication of wtPIV3 approached 7.5 log 10
pfu/mL by day 6 pi, while the attenuated virus replicated maximally
to 5.5 log 10 pfu/ml on day six. Reduced replication in attenuated
viruses may be due to a slower progression from layer to layer in
the 3D HBE tissue-like assemblies. Inceased secretion of at least
2-fold of cytokines, chemokines and colony stimulating factors was
found for interleukin-1, -4, -6, and -8, for RANTES, MIP-1a, MIP-1,
and G-CSF.
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T. J. Physiological and Molecular Genetic Effects of Time-Varying
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(1988). [0120] 7. Goodwin et al. Proc Soc Exp Biol Med 202:181-192
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[0124] While the present invention is described with reference to
one or more particular embodiments, those skilled in the art will
recognize that many changes may be made thereto without departing
from the spirit and scope of the present invention. Each of these
embodiments and obvious variations thereof is contemplated as
falling within the spirit and scope of the claimed invention set
forth in the following claims.
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