Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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PLURIPOTENT STEM CELL EXPANSION AND PASSAGE USING A STIRRED TANK
BIOREACTOR
BACKGROUND
This disclosure relates generally to expansion and passaging of cells and/or
cell aggregates using
a stirred tank bioreactor.
A need for large scale pluripotent stem cell culture is emerging for
applications in pluripotent
stem cell banking (e.g., for induced pluripotent stem cells), commercial
production of cells (e.g.,
GE's CytivaTM cardiomyocytes), and cell expansion for clinical trials.
Advances in feeder-free
pluripotent stem cell culture have enabled large scale cell expansion in
flasks, on microcarriers
(150 to 250 microns in diameter) or on macro carriers (-6 mm in diameter) in
bioreactors. The
use of suspension culture avoids some of the challenges that occur when
culturing pluripotent
cells on traditional microcarriers including inefficient seeding and release
of cells from carriers,
physical separation of microcarriers and cells during harvest, and formation
of cell-carrier
clumping that can lead to phenotypic changes in the cells. Typically,
perfusion is used for
suspension cultures in bioreactors.
However one challenge in perfusion/suspension culture is how to retain the
cells in the
bioreactor. Prior art provides some basic separation techniques --1)
filtration, 2) gravity
sedimentation, and 3) centrifugation. Filtration methods require some means to
keep the filter
from clogging over the required weeks of operation. A problem with gravity
sedimentation is
the varying sedimentation characteristics of different cells, the difficulty
in scale-up to industrial
systems, and difficulty in maintaining sterility. Similarly, centrifugation is
routinely used in
open cell culture but has found limited application in fully closed system
cell culture due to
concerns regarding sterility.
There is a need in the field for techniques which reduce human intervention
and cross-
contamination during the process of culturing cells, including pluripotent
stem cells and/or
differentiated human cells.
BRIEF DESCRIPTION
Described herein are improved methods for culturing cells, including
pluripotent stem cells
and/or differentiated human cells.
Provided herein are methods for expansion of cell aggregates in a closed
system comprising
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a cell culture vessel;
aggregate formation in the vessel;
automated perfusion of cell aggregates in the vessel;
gravity settling of cell aggregates during the perfusion; and
aggregate harvest and passaging in the closed system.
Also provided herein are closed systems for use in expansion of cell
aggregates using the
methods described herein.
DRAWINGS
These and other features, aspects, and advantages of the present invention
will become better
understood when the following detailed description is read with reference to
the accompanying
drawings in which like characters represent like parts throughout the
drawings, wherein:
FIG. 1 shows a diagram of the tubing assembly for gravity settling and medium
exchange in
stirred taffl( bioreactors in which spent medium is being removed from the
bioreactor in a
vertical orientation.
FIG. 2 shows a diagram of the tubing assembly for gravity settling and medium
exchange in
stirred taffl( bioreactors in which fresh medium is being added to the
bioreactor in a vertical
orientation.
FIG. 3 shows a diagram of the tubing assembly for gravity settling and medium
exchange in
stirred taffl( bioreactors in which spent medium is being removed from the
bioreactor in a slanted
orientation.
FIG. 4 shows a diagram of the tubing assembly for gravity settling and medium
exchange in
stirred tank bioreactors in which fresh medium is being added to the
bioreactor in a slanted
orientation.
FIG. 5 shows a diagram of the 10 mm slicer grid structure with thickness of
100um to 300 um.
FIG. 6 shows a diagram of the square grid slicer with 100um spacing between
walls and 30 um
wall thickness.
FIG. 7 shows a diagram of the hexagon grid slicer with 100um spacing between
walls and 30
um wall thickness.
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FIG. 8 shows a diagram of a method for closed system processing of aggregates
through the
slicer. A circulation loop driven by a pump and an in line conical bag
suspends and distributes
the aggregates. Tubing leading to the slicer is connected to the main
circulation loop and a
portion of the cell aggregates is delivered to the slicer through a second
pump operating at a
lower speed.
FIG. 9 shows images of the morphology of sliced aggregates of CT2 human
embryonic stem
cells.
FIG. 10 shows expansion rate comparison after enzymatic passaging or
mechanical passaging
with the slicer on CT2 human embryonic stem cell aggregates seeded at 4x10'5
cells per mL.
FIG. 11 shows expansion rate comparison after enzymatic passaging or
mechanical passaging
with the slicer on CT2 human embryonic stem cell aggregates seeded at 1.5x10A6
cells per mL.
(1) CT-2 passaged with Accutase0 + ROCK inhibitor; (2) CT-2 passaged with
square grid +
ROCK inhibitor; (3) CT-2 passaged with hexagon grid + ROCK inhibitor; (4) CT-2
passaged
with square grid + ROCK inhibitor; (5) CT-2 passaged with hexagon grid + ROCK
inhibitor.
FIG. 12 shows CT2 human embryonic stem cell aggregate morphology in the
stirred tank
reactor.
DETAILED DESCRIPTION
"A" or "an" means herein one or more than one; at least one. Where the plural
form is used
herein, it generally includes the singular.
As used herein "perfusion" refers to the process of keeping culture cells
alive by continuously
feeding the cells with fresh media and removing spent media while keeping
cells in culture.
"Aggregate" refers to an association of cells in which the association is
caused by cell-cell
interaction rather than adherence to a substrate. In an aggregate, two or more
cells associate with
each other by biologic attachments to one another. This can be through surface
proteins, such as
extracellular matrix proteins. In one embodiment, cells can be initially grown
on a substrate
where some cells associate with (adhere to) the substrate but further growth
forms cell-cell
associations (aggregation) that do not depend on association (adherence) of
the further-grown
cells with the substrate. In another embodiment, cells spontaneously associate
in suspension to
form cell-cell attachments independent of any adherence to a surface. A
cellular feeder layer is
also considered a substrate. So attachment of cells to a feeder layer is also
a form of adherent
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culture (not an aggregate) since attachment of the cells is not to each other
but to the cells in the
feeder layer.
"Expansion" refers to the proliferation of cells with or without
differentiation and may include
no passaging, one passage or more than one passage and/or serial passages. In
one embodiment,
expansion refers to proliferation of cells without differentiation and
includes one or more than
one passage and/or serial passages.
"Stem cell" means a cell that can undergo self-renewal (i.e., progeny with the
same
differentiation potential) and also produce progeny cells that are more
restricted in
differentiation potential. Within the context of the disclosure, a stem cell
would also encompass
a more differentiated cell that has de-differentiated, for example, by nuclear
transfer, by fusion
with a more primitive stem cell, by introduction of specific transcription
factors, or by culture
under specific conditions. A "pluripotent stem cell" can potentially produce
any cell or tissue
the body needs to repair itself Pluripotent stem cells are also able to self-
renew, and can
perpetually create more copies of themselves. Pluripotent stem cells include
induced pluripotent
stem cells (iPSCs) and embryonic stem cells (ESCs).
"Culture vessel" includes disposable and non-disposable plasticware, bags
and/or containers
and/or bioreactors. The term includes single-use plasticware, bags and/or
containers and/or
bioreactors and multiple-use plasticware, bags and/or containers and/or
bioreactors.
"Closed system" refers to a culture vessel and accessory components that have
been pre-
sterilized while closed and/or sealed and retains integrity and/or sterility.
The vessels and
components are utilized without breach of the integrity of the system, permit
fluid transfers in
and/or out while maintaining asepsis, and are connectable to other closed
systems without loss
of integrity. A closed system bioreactor and/or vessel refers to a system in
which cells, cell
culture medium, chemicals and reagents are aseptically added, removed and/or
manipulated
without breach of integrity of the system (e.g., by opening the cap of a tube
or lifting the lid off
a cell culture plate or dish). Single-use or multiple-use bags and/or
containers and/or bioreactors
in a closed system are added onto or into the closed system for example by
sterile tube welding
at the site of the vessel or bioreactor.
A "subject" is a vertebrate, preferably a mammal, more preferably a human.
Mammals include,
but are not limited to, humans, farm animals, sport animals, and pets.
Subjects in need of
treatment by methods of the present invention include those suffering from a
loss of function as
a result of physical or disease-related damage.
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The term "therapeutically effective amount" refers to the amount determined to
produce any
therapeutic response in a mammal. For example, effective amounts of the
therapeutic cells or
cell-associated agents may prolong the survivability of the patients.
Alternatively, said
treatment may be prophylactic and prevent and/or inhibit overt clinical
symptoms. Treatments
that are therapeutically effective within the meaning of the term as used
herein, include
treatments that improve a subject's quality of life even if they do not
improve the disease
outcome per se. Such therapeutically effective amounts are ascertained by one
of ordinary skill
in the art through routine application to subject populations such as in
clinical and pre-clinical
trials. Thus, to "treat" means to deliver such an amount.
"Treat," "treating" or "treatment" are used broadly in relation to the
invention and each such
term encompasses, among others, ameliorating, inhibiting, or curing a
deficiency, dysfunction,
disease, or other deleterious process, including those that interfere with
and/or result from a
therapy.
Large scale pluripotent stem cell culture is needed for pluripotent stem cell
banking (e.g., for
induced pluripotent stem cells), commercial production of cells (e.g., GE's
CytivaTM
cardiomyocytes), and/or cell expansion for clinical trials. Pluritpotent stem
cells may be
induced pluripotent cell (iPS cells), "true" embryonic stem cell (ES cells)
derived from embryos,
embryonic stem cells made by somatic cell nuclear transfer (ntES cells), or
embryonic stem cells
from unfertilized eggs (parthenogenesis embryonic stem cells, or pES cells).
Large scale cell
feeder-free embryonic stem cell expansion in flasks is labor intensive, space
prohibitive and
separated populations may exhibit phenotypic drift. Therefore, there have been
attempts in the
field for developing alternative approaches for large scale pluripotent stem
cell culture; e.g.,
CellSTACK0 (Corning), Cell Factory (Nunc0) and bioreactors (microcarriers or
suspension
culture).
There are many types of bioreactors used for cell culture, including but not
limited to stirred
tank reactors, spinner flasks, orbital shakers, rocking motion, and paddle
wheel. Those skilled in
the art will recognize that there are also alternative approaches for cell
culture in bioreactors.
Suspension aggregate culture of pluripotent stem cells in an impeller stirred
tank bioreactor
system has been demonstrated (Chen, VC et.al, Stem Cell Res. 2012 May;8(3):388-
402) which
obviates the need for any substrate or carriers in the bioreactor culture. In
the Chen method for
passaging cells from suspension culture, aggregates were harvested by
centrifugation. By
contrast, the methods provided herein allow for closed system expansion
(including seed,
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perfusion, passage and harvest) of pluripotent stem cells and/or pluripotent
stem cell aggregates
in a closed system using a stirred tank bioreactor. Further, the methods
described herein also
allow for suspension and/or non-adherent cultures of pluripotent stem cells in
closed systems
without the use of membrane filters and/or centrifugation and/or enzyme
digestion, which
allows for maintenance of sterility in closed systems, reduces costs (e.g.,
for setting up
centrifuges) and also reduces human intervention which assists in reducing
cross contamination.
Also contemplated within the scope of embodiments presented herein is the use
of the present
methods in combination with additional passages of the cells or cell
aggregates which may
include the use of membrane filters and/or centrifugation and the like and may
include the use of
enzyme digestion for dissociation of aggregates in the additional passages of
the cells or cell
aggregates.
Previously described methods for expansion of pluripotent stem cells in
stirred tank bioreactors
(e.g., Niebruegge et al., Tissue Engineering: Part A, 2008, Volume 14, Issue
10, Pages 1591-
1601) disclose expansion of mouse stem cells. Further such previously
described methods are
focused on sustaining the stem cell culture for a sufficient length of time
and, generally,
consequential differentiation of the stem cells. By contrast, the methods
described herein allow
for expansion of human stem cells, followed by dissociation of aggregates and
passaging of the
disassociated cells so that the cells retain their pluripotency through
expansion and serial
passages. In addition, the methods of expansion and passage described herein
are carried out in
a closed system which ensures sterility during the production process.
Typically, previously
described stem cell expansion methods employ Rho-associated protein kinase
inhibitors (ROCK
inhibitors) for sustaining the cell culture during enzymatic passaging. A
drawback of employing
ROCK inhibitors for stem cell expansion and passage is that the ROCK
inhibitors affect
chromosomal stability, possibly triggering karyotypic abnormalities and
genetic drift of the stem
cells. By contrast, the expansion and passaging of stem cells described herein
is carried out in
the absence of ROCK inhibitors thereby reducing karyotypic abnormalities and
genetic drift and
allowing for retention of pluripotency during the passaging of the stem cells.
The methods described herein rely on gravity settling combined with automated
perfusion in
closed systems. Additionally, in some embodiments, the methods described
herein comprise the
use of a slicer thereby allowing for enzyme free dissociation of cell
aggregates between
successive passages of cells. Certain previously described lamellar gravity
settlers require the
cells or cell aggregates to roll down inclined planes/tubes. The drawbacks of
such lamellar
gravity settlers for suspension aggregate applications include: the cell
aggregates are subject to
shear stress which reduces the efficiency of the expansion process, the large
cell loss due to
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incomplete settling efficiency, and aggregate association/adherence to the
gravity settler.
Lamellar gravity settlers have been used to separate and discard cell
aggregates from the cell
population. By contrast, the methods described herein separate and discard the
single cells (low
viability stem cells) while retaining aggregates for subsequent passaging.
Other previously described methods for medium exchange temporarily pause
agitation, thereby
allowing aggregates to gravity settle in the stirred taffl( bioreactor. After
the aggregates settle, a
portion of the spent medium can be removed from the bioreactor and replaced
with fresh
medium. A risk of this approach is the potential for aggregate conglomeration
while the
aggregates are concentrated together at the bottom surface.
Accordingly, described herein are methods for cell aggregate expansion,
including human
pluripotent stem cell expansion, in stirred taffl( bioreactors. The continuous
or discontinuous
stirring of the culture fluid provides mixing and aeration, resulting in a
robust environment for
cell growth. The method employs culture vessels of varying sizes, providing
ease of operation
and protection against cross-contamination. Sensors are available for
continuous monitoring of
dissolved oxygen, pH and medium components such as lactate and glucose, with
real time
controls and data storage. The platform software provides the ability to
perform continuous or
discontinuous perfusion/ medium exchange in a closed system.
Typically, during perfusion, there are different ways to keep the cells in
culture while removing
spent media. One way is to keep the cells in the bioreactor by using capillary
fibers or
membranes with pore sizes smaller than the size of the cell. Another method is
to utilize a "lily
pad" floating filter that keeps the cells in the bioreactor while allowing the
media to be removed.
Another method is the use of a centrifuge to separate cells and return them to
the bioreactor.
Yet another method uses a physical approach such as acoustics to trap cells in
the cell culture
vessel or associated tubing while spent medium is removed.
By contrast, the methods described herein rely on gravity settling of cell
aggregates which
allows for removal of spent media without the use of filtration systems which
are typically used
to keep the cells in the bioreactor while concomitantly allowing the media to
be removed. An
advantage to this method is the loss of single cells, which are predominantly
non-viable in
pluripotent stem cell cultures, and maintenance of aggregates, thereby
increasing the overall
quality and viability of the culture.
By continuously removing spent media and replacing it with new media, nutrient
levels are
maintained for optimal growing conditions and cell waste product is removed to
avoid toxicity.
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When perfusion is carried out in a closed system using the methods described
herein, the
possibility of contamination is reduced. Advantageously, the closed systems
and cell culture
methods described herein utilize gravity-settling of cell aggregates thereby
allowing for
membrane-less perfusion which allows for reduction in losses due to adhesion
of cells to
filtration membranes and/or damage to cells due to shear during the filtration
process.
The methods described herein are preferably employed in closed systems to
minimize risk of
culture contamination and cell cross-contamination and allow for reaching high
viabilities and
high cell densities with confidence. The methods described herein are designed
for ease-of-use
and reliability.
Provided herein are methods which describe pluripotent stem cell expansion as
suspension
aggregates in a stirred taffl( system, with serial passage. Also provided
herein are examples
showing that the use of a stirred taffl( reactor in combination with a slicer
for passaging is
advantageous for expansion and serial passage of pluripotent stem cells in a
unified closed
system, specifically in reductions of time, reagents and labor.
Also provided herein is a specific assembly of tubing connected to culture
vessel (e.g., a stirred
taffl( bioreactor) that interacts with computer controlled peristaltic pumps
to drive automated
medium exchange. In one embodiment, the tubing is shaped like a T, with a
lower vertical piece
of tubing, a branch point, and two additional lengths of tubing connected at
the branch point.
The additional tubing is placed onto peristaltic pumps that are controlled by
software. A slow
harvest rate is used to draw off medium. The vertical nature of the tubing
allows aggregates to
gravity settle at rates that exceed the flow rate of the removed medium. The
net effect is that
aggregates remain in the optimal cell culture conditions in the vessel (e.g.,
a stirred tank
bioreactor) during the medium removal step. Medium removal can be continuous
e.g., for up to
8 hours, up to 4 hours, and the like, but medium removal can be performed for
much shorter or
longer lengths of time. Following the medium removal step, fresh medium is
rapidly added to
the vessel (e.g., the stirred tank bioreactor) over seconds to a few minutes
or over any suitable
length of time. The cycle of medium removal/rapid medium addition is repeated
for the desired
length of cell culture. In alternate instances, perfusion may be discontinuous
and such
embodiments are also contemplated within the scope of embodiments presented
herein.
The automated perfusion design described herein is inherently low cost, is
fully compatible with
culture vessels including stirred tank reactors, can be adjusted to be fully
compatible with any
other culture vessel, and does not require any filters which would add cost
and increase the risk
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for fouling and reduce performance. Further the automated perfusion described
herein does not
comprise moving parts or electronics which would increase complexity, cost,
and risk for
failure.
Provided herein is a method for expansion of cell aggregates in a closed
system comprising
providing a cell culture vessel;
aggregate formation in the vessel;
automated perfusion of cell aggregates in the vessel;
gravity settling of cell aggregates during the perfusion; and
aggregate harvest and passaging in the closed system.
In some embodiments, the passaging in the closed system is carried out in the
presence of a
Rho-associated protein kinase (ROCK) inhibitor (e.g., a concentration of the
ROCK inhibitor in
the cell culture medium in the vessel is about 10 micromolar). In other
embodiments of the
method described above, the passaging in the closed system is carried out
substantially in the
absence of an agent which maintains the viability of passaged, monodispersed
or disaggregated
pluripotent cells. In some of such embodiments, the agent which maintains the
viability of
pluripotent cells is a Rho-associated protein kinase (ROCK) inhibitor. In some
embodiments of
the method described above, the passaging in the closed system is carried out
in the absence of a
ROCK inhibitor. In some of such embodiments, the ROCK inhibitor is Y27632. As
used
herein, in one embodiment, "substantially in the absence of an agent which
maintains the
viability of passaged, monodispersed or disaggregated pluripotent cells" means
there is no agent
added to the cell culture medium to maintain the viability of passaged,
monodispersed or
disaggregated pluripotent cells. In another embodiment, "substantially in the
absence of an
agent which maintains the viability of passaged, monodispersed or
disaggregated pluripotent
cells" means that the concentration of the agent in the cell culture medium
present in the vessel
is less than 1 micromolar, or less than 5 micromolar or less than 10
micromolar.
In some embodiments, the cell aggregates which are expanded are of plant,
animal, insect or
microbial origin. In some embodiments, the cell aggregates which are expanded
comprise
human pluripotent stem cells or differentiated human cells or a combination
thereof. In some
embodiments, the cell aggregates which are expanded comprise human pluripotent
stem cells.
In some embodiments, the cell culture vessel is a stirred tank bioreactor. In
some embodiments,
said automated perfusion is carried out in the closed system without human
intervention.
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In some embodiments, the gravity settling chamber is at least 1 cm in length
and positioned in
an orientation that drives gravity settling of cell aggregates.
In some embodiments, the automated perfusion is conducted at a rate that
allows gravity settling
of cell aggregates of about 100 micron to about 800 micron diameter.
In some embodiments, the methods described above further comprise one or more
additional
expansions of the cell aggregates and/or progeny thereof by conducting serial
passages. In some
of such embodiments, the one or more of the additional expansions of the cell
aggregates and/or
progeny thereof, are carried out by passaging into a second vessel having a
membrane filter.
In some embodiments, the methods described above comprise one or more
additional
expansions of the cell aggregates and/or progeny thereof where the one or more
additional
passages are conducted in the same vessel. In other embodiments, the methods
described above
comprise one or more additional expansions of the cell aggregates and/or
progeny thereof where
the one or more additional passages are conducted in a second vessel in the
absence of a
membrane filter (e.g., by gravity settling of cell aggregates) in the culture
vessel. In other
instances, the methods described above comprise one or more additional
expansions of the cell
aggregates and/or progeny thereof where the one or more additional passages
are conducted in a
second vessel having a membrane filter (e.g., a floating membrane filter).
In such embodiments, the additional expansions and/or passages may be carried
out in any
order. By way of example, an initial passage may be carried out under membrane-
free
conditions (e.g., by gravity settling of cell aggregates) in the culture
vessel, followed by one or
more additional expansions carried out in a culture vessel having a membrane
filter. As an
alternate example, one or more initial expansions and/or passages may be
carried out in culture
vessels which comprise membrane filters, followed by subsequent expansions
and/or passages
under membrane-free filtration conditions (e.g., by gravity settling of cell
aggregates).
Accordingly any sequence of expansions and/or passages comprising membrane-
filtration or
membrane-free filtration is contemplated within the scope of embodiments
described herein
where said sequence includes at least one expansion under the present membrane-
free
conditions (e.g., by gravity settling of cell aggregates).
In some embodiments, serial passaging of cell aggregates is enabled by enzyme-
free passaging
using slicer grids in the closed system. In some of such embodiments, the cell
aggregates are
dissociated with a slicer grid having blades separated by a distance of about
20 to about 500
microns. In some other embodiments, the cell aggregates are dissociated with a
slicer grid
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having blades separated by a distance of about 100 microns. In some of such
embodiments, the
cell aggregates are dissociated with a slicer grid in line with tubing and a
device for mixing of
cell aggregates. In other words, in some embodiments, the cell aggregates are
mixed in e.g., a
conical bag, shown in FIG. 8, prior to dissociation with a slicer and the
mixing bag is typically
placed between the culture vessel and the slicer in the closed system.
The cell aggregate concentration used to pass through the slicer influences
the recovery of sliced
cell aggregates at high viability. Unexpectedly, it was found that slicing of
cell aggregate
concentrations below about 3 x 10^6 cells per mL produced higher viability
samples with higher
recovery than cell concentrations greater than about 3 x 10^6 cells per mL
using a slicer
geometry shown in FIG. 5-7. Those skilled in the art will recognize that
alternative slicer
geometries will modify the threshold concentration that provides relatively
higher viability and
recovery. Fouling was minimized by maintaining a uniform suspension of
aggregates in the
flow stream, for example by using the mixing device shown in Figure 8,
resulting in higher cell
viability and recovery. Advantageously, the use of a slicer obviates the need
for the Y27632
ROCK inhibitor during expansion of sliced pluripotent stem cell aggregates,
unlike
enzymatically passaged pluripotent stem cell aggregates which generally
requires agents such as
the Y27632 ROCK inhibitor to maintain the viability of single pluripotent
cells.
In one group of embodiments the slicer is coated with a hydrophobic material.
In some
embodiments, the slicer comprises a hydrophobic material.
Also contemplated are certain other embodiments, wherein serial passaging of
the cell
aggregates is enabled by disassociation of the cell aggregates in the closed
system vessel in the
presence of an enzyme. In such embodiments, additional expansion or passaging
may be carried
out in the presence of a ROCK inhibitor.
In some embodiments of the methods described above, during the expansions
and/or passages,
the average diameter of each expanded cell aggregate is no more than about 800
micron in size.
In some embodiments of the methods described above, during the expansions
and/or passages,
the average diameter of each expanded cell aggregate is no more than about 500
micron in size.
In some embodiments of the methods described above, during the expansions
and/or passages,
the average diameter of each expanded cell aggregate is no more than about 400
micron in size.
In some embodiments of the methods described above, during the expansions
and/or passages,
the average diameter of each expanded cell aggregate is no more than about 300
micron in size.
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In some embodiments, the volume of the culture vessel is from about 50 mL to
about 100 L. In
some embodiments, the volume of the culture vessel is from about 50 mL to
about 50 L. In
some embodiments, the volume of the culture vessel is from about 100 mL to
about 10 L. In
some embodiments, the volume of the culture vessel is from about 100 mL to
about 5 L. In
some embodiments, the volume of the culture vessel is from about 150 mL to
about 1 L. In
some embodiments, the volume of the culture vessel is from about 50 mL to
about 20 L. In
some embodiments, the volume of the culture vessel is from about 200 mL to
about 2 L or
greater than 2 L.
Further provided herein is a method for passaging cell aggregates wherein cell
aggregates are
reduced in size by a slicer grid associated with a bioreactor in a closed
system. In one group of
embodiments for any method described herein, the cell aggregates are passaged
in volumes
exceeding 100 mL. In other words, passaging has generally been carried out in
smaller volumes
of culture medium and with lower cell counts. By contrast, the present methods
allow for use of
large volumes of culture medium in closed systems thereby allowing for
passaging of cell
aggregates in bioreactors and/or on industrial scale. The use of a slicer grid
in combination with
a bioreactor in a closed system for cell aggregate passaging in large volumes
e.g., exceeding 100
ml, has not been disclosed in the art prior to this disclosure. In another
embodiment, the cell
aggregates are passaged in volumes exceeding 250 mL, 500 mL, 1L, 2L or 5L. In
one
embodiment, the slicer grid is a polygonal slicer grid. In one instance, said
passaging of cell
aggregates in volumes exceeding 100 mL is performed without the addition of a
ROCK
inhibitor (e.g., Y27632) to the medium.
In some embodiments of the method for passaging cells described above, the
cell aggregates are
dissociated with a slicer grid having blades separated by a distance of about
20 to about 500
microns. In some embodiments of the method for passaging cells described
above, the cell
aggregates are dissociated with a slicer grid having blades separated by a
distance of about 100
microns. In some of such embodiments, the cell aggregates are dissociated with
a slicer grid in
line with tubing and a device for mixing of cell aggregates. In certain
instances, the slicer is
coated with a hydrophobic material. In other instances, the slicer comprises a
hydrophobic
material.
In some embodiments of the method for passaging cells described above, the
average diameter
of each cell aggregate prior to passaging is no more than about 800 micron in
size. In some
embodiments of the method for passaging cells described above, the average
diameter of each
cell aggregate prior to passaging is no more than about 500 micron in size. In
some
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embodiments of the method for passaging cells described above, the average
diameter of each
cell aggregate prior to passaging is no more than about 400 micron in size. In
some
embodiments of the method for passaging cells described above, the average
diameter of each
cell aggregate prior to passaging is no more than about 300 micron in size.
In another aspect, provided herein is a method for expansion of cell
aggregates in a closed
system comprising
providing a stirred tank bioreactor vessel;
aggregate formation in the vessel;
automated perfusion of cell aggregates in the vessel;
gravity settling of cell aggregates during the perfusion;
aggregate harvesting and enzyme-free slicing of cell aggregates; and
passaging in the closed system.
In some embodiments of the method described above, the passaging in the closed
system is
carried out in the presence of a ROCK inhibitor (e.g., a concentration of the
ROCK inhibitor in
the cell culture medium in the vessel is about 10 micromolar). In other
embodiments of the
method described above, the passaging in the closed system is carried out
substantially in the
absence of an agent which maintains the viability of passaged, monodispersed
or disaggregated
pluripotent cells. In some of such embodiments, the agent which maintains the
viability of
pluripotent cells is a Rho-associated protein kinase (ROCK) inhibitor. In some
embodiments of
the method described above, the passaging in the closed system is carried out
in the absence of a
ROCK inhibitor. In some of such embodiments, the ROCK inhibitor is Y27632. As
used
herein, in one embodiment, "substantially in the absence of an agent which
maintains the
viability of passaged, monodispersed or disaggregated pluripotent cells" means
there is no agent
added to the cell culture medium to maintain the viability of passaged,
monodispersed or
disaggregated pluripotent cells. In another embodiment, "substantially in the
absence of an
agent which maintains the viability of passaged, monodispersed or
disaggregated pluripotent
cells" means that the concentration of the agent in the cell culture medium
present in the vessel
is less than 1 micromolar, or less than 5 micromolar or less than 10
micromolar
Accordingly, the methods provided herein enable several workflows including
and not limited to
(1) Aggregate formation in bioreactors including from these sources:
enzymatically dissociated
aggregates (e.g., AccutaseTM), cryopreserved stocks, and/or mechanically
sliced aggregates
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(e.g., polygonal slicer grid); (2) Expansion method in stirred systems: non-
perfusion bioreactor
with tubing assembly for gravity settling, and/or perfusion bioreactor; and
(3) Serial Passaging:
enzyme added to aggregates in cell culture vessel (e.g., AccutaseTM in
bioreactor), enzyme
added to aggregates outside of cell culture vessel and/or mechanical passage
using polygonal
slicer grid.
Further, the methods described herein are suitable for use in a variety of
bioreactors that have
been used for suspension culture of cells including and not limited to stirred
suspension
bioreactors, rocking motion bioreactors, spinner flasks, orbital motion
bioreactors, rotary motion
bioreactors, and tangential fluid flow bioreactors. The methods described
herein, including filter
free gravity settling for perfusion and enzymatic-free passaging using a
slicer, are compatible
with all such bioreactor systems and enable closed system aggregate formation,
perfusion,
expansion, harvest and passaging in such bioreactors.
Contemplated within the scope of embodiments provided herein is the use of the
methods
described herein for generating banks of cells, for generating expanded cell
aggregates for
research applications, for therapeutic and/or diagnostic testing (e.g., drug
testing, toxicology or
quality control assays in clinical trials), and/or for treatment of patients.
Provided herein are
methods comprising administering to subjects in need thereof a pharmaceutical
composition
comprising a pharmaceutically-acceptable carrier and at least one cell and/or
cell aggregate
obtained from the methods described herein.
Also provided herein is a method of treating a disorder in a subject in need
of treatment by
administering a therapeutically effective amount of the cells and/or
aggregates produced in the
methods above to the subject in need thereof. The methods further include a
method of treating
a disorder in a subject in need of treatment by administering a
therapeutically effective amount
of a pharmaceutical composition comprising a pharmaceutically-acceptable
carrier and the cells
and/or aggregates produced in the methods above. It will be understood that
the methods
described herein are applicable to pluripotent stem cells and also
differentiated cells.
In some embodiments the purity and/or homogeneity of the expanded cells
obtained from the
methods described herein and/or for administration to a subject is about 100%
(substantially
homogeneous). In other embodiments the purity and/or homogeneity of the
expanded cells
obtained from the methods described herein and/or for administration to a
subject is 95% to
100%. In some embodiments the purity and/or homogeneity of the expanded cells
obtained from
the methods described herein and/or for administration to a subject is 85% to
95%. In the case of
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admixtures with other cells, the percentage can be about 10%- 15%, 15%-20%,
20%-25%, 25%-
30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 60%-70%, 70%-80%, 80%-90%, or 90%-
95%.
The choice of formulation for administering the cells for a given application
will depend on a
variety of factors. Prominent among these will be the species of subject, the
nature of the
condition being treated, its state and distribution in the subject, the nature
of other therapies and
agents that are being administered, the optimum route for administration,
survivability via the
route, the dosing regimen, and other factors that will be apparent to those
skilled in the art. For
instance, the choice of suitable carriers and other additives will depend on
the exact route of
administration and the nature of the particular dosage form. Final
formulations of the aqueous
suspension of cells/medium will typically involve adjusting the ionic strength
of the suspension
to isotonicity (i.e., about 0.1 to 0.2) and to physiological pH (i.e., about
pH 6.8 to 7.5). The final
formulation will also typically contain a fluid lubricant.
In some embodiments, cells are formulated in a unit dosage injectable form,
such as a solution,
suspension, or emulsion. Pharmaceutical formulations suitable for injection of
cells typically are
sterile aqueous solutions and dispersions. Carriers for injectable
formulations can be a solvent or
dispersing medium containing, for example, water, saline, phosphate buffered
saline, polyol (for
example, glycerol, propylene glycol, liquid polyethylene glycol, and the
like), and suitable
mixtures thereof The skilled artisan can readily determine the amount of cells
and optional
additives, vehicles, and/or carrier in compositions to be administered in
methods of the
invention.
Compositions can be administered in dosages and by techniques well known to
those skilled in
the medical and veterinary arts taking into consideration such factors as the
age, sex, weight,
and condition of the particular patient, and the formulation that will be
administered (e.g., solid
vs. liquid).
It is to be appreciated that a single dose may be delivered all at once,
fractionally, or
continuously over a period of time. The entire dose also may be delivered to a
single location or
spread fractionally over several locations.
In various embodiments, cells may be administered in an initial dose, and
thereafter maintained
by further administration. Cells may be administered by one method initially,
and thereafter
administered by the same method or one or more different methods. The levels
can be
maintained by the ongoing administration of the cells. Various embodiments
administer the cells
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either initially or to maintain their level in the subject or both by
intravenous injection. In a
variety of embodiments, other forms of administration are used, dependent upon
the patient's
condition and other factors, discussed elsewhere herein. Suitable regimens for
initial
administration and further doses or for sequential administrations may all be
the same or may be
variable. Appropriate regimens can be ascertained by the skilled artisan, from
this disclosure,
the documents cited herein, and the knowledge in the art. The dose, frequency,
and duration of
treatment will depend on many factors, including the nature of the disease,
the subject, and other
therapies that may be co-administered. In any of the embodiments described
herein, the cells
may be differentiated or non-differentiated. In any of the embodiments
described herein, the
cells may be dissociated or aggregates. In any of the embodiments described
herein the cells
and/or cell aggregates may comprise a combination of pluripotent stem cells
and progeny
thereof.
EXAMPLES
Material and Methods
Materials: Aggregates were cultured in a GE prototype stirred tank reactor
depicted in Figures
3-4. Hyclone Labtainer bags were used to hold fresh medium for feed as well as
to collect spent
medium. AccutaseTM was purchased from MP Biomedical (CA, USA); mTeSRTM1 medium
was purchased from STEMCELLTM Technology Inc. (Vancouver, BC, Canada). Y-27632
(ROCK Inhibitor) was purchased from Sigma Aldrich (St. Louis, MO). CT2 hESC
were
obtained from Ren-He Xu at the University of Connecticut Health Center. The
polygonal grid
slicers and the tubing assembly for gravity settling used for passaging were
manufactured
specifically for this application and were not purchased through a commercial
vendor.
Methods: Human embryonic stem cells were adapted from MatrigelTM to suspension
aggregates for greater than 5 passages prior to stirred tank reactor
experiments. Stock cells were
confirmed to be karyotypically normal. Serial passaging was performed using
AccutaseTM to
reduce aggregates to small clusters and single cells which reformed suspension
aggregates after
seeding. Cell counts and viability were determined using a Nucleocounter0
NC2OOTM
(Chemometec, Denmark).
The human embryonic stem cell line CT2 was suspension adapted from feeder-free
cell stocks
and maintained for at least 5 passages at small scale prior to bioreactor
culture. Enzymatic
passaging using AccutaseTM produced a population of single cells and small (<5
cell) clusters.
Aggregates formed 2-12 hours after addition of single cells/small clumps to
the vessel. The
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cells formed aggregates between about 50 and 200 um diameter. It was normal to
obtain a
distribution of aggregate diameters 50 um above and below the mean aggregate
diameter. The
majority of aggregates fell within that size range, however there were on
occasion some larger
aggregates of roughly 200 to 400 um that formed. Conditions that favor smaller
aggregates are
preferred as nutrient availability can be limited in larger aggregates, and
the smaller aggregates
provide a greater relative expansion in the culture.
The preferred conditions provide spherical aggregates with minimal clumping.
It is important to
balance the level of agitation in the bioreactor, as too much agitation will
lead to shearing
including deformation of aggregates and producing excessive numbers of non-
aggregated single
cells. Too little agitation will lead to clumping of aggregates.
Not all pluripotent cell lines prefer the same culture conditions. The
following parameters were
used for PSC expansion, and those skilled in the art will recognize that other
conditions will also
provide PSC expansion in similar vessels: Temperature 37 degrees C, CO2 level
5%, ambient
02 (-21%) or a reduced 02 level, continuous or discontinuous stir speed of 40
rpm.
Tubing assembly for gravity settling and medium exchange in non-perfusion
bags:
The conceptual construct of the tubing assembly on a stirred tank bioreactor
is shown in FIG. 1-
4. The assembly provides the following functions including but not limited to
the following:
(1) removal of cell/ cell culture medium mixture, (2) cell aggregate
separation from outgoing
cell culture medium, (3) cell culture medium addition, and (4) cell culture
medium removal.
Removal of cell aggregate/ cell culture medium mixture is accomplished through
the use of a
dip tube. The dip tube should be of sufficient length/orientation such that
cells/media can be
removed from the bioreactor while it is installed and in operation. Cell
aggregate separation
from outgoing media is achieved by the introduction of a gravity settling
chamber with
sufficient length (height) and diameter to ensure adequate gravity setting
during media removal.
The design of this chamber is not limited to a large diameter tube; a tortuous
path may also be
integrated if necessary for satisfactory cell aggregate separation. One or
more than one tubing
assemblies operating in parallel could be associated with the bioreactor to
increase the rate of
medium removal/addition. Tubing for fluid addition/removal needs to be of
adequate length to
ensure attainment of connections to media/waste containers. Fluid removal is
achieved by
pulling out the medium through the fluid removal path while keeping the fluid
addition path
closed. Fluid addition is achieved by instilling fresh medium through the
fluid addition path
while keeping the fluid removal path closed. The conceptual design in Figures
1-4 depicts a
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discontinuous perfusion method in which there is alternating medium removal
and medium
addition. Another embodiment of the concept is a continuous perfusion method
in which
separate tubing is used for filterless medium removal and medium addition.
For discontinuous perfusion, the software controls regulate the removal of a
specific amount of
spent medium and the addition of fresh medium. This approach is typically
independent of the
vessel weight. Preferably, a predefined volume of spent medium is removed
followed by bolus
addition of a volume of fresh medium, according to a pre-defined feeding
schedule. For
example, the feeding schedule could be set to remove 50 mL of spent medium
every 2 hours,
followed by an addition of 50 mL of fresh medium.
Filterless perfusion from stirred tank bioreactor. CT2 human embryonic stem
cells at about
600,000 cells per mL (about 97% viability) were cultured as suspension
aggregates in 250 mL in
the GE prototype stirred tank bioreactor, and agitated at about 40 rpm. In
other experiments,
suspension aggregates were agitated at between 40 and 75 rpm. The aggregate
sizes in the
bioreactor ranged from about 100 um to about 300 um (Figure 12). The gravity
settling chamber
was inclined at about a 45 degree angle and ran along the inner edge of the
bioreactor. The
tubing inner diameter was 3/32". The design of the filterless perfusion
assembly was depicted as
in figures 3 and 4, and enabled both spent medium removal and fresh medium
addition. Fresh
medium addition in the same tubing dispaced any aggregates retained in the
tubing back into the
main bioreactor chamber. About 150 mL/day was exchanged using flow rates of
0.2m1/min to
0.4 mL/min to remove medium from the bioreactor and 8 mL/min for fresh medium
addition.
Medium exchange was not continuous; instead 30 mL was exchanged 5 times per
day. At the
end of the culture, only 13 million cells (predominantly single cells) were
found in the waste at
45% viability demonstrating very little loss of cells in the waste.
Slicer design:
The slicer can be composed of a variety of biocompatible materials. The
material must be
amenable to sterilization, and have mechanical strength that allows it to
withstand the stress
experienced during flow of the cellular samples. The two materials tested for
pluripotent stem
cell aggregate passaging were nickel alloy and silicon. Those skilled in the
art will recognize
that other materials have properties that enable the desired slicer
performance for aggregate
passaging. The slicer is designed with a polygonal grid-like pattern, for
example a square or
hexagonal grid, with spacing between the walls of the grid between 50 microns
and 400
microns. In some experiments, the slicer was coated with a hydrophobic
material to reduce
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shearing and fouling. For the pluripotent stem cell aggregate passaging
experiments described
below, square and hexagonal grids with 100 um spacing were used. The slicer
was mounted in
line with tubing that permitted the sterile flow of aggregates through the
tubing and across the
slicer in a closed system. The slicer may be integrated into the closed system
by various
fastening mechanisms including, but not limited to adhesive, molten polymer
flow, or clamping.
In a preferred method, aggregates are maintained in suspension via a
circulation loop driven by
a pump and an in line conical bag (FIG. 8). Tubing leading to the slicer is
connected to the main
circulation loop, and a fraction of the cell aggregates in the circulation
loop is delivered to the
slicer through a second pump operating at a lower speed than the pump
controlling the
circulation loop. The sliced aggregates can be collected in a separate vessel,
or reintroduced
into the same vessel.
Slicer performance during cellular aggregate passaging:
Aggregates were passed across the slicer in a flow stream consisting of 100 mL
to 1L volumes.
A benefit of the slicer compared to enzymatic passaging is a reduction in
time, labor and
reagents. Successful slicing down to roughly 100um dimension was achieved by
one or more
passes through the slicer in a unidirectional or bidirectional flow. The flow
rate was controlled
to minimize shear. Aggregate slicing performance for size reduction,
maintenance of cell
viability and subsequent expansion were demonstrated on pluripotent stem cell
aggregates
passed through the slicer at flow rates of 15 to 150 mL/min. Those skilled in
the art will
recognize that good performance can also be achieved at other flow rates. The
cell aggregate
concentration used to pass through the slicer was determined to influence the
recovery of sliced
cell aggregates at high viability. Fouling of the slicer can result in reduced
cell recovery and
viability. It was determined that slicing of cell aggregate concentrations
below about 3 x 10^6
cells per mL produced higher viability samples with higher recovery than cell
concentrations
greater than about 3 x 10^6 cells per mL. Fouling was minimized by maintaining
a uniform
suspension of aggregates in the flow stream, for example by using the mixing
device shown in
Figure 8, resulting in higher cell viability and recovery. Sample images of
sliced aggregates are
shown in FIG. 9. The aggregate morphology after slicing includes irregular
shapes, cuboidal
shapes and spherical shapes. For 100 um slicers, at least one dimension of the
aggregate is
reduced to roughly 100um diameter. Sliced aggregates were cultured in mTeSR1
and optionally
1 to 10 uM Y27632 ROCK inhibitor and allowed to expand. Y27632 ROCK inhibitor
was not
required for expansion of sliced pluripotent stem cell aggregates, unlike
enzymatically passaged
pluripotent stem cell aggregates which generally requires agents such as
Y27632 ROCK
inhibitor to maintain the viability of single pluripotent cells. The
morphology of the sliced
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aggregates rapidly reformed a spherical shape under culture conditions. The
expansion rate of
sliced aggregates was similar to the expansion rate of enzymatically passaged
cells (Figures 10
and 11). Aggregates can be passaged by the slicer without PBS wash required
for enzymatic
passaging, therefore passaging by slicing takes less time and less overall
effort than enzymatic
passaging. Those skilled in the art will recognize that the slicer function
and performance is not
dependent upon the Xuri Cellbag bioreactor platform, and is compatible with
other types of
bioreactors including but not limited to other rocking motion, spinning motion
or orbital motion
platforms or stirred taffl( bioreactors.
While only certain features of the invention have been illustrated and
described herein, many
modifications and changes will occur to those skilled in the art. It is,
therefore, to be understood
that the appended claims are intended to cover all such modifications and
changes as fall within
the true spirit of the invention.
