Note: Descriptions are shown in the official language in which they were submitted.
CA 02595744 2007-08-01
. . _
t
METHOD OF PROVIDING READILY AVAILABLE CELLULAR MATERIAL DERIVED FROM CORD
BLOOD, AND A COMPOSITION THEREOF
FIELD OF THE INVENTION
The present invention is directed to cord blood stem cells prepared in a TVEMF-
bioreactor, and to the process for such preparation, compositions thereof, and
methods of
treating a mammal with the cells or compositions.
BACKGROUND OF THE INVENTION
Regeneration of human tissue has long been a desire of the medical community.
Thus
far, repair of human tissue has been accomplished largely by transplantations
of like tissue
from a donor. Beginning essentially with the kidney transplant from one of the
Herrick twins
to the other and later made world famous by South African Doctor Christian
Barnard's
transplant of a heart from Denise Darval to Louis Washkansky on December 3,
1967, tissue
transplantation became a widely accepted method of extending life in terminal
patients.
Transplantation of human tissue, from its first use, encountered major
problems,
primarily tissue rejection due to the body's natural immune system. This often
caused the use
of tissue transplantation to have a limited prolongation of life (Washkansky
lived only 18
days past the surgery).
In order to overcome the problem of the body's immune system, numerous anti-
drugs (e.g. Imuran, Cyclosporine) were soon developed to suppress the immune
rejection
system and thus prolong the use of the tissue prior to rejection. However, the
rejection
problem has continued creating the need for an alternative to tissue
transplantation.
Bone marrow transplantation was also used, and is still the procedure of
choice for
treatment of some illnesses, such as leukemia, to repair certain tissues such
as bone marrow,
but bone marrow transplantation also has problems. It requires a match from a
donor (found
less than 50% of the time); it is painful, expensive, and risky. Consequently,
an alternative to
bone marrow transplantation is highly desirable, Transplantation of tissue
stem cells such as
the transplantation of liver stem cells found in U.S. Patent No. 6,129,911
have similar
limitations rendering their widespread use questionable.
In recent years, researchers have experimented with the use of pluripotent
embryonic
stem cells as an alternative to tissue transplant. The theory behind the use
of embryonic stem
cells has been that they can theoretically be utilized to regenerate virtually
any tissue in the
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CA 02595744 2007-08-01
~ body. The use of embryonic stem cells for tissue regeneration, however, has
also encountered
problems. Among the more serious of these problems are that transplanted
embryonic stem
cells have limited controllability, they sometimes grow into tumors, and the
human embryonic
stem cells that are available for research would be rejected by a patient's
immune system
(Nature, June 17, 2002: Pearson, "Stem Cell Hopes Double", news@nature.com,
published
online:21 June 2002). Further, widespread use of embryonic stem cells is so
burdened with
ethical, moral, and political concerns that its widespread use remains
questionable.
Cord blood has been the focus of several areas of research. The pluripotent
nature of
stem cells was first discovered from an adult stem cell found in bone marrow.
Verfaille, C.M.
et al., Pluripotency of mesenchymal stem cells derived from adult marrow.
Nature 417,
published online 20 June; doi:10.1038/nature00900, (2002) cited by Pearson, H.
Stem cell
hopes double. news@nature.com, published online:21 June 2002; doi:
10.1038/news020617- ~
11. Pluripotent CD34+ stem cells and oligopotent lymphoid progenitor cells
have been found
in umbilical cord blood and have been shown to differentiate into cell types
such as lymphoid
natural killer cells. Perez S.A. et al., A novel myeloid-like NK cell
progenitor in human
umbilical cord blood. Blood 101(9):3444-50 (May 1, 2003). In addition, B cell
progenitors
have been discovered to reside not only in bone marrow, but also in cord
blood. Sanz E. et
al., Human cord blood CD34+Pax-5+ B-cell progenitors: single-cell analyses of
their gene
expression profiles. Blood 101(9):4324-30 (May 1, 2003).
Boyse et al., U.S. Pat. No. 6,569,427 B1, discloses the cryopreservation and
usefulness of cryopreserved fetal or neonatal blood in the treatment or
prevention of various
diseases and disorders such as anemias, malignancies, autoimmune disorders,
and various
immune dysfunctions an deficiencies. Boyse also discloses the use of
hematopoietic
reconstitution in gene therapy with the use of a heterologous gene sequence.
The Boyse
disclosure stops short, however, of expansion of cells for therapeutic uses.
CorCell, a cord
blood bank, provides statistics on expansion, cryopreservation, and
transplantation of
umbilical cord blood stem cells. "Expansion of Umbilical Cord Blood Stem
Cells",
Information Sheet Umbilical Cord Blood, CorCell, Inc. (2003). One expansion
process
discloses utilizing a bioreactor with a central collagen based matrix.
Research Center Julich:
Blood Stem Cells from the Bioreactor. Press release May 17, 2001. In addition,
freezing
cord blood cells before or after expansion has been shown to have no effect on
the expansion
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CA 02595744 2007-08-01
capabilities of stem cells. Lazzari, L. et al., Evaluation of the effect of
cryopreservation on ex
vivo expansion of hematopoitic progenitors from cord blood. Bone Marrow Trans.
28:693-
698(2001).
Cord blood has been found to provide better reconstitution. of the
hematopoietic
reservoir as compared to bone marrow. Frassoni F. et al., Cord blood
transplantation provides
better reconstitution of hematopoeitic reservoir as compared to bone marrow
transplantation.
Blood (April 3, 2003). See also First Unrelated Stem Cell Transplant Performed
in Atlanta
December 12, 1998- 1 year update. Bone Marrow and Cord Blood'Stem Cell
Transplant, The
Sickle Cell Information Center (1999).
Research continues in an effort to elucidate the molecular mechanisms involved
in the
expansion of stem cells. For example, the CorCell article discloses that a
signal molecule
named Delta-1 aids in the developmeint of cord blood stem cells. Ohishi K. et
al.: Delta-1
enhances marrow and thymus repopulating ability of human CD34+/CD38- cord
blood cells.
Clin. Invest. 110:1165-1174 (2002).
Throughout this application, the term "cord blood cells" means blood cells
derived
from the umbilical cord and/or the placenta of a fetus or infant.
Although cord blood cells are defined as adult, or somatic, stem cells,
several factors
make cord blood cells, and in particular cord blood stem cells, unique.
First, cord blood is primitive. Cord blood stem cells are young; they may have
more
plasticity than older cells, meaning they can give rise to a greater variety
of specialized cells.
They are also more likely to be healthier cells because they have had fewer
opportunities to be
affected by damaging environmental toxins that may change DNA. Furthermore,
because
they are young, cord blood stem cells can better integrate into the recipient
patient and are less
likely to cause graft vs. host disease (GvHD) or cell rejection: Also because
they are young,
cord blood stem cells may be considered a little less stable than adult-aged
peripheral blood
stem cells, for instance because the cord blood stem cells are still
relatively new and have
been in a very protected environment. Cord blood stem cells may therefore be
more
susceptible to damage, for instance from cryopreservation, than more aged stem
cells.
Second, cord blood is stem cell-rich. Cord blood contains white blood cells
(including
mononuclear cells; for the purposes of this invention, a mononuclear cell is a
cells having
on.ly one nucleus) and red blood cells. Typically, approximately 1-2% of cord
blood
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CA 02595744 2007-08-01
=5 mononuclear cells are stem cells. This makes cord blood one of the richest
sources of stem
cells. Cord blood collected from a Caesarean section is typically even a
little richer in stem
cells than cord blood collected immediately after vaginal birth. It is also
easier to isolate stem
cells in cord blood as opposed to other tissues. While adult stem cells can be
found in
numerous mature tissues, they are found in lesser quantities and are harder to
locate.
Third, and finally, cord blood is an available source of stem cells. Adult
stem cell
transplants from body tissue such as bone marrow are not readily available.
Cord blood
banking provides a source of readily available stem cells. A cord blood
collection from a
typical human infant immediately after birth will typically yield 50 to 100 ml
cord blood.
The umbilical cord, which contains cord blood, is the cord that connects a
fetus to a
maternal placenta, providing nutrients and removing wastes. The umbilical cord
is a cordlike
structure about 22 in. (56 cm) long, extending from the abdominal wall of the
fetus to the
maternal placenta.
The main function of the umbilical cord is to carry nourishment and oxygen
from the }
placenta to the fetus and return waste products to the placenta from the
fetus. Essentially, the
umbilical cord is a cord like structure formed by, and integral with, the
fetus' membrane at
one end, with the other end terminating in the placenta. Enclosed within the
cord is a mucoid
jelly which houses one vein which carries oxygenated blood to the fetus and
two arteries
which carry un-oxygenated blood away from the fetus.
Blood is carried from the fetus along the umbilical cord and into the
placenta. In the
placenta in vivo, cord blood is brought into close proximity with the mother's
blood 'such that
oxygen, nutrients, and antibodies diffuse from the mother's blood into the
cord blood. Waste
materials from the fetus pass into the mother's blood, via the two un-
oxygenated arteries. The
cord blood, which has been enriched with nutrients, oxygenated, and cleaned of
waste, is then
carried back to the fetus by the vein that carries oxygenated blood through
the umbilical cord.
After birth, the umbilical cord is clarnped off and cut. The stump that is
attached to the
infant after the cord is cut off, eventually withers and drops off, leaving
the scar known as the
navel.
Because cord blood is especially rich in stem aells some parents choose to
save it in
special cord blood banks. The cord blood stem cells contained therein can be
used in case of
future need as a transplant alternative to bone marrow. Studies have shown
that even people
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not related to the cord blood donor (genetically mismatched) may benefit from
transplants of
cord blood in combating leukemia and other cancers without eliciting an immune
reaction
rejecting the cord blood cells.
There are two.typical.ways of collecting cord blood; blood bag collection and
syringe
collection. Blood bag collection involves a health care provider inserting a
needle into the
umbilical vein and, with the assistance of gravity, draining the blood into a
bag. Once the {
blood has stopped flowing, the bag will be sealed and labeled by the health
care provider.
This method is usually done before the placenta is delivered.
Syringe collection is similar to blood bag collection except that the cord
blood is
drawn into syringes containing anticoagulants (a substance that prevents the
blood from
clotting). The blood is stored in the syringes instead of in blood bags. This
method can be
done before or after the placenta is delivered. It is thought to be a more
reliable way of
collecting blood than blood bag collection. It also allows for more blood to
be collected than
is possible with blood bag collection. Regardless of which method is utilized,
or whether
another process for collecting cord blood is utilized, the whole process of
collection may take
as little as five minutes to perform, or even less. Preferably, the cord blood
is collected within
10 to 15 minutes after birth. Waiting longer than this may result in less cord
blood being
collected, and therefore, fewer cord blood stem cells collected.
In the case of cord blood banking, or storage, once the cord blood arrives at
the
storage facility the cord blood is tested to make sure it does not carry any
infectious or genetic
diseases, like hepatitis, HIV/AIDS, leukemia, or an immune disorder. If there
are any such
problems with the cord blood, it may either be considered unsuitable for
storage, or, in some
instances, the blood may still be stored with the associated risks noted. If
the blood is needed
in the future parents can assess whether or not the need for the cord blood
stem cells
outweighs the associated risks carried with the cord blood.
Cord blood that will be stored typically goes through a series of processing
before
being banked. First, the cord blood is separated into its parts; white blood
cells, red blood
cells, and plasma. This is either done in a centrifuge (an apparatus that
spins the container of
blood until the blood is divided) or by sedimentation (the process of
injecting sediment into
the container of blood causing the blood to separate). Second, once the cord
blood is divided
with the red blood cells (RBC) on the bottom, white blood cells (WBC) in the
middle, and the
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plasma on top, the white blood cells are removed for storage. The middle
layer, also known as
the "buffy coat" contains the cord blood stem cells of interest; the other
parts of the blood are
not needed. For some banks, this will be the extent of their processing.
However, other banks
will go on to process the buffy coat by removing the mononuclear cells (in
this case, a subset ~of white blood cells) from the WBC. While not everyone
agrees with this method, there is less
to store and less cryogenic nitrogen is needed to store the cells. 3 j
It is preferable to remove the RBC from the cord blood sample. While people
may
have the same HLA type (which is needed for the transplanting of stem cells),
they may not
have the same blood type. By removing the RBC, adverse reactions to a stem
cell transplant
can be minimized. By eliminating the RBC, therefore, the stem cell sample has
a better
chance of being compatible with more people.RBC can also burst when they are
thawed,
releasing free hemoglobin. This type of hemoglobin can seriously affect the
kidneys of people
receiving a transplant. Additionally, the viability of the stem cells are
reduced when RBC
rupture.
Prior to the cells being frozen, they may be expanded (that is, increased in
number, not
size).
Once the RBC's are removed the cells will begin to be preserved and frozen for
long-
term storage.However, this must be done slowly and carefully in order not to.
damage the stem
cells. Before the blood cells are frozen, they are first mixed in a solution
to help prevent them
from being damaged while frozen. This solution is referred to as the
cryopreservative,
cryopreservation solvent or cryoprotectant. Once the expanded cells are in
cryopreservative,
they are slowly frozen so as to guard the cells against damage.
Once frozen (generally to a temperature of about -196 C), the cells are
transferred to a
permanent storage freezer. While in this freezer, they will remain frozen in
either liquid or
vapor nitrogen. Different types of freezers are commonly used to preserve cord
blood stem
cells. One type is the "BioArchive" freezer. This machine not only freezes the
blood, but also
inventories it and manages up to 3,626 blood bags. It has a robotic arm that
will retrieve the
specified blood sample when required. This ensures that no other samples are
disturbed or
exposed to warmer temperatures. Other types presently commercially available
include, but
are not limited to, Sanyo Model MDF-1155ATN-152C and Model MDF-2136 ATN-135C,
and Princeton CryoTech TEC 2000.
6
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CA 02595744 2007-08-01
=
S Expansion of the cord blood stem cells may take several days. In a situation
where it
is important to have an imrnediate supply of cord blood stem cells, such as a
life-or-death
situation or in the case of a traumatic injury, especially if research needs
to be accomplished prior to reintroduction of the cells, several days may not
be available to await for the
expansion of the cord blood stem cells. It is particularly desirable,
therefore, to have such
expanded cord blood stem cells available from birth forward in anticipation of
an emergency
where every minute in delaying treatment can mean the difference in life or
death.
There is a need, therefore, to provide a method and process of repairing human
tissue
that is not based on organ transplantation, bone marrow transplantation, or
embryonic stem
cells, and yet provides a composition of expanded cord blood stem cells,
unlikely to elicit an
immune response, for use in a matter of hours rather than days.
SUMMARY OF THE INVENTION
The present invention relates in part to cord blood stem cells from a mammal,
preferably
human, wherein said cord blood stem cells are TVEMF-expanded in a rotating
TVEMF-
bioreactor. The present invention also relates to cord blood stem cells from a
mammal,
preferably human. The invention also relates to compositions comprising these
cells, with other
components added as desired, including pharmaceutically acceptable carriers,
cryopreservatives,
and cell culture media.
The present invention also relates to a method of treating a mammal with the
cord blood
stem cells and cord blood stem cell compositions of the present invention.
Such treatment may 25 be for tissue repair and regeneration, to treat a
disease, or any other uses discussed throughout
this application. Also comprised herein is a composition-and method for the
treatment of any of
the diseases defined herein, or for the repair of tissue or organ, comprising
the cord blood stem
cell compositions of the present invention. Also comprised herein is use of a
composition of the
present invention for the preparation of a medicament for the treatment of any
of the diseases
discussed herein, or for the repair or regeneration of tissue as described
herein.
BRIEF DESCRIPTION OF THE DRAWINGS
In the drawings,
Figure 1 schematically illustrates a preferred embodiment of a culture carrier
flow loop of a
bioreactor;
7
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Figure 2 is an elevated side view of a preferred embodiment of a TVEMF-
bioreactor of the
invention;
Figure 3 is a side perspective of a preferred embodiment of the TVEMF-
bioreactor of Figure 2;
Figure 4 is a vertical cross sectional view of a preferred embodiment of a
TVEMF- bioreactor;
Figure 5 is a vertical cross sectional view of a TVEMF- bioreactor;
Figure 6 is an elevated side view of a time varying electromagnetic force
device that can house,
and provide a time varying electromagnetic force to, a bioreactor;
Figure 7 is a front view of the device shown in Figure 6;
Figure 8 is a front view of the device shown in Figure 6, further showing a
bioreactor therein, Figure 9 is the orbital path of a typical cell in a non-
rotating reference frame; ;
Figure 10 is a graph of the magnitude of deviation of a cell per revolution;
Figure 11 is a representative cell path as observed in a rotating reference
frame of the culture
medium;
Figure 12 illustrates the expansion pattern of total nucleated cells in a
rotating bioreactor versus a
dynamic moving culture;
.
Figure 13 illustrates the expansion pattern of CD133+ cells in a rotating
bioreactor versus a
dynamic moving culture; and
Figure 14 illustrates the expansion pattern of CD34+ cells in a rotating
bioreactor versus a
dynamic moving culture.
DETAILED DESCRIPTION OF THE DRAWINGS
In the simplest terms, a rotating TVEMF- bioreactor comprises a cell culture
chamber
and a time varying electromagnetic force source. In operation, a cord blood
mixture is place into
the cell culture chamber. The cell culture chamber is filled so as to create a
three-dimensional
environment wherein each individual non-adherent cord blood cell is sus nded.
The cell
1~
culture chamber is rotated in one direction, 360 degrees, over a period of
time during which a
time varying electromagnetic force is generated in the chamber by the time
varying
electromagnetic force source. During their time in the rotating TVEMF-
bioreactor, the cells are
suspended in discrete microenvironments in the essentially quiescent three-
dimensional
enviroxument created therein. Upon completion of the time, the expanded cord
blood mixture is
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CA 02595744 2007-08-01
. .
t t
L T #
. ,5 removed from the chamber. In a more complex TVEMF- bioreactor system, the
time varying
electromagnetic force source can be integral to the TVEMF- bioreactor, as
illustrated in Figures ~
. 2-5, but can also be adjacent to a bioreactor as in Figures 6-8.
Furthernaore, a fluid carrier, which
provides sustenance to the cells, can be periodically refreshed and removed.
Preferred TVEMF-
are described herein.
bioreactors
Referring now to Figure 1, illustrated is a preferred embodiment of a culture
carrier flow
loop 1 in an overall bioreactor culture system for growing mammalian cells
having a cell culture
chamber 19, preferably a rotating cell culture chamber, an oxygenator 21, an
apparatus for '.
facilitating the directional flow of the culture carrier, preferably by the
use of a main pump 15,
and a supply manifold 17 for the selective input of such culture carrier
requirements as, but not
limited to, nutrients 3, buffers 5, fresh medium 7, cytokines 9, growth
factors 11, and hormones
13. In this preferred embodiment, the main pump 15 provides fresh fluid
carrier to the
oxygenator 21 where the fluid ca.rrier is oxygenated and passed through the
cell culture chamber
19. The waste in the spent fluid carcier from the cell culture chamber 19 is
removed and
delivered to the waste 18 and the remaining cell culture carrier is returned
to the manifold 17 I~
where it receives a fresh charge, as necessary, before recycling by the pump
15 through the
oxygenator 21 to the cell culture chamber 19.
In the culture carrier flow loop 1, the culture carrier is circulated through
the living cell
culture in the chamber 19 and around the culture carrier flow loop 1, as shown
in Figure 1, In
this loop 1, adjustments are made in response to chemical sensors (not shown)
that maintain
constant conditions within the cell culture reactor chamber 19. Controlling
carbon dioxide
pressures and introducing acids or bases corrects pH. Oxygen, nitrogen, and
carbon dioxide are
dissolved in a gas exchange system (not shown) in order to support cell
respiration. The closed
loop I adds oxygen and removes carbon dioxide firom a circulating gas
capacitance. Although
Figure 1 is one preferred embodiment of a culture carrier flow loop that may
be used in the
present invention, the invention is not intended to be so limited. The input
of culture carrier
requirements such as, but not limited to, oxygen, nutrients, buffers, fresh
medium, cytokines,
growth factors, and hormones into a bioreactor can also be performed manually,
automatically,
or by other control means, as can be the control and removal of waste and
carbon dioxide.
Figures 2 and 3 illustrate a preferred embodiment of a TVEMF- bioreactor 10
with an
integral time varying electromagnetic force source. Figure 4 is a cross
section of a rotatable
9
.
CA 02595744 2007-08-01
{
.5 TVEMF-bioreactor 10 for use in the present invention in a preferred form.
The TVEMF-
of Figure 4 is illustrated with an integral tirne varying electromagnetic
force
bioreactor
source. Figure 5 also illustrates a preferred embodiment of a TVEMF-
bioreactor with an
integral time varying electromagnetic force -source. Figures 6-8 show a
rotating bioreactor with
an adjacent time varying electromagnetic force source.
10 Turning now to Figure 2, illustrated in Figure 2 is an elevated side view
of a preferred
embodiment of a TVEMF-bioreactor 10 of the present invention. Figure 2
comprises a motor
housing 111 supported by a base 112. A motor 113 is attached inside the motor
housing 111 and
connected by a first wire 114 and a second wire 115 to a control box 116 that
has a control
means therein whereby the speed of the motor 113 can be incrementally
controlled by turning the
control knob 117. The motor housing 111 has a motor 113 inside set so that a
motor shaft 118
extends through the housing 111 with the motor shaft 118 being longitudinal so
that the center of
the shaft 118 is parallel to the plane of the earth at the location of a
longitudinal chamber 119,
preferably made of a transparent material including, but not limited to,
plastic.
In this preferred embodiment, the longitudinal chamber 119 is connected to the
shaft 118
so that in operation the chamber 119 rotates about its longitudinal axis with
the longitudinal axis
parallel to the plane of the earth. The chamber 119 is wound with a wire coil.
120. The size of the
wire coil 120 and number of times it is wound are such that when a square wave
current
preferably of from 0.1mA to 1000mA is supplied to the wire coil 120, a time
varying
electromagnetic force preferably of from 0.05 gauss to 6 gauss is generated
within the chamber
119. The wire coil 120 is connected to a first ring 121 and a second ring 122
at the end of the
shaft 118 by wires 123 and 124. These rings 121, 122 are then contacted by a
first
electromagnetic delivery wire 125 and a second electromagnetic delivery wire
128 in such a
manner that the chamber 119 can rotate while the current is constantly
supplied to the coil 120.
An electromagnetic generating device 126 is connected to the wires 125, 128.
The
electromagnetic generating device 126 supplies a square wave to the wires 125,
128 and coi1120
by adjusting its output by turning an electromagnetic generating device knob
127.
Figure 3 is a side perspective view of the TVEMF-bioreactor 10 shown in Figure
2 that
may be used in the present invention.
Turning now to the rotating TVEMF-bioreactor 10 illustrated in Figure 4 with a
culture
chamber 230 which is preferably transparent and adapted to contain a cord
blood mixture
CA 02595744 2007-08-01
.5 therein, fnrther comprising an outer housing 220 which includes a first 290
and second 291
cylindrically shaped transverse end cap member having facing first 228 and
second 229 end
surfaces arranged to receive an inner cylindrical tubular glass member 293 and
an outer tubular
glass member 294. Suitable pressure seals are provided. Between the inner 293
and outer 294
tubular members is an annular wire heater 296 which is utilized for obtaining
the proper
incubation temperatures for cell growth. The wire heater 296 can also be used
as a time varying
electromagnetic force device to supply a time varying electric field to the
culture chamber 230
or, as depicted in Figure 5, a separate wire coi1144 can be used to supply a
time varying
electromagnetic force. The first end cap member 290 and second end cap member
291 have inner
curved surfaces adjoining the end surfaces 228, 229 for promoting smoother
flow of the mixture
within the chamber 230. The first end cap member 290, and second end cap
member 291 have a
first central fluid transfer journai member 292 and second central fluid
transfer journal member
295, respectively, that are rotatably received respectively on an input shaft
223 and an output
shaft 225. Each transfer journal member 294, 295 has a flange to seat in a
recessed counter bore
in an end cap member 290, 291 and is attached by a first lock washer and ring
297, and second
lock washer and ring 298 against longitudinal motion relative to a shaft 223,
225. Each journal
member 294, 295 has an iratermediate annular recess that is connected to
longitudinally
extending, circumferentially arranged passages. Each annular recess in a
journal member 292,
295 is coupled by a first radially disposed passage 278 and second radially
disposed passage 279
in an end cap member 290 and 291, respectively, to first input coupling 203
and second input
coupling 204. Carrier in a radial passage 278 or 279 flows through a first
annular recess and the
longitudinal passages in a journal member 294 or 295 to permit access carrier
through a journal
member 292, 295 to each end of the j ournal 292, 295 where the access is
circumferential about a
shaft 223, 225.
Attached to the end cap members 290 and 291 are a first tubular bearing
housing 205,
and second tubular bearing housing 206 containing ball bearings which
relatively support the
outer housing 220 on the input 223 and output 225 shafts. The first bearing
housing 205 has an
attached first sprocket gear 210 for providing a rotative drive for the outer
housing 220 in a
rotative direction about the input 223 and output 225 shafts and the
longitudinal axis 221. The
first bearing housing 205, and second bearing housing 206 also have provisions
for electrical
take out of the wire heater 296 and any other sensor.
11
. !.
CA 02595744 2007-08-01
.5 The inner filter assembly 235 includes inner 215 and outer 216 tubular
members having
perforations or apertures along their lengths and have a first 217 and second
218 inner filter
assembly end cap member with perforations. The inner tubular member 215 is
constructed in two
pieces with an interlocking centrally located coupling section and each piece
attached to an end
cap 217 or 218. The outer tubular member 216 is mounted between the first 217
and second
inner filter assembly end caps .
The end cap members 217, 218 are respectively rotatably supported on the input
shaft
223 and the output shaft 225. The inner member 215 is rotatively attached to
the output shaft 225
by a pin and an interfitting groove 219. A polyester cloth 224 with a ten-
micron weave is
disposed over the outer surface of the outer member 216 and attached to 0-
rings at eitlaer end.
Because the inner member 215 is attached by a coupl'zng pin to a slot in the
output drive shaft
225, the output drive shaft 225 can rotate the inner member 215. The inner
member 215 is
~=
coupled by the first 217 and second 218 end caps that support the outer member
216. The output
shaft 225 is extended through bearings in a first stationary housing 240 and
is coupled to a first
sprocket gear 241. As illustrated, the output shaft 225 has a tubular bore 222
that extends from a
first port or passageway 289 in the first stationary housing 2401ocated
between seals to the inner
member 215 so that a flow of fluid carrier can be exited from the inner member
215 through the
stationary housing 240.
Between the first 217 and second 218 end caps for the inner member 235 and the
journals
292, 295 in the outer housing 220, are a first 227 and second 226 hub for the
blade members 50a
and 50b. The second hub 226 on the input shaft 223 is coupled to the input
shaft 223 by a pin
231 so that the second hub 226 rotates with the input shaft 223. Each hub 227,
226 has axially
extending passageways for the transmittal of carrier through a hub,
The input shaft 223 extends through bearings in the second stationary housing
260 for
rotatable support of the input shaft 223. A second longitudinal passageway 267
extends through
the input shaft 223 to a location intermediate of retaining washers and rings
that are disposed in a
second annular recess 232 between the faceplate and the housing 260. A third
radial passageway
272 in the second end cap member 291 permits fluid carrier in the recess to
exit from the second
end cap member 291. While not shown, the third passageway 272 connects through
piping and a
Y joint to each of the passages 278 and 279.
12
CA 02595744 2007-08-01
. =
. A sample port is shown in Figure. 4, where a first bore 237 extending along
a first axis
intersects a corner 233 of the chamber 230 and forms a restricted opening 234.
The bore 237
has a counter bore and a threaded ring at one end to threadedly receive a
cylindrical valve
member 236. The valve member 236 has a complimentarily formed tip to engage
the opening
234 and protrude slightly into the interior of the chamber 230. An 0-ring 243
on the valve
member 236 provides a seal. A second bore 244 along a second axis intersects
the first bore 237
at a location between the 0-ring 243 and the opening 234. An elastomer or
plastic stopper 245
closes the second bore 244 and can be entered with a hypodermic syringe for
removing a sample.
To remove a sample, the valve member 236 is backed off to access the opening
234 and the bore
244. A syringe can then be used to extract a sample and the opening 234 can be
reclosed. No
outside contamination reaches the interior of the TVEMF-bioreactor 10.
In operation, carrier is input to the second port or passageway 266 to the
shaft
passageway and thence to the first radially disposed 278 and second radially
disposed
passageways 279 via the third radial passageway 272. When the carrier enters
the chamber 230
via the longitudinal passages in the journals 292, 294 the carrier impinges on
an end surface 228,
2-29 of the hubs 227, 226 and- is dispersed radially as well as axially
through the passageways in
the hubs 227, 226. Carrier passing through the hubs 227, 226 impinges on the
end cap members
217, 218 and is dispersed radially. The flow of entry fluid carrier is thus
radially outward away
from the longitudinal axis 221 and flows in a toroidal fashion from each end
to exit through the
polyester cloth 224 and openings in filter assembly 235 to exit via the
passageways 266 and 289.
By controlling the rotational speed and direction of rotation of the outer
housing 220, chamber
230, and inner filter assembly 235 any desired type of carrier action can be
obtained. Of major
importance, however, is the fact that a clinostat operation can be obtained
together with a
continuous supply of fresh fluid carrier.
If a time varying electromagnetic force is not applied using the integral
annular wire
heater 296, it can be applied by another preferred time varying
electromagnetic force source. For
instance, Figures 6-8 illustrate a time varying electromagnetic force device
140 which provides
an electromagnetic force to a cell culture in a bioreactor which does not have
an integral time
varying electromagnetic force, but rather has an adjacent time varying
electromagnetic force
device. Specifically, Figure 6 is a preferred embodiment of a time varying
electromagnetic force
device 140. Figure 6 is an elevated side perspective of the device 140 which
comprises a support
13
=
CA 02595744 2007-08-01
. ;.
base 145, a cylinder coil support 146 supported on the base 145 with a wire
coil 147 wrapped {
around the support 146. Figure 7 is a front perspective of the time varying
electromagnetic force
device 140 illustrated in Figure 6. Figure 8 is a front perspective of the
time varying
electromagnetic force device 140, which illustrates that in operation, an
entire bioreactor 148 is
inserted into a cylinder coil support 146 which is supported by a support base
145 and which is
wound by a wire coil 147. Since the time varying electromagnetic force device
140 is adjacent
to the bioreactor 148, the time varying electromagnetic force device 140 can
be reused. In
addition, since the time varying electromagnetic force device 140 is adjacent
to the bioreactor
148, the device 140 can be used to generate an electromagnetic force in all
types of bioreactors,
preferably rotating.
Furthermore, in operation the present invention contemplates that an
electromagnetic
generating device is turned on and adjusted so that the output generates the
desired
electromagnetic field in the cord blood mixture-containing chamber. The size
of the electrically conductive coil, and number of times it is wound around
the culture chamber of the rotatable
TVEMF bioreactor, are such that when a TVEMF is supplied to the electrically
conductive coil a
TVEMF is generated within the three-dimensional culture in the culture chamber
of the TVEMF
bioreactor. The TVEMF is preferably selected from one of the following: (1) a
TVEMF with a
force amplitude less than 100 gauss and slew rate greater than 1000 gauss per
second, (2) a
TVEMF with a low force axnpiitude bipolar square wave at a frequency less than
100 Hz., (3) a
TVEMF with a low force amplitude square wave with less than 100% duty cycle,
(4) a TVEMF
with slew rates greater than 1000 gauss per second for duration pulses less
than I ms., (5) a
TVEMF with slew rate bipolar delta function-like pulses with a duty cycle less
than 1 /a, (6) a
TVEMF with a force amplitude less than 100 gauss peak-to-peak and slew rate
bipolar delta
function-like pulses and where the duty cycle is less than 1%, (7) a TVEMF
applied using a
solenoid coil to create uniform force strength throughout the cell mixture,
(8) and a TVEMF
applied utilizing a flux concentrator to provide spatial gradients of magnetic
flux and magnetic
flux focusing within the cell mixture. The range of frequency in oscillating
electromagnetic
force strength is a parameter that may be selected for achieving the desired
stimulation of the
cells in the three-dimensional culture. However, these parameters are not
meant to be limiting to
the TVEMF of the present invention, and as such may vary based on other
aspects of this
14
1
CA 02595744 2007-08-01
=
invention. TVEMF may be measured for instance by standard equipment such as an
EN131 Cell
Sensor Gauss Meter.
As various changes could be made in rotating bioreactors subjected to a time
varying
electromagnetic force as are contemplated in the present invention, without
departing from the
scope of the invention, it is intended that all matter contained in the above
description be
interpreted as illustrative and not limiting.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF
THE INVENTION
The following definitions are meant to aid in the description and
understanding of the
defined terms in the context of the present invention. The definitions are not
meant to limit these
terms to less than is described throughout this application. Furthermore,
several definitions are } f
included relating to TVEMF - all of the defmitions in this regard should be
considered to
complement each other, and not construed against each other.
As used throughout this application, the term "adult stem cell" refers to a
pluripotent,
totipotent, and/or multipotent cell that is undifferentiated and that may give
rise to more
undifferentiated cells and is also capable to giving rise to differentiated
cells, but only if directed
to. With regard-to the present invention, an adult stem cell is preferably a
CD133+ cell, more
preferably a CD34+, and most preferably a non-terminally differentiated cord
blood stem cell.
As used throughout this application, the term "cord blood" refers to blood
from the
umbilical cord and/or placenta of a fetus or infant. Cord blood is one of the
richest sources of
stem cells known. The term "cord" is not intended in any way to limit the term
"cord blood" of
this invention.to blood from the umbilical cord; as explained throughout the
application, the
blood of a fetus' or infant's placenta is confluent with the blood of the
umbilical cord. For the
purposes of the present invention, there is=no reason to distinguish between
blood located at
different parts of the same circulatory loop.
As used throughout this application, the term "cord blood cell" refers to a
cell from cord
blood. Cord blood cells capable of replication may undergo TVEMF-expansion in
a TVEMF-
bioreactor, and may be present in compositions of the present invention.
As used throughout this application, the term "cord blood stem cell" refers to
an adult
stem cell from cord blood. Cord blood stem cells are adult stem cells, also
known as somatic
stem cells, and are not embryonic stem cells derived directly from an embryo.
Preferably, a cord
CA 02595744 2007-08-01
- . ... . - _ . . .. .
=5 blood stem cell of the present invention is a CD34+ce11, more preferably a
CD 133+ cell, and
most preferably a non-terminally differentiated cord blood stem cell.
As used throughout this application, the term "cord blood stem cell
composition" refers
to cord blood stem cells of the present invention, after TVEMF-expansion and
wherein the
TVEMF-expanded cord blood stem cells retain some characteristics of the same
cells before
expansion, but also have a unique phenotype as a result of expansion in a
rotating TVEMF-
bioreactor, due to the up or down regulation of genes. The non-turbulent
culture environment of
the TVEMF-bioreactor maintains the cells three-dimensional geometry and cell-
to-cell geometry
and cell-to-cell support substantially similar to that in naturally-occurring
cord blood stem cells,.
With the cord blood stem cells is a carrier of some sort, whether a
pharmaceutically acceptable
carrier, plasma, blood, albumin, cell culture medium, growth factor, copper
chelating agent,
hormone, buffer, cryopreservative, or some other substance. Reference to
naturally-occurring
cord blood is preferably to compare cord blood stem cells of the present
invention with the same
cells in vivo, their original cord blood source. However, if such a comparison
is not available,
then naturally-occurring cord blood may refer to average or typical
characteristics of cord blood,
preferably of the same mammalian species as the source of the cord blood stem
cells of this
invention.
As used throughout this application, the term "cord blood mixture" refers to a
mixture of
cord blood cells with a substance that allows the cells to expand, such as a
medium for growth of
cells, that will be placed in a TVEMF-bioreactor (for instance in a cell
culture chamber). The
cord blood cells may be present in the cord blood mixture simply by mixing
whole cord blood
with a substance such as a cell culture medium. Also, the cord blood mixture
may be made with
a cellular preparation from cord blood, as described throughout this
application, containing cord
blood stem cells. Preferably, the cord blood mixture comprises CD34+, CDI33+,
and/or non-
terminally differentiated cord blood stem cells and Dulbecco's medium (DMEM).
Preferably, at
least half of the cord blood mixture is a cell culture medium such as DMEM.
As used throughout this application, the term "TVEMF" refers to "Time Varying
Electromagnetic Force".
As used throughout this application, the term "TVEMF-bioreactor" refers to a
rotating
bioreactor to which TVEMF is applied, as described more fully in the
Description of the
Drawings, above. The TVEMF applied to a bioreactor is preferably as disclosed
herein. See for
16
CA 02595744 2007-08-01
= 5 instance Figures 2, 3, 4 and 5 herein for examples (not meant to be
limiting) of a TVEMF-
bioreactor. In a simple embodiment, a TVEMF-bioreactor of the present
invention provides for
the rotation of an enclosed cord blood mixture at an appropriate TVEMF and
allows the cord
blood cells (preferably cord blood stern cells) therein to expand. Preferably,
a TVEMF-
allows for the exchange of growth medium (preferably with additives) and for
bioreactor
oxygenation of the cord blood mixture. The TVEMF-bioreactor provides a
mechanism for
expanding cells for several days or more. The TVEMF-bioreactor subjects cells
in the bioreactor
to TVEMF, so that TVEMF is passed through the cells, thus undergoing TVEMF-
expansion.
As used throughout this application, the term "TVEMF-expanded cord blood
cells" refers
to cord blood cells increased in number per volume (ie concentration) after
being placed in a
TVEMF-bioreactor and subjected to a TVEMF. The increase in number of cells per
volume is
the result of cell replication in the TVEMF-bioreactor, so that the total
number of cells in the
bioreactor increase. The increase in number of cells per volume is expressly
not due to a simple
reduction in volume of fluid, for instance, reducing the volume of blood from
70 ml to 10 ml and
thereby increasing the number of cells per ml. ~..
As used throughout this application, the term "TVEMF-expanding" refers to the
step of
cells in a TVEMF-bioreactor replicating (splitting and growing) in the
presence of TVEMF iri a
TVEMF-(rotating) bioreactor. Cord blood stem cells (preferably CD34+more
preferably
CD 133+; and most preferably non-terminally differentiated stem cells)
preferably replicate
without undergoing further differentiation.
As used throughout this application, the term "TVEMF-expansion" refers to the
process of increasing the number of cord blood cells in a TVEMF-bioreactor,
prefetably cord
blood stem cells, by subjecting the cells to a TVEMF. Preferably, the increase
in number of
cord blood cells, preferably cord blood stem cells, is at least 7 times the
number of cord
blood stem cells that were placed into the TVEMF-bioreactor for expansion. The
expansion
of cord blood stem cells in a TVEMF-biozeactor according to the present
invention provides
for cord blood stem cells that maintain, or have essentially the same, three-
dimensional
geometry and cell-to-cell support and cell-to-cell geometry as cord blood stem
cells prior to
TVEMF-expansion, and also have a unique phenotypic expression due to the three-
dimensional culture in the TVEMF-bioreactor. Othex aspects of TVEMF-expansion
may also
provide the exceptional characteristics of the cord blood stem cells of the
present invention.
17
CA 02595744 2007-08-01
.5 Not to be bound by theory, TVEMF-expansion not only provides for high
concentrations of
cord blood stem cells that maintain their tluee-dimensional geometry and cell-
to-cell support.
TVEMF may affect some properties of stem cells during TVEMF-expansion, for
instance up-
of genes promoting growt-,or down regulation of genes preventing growth.
regulation
Overall, TVEMF-expansion results in promoting growth but not differentiation
overall.
Some genes that are up regulated may preferably include, but are not limited
to, those
coding for membrane proteins such as proteoglycan 3, CYPIBI, IL9R, HBAI, and
RHAG;
coding for cytoskeletal proteins such as SPTA1, ANKI; enzymes such as NCALD,
LSS,
PDE4B, SPUB, ELA2, HOD, ADAMDECI, HMGCSI, COVA1, and PFKB4;
nuclear/transcription factors such as Pirin; and others such as S I00A8, A9.
Some genes that
are down regulated may preferably include, but are not Iimited to, membrane
proteins such as
IL2R, IL17 R, EV127, TGFR3, FCGRIA, MRCI, CCRI, CRL4, FER1L3, EMP1, and
THBD; transport proteins such as ABCIA and ABCGI; glycoproteins/cell surface
proteins
such as Versican, CD1c, CD 14, areg, z39iG, hml2, and CLECSF5; cytoskeletal
transduction
proteins such as SKGI; secreted proteins such as SCYA3, gro3, and galectin3;
nuclear
transcription factors sucha s KRML, LOCS 1713, KLF4, and EGR1; and HMOX1 and
BPHL.
Preferably, the up regulated genes are up regulated up to 2 fold, and
preferably the down
regulated genes are down regulated up.to four fold. }
As used throughout this application, the term "TVEMF-expanded cell" refers to
a cell
that has been subjected to the process of TVEMF-expansion. A TVEMF-expanded
cell retains
some core properties of the same cell in vivo, but also has a unique
phenotypic expression as a
result of the TVEMF-expansion process including suspension in the rotating
TVEMF-bioreactor.
As used throughout this application, the term "toxic substance" or related
terms may refer
to substances that are toxic to a cell, preferably a cord blood stem cell; or
to a patient. In
particular, the term toxic substance refers to dead cells, macrophages, as
well as substances that
may be unique or unusual in cord blood (for instance, sickle cells, naaternal
blood or maternal
urine or other tissue or waste). Other toxic substances are discussed
throughout this application.
Removal of these substances from blood is well-known in the art.
Other statements referring to the above-defined terms or other terms used
throughout
this application are not meant to be limited by the above defuutions, and may
contribute to the
definitions. Information relating to various aspects of this invention is
provided throughout
18
CA 02595744 2007-08-01
- j.
S this application, and is not meant to be limited only to the section to
which it is contained, but
is meant to contribute to an understanding of the invention as a whole.
The present invention is related to providing a rapidly available source of
TVEMF-
expanded expanded cord blood stem cells for repairing, replenishing and
regenerating tissue in humans.
This invention may be more fully described by the preferred embodiment(s) as
hereinafter
described, but is not intended to be limited thereto.
Operative Method - Preparing a cord blood mixture to be TVEMF-expanded
In a preferred embodiment of this invention, a method is described for
preparing
TVEMF-expanded cord blood stem cells that can assist the body in repairing,
replacing and
regenerating tissue or be useful in research.
Cord blood is collected from a mammal, preferably a primate mammal, and more
preferably a human, for instance as described throughout this application, and
preferably
according to the syringe method. Cord blood may also be collected in utero,
for instance in
life-threatening situations or extreme situations where a defect (for instance
an ear defect) is
apparent during the third trimester of pregnancy, so that cord blood stem
cells may be expanded and readily available if needed at birth or soon after
birth of the infant. Cord blood
in utero would only be removed in an amount that would not be threatening to
the unborn
infant. The collection of cord blood according to this invention is not meant
to be limiting,
but can also include for instance other means of directly collecting mammalian
cord blood, or
indirectly collecting blood for instance by acquiring the blood from a
commercial or other
source, including for instance cryopreserved blood from a "blood bank".
Preferably, red blood cells are removed from the cord blood and the remaining
cells
including cord blood stem cells are placed with an appropriate media in a
TVEMF-bioreactor
(see "cord blood mixture") such as that described herein. In a more preferred
embodiment of i
this invention, only the "buffy coat" (which includes cord blood stem cells,
as discussed
throughout this application) described above is placed in the TVEMF-
bioreactor. Other
11
embodiments include removing other non-stem cells and components of the cord
blood, to
prepare different cord blood preparation(s). Such a cord blood preparation may
preferably
have, as the only remaining cord blood component, non-terminaily
differentiated cells, more
preferably CD34+ cord blood stem cells, and most preferably CD133+ cord blood
stem cells.
19
CA 02595744 2007-08-01
,5 Removal of non-stem cell types of cord blood cells may be achieved through
negative
separation techniques, such as but not limited to sedimentation and
centrifugation. Many
negative separation methods are well-known in the art. However, positive
selection
techniques may also be used, and are prefeffed in this invention. Methods for
removing -
various components of the blood and positively selecting for, but not limited
to, CD34+
and/or other markers such as CD 133+, are known in the art, and may be used so
long as they
do not lyse or otherwise irreversibly harm the desired cord blood stem cells.
For instance, an
affinity method selective for CD34+ may be used. Preferably, a"buffy coat" as
described
above is prepared from cord blood, and the cord blood stem cells therein
separated from the
buffy coat for TVEMF-expansion.
The collected cord blood, or desired cellular parts as discussed above, must
be placed
into a TVEMF-bioreactor for TVEMF-expansion to occur. As discussed above, the
term
"cord blood mixture" comprises a mixture of cord blood (or desired cellular
part, for instance cord blood without red blood cells, or preferably cord
blood stem cells isolated from cord
blood) with a substance that allows the cells to expand, such as a medium for
growth of cells,
that will be placed in a TVEMF-bioreactor. Cell culture media, media that
allow cells to
grow and expand, are well-known in the art. Preferably, the substance that
aliows the cells to
expand is cell culture media, more preferably Dulbecco's medium. The
components of the
cell media must, of course, not kill or damage cells. Other components may
also be added to
the cord blood mixture prior to or during TVEMF-expansion., For instance, the
cord blood
may be placed in the bioreactor with Dulbecco's medium and further
supplemented with 5%
(or some other desired amount, for instance in the range of about 1% to about
I0%) of human
serum albumin. Other additives to the cord blood mixture, including but not
limited to growth
factor, copper chelating agent, cytokine, hormone and other substances that
may enhance
TVEMF-expansion may also be added to the cord blood outside or inside the
bioreactor
before being placed in the bioreactor. Preferably, the entire volume of a cord
blood collection
from one individual (preferably human cord blood in an amount of about 10 ml
to about 100
ml, i-nore preferably 50 mi to about 100 mi cord blood) is mixed with from
about 25 ml to
about 100 ml Dulbecco's medium (DMEM) and supplemented with 5% human serum
albumin so that the total volume of the cord blood mixture is about 75 to
about 200 ml when
placed in the bioreactor. As a general rule, the more cord blood that may be
collected, the
CA 02595744 2007-08-01
,5 better; if a collection from one individual results in more than 100 ml,
the use of all of that
valuable cord blood is preferred. Where a larger volume is available, for
instance by pooling
cord blood, more than one dose may be preferred. The use of a perfusion TVEMF-
bioreactor
is particularly useful when cord blood collections are pooled and TVEMF-
expanded together.
The term "placed into a TVEMF-bioreactor" is not meant to be limiting - the
cord
blood mixture may be made entirely outside of the bioreactor and then the
mixture placed
inside the bioreactor. Also, the cord blood mixture may be entirely mixed
inside the
bioreactor. For instance, the cord blood may be placed in the bioreactor with
Dulbecco's
medium and supplemented with 5% human serum albumin either already in the
bioreactor,
added simultaneously to the bioreactor, or added after the cord blood to the
bioreactor.
A preferred cord blood mixture of the present invention comprises the
following:
CD34+ stem cells isolated from the buffy coat of a cord blood sample collected
from one
infant at C-section; and Dulbecco's medium which, with the CD34+ cells, is
about 200 ml
total volume. Even more preferably, G-CSF (Granulocyte-Colony Stimulating
Factor) is t
included in the cord blood mixture. Preferably, G-CSF is present in an amount
sufficient to
stimulate TVEMF-expan.sion of cord blood stem cells. Even more preferably, the
amount of
G-CSF present in the cord blood mixture prior to TVEMF-expansion is about 25
to about 200
ng/ml mixture, more preferably about 50 to about 150 ng/ml, and even more
preferably about
100 ng/ml.
Operative Method-TVEMF-expansion and Preparation of a TVEMF-expanded cord
blood
stem ccll composition
In use, the rotation of a TVEMF-bioreactor provides a stabilized culture
environment into
which cells may be introduced, suspended, maintained, and expanded with
improved retention of
delicate three-dimensional structural integrity by simultaneously minimizuig
the fluid shear
stress, providing three-dimensional freedom for cell and substrate spatial
orientation, and
increasing localization of cells in a particular spatial region for the
duration of the expansion
(hereinafter referred to as "three criteria "). The rotating TVEMF-bioreactor
also provides these
three criteria, and at the same time, exposes the cells to a TVEMF. Of
particular interest to the
present invention is the dimension of the culture chamber, the sedimentation
rate of the cells, the
rotation rate, the external gravitational field, and the TVEMF.
21
CA 02595744 2007-08-01
.5 The stabilized culture environment referred to in the operation of present
invention is that
condition in the culture medium, particularly the fluid velocity gradients,
prior to introduction of
cells, which will support a nearly uniform suspension of cells upon their
introduction thereby
creating a three-dimensional culture upon addition of the cells. In a
preferred embodiment, the
culture medium is initially stabilized into a near solid body horizontal
rotation 360 degrees about ~
an axis within the confines of a similarly rotating chamber wall of a
rotatable TVEMF
bioreactor. The rotating continues in the same direction about the axis. The
chamber walls are
set in motion relative to the culture medium so as to initially introduce
essentially no fluid stress
shear field therein. Cells are introduced to, and move through, the culture
medium in the
stabilized culture environment thus creating a three-dimensional culture. The
ceIls move under
the influence of gravity, centrifugal, and coriolus forces, and the presence
of cells within the
culture medium of the three-dimensional culture induces secondary effects to
the culture
medium. The motion of the culture medium with respect to the culture chamber,
fluid shear
stress, and other fluid motions, is due to the presence of these cells within
the culture medium.
In most cases the cells with which the stabilized culture environment is
primed sediment
at a slow rate preferably under 0.1 centimeter per second. It is therefore
possible, at this early
stage of the three-dimensional culture, to select from a broad range of
rotational rates (preferably
of from about 2 to about 30 RPM) and chamber diameters (preferably of from
about 0.5 to about
36 inches). Preferably, the slowest rotational rate is advantageous because it
minimizes
equipment wear and other logistics associated with handling the three-
dimensional culture. The
preferred speed of the present invention is of from 5 to 120 RPM, and more
preferably from 10
to 30 RPM.
Not to be bound by theory, rotation about a substantially horizontal axis with
respect to
the external gravity vector at an angular rate optimizes the orbital path of
cells suspended within
the three-dimensional culture. The progress of the three-dimensional culture
is preferably
assessed by a visual, manual, or automatic determination. An increase in the
density of cells
may require appropriate adjustment of the rotation speed in order to optimize
the particular
paths. An increase in density is related to an increase in the number of cells
in the culture
chamber The rotation of the culture chamber optimally controls collision
frequencies, collision
intensities, and localization of the cells in relation to other cells and also
the limiting boundaries
of the culture chamber of the rotatable TVEMF bioreactor. In order to control
the rotation, if the
22
CA 02595744 2007-08-01
-5 cells are observed to excessively distort inwards on the downward side and
outwards on the
upwards side then the revolutions per minute ("RPM") may preferably be
increased. If the cells
are observed to centrifugate excessively to the outer walls then the RPM may
preferably be
reduced. Optimally, the zero-head space of the three-dimensional culture
provides a space
wherein cells may preferably be distributed throughout the volume of culture
medium effectively
. ~,
utilizing the fuil culture chamber capacity. The cell sedimentation rate and
the external gravitations field place a lower limit on the
fluid shear stress obtainable, even within the operating range of the present
invention, due to
gravitationally induced drift of the cells through the culture medium of the
three-dimensional
culture. Calculations and measurements place this minimum fluid shear stress
very nearly to that
resulting from the cells' terminal sedimentation velocity (through the culture
medium) for the
external gravity field strength. Centrifugal and coriolis induced motion
[classical angular
kinematics provide the following equation relating the Coriolis force to an
object's mass (m), its
velocity in a rotating frame (vr) and the angular velocity of the rotating
frame of reference (El):
pcorioti5 = -2 m (w x v,)] along with secondary effects due to cell and
culture medium interactions,
act to further degrade the fluid shear stress level as the cells expand.
Not to be bound by theory, but an environment that is substantially similar to
microgravity may be obtained in the rotating TVEMF-bioreactor. In order to
obtain the minimal
fluid shear stress level it is preferable that the culture chamber be rotated
at substantially the
same rate as the culture medium. Not to be bound by theory, but this minimizes
the fluid
velocity gradient induced upon the three-dimensional culture. It is
advantageous to control the
rate of expansion in order to maintain the cell density (and associated
sedimentation rate) within
a range for which the rate of expansion is able to satisfy the three criteria.
In addition, transient
disruptions of the expansion process are permitted and tolerated for, among
other reasons,
logistical purposes during initial system priming, sample acquisition, system
maintenance, and
culture ternnination.
Rotating cells about an axis substantially perpendicutar to gravity can
produce a variety
of sedimentation rates, ali of which according to the present invention remain
spatially localized
in distinct regions for extended periods of time ranging from seconds (when
sedimentation
characteristics are large) to hours or days (when sedimentation differences
are small). Not to be
bound by theory, but this allows these cells sufficient time to interact and
associate as necessary
23
I
~
CA 02595744 2007-08-01
-5 with each other in a three-dimensional culture. Preferably, cells undergo
expansion for at least 4
days, more preferably from about 7 days to about 14 days, most preferably from
about 7 days to
about 10 days, even more preferably about 7 days. TVEMF-expansion may continue
in a s
TVEMF-bioreactor for up to 160 days. While TVEMF-expansion may occur for even
longer
than 160 days, such a lengthy expansion is not a preferred embodiment of the
present invention.
Preferably, TVEMF-expansion may continue in a rotatable TVEMF bioreactor to
produce a
number of cells that is at least 7 times the original number of cells that
were placed in the
rotatable TVEMF bioreactor.
Culture chamber dimensions also influence the path of cells in the three-
dimensional
culture of the present invention. A culture chamber diameter is preferably
chosen which has the
appropriate volume, preferably of from about 15m1 to about 2L for the intended
three-
dimensional culture and which will allow a sufficient seeding density of
cells. Not to be bound
by theory, but the outward cells drift due to centrifugal force is exaggerated
at higher culture
chamber radii and for rapidly sedimenting cells.
The path of the celis in the three-dimensional culture has been andytically
calculated
incorporating the cell motion resulting from gravity, centrifugation, and
coriolus effects. A
computer simulation of these governing equations allows the operator to model
the process and
select parameters acceptable (or optimal) for the particular planned three-
dimensional culture.
Figure 9 shows the typical shape of the cell orbit as observed from the
external (non-rotating)
reference frame. Figure 10 is a graph of the radial deviation of a cell from
the ideal circular
streamline plotted as a function of RPM (for a typical cell sedimenting at 0.5
cm per second
terminal velocity). This graph (Figure 10) shows the decreasing amplitude of
the sinusoidally
varying radial cells deviation as induced by gravitational sedimentation.
Figure 10 also shows
increasing radial cell deviation (per revolution) due to centrifugation as RPM
is increased. These
opposing constraints influence carefully choosing the optimal RPM to
preferably minimize cell
impact with, or accumulation at, the chamber walls. A family of curves is
generated which is
increasingly restrictive, in terms of workable RPM selections, as the external
gravity field
strength is increased or the cell sedimentation rate is increased. This family
of curves, or
preferably the computer model which solves these governing orbit equations, is
preferably
utilized to select the optimal RPM and chamber dimensions for the expansion of
cells of a given
sedimentation rate in a given external gravity field strength. Not to be bound
by theory, but as a
24
E=-
CA 02595744 2007-08-01
S typical three-dimensional culture is expanded the number of cells and
therefore the cell density
effects the sedimentation rate, and therefore, the rotation rate may
preferably be adjusted to
optinnize the same.
In the three-dimensional culture, the cell orbit (Figure 9) from the rotating
reference
franie of the culture medium is seen to move in a nearly circular path under
the influence of the
rotating gravity vector (Figure 11). Not to be bound by theory, but the two
pseudo forces,
coriolis and centrifugal, result from the rotating (accelerated) reference
frame and cause
distortion of the otherwise nearly circular path. Higher gravity levels and
higher cell
sedimentation rates produce larger radius circular paths which correspond to
larger trajectory
deviations from the ideal circular orbit as seen in the non- rotating
reference frame. In the
rotating reference frame it is thought, not to be bound by theory, that cells
of differing
sedimentation rates will remain spatially localized near each other for long
periods of time with
greatly reduced net cumulative separation than if the gravity vector were not
rotated; the cells are
sedimenting, but in a small circle (as observed in the rotating reference
frame). Thus, in
operation the present invention provides cells of differing sedimentation
properties with
sufficient time to interact mechanically and through soluble chemical signals
thereby effecting
their cell-to-cell interactions including geometry and support. In operation,
the present invention
provides for sedimentation rates of preferably from about 0 cm/second up to 10
cm/second.
Furthermore, in operation the culture chamber of the present invention has at
least one
aperture preferably for the input of fresh culture medium and a cell mixture
and the removal of a
volume of spent culture medium containing metabolic waste, but not limited
thereto. Preferably,
the exchange of culture medium can also be via a culture medium loop wherein
fresh or recycled
culture medium may be moved within the culture chamber preferably at a rate
sufficient to
support metabolic gas exchange, nutrient delivery, and metabolic waste product
removal. This
may slightly degrade the otherwise quiescent three-dimensional culture. It is
preferable,
therefore, to introduce a mechanism for the support of preferred components
including, but not
limited to, respiratory gas exchange, nutrient delivery, growth factor
delivery to the culture
medium of the three-dimensional culture, and also a mechanism for metabolic
waste product
removal in order to provide a long term three-dimensional culture able to
support significant
metabolic loads for periods of hours to months.
CA 02595744 2007-08-01
}
It is expected that expansion in a rotating TVEMF-bioreactor provides a unique
environment that effects the cell phenotype, as gauged by RNA expression
levels. The cells
adapt to the unique three-dimensional environment in which they are suspended.
Cells expanded
in the three-dimensional environment of a rotating TVEMF-bioreactor express
different gene
expression patterns, and therefore, different. membrane and surface protein
configurations, and
different cytoskeletal details.
During the time that the cells are in the TVEMF-bioreactor, they are
preferably fed
nutrients and fresh media (DMEM and 5% human serum albumin), exposed to
hormones,
cytokines, and/or growth factors (preferably G-CSF); and toxic materials are
removed. The
toxic materials removed from cord blood cells in a TVEMF-bioreactor include
the toxic
granular material of dying cells and the toxic material of granulocytes and
macrophages.
Preferably, TVEMF-expansion is carried out in a TVEMF-bioreactor at a
temperature
of about 26 C to about 41 C, and more preferably, at a temperature of about
37C.
One method of monitoring the overall expansion of cells undergoing TVEMF-
is by visual inspection. Cord blood stem cells are typically dark red in
color. Once ~
expansion
the bioreactor begins to rotate and the TVEMF is applied, the cells that are
distributed
throughout the full volume of inedia preferably cluster in the center of the
bioreactor vessel as
they become greater in number (denser), with the medium surrounding the
colored cluster of
cells. Oxygenation and other nutrient additions often do not cloud the ability
to visualize the
,..
cell cluster through a visualization (typically clear plastic) window built
into the bioreactor.
Formation of the cluster is important for helping the stem cells maintain
their three-
dimensional geometry and cell-to-cell support and cell-to-cell geometry; if
the cluster appears
to scatter and cells begin to contact the wall of the bioreactor vessel, the
rotational speed is
increased (manually or automatically) so that the centralized cluster of cells
may form again.
A measurement of the visualizable diameter of the cell cluster taken soon
after formation may
be compared with later cluster diameters, to indicate the approximate number
increase in cells
in the TVEMF-bioreactor. Measurement of the increase in the number of cells
during
TVEMF expansion may also be taken in a number of ways, as known in the art. An
automatic sensor could also be included in the TVEMF-bioreactor to monitor and
measure the
increase in cluster size.
26
CA 02595744 2007-08-01
.. . .. ..
The TVEMF-expansion process may be carefully monitored, for instance by a
laboratory expert, who will check cell cluster formation to ensure the cells
remain clustered
inside the bioreactor and will increase the rotation of the bioreactor when
the cell cluster
begins to scatter. An automatic system for monitoring the cell cluster and
viscosity of the
cord blood mixture inside the bioreactor may also monitor the cell clusters. A
change in the
viscosity of the cell cluster may become apparent about 2 days after beginning
the TVEMF-
expansion process, and the rotational speed of the TVEMF-bioreactor may be
increased around that time. The TVEMF-bioreactor speed may vary throughout
TVEMF-expansion.
Preferably, the rotational speed is timely adjusted so that the cells
undergoing TVEMF-
expansion do not contact the sides of the TVEMF-bioreactor vessel.
Also, the laboratory expert may, for instance once a day, or once every two
days,
manually (for instance with a syringe) insert fresh media and preferably other
desired
additives such as nutrients and growth factors, as discussed above, intc5 the
bioreactor, and
E.
draw off the old media containing cell wastes and toxins. Also, fresh media
and other
additives may be automatically pumped into the TVEMF-bioreactor during TVEMF-
expansion, and wastes automatically removed.
Cord blood stem cells may increase to at least seven times their original
number about
7 to about 14 days after being placed in the TVEMF-bioreactor and TVEMF-
expanded.
Preferably, the TVEMF-expansion lasts about 7 to 10 days, and more preferably
about 7 days.
Measurement of the number of stem cells does not need to be taken during TVEMF-
expansion therefore. As indicated above and throughout this application, TVEMF-
expanded
cord blood stem cells of the present invention have essentially the same three-
dimensional
geometry and cell-to-cell support and cell-to-cell geometry as naturally-
occurring, non-
TVEMF-expanded cord blood stem cells due to the essentially non-turbulent and
low shear
stress culture regime. The TVEM.F-expanded cord blood cell retains fundamental
properties
of the non-TVEMF-expanded cord blood cells. The gentle free drifting of the
cells through
soluble molecular species which control cell function and are substrates and
products of cell
metabolism allows the rotating TVEMF-bioreactor systems to produce a unique
living
product cell in terms of transcribed RNA pattern coding for multiple cell
structural and
functional proteins and cell sub organelles.
27
CA 02595744 2007-08-01
_. . . _ . !
t..
Another embodiment of the present invention relates to an ex vivo mammalian
cord
blood stem cell composition that functions to assist a body system or tissue
to repair, replenish
and regenerate tissue, for example, the tissues described throughout this
application. The
composition comprises TVEMF-expanded cord blood stem cells. The cord blood
cells in the
composition are preferably expanded to at least seven times the number that
were placed in the
culture chamber of the TVEMF-bioreactor. For instance, preferably, if a number
X of cord
blood stem cells was placed in a certain volume into a TVEMF-bioreactor, then
after TVEMF-
expansion, the number of cord blood stem cells from that same volume of cord
blood stem cells
place into the TVEMF-bioreactor will be at least 7X. While this at-least-seven-
times-expansion
is not necessary for this invention to work, this expansion is preferred for
therapeutic purposes.
For instance, the TVEMF-expanded cells may be only in=amount of 2 times the
number of cord
blood stem cells in the naturally-occuring cord blood, if desired. Preferably,
TVEMF-expanded
cells are in a range of about 4 times to about 25 times the number per volume
of cord blood stem ~.
cells in naturally-occurring cord blood. In another preferred embodiment, the
TVEMF-expanded
cells number in an amount that is at least one cell more than the number that
were placed in the
culture chamber of the TVEMF-bioreactor. In this embodiment, the phenotypic
expression of
the cells after TVEMF-expansion is the preferred focus for repairing a body
function or tissue.
The present invention is also directed to a composition comprising cord blood
stem cells
from a mammal, wherein said cord blood stem cells are expanded in a rotating
TVEMF-
while suspending the cells therein to up or down regulate genes as effected by
the
bioreactor
cells environment, interactions, and three-dimensional geometry. A composition
of the present
invention may include a pharmaceutically acceptable carrier; plasma, blood,
albumin, cell culture
medium, growth factor, copper chelating agent, hormone, buffer or
cryopxeservative.
"Pharmaceutically acceptable carrier" means an agent that will allow the
introduction of the stem
cells into a mammal, preferably a human. Such carrier may include substances
mentioned
herein, including in particular any substances that may be used for blood
transfusion, for instance
blood, plasma, albumin, preferably from the mammal to which the composition
will be
introduced. The term "introduction" of a composition to a mammal is meant to
refer to
"administration" of a composition to an animal. "Acceptable carrier" generally
refers to any
substance the cord blood stem cells of the present invention may survive in,
ie that is not toxic to
the cells, whether after TVEMF-expansion, prior to or after cryopreservation,
prior to
28
CA 02595744 2007-08-01
_
.5 introduction (administration) into a mammal. Such carriers are well known
in the art, and may
include a wide variety of substances, including substances described for such
a purpose
throughout this application. For instance, plasma, blood, albumin, cell
culture medium, buffer
and cryopreservative are all acceptable carriers of this invention. The
desired carrier may
depend in part on the desired use
TVEMF-expanded cord blood stem cells have essentially the same, or maintain,
the
three-dimensional geometry and the cell-to-cell support and cell-to-cell
geometry as the cord
blood from which they originated, The composition comprises TVEMF-expanded
cord blood
stem cells, preferably in a suspension of Dulbecco's medium or in a solution
ready for
cryopreservation. The composition is preferably free of toxic granular
material, for example,
dying cells and the toxic material or content of granulocytes and macrophages.
The
composition may be a cryopreserved composition comprising TVEMF-expanded cord
blood '
stem cells by decreasing the temperature of the composition to a temperature
of from -120 C
to -196 C and maintaining the cryopreserved composition at that temperature
range until
. ~.
needed for therapeutic or other use. As discussed below, preferably, as much
toxic material
as is possible is removed from the composition prior to cryopreservation.
Another embodiment of the present invention relates to a method of
regenerating
tissue and/or treating diseases such as auto-immune diseases (as discussed
above) with a
composition of TVEMF-cord blood stem cells, either having undergone
cryopreservation or
soon after TVEMF-expansion is complete. The cells may be introduced into a
mammalian
body, preferably human, for instance injected intravenously, directly into the
tissue to be
repaired, into the abdominal cavity, attaching to the peritoneum/peritoneal
cavity, allowing
the body's natural system to repair and regenerate the tissue. Preferably, the
composition
introduced into the mammalian body is free of toxic material and other
materials that may
cause an adverse reaction to the administered TVEMF-expanded cord blood stem
cells. The
method (and composition) can potentially be used to repair a mammalian,
preferably human,
vital organ and other tissue, with such potential use including but not
limited to liver tissue,
hear'L tissue, hematopoietic tissue, blood vessels, skin tissue, muscle
tissue, gut tissue,
pancreatic tissue, central nervous system cells, bone, cartilage tissue,
connective tissue,
pulmonary tissue, spleen tissue, brain tissue and other body tissue. The cells
are readily
29
CA 02595744 2007-08-01
available for treatment or research where such treatment or research requires
the individual's
blood cells, especially if a disease has occurred and cells free of the
disease are needed.
A TVEMF-expanded cord blood stem cell composition of the present invention
should
be introduced into a mammal, preferably a human, in an amount sufficient to
achieve tissue
repair or regeneration, or to- treat a desired disease or condition.
Preferably, at least 20 ml of a
TVEMF-expanded cord blood stem cell composition having 107 to 109 stem cells
per ml is
used for any treatment, preferably all at once, in particular where a
traumatic injury has
occurred and immediate tissue repair needed. This amount is particularly
preferred in a 75-80
kg human. The amount of TVEMF-expanded cord blood stem cells in a composition
being
introduced into the source mammal is inherently related to the number of cells
present in the
source cord blood material (for instance, the amount of stem cells present in
one infant's cord
blood). A preferred range of TVEMF-expanded cord blood stem cells introduced
into a {
patient may be, for instance, about 10 ml to about 50 ml of a TVEMF-expanded
cord blood
stem cell composition having 107 to 109 stem cells per ml, or potentially even
more. While it
is understood that a high concentration of any substance, administered to a
mammal, may be
toxic or even lethal, it is unlikely that introducing all of a mammal's cord
blood stem cells, for
instance after TVEMF-expansion, will cause an overdose in. TVEMF-expanded cord
blood
stem cells. Where cord blood from several donors is used, the number of cord
blood stem
cells introduced into a mammal may be higher. Therefore, it should be realized
that the
TVEMF-expanded cells may be introduced to the mammal from an allogeneic source
or an
autologous source. Also, the dosage of TVEMF-cells that may be introduced to
the patient is
not limited by the amount of cord blood provided from collection from one
individual;
multiple administrations, for instance once a day or twice a day, or once a
week, or other
administration time frames, may more easily be used. Also, where a tissue is
to be treated, the
type of tissue may warrant-the use of as many TVEMF-expanded cord blood stem
cells as are I
available. For instance, liver is easiest to treat.
Example- Quaiitative and Quantitative comparison between a Rotating Bioreactor
and a
Dynamic Moving Culture.
An experiment was conducted to demonstrate the qualitative differences between
two
cultures and the differences in the rates of expansion. To illustrate the
differences a
, i.
CA 02595744 2007-08-01
comparison was made between gene expression levels as assayed by abundance of
mRNA
transcripts in two samples of blood stem cells cultured in two different
methods: (A) shaken
Petri plate (dynamic moving culture) (B) rotating bioreactor. The cultures
were set up, refed,
harvested and otherwise manipulated in the identical manner. The test was
documented using
techniques well accepted in the art including Affymetrix Gene Array to prove
the differences
in genetic expression levels. All conditions and manipulations were the satne
for the two
cultures except for the type of culture vessel in which they were expanded.
Culture A serves as the baseline on which to determine increase or decrease of
i~
transcript levels in culture B. There are several differences in membrane
composition
between the 2 cultures, as far as cell surface receptors are concerned. In
addition, several of i
the other genes that are altered in the rotating bioreactor culture (mostly
the `decreased' ones)
have a role in innate and adaptive immunity. Also, some transcripts of genes
involved in ceil-
to-cell contacts and cytoskeletal structures are significantly changed. Some
of the altered
genes are involved in cell proliferation.
Below is a summary of the most relevant functions of a subset of the array
data. Included
in this summary are only those genes that show at least a 200% (1-fold)
difference in expression
levels between samples, either decreased (1) or increased (TZ). The data are
fiuther clustered
based on cellutar localization and/or function.
"Decreased" Genes(Range of change is 4-to-1 fold)
A. MEMBRANE PROTEINS
1. Receptors
- IL2R: aka CD25, expressed in regulatory T cells and macrophages and
activated T-
and B-cells; involved in cytokine-cytolune receptor interactions and role in
cell proliferation
- If.17R: receptor for IL17, and essential cytokine that acts as an immune
response
modulator
- EV127: truncated precursor of IL17 receptor homolog
- TGFR3: (aka beta-glycan) also has a soluble form; involved in cell
differentiation, cell
cycle progression, migration, adhesion, ECM production
- FCGRIa: (aka CD64, human Fc- receptor) expressed in macrophages/monocytes,
neutrophils; involved in phagocytosis, the immune response and cell signal
transduction
31
CA 02595744 2007-08-01
- MRCI: (aka CD206; Mannose Receptor; lectin-family) expressed in macrophages/
monocytes (where expression increasing during culture), and dendrtitic cells;
involved in
innate and adaptive immunity - CCRI :(chemokine receptor, aka CD 191, MIP I
receptor, RANTES receptor);
multipass protein expressed in several hematopoietic cells that transduces a
signal in response
to several chemokines by increasing intracellular calcium ions level;
responsible for affecting
stem cell proliferation; role in cell adhesion, inflammation and immune
response
- CRL4: putative cytokine receptor precursor with role in signal transduction
and {
proliferation
- FER1L3: (myoferlin) single-pass protein at nuclear and plasma membranes;
involved
in membrane regeneration and repair; expressed in cardiac and skeletal muscle
- EMP 1: (aka TMP) mutli-pass protein of claudin family involved in formation
of tight
junctions, and cell-to-cell contact
- THBD: (thrombomodulin aka CD141); single pass endothelial cell receptor with
lectin
and EGF-like domains; complexes with thrombin to activate the coagulation
cascade (factor
Va and V111a)
2. Transporters
- ABCAI: multipass protein involved in cholesterol trafficking (efflux);
expressed in
macrophages and keratinocytes
- ABCGI: multi-pass transporter involved in macrophage lipid homeostasis;
expressed
in intracellular compartments of macrophages mostly; found in the endoplasmic
reticulum
membrane and Golgi apparatus;
3. Glycoproteins/Cell Surface
- Versican (aka CSPG2, chondroitin sulfate proteoglycan 2); involved in
maintaining
ECM integrity, and has a role in cell proliferation, migration, and cell-cell
adhesion (also
interacts with tenascinR)
- CD 1 c: expressed in activated Tcells; involved in mounting immune response
- CD 14: cell surface marker expressed in monocytes/macrophages
32
CA 02595744 2007-08-01
. . . . . . . .. .
- AREG: (amphiregulin) involved in cell-to-cell signaling and proliferation;
growth-
glycoprotein. Inhibits growth of several human carcinoma cells in culture and
modulating
stimulates proliferation of human fibroblasts and certain other tumor cells
- Z391g: a membrane spanning immunoglobulin with a role in mounting the immune
response; expressed in monocytes and dendritic cells
- HML2: (aka CLEC10A, CD301) single pass lectin expressed in macrophages;
Probable role in regulating adaptive and innate immune responses. Binds in a
calcium-
dependent manner to terminal galactose and N-acetylgalactosamine units, linked
to serine or
threonine.
- CLECSF5: single pass myeloid lectin; involved in proinflammatory activation
of
myeloid cells via TYROBP-mediated signaling in a calcium-dependent rnanner
B. CYTOSOLIC/SIGNAL TRANSDUCTION:
- SKGl : expressed in granulocytes; has a role in response to oxidative stress
and in
cellular communication; part of the proteasome - ubiquitin pathway 20
C.SECRETED
- SCYA3 (aka CCL3, MIP1): secreted by macrophages/monocytes; soluble monokine
with inflammatory and chemokinetic properties involved in mediating the
inflammatory
response; a major HIV-suppressive factor produced by CD8+ T-cells.
- GRO3: (aka CKCL3, MIP2); secreted by PB monocytes; chemokine with
chemotactic
activity for neutrophils and a role in inflammation and immunity
- Galectin3: soluble protein secreted by macrophages/monocytes; can bind the
ECM to
activate cells or restrain mobility; involved in other processes including
inflammation,
neoplastic transformation, and innate and acquired immunity by binding IgE;
also has a
nuclear form; inhibited by MMP9.
D. NUCLEAR/TRANSCRIPTt ION FAC T ORS
- KRML; LOC51713; KLF4: three gene members of Kreisler/Krox family of nuclear
transcription factors involved in bone and inner ear morphogenesis, epithelial
cell
differentiation and/or development of the skeleton and kidney
33
CA 02595744 2007-08-01
.. . . . . . . . , . . . ~
=5 - EGRl: (aka KROX24) expressed in lymphocytes and lymphoid organs; involved
in
macrophage differentiation, and inflammation/apoptosis pathways; activates
genes in
d'zfferentiation
E. ENZYMES
- HMOXI: (heme oxygenase) microsomal (ER); highly expressed in spleen;
involved in
heme turnover; ubiquitously expressed following induction by several stresses,
potent anti-
proteins whenever oxidation injury takes place
inflammatory
- BPHL: mitochondrial serine hydrolase that catalyzes the hydrolytic
activation of
amino acid ester prodrugs of nucleoside analogs; may play a role in
detoxification processes
"Increased" Genes (range of change is 2-to-I fold)
A. MEMBRANE PROTEINS
~ Proteoglycan 3: expressed in eosinophils and granulocytes, highly expressed
in bone
marrow; involved in immune response, neutrophil activation and release of IL8
and histamine
- CYP 1 B 1: Cytochromes P450 ate a group of heme-thiolate monooxygenases
involved
in an NADPH-dependent electron transport pathway. It oxidizes a variety of
structurally
unrelated compounds, including steroids, fatty acids, and xenobiotics
- IL9R: single pass interleukin receptor, involved in cell proliferation and
signaling,
expressed in hemaotpoietic cells
- HBAI :(CD3I) binds heme and iron involved in oxygen transport, specific to
RBCs
- RHAG (aka CD241) expressed in erythrocytes, Rh blood group protein multipass
protein anmmon.ium transporter; binds ankyrin, a component of the RBC
cytoskeleton
B. CYTOSKELETAL PRTOEINS I~
- SPTA1; ANKI : both proteins are located on cytoplasmic face of plasma
membrane of
erythrocytes (RBC) and act to anchor transmembrane proteins to the
cytoskeleton; together
with actin and other proteins they form the RBC cytoskeleton superstructure
and are
responsible for keeping its shape
- NCALD: neurocalcin; cytosolic; involved in vesicle-mediated transport; binds
actin,
tubulin and clathrin; can bind Ca2+; expressed in neural tissues and testes
34
! t{{{
CA 02595744 2007-08-01
C. ENZYMES (Cytosollc)
- LSS: cholesterol metabolism-steroid biosynthesis
- PDE4B: involved in anti-inflammatory response, high in CNS; purine
metabolism
- SPUVE: a secreted serine protease (unknown function)
- ELA2: serine protease expressed in leukocytes/neutrophoils, involved in
protein
hydrolysis including elastin; serves to modify the function of NK cells,
monocytes and
granulocytes; inhibits chemotaxis in anti-inflammatory response, high in BM
- HGD: iron binding oxygenase involved in tyrosine metab and phenylalalnine
catabolism
- ADAMDECI: expressed in macrophages; a secreted zinc binding serum protease
involved in immune response; up-regulated during primary monocyte to
macrophage and/or
dendritic cell differentiation
- HMGCSI: soluble co-enzyme A synthase involved in cholesterol biosynthesis
- COVAI hydroquinone oxidase (X-linked) extracellular and trans plasma
membrane
associated (secreted factor) has copper as a cofactor has several properties
associated with
prions; naturally is glycosylated ; involved ultradian rhythm maintenance,
cell growth
regulation, electron transport !.
- PFKB4: glycolytic enzyme
~.!
D. NUCLEAR/TRANSCRtPTION FACTORS
- Pirin: iron-binding nuclear transcription factor; DNA replica.tion and
transactivation
(X-linked); interacts with SMAD signaling cascade
E. OTHER
- S 100A8, A9: secreted, calcium binding proteins (isoforms A8, A9 expressed
in
epithelial cells) expressed by monocytes/macrophages and granulocytes as part
of the
inflammatory response; inhibitor of protein kinases. Also expressed in
epithelial cells
constitutively or induced during dermatoses. May interact with components of
the
intermediate filaments in monocytes and epithelial cells; highly expressed in
bone marrow
. ~a
II
CA 02595744 2007-08-01
Figures 12, 13, and 14 illustrate that the cells in a rotating bioreactor
expand to a significantly
greater number than cells in a dynamic moving culture. The expansion of CD133+
cells, total
nucleated cells and CD34+ cells were analyzed.
These results demonstrate that cells expanded in a rotating system, such as a
TVEMF-
bioreactor are quantitatively and qualitatively unique. The non-turbulent
regime in the
rotating TVEMF-bioreactor allows the cells to expand in a low shear
environment so that the
input cell is not disturbed as much as it would be in other three-dimensional
systems.
However, as a result of the TVEMF-expansion process, the TVEMF-expanded cord
blood
stem cells have a unique phenotypic expression to support their suspension in
the three-
dimensional environment. That expression is fostered and maintained without
differentiation
and over a high rate of expansion.
Operative Method - Cryopreservation
As mentioned above, cord blood is collected for instance during the birth
(even more
preferably at a Caesarean section) of a baby mammal, preferably a human
infant. Red blood
cells are preferably removed from the cord blood. The cord blood stem cells
(with other cells
and media as desired) are placed in a TVEMF-bioreactor, subjected to a time
varying
electromagnetic force and expanded, After expansion, the cells may be
cryogenically
preserved. Further details relating to cryopreservation of a TVEMF-expanded
cord blood
stem cell composition are provided herein and in particular below.
After TVEMF-expansion, the TVEMF-expanded cells, including TVEMF-expanded
cord blood stem cells, are preferably transferred into at least one
cryopreservation container
containing at least one cryoprotective agent. The TVEMF-expanded cord blood
stem cells are
preferably first washed with a solution (for instance, a buffer solution or
the desired
cryopreservative solution) to remove media and other components present during
TVEMF-
expansion, expansion, and then put in a solution that allows for
cryopreservation of the cells. The cells
are transferred to an appropriate cryogenic container and the container
decreased in
temperature to generally from -120 C to -196 C, preferably about -130 to about
-150 C, and
maintained at that temperature. When needed, the temperature of the cells (ie
the temperature
of the cryogenic container) is raised to a temperature compatible with
introduction into the
= 36
=
CA 02595744 2007-08-01
human body (generally from around room temperature to around body
temperature), and the
TVEMF-expanded cells are introduced into a mammalian body, preferably human,
for
instance as discussed above.
Freezing cells is ordinarily destructive. On cooling, water within the cell
freezes.
Injury then occurs by osmotic effects on the cell membrane, cell dehydration,
solute 10 concentratton, and ice crystal formation. As ice forms outside the
cell, available water is
removed from solution and withdrawn from the cell, causing osmotic dehydration
and raised
solute concentration that eventually destroys the cell. (For a discussion, sce
Mazur, P., 1977,
Cryobiology 14:251-272.)
Different materials have different freezing points. Preferably, a cord blood
stem cell
composition ready for cryopreservation contains as few contaminating
substances as possible, to
minimize cell wall damage from the crystallizaton and freezing process.
These injurious effects can be circumvented by (a) use of a cryoprotective
agent, (b)
control of the freezing rate, and (c) storage at a temperature sufficiently
low to mininnize
degradative reactions.
The inclusion of cryopreservation agents is preferred in the present
invention.
Cryoprotective agents which can be used include but are not limited to a
sufficient amount of
dimethyl sulfoxide (DMSO) (Lovelock, J. E. and Bishop, M. W. H., 1959, Nature
183:1394-
1395; =
Ashwood-Smith, M. J., 1961, Nature 190:1204-1205), glycerol,
polyvinylpyrrolidine
(R.infret, A. P., 1960, Ann. N.Y. Acad. Sci. 85:576), polyethylene glycol
(Sloviter, H. A. and
Ravdin, R. G., 1962, Nature 196:548), albumin, dextran, sucrose, ethylene
glycol, i-erythritol, D-
ribitol, D-mannitol (Rowe, A. W., et a1.,1962, Fed. Proc. 21:157), D-sorbitol,
i-inositol, D-
lactose, choline chloride (Bender, M. A., et al., 1960, J. Appl. Physiol.
15:520), amino acid-
glucose solutions or amino acids (Phan The Tran and Bender, M. A., 1960, Exp.
Cell Res.
20:651), methanol, acetamide, glycerol monoacetate (Lovelock, J. E., 1954,
Biochem. J. 56:265),
and inorganic salts (Phan The Tran and Bender, M. A., 1960, Proc. Soc. Exp.
Biol. Med.
104:388; Phan The Tran and Bender, M. A., 1961, in Radiobiology, Proceedings
of the Third
Australian Conference on Radiobiology, Ilbery, P. L. T., ed., Butterworth,
London, p. 59). In a
preferred embodiment, DMSO is used. DMSO, a liquid, is nontoxic to cells in
low
concentration. Being a small molecule, DMSO freely permeates the cell and
protects intracellular
organelles by combining with water to modify its freezability and prevent
damage from ice
37 ~ I
= ~I
. f~
CA 02595744 2007-08-01
. . .. .
;. ;
-5 formation. Adding plasma (ie, to a concentration of 20-25%) can augment the
protective effect of
DMSO. After addition of DMSO, cells should be kept at 0 C until freezing,
since DMSO
concentrations of about 1% are toxic at temperatures above 4 C. My selected
preferred
cryoprotective agents are, in combination with TVEMF-expanded cord blood stem
cells, 20 to
40% dimethyl sulfoxide solution in 60 to 80% amino acid-glucose solution, or
15 to 25%
hydroxyethyl starch solution, or 4 to 6% glycerol, 3 to 5% glucose, 6 to 10%
dextran T10, or 15
to 25% polyethylene glycol or 75 to 85% amino acid-glucose solution.
While other substances, other than cord blood cells and a cryoprotective
agent, may be
present in the present invention, preferably cryopreservation of a TVEMF-
expanded cord blood
stem cell composition of the present invention occurs with as few other
substances as possible,
for instance for reasons such as those discussed regarding the mechanism of
freezing, above.
Preferably, a TVEMF-expanded cord blood stem cell composition of the present
invention is cooled to a temperature in the range of about -120C to about -196
C, preferably
about -130C to about -196C, and even more preferably about -130C to about -
150C.
A controlled slow cooling rate is critical. Different cryoprotective agents
(Rapatz, G.,
et al., 1968, Cryobiology 5(1):18-25) and different cell types have different
optimal cooling
rates (see e.g. Rowe, A. W. and Rinfret, A. P., 1962, Blood 20:636; Rowe, A.
W., 1966,
Cryobiology 3(1):12-18; Lewis, J. P,, et al., 1967, Transfusion 7(1):17-32;
and Mazur, P.,
1970, Science 168:939-949 for effects of cooling velocity on survival of
peripheral cells (and
on their transplantation potential)). The heat of fusion phase where water
turns to ice should
be minimal. The cooling procedure can be carried out by use of, e.g., a
programmable
freezing device or a methanol bath procedure.
Programmable freezing apparatuses allow determination of optimal cooling rates
and
facilitate standard reproducible cooling. Programmable controlled-rate
freezers such as
Cryomed or Planar permit tuning of the freezing regimen to the desired cooling
rate curve.
Other acceptable freezers may be, for example, Sanyo Modl MDF-1155ATN-152C and
Model MDF-2136ATN -135C, Princeton CryoTech TEC 2000. For example, for
peripheral
blood cells or CD34+ cells in 10% DMSO and 20% plasma, the optimal rate is 1
to 3 C
/minute from 0 C to -200 C.
In a preferred embodiment, this cooling rate can be used for the cells of the
invention.
The cryogenic container holding the cells must be stable at cryogenic
temperatures and allow
38
CA 02595744 2007-08-01
. _ , . . . . . - . {
= {
-
{
for rapid heat transfer for effective control of both freezing and thawing.
Sealed plastic vials
(e.g., Nunc, Wheaton cryules) or glass ampules can be used for multiple small
amounts. (1-2
ml), while larger volumes (100-200 mi) can be frozen in polyolefin bags (e.g.,
Delmed) held
between metal plates for better heat transfer during cooling. (Bags of bone
marrow cells have
been successfully frozen by placing them in -80 C freezers that, fortuitously,
gives a cooling
rate of approximately 3 C /minute).
In an alternative embodiment, the methanol bath method of cooling can be used.
The
methanol bath method is well suited to routine cryopreservation of multiple
small items on a
large scale. The method does not require manual control of the freezing rate
nor a recorder to
monitor the rate. In a preferred aspect, DMSO-treated cells are precooled on
ice and
transferred to a tray containing chilled methanol that is placed, in turn, in
a mechanical
refrigerator (e.g., Harris or Revco) at -130 C Thermocouple measurements of
the methanol
bath and the samples indicate the desired cooling rate of I to 3 C /minute.
After at least two
hours, the specimens have reached a temperature of -80 C and can be placed
directly into
liquid nitrogen (-196 C) for permanent storage.
After thorough freezing, TVEMF-expanded stem cells can be rapidly transferred
to a
long-term cryogenic storage vessel. In a preferred embodiment, samples can be
cryogenically
stored in liquid nitrogen (-196 C) or its vapor (-I65 C). The storage
temperature should be
below -120 C, preferably below -130 C. Such storage is greatly facilitated by
the availability
of highly efficient liquid nitrogen refrigerators, which resemble large
Thermos containers
with an extremely low vacuum and internal super insulation, such that heat
leakage and
nitrogen losses are kept to an absolute minimum.
The preferred apparatus and procedure for the cryopreservation of the cells is
that
manufactured by Thermogenesis Corp., Rancho Cordovo, CA, utilizing their
procedure for
lowering the cell temperature to below -130 C. The cells are held in a
Thermogenesis plasma
bag during freezing and storage.
After the temperature of the TVEMF-expanded cord blood stem cell composition
is
reduced to below -120 C, preferably below -130 C, they may be held in an
apparatus such as
a Thermogenesis freezer. Their temperature is maintained at a temperature of
about -120 C
to -196 C, preferably -130 C to -150 C. The temperature of a cryopreserved
TVEMF-
.
39
CA 02595744 2007-08-01
expanded cord blood stem cell composition of the present invention should not
be about -
120C for a prolonged period of time.
A cryopreserved TVEMF-expanded cord blood stem cell composition according to
the
present invention may be frozen for an indefinite period of time, to be thawed
when needed.
For instance, a composition may be frozen for up to 18 years. Even longer time
periods may 10 work, perhaps even as long as the lifetime of an infant donor.
When needed, bags with the cells therein may be placed in a thawing system
such as a
Thermogenesis Plasma Thawer or other apparatus in the Thermoline Thawer
series. The
temperature of the cryopreserved composition is raised to room temperature. In
another
preferred method of thawing the cells mixed with a cryoprotective agent, bags
having a
cryopreserved TVEMF-expanded cord blood stem cell composition of the present
invention,
stored in liquid nitrogen, may be placed in the gas phase of liquid nitrogen
for 15 minutes,
exposed to ambient air room temperature for 5 minutes, and finally thawed in a
37 C water
bath as rapidly as possible. The thawed bags are immediately diluted with an
equal volume of
a solution containing 2.5% (weight/volume) human serum albumin and 5%
(weight/volume)
Dextran 40 (Solplex 40; Sifra, Verona, Italy) in isotonic salt solution and
subsequently
centrifuged at 400 g for ten minutes. The supematant is removed and the
sedimented cells are
resuspended in fresh albumin/Dextran solution. See Rubinstein, P. et al.,
Processing and 'i.
cryopreservation of placental/umbilical cord blood for unrelated bone marrow
reconstitution.
Proc. Natd. Acad. Sci. 92:10119-1012 (1995) for Removal of Hypertonic
Cryoprotectant; a
variation on this preferred method of thawing cells can be found in Lazzari,
L. et al.,
Evaluation of the effect of cryopreservation on ex vivo expansion of
hematopoietic
progenitors from cord blood. Bone Marrow-Trans. 28:693-698 (2001).
After the cells are raised in temperature to room temperature, they are
available for
research or regeneration therapy. The thawed TVEMF-expanded cord blood stem
cell
composition may be introduced directly into a mammal, preferably human, or
used in its
thawed form in desired research. Various additives may be added to the thawed
compositions
(or to a non-cryopreserved TVEMF-expanded cord blood stem cell composition)
prior to
introduction into a mammalian body, preferably soon to immediately prior to
such
introduction. Such additives include but are not limited to a growth factor, a
copper chelating
agent, a cytokine, a hormone, a suitable buffer or diluent. Preferably, G-CSF
is added. Even
CA 02595744 2007-08-01
.S more preferably, for humans, G-CSF is added in an amount of about 20 to
about 40 micrograms/kg body weight, and even more preferably in an amount of
about 30
micrograms/kg body weight. Also, prior to introduction, the TVEMF-expanded
cord blood
stem cell composition may be mixed with the mammal's own, or a suitable
donor's, plasma,
blood or albumin, or other materials that may accompany blood transfusions.
The thawed
cord blood stem cells can be used for instance to test to see if there is an
adverse reaction to a
pharmaceutical that is desired to be used for treatment or they can be used
for treatment.
While the FDA has not approved use of expanded cord blood stem cells for
regeneration of tissue in the United States, such approval appears to be
imminent. Since the
collection of cord blood can only be accomplished within a short time period
of birth, if they
are going to be collected for future use, they must be collected and expanded
and stored for
later research and possible later uses. Direct injection of a sufficient
amount of expanded cord
blood stem cells should be able to be used to regenerate vital organs such as
the heart, liver,
pancreas, skin, muscle, gut, spleen, brain, etc.
TVEMF-expansion may occur after thawing of already cryopreserved, non-
expanded,
or non-TVEMF-expanded, cord blood stem cells. Many cord blood banks, for
instance, have
cryopreserved compositions comprising cord blood stem cells in frozen storage,
in case such
is needed at some point in time. Such compositions may be thawed according to
conventional
methods and then TVEMF-expanded as described herein, including variations in
the TVEMF-
process as described herein. Thereafter, such TVEMF-expanded cord blood stem
cells are
considered to be compositions of the present invention, as described above.
TVEMF-
expansion prior to cryopreserving is preferred, for instance as if a traumatic
injury occurs, a
patient's cord blood stem cells have already been expanded and do not require
precious extra
days to prepare.
Also, while not preferred, it should be noted that TVEMF-expanded cord blood
stem
cells of the present invention may be cryopreserved, and then thawed, and then
if not used,
cryopreserved again.
Also, it is to be understood that the TVEMF-expanded cord blood stem cells of
the
present application may be introduced into a mammal, preferably the source
mammal
(mammal that is the source of the cord blood), after TVEMF-expansion, with or
without
cryopreservation.
41
CA 02595744 2007-08-01
{
Thawed TVENIF-expanded cord blood stem cells may be used for research for {
possible cures for the following diseases:
1. Diseases resulting from a failure or dysfunction of normal blood cell
production and
maturation, hyperproliferative stem cell disorders, aplastic anemia,
pancytopenia,
thrombocytopenia, red cell aplasia, Blackfan-Diamond syndrome due to drugs,
radiation, or
infection, idiopathic;
II. Hematopoietic malignancies, acute lymphoblastic (lymphocytic) leukemia,
chronic
lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous
leukemia, acute
malignant mYelosclerosis, multiple myeloma, polycythemia vera, agnogenic
myelometaplasia, Waldenstrom's macroglobulinemia, Hodgkin's lymphoma, non-
Hodgkins's lymphoma;
III. Irnmunosuppression in patients with malignant, solid tumors, malignant
melanoma,
carcinoma of the stomach, ovarian carcinoma, breast carcinoma, small cell
lung, carcinoma,
retinoblastoma, testicular carcinoma, glioblastoma, rhabdomyosarcoma,
neuroblastoma, Ewing's
sarcoma, lymphoma; IV. Autoimmune diseases, rheumatoid arEhritis, diabetes
type I, chronic hepatitis, multiple
sclerosis, and systemic lupus erythematosus;
V. Genetic (congenital) disorders, anemias, familial aplastic, Fanconi's
syndrome,
Bloom's syndrome, pure red cell aplasia (PRCA), dyskeratosis congenital,
Blackfan-
syndrome, congenital dyserythropoietic syndromes I-IV, Chwachmann-Diamond
Diamond
syndrome, dihydrofolate reductase deficiencies, formamino transferase
deficiency,
Lesch-Nyhan syndrome, congenital spherocytosis, congenital elliptocytosis,
congenital
stomatocytosis, congenital Rh null disease, paroxysmal nocturnal
hemoglobinuria, G6PD
(glucose-6-phosphate dehydrogenase), variants 1,2,3, pyruvate kinase
deficiency,
congenital erythropoietin sensitivity, deficiency, sickle cell disease and
trait, thalassemia
alpha, beta, gamma met-hemoglobinemia, congenital disorders of immunity,
severe combined
immunodeficiency disease, (SCID), bare lymphocyte syndrome, ionophore-
responsive
combined, immunodeficiency, combined immunodeficiency with a capping
abnormality,
nucleoside phosphorylase deficiency, granulocyte actin deficiency, infantile
agranulocytosis,
Gaucher's disease, adenosine deaminase deficiency, Kostmann's syndrome,
reticular dysgenesis,
congenital leukocyte dysfunction syndromes; and
42
CA 02595744 2007-08-01
VI. Others including osteopetrosis, myelosclerosis, acquired hemolytic
anemias, acquired
immunodeficiencies, infectious disorders causing primary or secondary
imumunodeficiencies,
bacterial infections (e.g., Brucellosis, Listerosis, tuberculosis, leprosy),
parasitic infections (e.g.,
malaria, Leishmaniasis), fungal infections, disorders involving disproportions
in lymphoid cell
=
sets and impaired immune functions due to aging phag~ocyte disorders,
Kostmann's
agranulocytosis, chronic granulomatous disease, Chediak-Higachi syndrome,
neutrophil actin
deficiency, neutrophil membrane GP-180 deficiency, metabolic storage diseases,
mucopolysaccharidoses, anucolipidoses, miscellaneous disorders involving
immune mechanisms, Wiskott-Aldrich Syndrome, alpha l.-antitrypsin deficiency.
During the entire process of expansion, preservation, and thawing, cord blood
stem cells
of the present invention maintain the phenotypic characteristics maintained,
fostered, and
developed as a result of the TVEMF-expansion.
While preferred embodiments have been herein described, those skilled in the
art will
understand the present invention to include various changes and modifications.
The scope of the
invention is not intended to be limited to the above-described embodiments.
3 43 .
