CELL CARRIER, ASSOCIATED METHODS FOR
MAKING CELL CARRIER AND CULTURING
CELLS USING THE SAME
FIELD
[0001] The invention relates to cell carriers, and associated methods for making
and using the cell carriers. More particularly, the invention relates to polymer based
cell carriers for cell growth.
BACKGROUND
[0002] Adherent cells have conventionally been grown on glass surfaces or on
polymer substrates. Surfaces for cell culture are often pre-treated to enhance cell
adhesion and proliferation. A wide variety of static culture vessels is available for
adherent cell culture in the laboratory. While static culture vessels such as T-flasks,
Cell Factory (Nunc) or Cell Stack® (Corning®) units do allow for some scale-up of
adherent cell culture, they become limiting at larger scales as they are labor-intensive,
subject to variability due to manual processing, and limited in volumetric productivity
(e.g. cell yield per volume of incubator space).
[0003] Cell culture using bioreactors has long been practiced as the preferred
scale-up method for cell culture. The use of microcarriers for adherent cell culture is
common in industrial practice, such as in bioprocessing. Microcarrier beads have
been developed to provide increased surface area for cell attachment, and to enable
high-density adherent cell culture on an industrial scale. Typical bioreactor vessels
employ some means of agitation, such as internal impellers, rocking or shaking
mechanisms to suspend the cells and allow mass transfer of nutrients, oxygen and
metabolic waste products. However, agitation can subject cells to high degrees of
flow-induced stress that can damage cells, especially sensitive ones such as certain
mammalian cell lines and primary cells. Flow-induced stresses in bioreactors can
arise due to relative motion of the liquid medium with respect to vessel walls,
impellers, or other vessel components, and due to relative motion of carriers withrespect to the medium. Cells may also be damaged in bioreactor vessels with internal
moving parts if microcarriers with cells collide with one another, or with vessel
components, or agitator components. Therefore, carriers that protect cells from
agitation-induced damage are desired. Certain carrier designs (e.g. macro porous
beads, nonwoven fibrous mats) do offer protection for cells; however, cell
visualization and cell recovery from such carriers is difficult.
[0004] Therefore, there is a need for a carrier for adherent cell growth that protects
cells from the effects of agitation or shear stress and yet facilitates cell expansion,
visualization and release. Efficient cell expansion is particularly important for high
yield industrial scale cell culture processes for adherent cells, including such shear-
sensitive cells as Mesenchymal Stromal Cells (MSCs), which are currently expanded
in static culture vessels. Therefore, the development of cell culture carriers that
facilitate cell attachment, proliferation and release, and that reduce shear forces on
cells is highly desired.
BRIEF DESCRIPTION
[0005] The invention relates to carriers for cell culture and methods of making and
using the carriers. One or more embodiments of the carrier for cell culture comprise
one or more indentations.
[0006] One embodiment of a carrier for growing adherent cells, comprises one or
more outer surfaces; and one or more structured indentations on one or more of the
outer surfaces, wherein the carrier has a length at least about 0.2 mm, a width at least
about 0.2 mm, and a height in a range from about 0.05 mm to 1.2 mm and each of the
structured indentations has a major axis in a range from about 0.1 mm to 0.5 mm,
minor axis in a range from about 0.1 mm to 0.5 mm and depth in a range from about
0.025 mm to about 0.5 mm.
[0007] A carrier for growing adherent cells, comprises one or more outer surfaces;
and a single structured indentation on at least one surface, wherein the single
structured indentation has a length of at least about 1 mm, a width of at least aboutlmm, a height in a range from about 1mm to 10mm, and a wall-thickness of the
carrier in a range from about 0.05 mm to 2 mm.
[0008] A method of culturing adherent cells, comprises providing a carrier
comprising one or more outer surfaces; and one or more structured indentations on
one or more of the outer surfaces, wherein the carrier has a length at least about 0.2
mm, a width at least about 0.2 mm, and a height in a range from about 0.05 mm to
1.2mm and each of the structured indentations has a major axis in a range from about
0 .1mm to 0.5 mm, minor axis in a range from about 0 .1mm to 0.5 mm and depth in a
range from about 0.025 mm to about 0.5 mm, and growing the cells on the carrier.
[0009] One example of a method of making a carrier for growing cells comprises
a) providing a flat polymer film, b) forming on the flat polymer film, on one or more
sides, one or more structured indentations, c) imparting a surface treatment to the film
comprising one or more of corona discharge treatment, gas plasma treatment,
chemical functionalization or coating; and d) cutting the treated polymer film into a
plurality of portions.
[0010] Another example of a method of making a carrier for growing cells,
comprises a) providing one or more flat polymer films, b) forming on the flat polymer
film, on one or more sides, one or more structured indentations, c) cutting the polymer
films into plurality of portions; and d) imparting a treatment to the portions
comprising one or more of corona discharge treatment, gas plasma treatment,
chemical functionalization or coating.
[0011] Another example of a method of making a carrier for growing cells,
comprises a) providing two flat polymer films, b) forming on the two flat polymer
films a plurality of structured indentations on at least one surface of each of the two
films, c) laminating the two polymer films together (with flat surfaces joined to each
other) to form a laminated polymer film with two outwardly facing surfaces
comprising a plurality of the structured indentations, d) cutting the laminated polymer
film into a plurality of portions, and e) imparting a treatment to the portionscomprising one or more of corona discharge treatment, gas plasma treatment,
chemical functionalization or coating.
DRAWINGS
[0012] 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:
[0013] FIG. 1A is an image of a carrier of the invention comprising a plurality of
indentations showing dimensions of the carrier. FIG. IB is an image of the same
carrier showing dimensions of each indentation.
[0014] FIG. 2A is an image of a carrier of the invention comprising one
indentation on one side of the base. FIG. 2B is an image of a carrier of the invention
comprising one indentation each on two opposing sides of the base. FIG. 2C is a
scanning electron microscope (SEM) image of a carrier of the invention comprising a
plurality of indentations on one side of the base. FIG. 2D is an SEM image of a
carrier of the invention comprising a plurality of indentations on both sides of the
base.
[0015] FIG. 3 is a process flow diagram of an example of methods of making
carriers of the invention on a small scale in batch mode.
[0016] FIG. 4A is an SEM image of a carrier of the invention with roughly
rectangular wall cross-section, and FIG. 4B is an image of a carrier of the invention
having an alternate wall shape (roughly triangular wall cross-section).
[0017] FIG. 5A is a lOOx fluorescence microscopy image of hMSC grown on the
carrier of the invention illustrating cell growth on top and bottom of the carrier
surfaces at day 1, day 4, day 7, and day 9 . FIG. 5B is a graph showing growth of
hMSCs on the carriers of the invention in spinner flasks or STRs and comparison to
their growth in TCPS under static conditions.[0018] FIG. 6 is a graph illustrating the growth of hMSCs procured from various
sources on carriers of the invention in spinner flasks and on tissue culture treated plate
surface (TCPS) in static condition as a comparison.
[0019] FIG. 7 is a graph illustrating larger scale culture of hMSC in a spinner
flask.
[0020] FIG. 8A is a series of lOOx optical microscopy images of MDCK cells
grown on the carrier of the invention, illustrating cell growth on top and bottom of the
indentations of the carrier at 24 hours, 96 hours, and 168 hours. FIG. 8B is a graph
representing the luminescence signal for cultured MDCK cells grown on the carriers
of the invention and on flat carriers as a function of time.
[0021] FIG. 9A is a series of lOOx optical microscopy images of MRC-5 cells
grown on the carrier of the invention illustrating cell growth on top and bottom of the
indentations of the carrier at 24 hours, 96 hours, and 168 hours. FIG. 9B is a graph
representing the luminescence of cultured MRC-5 cells grown on the carriers of the
invention and on flat carriers as a function of time.
[0022] FIG. 1OA is a series of images of CHO cells grown on the carriers of the
invention imaged after 1 day, 4 days, 8 days, and post-trypsin treatment. FIG. 10B is
a graph showing CHO cells grown on the carriers of the invention under static
conditions, and in a spinner flask.
[0023] FIG. 11A is a fluorescence microscope image of hMSCs cultured on the
carriers of the invention in spinner flasks, after recovery and adipogenesis. FIG. 1IB
is a fluorescence microscope image of hMSCs used as control.
[0024] FIG. 12 is a graph showing calcium content of hMSCs after osteogenesis in
comparison to control cells.DETAILED DESCRIPTION
[0025] One or more of the embodiments of the invention relate to a carrier for
growing adherent cells, wherein the carrier is suspended in a bioreactor wherein the
carrier is useful for efficient cell adhesion, cell growth, and cell release. High yield of
cells is required in various applications involving cell culture, and this carrier may
meet that requirement.
[0026] To more clearly and concisely describe the subject matter of the claimed
invention, the following definitions are provided for specific terms, which are used in
the following description and the appended claims. Throughout the specification,
exemplification of specific terms should be considered as non-limiting examples.
[0027] The singular forms "a", "an" and "the" include plural referents unless the
context clearly dictates otherwise. Approximating language, as used herein
throughout the specification and claims, may be applied to modify any quantitative
representation that could permissibly vary without resulting in a change in the basic
function to which it is related. Accordingly, a value modified by a term such as
"about" is not to be limited to the precise value specified. In some instances, the
approximating language may correspond to the precision of an instrument for
measuring the value. Where necessary, ranges have been supplied, and those ranges
are inclusive of all sub-ranges there between.
[0028] A "carrier" or "carrier for growing cells", as referred to herein, is a support
for adhering and culturing cells. The carrier may have indentations on it. Suitable
materials of the carrier may include, but are not limited to, polymers, copolymers or
blends of polymers. The carrier may further be coated with a suitable coating
material for effective cell adherence and proliferation.
[0029] A "major axis", as referred to herein, is the longest dimension of each
indentation present on the carrier surface. For example, for a rectangular indentation,
length of the indentation is referred as the 'major axis'. A "minor axis", as referred to
herein, refers to a dimension other than the longest dimension, of each indentation
present on the carrier surface. For example, for a rectangular indentation, width of theindentation is referred as the 'minor axis'. For example, the major axis is same for a
square indentation as the length and width are same, as shown in FIG. IB, 14 and 16
respectively, the major axis is a diameter for a circular indentation as shown in FIG.
2B, 14, major axis is length for a rectangular indentation, and major axis is the major
axis of an elliptical indentation.
[0030] An "aspect ratio", as referred to herein, is a ratio of depth to major axis of
each structured indentation. For example, an aspect ratio for a circular indentation is
a ratio of depth to diameter.
[0031] Embodiments of the carrier in suspension comprise one or more outer
surfaces; wherein one or more of the outer surfaces of the carrier comprise one or
more structured indentations. The invention also comprises methods of making the
carrier, and methods and kits for culturing cells using the carriers for cell growth.
[0032] The carrier for growing adherent cells, comprises one or more outer
surfaces; and one or more structured indentations in one or more of the outer surfaces,
wherein the carrier 2, as shown in FIG. 1A, has a length 4 at least about 0.2 mm, a
width 6 at least about 0.2 mm, and a height 8 in a range from about 0.05 mm to 1.2
mm. In some embodiments, the carrier has a length 4 in a range from about 0.2 mm
to 5 mm, a width 6 in a range from about 0.2 mm to 5mm, and a height 8 in a range
from about 0.05 mm to 1.2 mm. In some embodiments, the carrier has a width and
length from about 0.2 to 25 mm. In some embodiments, the wall-thickness 10 of the
carrier is in a range from about 0.05 mm to 2 mm.
[0033] Embodiments of the structured indentations, as shown in FIG. IB, comprise
a depth 12, a major axis 14, and a minor axis 16, wherein the major axis 14 of an
indentation is in a range from about 0 .1mm to 0.5 mm, the minor axis 16 is in a range
from about 0.1 mm to 0.5 mm, and the depth 12 is in a range from about 0.025 mm to
about 0.5 mm. The wall-thickness 10 is in a range from about 0.05 mm to 2 mm. As
used herein the term, 'depth' of an indentation refers to the depth of the inner wall of
each indentation. As used herein, the term 'wall-thickness' refers to a thickness of a
single wall for a carrier with single indentation, or thickness of each of the multiplewalls for the carrier with a plurality of the structured indentations as shown in FIG.
IB. Each of the structured indentation has an aspect ratio in a range from about 0 .1 to
about 1.5.
[0034] In one embodiment, the carrier may comprise one indentation on at least
one surface of the carrier as shown in FIG. A. In this embodiment, the carrier is a
'cup' like structure on one outer surface of the base with a continuous wall
surrounding the base of the carrier. In an alternate embodiment, the carrier may
comprise one indentation on each of the surfaces of the carrier as shown in FIG. 2B.
In this embodiment, the carrier has two 'cup' like structures on opposing outer
surfaces of the base with a continuous wall surrounding the cups. This carrier may be
useful for specific cell culture conditions or for specific cell-types. The single carrier
(FIG 2A and 2B) has a length in a range from about 0.1 mm to 5 mm, a width in a
range from about 0.1 to 5 mm, and a height 8 in a range from about 1mm to 10mm,
and a wall-thickness 10 of the carrier in a range from about 0.05 mm to 2 mm. In
case of a single 'cup' (FIG. 2A) or two 'cups' on opposing sides of the base (FIG.
2B), has a length that is same as the major axis 14 as shown in FIG. 2A and 2B, a
width that is same as the minor axis 16, and the cup has a depth 12, as shown in FIG.
2A.
[0035] In some embodiments, the carrier comprises at least one surface for
growing adherent cells, wherein more than one structured indentation is present on the
surface, for example, the carrier has a plurality of structured indentations on one outer
surface of the base, as shown an SEM image in FIG. 2C. The carrier, in one
embodiment, comprises at least two outer surfaces. In this embodiment, more than
one structured indentation is formed on each of the outer surfaces, such as 18 and 20
are the structured indentations on the upper and lower surface respectively, as shown
in FIG. 2D. In this embodiment, the carrier has a plurality of indentations on
opposing outer surfaces of the base (FIG 2D).
[0036] In some embodiments, the carrier has a substantially planar disc-like
structure. As used herein, 'substantially planar disc', refers to a disc, which provides
a planar surface area for growing cells. The shape of the carrier may be polygonal. Inone or more embodiments, the shape of the carrier may vary, for example, the carrier
may have an overall perimeter that is circular, elliptical, triangular, rectangular,
square, pentagonal, or hexagonal shape.
[0037] The disc like-structure of the carrier may provide higher surface area per
unit volume for culturing cells, relative to, e.g. spherical structures. The carrier size
(both length, and width of 0.2 to 5 mm) may allow about 10 - 100X fold of hMSC
expansion per passage. Efficient separation of released (e.g. trypsinized) cells from
the carriers is facilitated due to the significant size difference between the cells (-15
micron) and the carriers (larger than 0.2 mm). Released cells may be separated from
the carriers via simple filtration, or separation of the supernatant after allowing the
carriers to settle.
[0038] The structured indentation has a wall that protrudes normal to the outer
surface of the carrier, as shown in FIGs. 1A, IB, 2A, and 2B. The wall height is
chosen to balance the various requirements of the carrier, for example, a lower wall
(i.e. shallow indentation) allows higher packing density of carriers per unit volume,
and therefore can provide higher cell yield per unit volume of reactor. Moreover,
transport of oxygen, nutrients and metabolic waste to/from the cells is facilitated at
lower wall height (i.e. shallower indentations). However, a higher wall (i.e. deeper
indentation) can offer higher degrees of protection from hydrodynamic forces arising
due to agitation inside the bioreactor. Further, a higher wall or deeper indentation can
provide a microenvironment that prevents dilution of any cell-secreted molecules.
This may be advantageous if cell-cell signaling or autocrine factors are a desired part
of the cell culture or processing operations. The desired range of the height of the
wall projected above the plane of the carrier is therefore optimized with these factors
in mind, in a range from 0.05 mm to 1.2 mm; in some embodiments from about 0.05
mm to about 0.5mm, or in some embodiments, from about 0.08mm to about 0.2mm.
[0039] The carriers are in suspension inside a bioreactor, comprising a fluid having
a convective motion that generates sufficient transport of nutrients and oxygen to
cells. The cells adhere to the surface of the structured indentations having a flat or
curved wall of sufficient height such that the effect of fluid-induced hydrodynamicstress on the cells is minimized. The carrier comprises an optimum depth of
indentations, balancing the needs of the cells providing access to nutrients and
metabolites, while protecting the cells from exposure to hydrodynamic shear
generated by fluid motion.
[0040] The structured indentations also form relief features on the carrier surface.
The relief feature may be present on one or more surfaces of the carriers, which
prevents the carriers from sticking to each other. Carrier sticking or clumping may be
issue with certain types of flat or smooth carriers. The relief features on the carrier
also serve to prevent the carriers from sticking to the inner walls of the reactor or
culture vessel, which facilitates cleaning the reactors / culture vessels between batches
of cell culture.
[0041] A cross sectional profile of each indentation may have, as non-limiting
examples, a polygonal, a circular, or an elliptical shape. Each of the polygonal
indentations may have, as non-limiting examples, a triangular, rectangular, square,
pentagonal or hexagonal shape. The dimension of the major axis and minor axis of
the indentations may be the same or different.
[0042] The carrier may be made of glass, polymer, ceramic, metal or a
combination thereof. In one embodiment, the carrier is made of a polymer or a
copolymer or a blend of polymers. The polymers may comprise, but are not limited
to synthetic and natural polymers such as, polyester including polyethylene
terephthalate (PET), polystyrene, polycarbonate, polyamide, polyurethane, olefin
polymer, dextran, silicone, or polyacrylate, or copolymer or blend of polymers
thereof. In one specific embodiment, the carrier is made of polystyrene.
[0043] The polymer may be transparent, which allows cell observation under an
optical microscope. In certain embodiments, the carrier has a substantially planar disc
shape, which facilitates cell visualization by preventing lensing effects. Refraction of
light can be a hindrance to visualization of cells on spherical carriers of certain
refractive index. Cell visualization is useful, for example, for culturing and
monitoring cells during vaccine production or stem cell expansion. In someembodiments, the polymer and surface treatment is substantially free of components
of animal origin. This is especially beneficial in therapeutic applications, e.g. in the
production of cells for cellular therapies. The polymer may be rigid at room
temperature / cell culture temperature, non-porous and may have non-swelling
properties in water, PBS or growth medium. The rigid, non-swelling, non-porous
properties of the polymer can facilitate cell release, for example, when using standard
trypsinization protocols.
[0044] The polymer-based carrier surfaces are optionally modified with functional
groups or coatings to enable better cell attachment and growth. In some
embodiments, a surface treatment is imparted to the embossed polymer film
comprising one or more of corona discharge treatment, gas plasma treatment,
chemical functionalization or coating. A variety of biomolecules may also be used to
modify surfaces of the carriers to enhance cell attachment. Non-limiting examples of
the biomolecules include collagen, fibronectin, vitronectin and laminin. In one
embodiment, the surfaces are modified with recombinant fibronectin to enhance
surface cytophilicity for better attachment of the cells. The surface modification may
result in a change, for example, in hydrophobicity or hydrophilicity.
[0045] In some embodiments, the surfaces are treated with corona discharge to
modify one or more surface properties of the carriers. In corona discharge treatment,
a current develops from an electrode with a high potential in a neutral gas, such as air.
Ionization of the gas generates a layer of plasma around the electrode. The ions
generated eventually pass the charge to nearby areas of lower potential, or recombine
to form neutral gas molecules. Surfaces of organic films such as polystyrene,
polyesters and others may be oxidized when exposed for a short time to the reactive
air plasma by corona discharge surface treatment. Corona discharge treatment can
increase the oxygen content on the polymer surface and improve the film wettability
by water.
[0046] The surface modification may be achieved via plasma treatment. In some
embodiments, the surface is treated with of plasma to modify the surface properties of
the carrier. Plasma treatment is carried out in a plasma reactor, which is a vacuumvessel with a gas at low pressure, typically 10 to 1000 mTorr. When a high frequency
electric field is generated in the reactor, a plasma is formed containing reactive
species like ions, free radicals and vacuum-UV photons. These species react with the
polymer surface and cause a chemical modification with various properties depending
on the nature of the gas and the plasma parameters. Gases such as oxygen, ammonia
and argon are typically used for modification of the surfaces and adhesion
improvement on polymer surfaces. In one embodiment, the polymer surface is
modified by oxygen-plasma treatment to increase the cytophilicity of the surface. The
surface functionality may also be altered via wet chemical methods such as oxidation
treatments using perchloric acid or permanganate or partial hydrolysis using strong
acids or bases.
[0047] A coating may also be applied on each of the surfaces to modify the surface
property of the carriers, e.g. hydrophobicity, hydrophilicity, or wettabilty. One index
of hydrophobicity/hydrophilicity is contact angle of a water droplet on the surface.
Contact angle can be measured by techniques well-known in the art. The water
contact angle for the coated carrier surface may be in a range from about 10°C to
about 90°C, or in some embodiments the water contact angle is from 30°C to 70°C.
The carrier surface may be modified, for example, to enhance cell release as well as
cell attachment. The coating may be made, for example, of a thermoresponsive
polymer, pH responsive polymer, or combination thereof. Thermoresponsive
polymers may include, but are not limited to, poly(N-isopropylacrylamide)
(PNIPAM), poly(di(ethyleneglycol)methylether methacrylate) (PDEGMA). pH
responsive polymers may include, but are not limited to, copolymers of acrylic acid,
dimethylaminoethylacrylate, and hydroxyethylacrylate. The coating may comprise
one or more layers. In some embodiments, where the coating comprises multiple
layers, the layers may be homogeneous or heterogeneous. For one example, one layer
may be made of thermoresponsive polymer, and another layer may be made of pH
responsive polymer. Thermoresponsive or pH responsive polymer coatings on the
surface can facilitate easy release of cultured cells from the carrier surface.
[0048] An example of a method of making a carrier for growing cells, comprises
providing a plurality of flat films and laminating the flat films to form a solid support.The solid support is subjected, to a method such as embossing, casting
thermoforming, or injection molding to form structured indentations. In some
embodiments, the solid support is embossed to form structured indentations and make
an embossed solid support, which is further treated with a plasma to form a plasma
treated embossed solid support, followed by cutting or dicing the plasma treated
embossed solid support to a plurality of portions or pieces to form a plurality of
carriers. In one example, the embossing of the solid support is performed using a
mold.
[0049] In one example, a process for making a carrier for growing cells is
generally illustrated in FIG. 3 . The process comprises two alternate methods, method
(1) and method (2). The method (1) 22 comprises the steps of preparing embossing
mold 24, and cutting a film from a roll 26, followed by embossing the film 28. The
embossed film is subjected to a plasma treatment 30 to form a plasma treated
embossed film. The embossed film is then optionally plasma treated on the other side
of the film for better uniformity of treatment 32. The plasma treated embossed film is
then diced or otherwise discretized into a plurality of carriers 34.
[0050] The method (2) also may comprise a method 36 comprising the steps of
preparing embossed mold 38, and cutting a film from a roll 40, followed by
embossing the film 42. The embossed film is cut or diced or otherwise discretized to
generate embossed pieces 44, which can then be sieved to a narrow size distribution
46. The carriers are then washed with a wash fluid such as water or a mixture of
water and isopropyl alcohol to remove fine particles, followed by drying 48. The
carriers are then subjected to a plasma treatment 50 in bulk accompanied by mixing to
ensure uniformity of surface treatment 52 to form plasma treated embossed carriers.
The methods (1) and (2) (as described above 22 and 36) can be modified to produce
carriers on large scale using roll-to-roll operations for some or all of the steps of
manufacturing. For example, the embossing or structure generation step can be
scaled-up into a roll-to-roll operation, and the plasma treatment operation can be done
in bulk in drum-style treaters, and the discretization can be done via roll-to-roll or
sheet-fed die cutting operations.[0051] Another example of a method for making the carriers comprises initially
providing two flat polymer films. The method further comprises forming one or more
structured indentations on the two flat polymer films individually on at least one
surface of each of the two films, such as by embossing to make two embossed
polymer films (embossed on one side each), and laminating the two embossed
polymer films together, back to back, to form a composite laminated embossed
polymer film, so that the outwardly facing surfaces comprise one or more of the
structured indentations. The laminated embossed polymer film may then be diced to
form a plurality of untreated carriers. The untreated carriers are then treated with a
plasma treatment to form a plurality of plasma treated carriers. To create structured
indentations, the flat polymer films may be alternatively be subjected to casting
thermoforming, or injection molding, or a bulk polymer may be made into a solution
and cast on a mold to form a film with the structured indentations.
[0052] The structured indentations may be formed in the carrier by one or more of
the following methods. In one example, a textured roll is used to make the structured
indentations on a heated polymer film in a roll-to-roll process. In another example, a
flat mold is prepared by cutting or machining the negative of the desired indentations
into a metal block. The metal block then may be used as-is or replicated first as a
positive and then as a negative, using, for example, a polymer casting process. The
negative mold can then be used in a batch-stamping or hot embossing process to
emboss the pattern into a polymer film. In another example, a mold thus formed can
be used in a solvent-casting process to make the polymer film with the structured
indentations. A polymer solution can be coated on to the mold or textured roll, and
dried and / or cured. The dried / cured film then peeled off to yield a film with the
desired structured indentations. Alternate methods such as thermoforming or
injection molding may also be used.
[0053] A cell culture system of the invention uses one or more of the carriers for
growing cells. In one embodiment, the cell culture system is a bioreactor, more
specifically, an agitated bioreactor. As mentioned herein, a bioreactor may refer to
any device or system that supports cell growth. In one aspect, a bioreactor may refer
to a device or a system for growing cells or tissues in the context of cell culture ortissue engineering. The bioreactor may employ agitation, generated by an internal
impeller or paddle, or via externally rocking, rolling or shaking the culture vessel, or
via bellows-induced motion of fluid. The bioreactor may, for example, be a reactor
with rocking or rolling motion, such asWave Bioreactor™, a stirred tank bioreactor, a
fluidized bed bioreactor, fixed bed bioreactor, a roller bottle or airlift bioreactor.
[0054] The Wave Bioreactor™ comprises a rocking platform supporting a vessel
containing a culture fluid, wherein the culture fluid comprises cells in a culture media.
The rocking motion of the platform induces mixing and mass transport in the culture
fluid. A stirred tank bioreactor generally comprises an impeller system and optionally
a sparging system to mix and aerate the culture. An airlift reactor relies on rising gas
bubbles to mix and aerate the culture medium. Hydrodynamic factors such as mass
transfer, mixing efficiency, and shear stress experienced by cells can be different in
the different types of bioreactors. In addition, the cell growth rate and quality of cells
may be influenced by operational differences between reactor types.
[0055] An example of a method of culturing adherent cells comprises providing
one or more carriers for growing cells in a bioreactor, adding culture medium, adding
an inoculum of cells to the carriers, allowing attachment of cells to the carriers,
suspending the carriers in the medium continuously or intermittently, and allowing the
cells to grow on the carriers. Cells may be grown in a culture flask prior to addition
to the carriers. Cells may be grown on the carriers after extraction, for example, from
blood, bone marrow or tissue section. In some other embodiments, the carriers may
be introduced into a spinner flask, a stacked culture flask, a stirred tank reactor, a
Wave bioreactor™ or any other in-vitro cell culture system.
[0056] Cultured cells may be detached or released from the carriers by a variety of
methods. The cells may be released, for example, by using a mechanical method, an
enzyme, a thermoresponsive polymer, a pH responsive polymer or a combination
thereof. The cell release by mechanical method includes cell scraping. The cells may
also be released by treating with proteolytic enzymes, such as trypsin. One non-
enzymatic method uses calcium chelators, such as EDTA. Other non-enzymatic
methods include, but are not limited to, physical methods that use ultrasound, whichgenerates bubbles that facilitate cell detachment. Cultured cells from carriers
comprising thermoresponsive polymers, such as poly-N-isopropylacrylamide
(PNIPAAm) may be released by cooling the carrier to a temperature below LCST.
[0057] The carriers of the invention may be used for growing various adherent
cells such as primary cells, stem cells and cell lines. In some embodiments, the
adherent cells are shear-sensitive cells such as hMSCs. The cells may be derived
from human tissue, for example, from adipose tissue, bone marrow or cord blood.
Culture and release of multipotent and pluripotent cells with high purity, high
efficiency and high yield are a current research and clinical need.
[0058] The carriers can be used in combination with a bioreactor or culture vessel,
to provide or enhance surface area for the attachment and growth of anchorage-
dependent cells. Some embodiments of the kit of the invention for culturing cells
comprise a disposable housing or vessel pre-loaded with one or more carriers. In one
embodiment, the carriers and the disposable housing or vessel may be provided
separately. In one embodiment, the housing may be reusable. The housing may be,
for example, a bag, a flask, a tank, a tube, a petridish or a bottle. The kit may further
comprise culture media suitable for cell growth. The kit may comprise cells in a
frozen condition and may further comprise a protocol for using the carriers.
EXAMPLE 1. Fabrication o f carrierfor growing cells
[0059] Method o f making a pattern master- pattern-master was prepared by cutting
grooves in a flat aluminum block using a dicing saw, which is outfitted with a resin-
bonded diamond blade. A set of parallel grooves (the term being interchangeably
used with 'indentations') was first cut in one direction, then a second set of parallel
grooves was cut perpendicular to the first set of grooves. Finally, an effort was made
to remove burrs that had formed in the first set of grooves during the cutting process.
After the grooves were completed, the aluminum block was cleaned to remove any
burrs on its surface. The pattern master determined the pattern geometry of the
embossed carriers.[0060] Formation o f first generation moldfrom the pattern master- first-generation
mold was then made from the pattern-master using a fiuorosilicone rubber, FSL 7661
(purchased from Momentive Performance Materials, Waterford, NY). To produce the
first-generation mold, the two part fiuorosilicone compound was mixed at a 1:1 ratio
according to directions from the manufacturer, using a Hauschild SpeedMixer. The
pattern-master was placed in a hollowed-out Teflon block and uncured fiuorosilicone
was applied, in excess, on the surface of the pattern master. A chrome-plated steel
plate was placed on top of the fiuorosilicone, and the fiuorosilicone was cured in a
heated hydraulic press at 4000 lb force and 170°C for 30 minutes. After cooling to
room temperature, the cured fiuorosilicone rubber-based first-generation mold was
removed from the pattern-master and cured overnight at 200°C in air.
[0061] Formation o f second generation moldfrom the pattern master- Two second-
generation molds were then prepared using a silicone rubber-molding compound,
RTV 664 (purchased from Momentive Performance Materials, Waterford, NY) from
the first-generation mold as mentioned above. The silicone compound was mixed at a
10:1 ratio according to directions from the manufacturer, using a Hauschild
SpeedMixer. The first-generation mold was placed inside a steel frame with the
patterned surface up and the silicone compound was dispensed, in excess, on the first-
generation mold. A flat stainless steel plate was placed on top of the silicone and the
silicone was cured in a heated hydraulic press at 1000 lb force and 120°C for 30
minutes. After cooling to room temperature, the cured silicone rubber second-
generation mold was removed from the fiuorosilicone first-generation mold.
[0062] Method o f making embossed polystyrene sheets- Multiple sheets of biaxially
oriented polystyrene film (Trycite 1003U, Dow Chemical Company) were placed in
between two second-generation molds with patterns facing in. The number of sheets
of film was chosen so that the volume of polystyrene was sufficient to fill the pattern
in the second-generation molds and still leave a small amount of polystyrene
separating the molds. The films were then embossed (28, FIG. 3) in a heated
hydraulic press with 1000 lb force and a temperature cycle that ramped up to 150°C
for 5 minutes and then cooled to below 60°C. The embossing process fused the
multiple sheets of film into a single monolithic structure that replicated the texture ofthe molds and pattern-master on both sides. The embossed polystyrene film was
removed from the molds after cooling to room temperature.
[0063] Chemical treatment o f the embossed film surface- To make the embossed
polystyrene film compatible with cell growth, the film was 0 2 plasma treated (30,
FIG. 3) using a Plasma Therm SLR vacuum plasma reactor as mentioned in FIG. 3 .
Plasma treatment was performed on each side of the embossed film for 1 minute at
100 mtorr pressure using 100 seem (Standard Cubic Centimeters per Minute) 0 2 flow
and 100 W forward radio frequency (RF) power in reactive ion etching (RIE) mode.
[0064] Dicing o f the film to generate carrier- Carriers for cell culture were prepared
from the plasma-treated embossed sheets either by manually cutting the film into
5mm x 5 mm pieces or 2 mm x 2 mm pieces, or by discretizing (44) and then sieving
(46) to select a particular size range, or by punching circular discs of the desired size.
[0065] Variants o f the carrierfabrication process- In some instances, a ceramic block
was used in place of the aluminum block to make the pattern-master. A pattern-
master was prepared by cutting grooves in a flat alumina block (99.6% alumina, fired,
20-25 µ polish from Acumet) using a dicing saw outfitted with a resin-bonded
diamond blade. A set of parallel grooves was first cut in one direction, and then a
second set of parallel grooves was cut perpendicular to the first set of grooves. The
geometry of the pattern master determined the pattern geometry of the eventual
embossed carriers. When the ceramic block was used, the first-generation mold was
prepared slightly differently. Instead of the Teflon block, a steel frame was used to
hold the ceramic pattern-master. The curing was performed at a higher temperature,
170°C for 15 minutes and then 200°C for 15 minutes. The rest of the procedure
remained the same as described above.
[0066] In some examples, the fluorosilicone first-generation molds were replaced
with RTV silicone first-generation molds. The procedure was modified as described
below. A first-generation mold was then made from the pattern-master using a
silicone rubber-molding compound, RTV 664 from Momentive Performance
Materials. To produce the first-generation mold, the silicone compound was mixed ata 10:1 ratio according to directions from the manufacturer, using a Hauschild
SpeedMixer. The pattern-master was placed in a hollowed-out Teflon block and
uncured silicone compound was applied, in excess, across the surface of the pattern
master. A chrome-plated steel plate was placed on top of the silicone, and the silicone
was cured in a heated hydraulic press at 1000 lb force and 120°C for 30 minutes.
After cooling to room temperature, the cured silicone rubber first-generation mold
was removed from the pattern-master. The first generation mold was coated with
(tridecafluoro-l,l,2,2-tetrahydrooctyl) trichlorosilane by vacuum deposition at 750
mtorr for 45 minutes prior to making any second-generation molds. Cell carriers of
different designs were made using the above fabrication procedures. The embossed
cell carriers of the invention may include carriers with alternate wall shape. For
example, carriers with rectangular shaped walls were made as shown in FIG. 4A, and
carrier with triangular shaped wall in cross-section were made, as shown in FIG. 4B.
EXAMPLE 2 Cell culture on the carrier and subsequent cell release
[0067] Cell carriers- carriers used for the following examples had a length and
width of 5mm, and a height of about 0.5mm. The carriers comprised a plurality of
structured indentations on each of the two outer surfaces. Each of the structured
indentations had a major axis and minor axis of 0.45mm each and a depth of 0.2mm.
[0068] Cell culture- carriers for cell culture were used to culture and release CHO
(Chinese Hamster Ovary, ATCC), MDCK (Madin-Darby Canine Kidney Cells,
ATCC), MRC-5 (human lung fibroblast, ATCC), and hMSCs (human mesenchymal
stem cells) cells. These cells were routinely cultured on polystyrene surfaces using
the following media: F-12K (EMEM, Invitrogen) and 10% FBS (fetal bovine serum);
and Eagle's minimum essential medium (EMEM, Invitrogen) and 10% FBS
supplemented with lOOU/mL penicillin-streptomycin (P/S, Invitrogen). Culture
methods were performed at 37°C, in a humidified atmosphere of 5% C0 2. Cells were
passaged by performing the steps of briefly rinsing the cell layer with PBS (phosphate
buffered saline) followed by addition of 3.0 ml of 0.25% (w/v) Trypsin and 0.53 mM
EDTA solution to the culture flask and observing the cells in an inverted microscope
until the cell layer is dispersed. Subsequently, 7 ml of complete growth medium wasadded to the cells and the media, and the cells were mixed by gently pipetting several
times. Appropriate aliquots of the cell suspension were transferred to new culture
vessels with fresh media.
[0069] Cells used in the following experiments were freshly pre-cultured and
harvested from cell culture flasks after growing in incubators at 37°C in a humidified,
5% C0 2 atmosphere. For static cell culture testing, pre-cultured cells were seeded at
2000 cells/cm2 in 24-well plates with lmL growth medium per well. Tissue culture
treated plates (TCPS surface, Nunc) and or non-adherent plates (from Corning®)
were used as control, wherein in the non-adherent plates (Corning®), disc-shaped
embossed polystyrene carriers of the invention were inserted so as to fit snugly into
the well. For cells grown under dynamic conditions on the carriers in stirred tank
reactors (STR), pre-cultured cells were also seeded at 2000 cells/cm2 in the carriers in
125mL disposable spinner flasks (Corning®). Cells and carriers were agitated at 60
rpm on spinner bases connected to timers to regulate the agitation cycle. Cells were
subjected to agitation continuously or intermittently. In intermittent conditions, for
example, the agitator was turned on for 1min, and off for 45 min per cycle.
[0070] Cell release b y trypsin- Cells were washed with PBS and harvested by trypsin-
EDTA (Invitrogen, -10 minutes), when the cells were about 80-90% confluent. The
trypsin was neutralized by addition of at least one volume of culture medium
containing 10% serum, after the cells were released from the growth surface. After
harvesting of the cells, cell number and cell viability were measured using a
NucleoCounter® automated cell counter (from ChemoMetec).
Qualitative and Quantitative estimation o f cell growth
[0071] Cell staining and imaging- Samples for imaging were fixed at room
temperature in 4% paraformaldehyde (PFA), which is freshly diluted in PBS from a
16% stock, stored in presence of argon in an amber glass vial. Once fixed, samples
were stored at 4°C until they were stained and imaged. Fixed cells were stained with
Hoechst 33342 dye (from Invitrogen) to highlight the nuclei and with phalloidin-
Alexa-568 (from Invitrogen) to visualize the cytoskeleton (actin) afterpermeabilization with 0.1% Triton X-100 detergent (Sigma). The stained cells were
imaged with an Nikon Eclipse TE2000-U inverted fluorescence microscope, wherein
the microscope was fitted with appropriate filter cubes and light source for the
fluorophores being used.
[0072] Cell viability measurement b y CellTiter-Glo®- Cell growth and morphology
was assessed at intervals by taking samples of carriers and either measuring total ATP
content or fixing and staining for fluorescence microscopy. Cell growth was assayed
by CellTiter-Glo® luminescent cell viability assay reagent from Promega, which
determines the number of viable cells in culture based on quantitation of the ATP
present, which signals the presence of metabolically active cells. The process
involves adding a single reagent (CellTiter-Glo®) directly to cells cultured in serum-
supplemented medium. The homogeneous reagent results in cell lysis and generation
of luminescent signal proportional to the amount of ATP present. The amount of
ATP is directly proportional to the number of cells present in the culture. The assay
relies on thermostable luciferase, which generates a stable 'glow type' luminescent
signal resulting from oxyluciferin catalysed by luciferase in presence of Mg+2, ATP,
and molecular oxygen. After 10 minutes of the cell lysis, 200µΙ aliquots of cell
lysate were transferred to an opaque 96-well plate, mixed gently and read in a
SpectraMax® luminescence microplate reader from Molecular devices to generate
readings for cell viability. Luminescence readings from this assay are proportional to
the number of viable cells present in the sample and so can be used to monitor the
progress of cell growth.
EXAMPLE 3-Characterization o f Human mesenchymal stromal cell (hMSC) growth
[0073] hMSCs- The hMSCs used for this experiment were purchased from Lonza
(Part number PT-2501) (Basel, Switzerland). The hMSCs were grown on the carrier
(interchangeably used herein as 'embossed carrier') in stirred tank reactors (STR).
FIG. 5A shows the growth of cells on day 1 (56, 58), day 4 (60, 62), day 7 (64, 66),
and day 9 (68, 70), clearly indicating an increase in cell count over time in culture.
Cells were observed to grow on both the top and the bottom of the indentations of the
embossed carrier. FIG. 5A further illustrates higher cell growth in the bottoms of theindentations than the tops. Cells were grown on embossed cell carriers in two
different types of spinner flasks, one from Corning, and one from Wheaton (Magna-
Flex®). Cells were grown on tissue culture polystyrene (TCPS) in static medium as a
positive control.
EXAMPLE 4- Quantitative estimation o f hMSC growth
[0074] The cell growth was monitored via CellTiter-Glo® measurements and
qualitatively via imaging. The growth rate of hMSCs on carriers in STR is
comparable with that on TCPS, as shown in FIG. 5B. Luminescence of the cells
represents cell number, and the luminescence increases with increasing cell count
over time (FIG. 5B). Therefore, robust growth of hMSC on the carriers of the
invention is demonstrated in FIG 5B for 11 days with a doubling time of -50 hours.
EXAMPLE 5- Characterization o f hMSC growthfrom various sources
[0075] To demonstrate that the carriers support the growth of hMSCs from a variety
of sources / donors, hMSCs were procured from 2 additional companies - (Poietics®
Lonza cells have been referenced in examples 3 and 4 Promocell (part number C-
12974) and Millipore (part number SCR108). FIG. 6 demonstrates robust hMSC cell
growth, wherein the cells were purchased from Promocell and Millipore, and cultured
in STR, and the growth is comparable to the cells grown on TCPS under static
conditions.
EXAMPLE 6- Characterization o f hMSC growth in larger scale culture
[0076] The hMSC culture was scaled up in 1L spinner flask with 500 ml of media.
FIG. 7 shows robust hMSC (Lonza, part number PT-2501) growth for 12 days with a
doubling time of about 85 hours.
EXAMPLE 8- Characterization o f Madin-Darby Canine Kidney (MDCK) Cell
growth
[0077] Madin-Darby Canine Kidney Cells (MDCK)- MDCK cell growth on carrier
comprising plurality of indentations (or 'embossed carrier') is shown in FIGs. 8A and8B. FIG. 8A shows MDCK cells growing at the top (72, 74, 76) and on the bottom
(78, 80, 82) of the indentations in the carriers. Cell counts increases with time from
24 hours to 168 hours. Quantitative measurement of MDCK growth is presented in
FIG.8B via CellTiter Glo® luminescence assay. As a comparative example, MDCK
cells were grown on flat polystyrene carriers (with no indentations) in an STR with
intermittent stirring. As controls, cell counts in static well plate (TCPS) and on the flat
carriers in static conditions are also shown in FIG. 8B. MDCK cell growth on
embossed carriers (marked as 'Embossed - STR' in FIG. 8B) was significantly higher
than that on flat polystyrene carriers (marked as 'Flat carrier-STR' in FIG. 8B), as
shown in FIG. 8B.
EXAMPLE 9- Characterization o f Human lungfibroblast cell growth
[0078] Human lung fibroblast cells (MRC-5)- Similar experiments were performed
with MRC-5 cells (FIG. 9A) as mentioned above. In FIG. 9A, the qualitative
measurement of MRC-5 cell growth at the top (84, 86, 88) and the bottom (90, 92, 94)
of the indentations of the carriers is presented. Cell counts increase with time, from
24 (84 and 90) hours to 168 hours (88 and 94). Quantitative measurement of cell
growth is presented in FIG.9B as CellTiterGlo® Luminescence (as described in above
example). As a comparative example, MRC-5 cells were grown on flat polystyrene
carriers (with no indentations) in an STR with intermittent stirring. As controls, cell
counts in static well plate (TCPS) and on the flat carriers in static conditions are also
shown in FIG. 9B. Cell growth on embossed cell carriers of the invention is greater
than that on flat polystyrene cell carriers, as shown in FIG. 9B.
EXAMPLE 10- Characterization o f Chinese Hamster Ovary (CHO) cell growth
[0079] Chinese Hamster Ovary (CHO)- Similar cell growth experiments were
performed using CHO cells on the carrier of the invention (FIG. 10A and 10B). Cells
were imaged after 1 day (96), 4 days (98), 8 days (100), and post-trypsin treatment
(102) as shown in FIG.10A. 0.2 X 106 cells were seeded on day 0 (zero). Cell counts
increased with time, and cell recovery after trypsin treatment was about 13 X 106 as
shown in FIG. 10A. The efficient release of CHO cells from the embossed carrier bystandard method of trypsinization is also shown in FIG. 10A. The embossed carrier
of the invention showed better cell release, compared to other commercially available
carriers (data not shown). CHO cell growth on embossed cell carriers in a spinner
flask, in a static well plate, and also on TCPS in static well plates is shown
quantitatively in FIG. 10B. The cell growth in these three different conditions were
comparable to each other (FIG. 10B), and CHO cells showed robust cell growth for 8
days with a doubling time of about 1 hours, with about 67 fold expansion as shown
in FIG. 10B.
Phenotypic and FunctionalAssays
[0080] In order to demonstrate that hMSCs grown on the carriers of the invention
maintain their phenotype after expansion in a bioreactor, phenotypic characterization
was done via adipogenic and osteogenic differentiation assays. Surface marker
profile was also characterized via flow cytomtery.
EXAMPLE 11- Adipogenic DifferentiationAssays
[0081] Adipogenesis-In order to demonstrate adipogenesis, MSCs were seeded at
2000-3 000/well in a 96-well plate with growth medium same as used above to grow
cells, Lonza MSCGM (part number PT-3001), with 3-6 wells per sample, and were
grown until the cells became 100% confluent. The growth medium was replaced with
adipogenic induction medium (Lonza, part number PT-3004) and incubated at 37°C
for 3-4 days. After that, the adipogenic induction medium was replaced with
maintenance or growth medium (Lonza) and incubated at 37°C for 3-4 days. These
two steps were repeated two more times. Maintenance medium was added and
replaced the medium on Day 3, and incubated for 7 days. The resulting fat cells were
stained with Nile Red. The MSCs were maintained in maintenance medium as a
negative control. The medium was removed and 4% paraformaldehyde (PFA) was
added to fix cells for 5 minutes. After 5 minutes, the PFA was removed and Nile Red
and Hoechst staining buffer were added, followed by incubation for 30-60mins.
Buffers were prepared using Nile Red stock at the ratio of 1:1000 in DMSO (lmg/ml
in DMSO), Hoechst buffer solution at the ratio of 1:1000, and 0 .1% of Triton X100 inphosphate buffered saline (PBS). The cells were rinsed with PBS twice by adding 0.2
ml PBS per well. The cells were imaged with Nikon Eclipse TE2000-U inverted
fluorescence microscope using DAPI and Cy3 stains. FIG. 11A shows red vacuoles
indicating accumulation of lipid, with respect to the control MSCs as shown in FIG.
11B. The accumulation of lipid in the vacuoles indicates that cells grown on these
carriers in bioreactor conditions retain the capability to differentiate into adipocytes
which is a property expected of MSCs. hMSCs from Lonza were used in this
experiment. Cells were grown as described in example 3 .
EXAMPLE 12- Osteogenic Differentiation Assays
[0082] Osteogenesis-The MSCs were seeded in 24-well tissue culture plate at a
concentration of 20-3Ok/well at three sets, and cells were cultured in growth medium
at 37°C overnight. The growth medium was replaced with osteogenesis induction
medium and the cells were fed with the same medium after every 3 days. This
procedure was continued for 2-3 weeks. The morphology change from spindle to
cubical shape was noted. The calcium content was measured by following method.
The cells were washed in Ca2+-free PBS, and ΙΟΟµΙ of 0.5M HCl added to dissolve
the cells homogenously, and 70µ1aliquots were added to a 96-well plate. ΙΟΟµΙ Color
Reagent was added followed by ΙΟΟµΙ Base Reagent (STANBIO, Cat#0 150-250,
Calcium Liquid Color). The reagents were mixed and incubated for lmin. The
optical density (O.D) was measured at 550nm within 60 minutes after mixing the
reagents. FIG. 12 shows the calcium content of differentiated osteogenic cells is
about 7 times higher than that of the control cells. The increased calcium content in
the differentiated cells indicates that the MSCs grown on these carriers retain the
ability to differentiate into an osteoblastic phenotype which is expected of cells
defined as MSCs.
EXAMPLE 13 Characterization o f MSCs b y Flow Cytometry analysis (FACS)
[0083] hMSCs are expected to express a number of surface protein markers and
simultaneously lack a number of others. Positive markers include CD105, CD90,
CD 166, CD29, CD44 and CD73 while negative markers include CD14, CD29, CD19,CD45 and CD3. For each marker, the assay contained at least 150,000 cells in test
tube. The cells were blocked in PBS and 10% normal goat serum (NGS) for 30 min
at 4°C. Antibodies were used for the following cell surface markers: CD105, CD90,
CD166, CD45, CD73, CD34, CD44, CD14, CD29, CD19 and CD31. hMSCs from
Millipore (part number SCR108) were grown in static T-flask (control) or a spinner
flask on the embossed carriers using conditions as described in example 3 . After
harvesting with trypsin, the cells were incubated with labeled antibody for 1 hour at
4°C in PBS and 1% NGS and then washed with PBS and 1% NGS by centrifugation
to pellet down the cells. The pellet was re-suspended in 150µΙ PBS and 1% NGS
buffer. The cells were analyzed by flow cytometer according to manufacturer's
instructions (Beckman Coulter- Cytomics FC500MPL). hMSCs and the
manufacturer's growth medium were sourced from Millipore and cells were grown as
described in example 5 .
[0084] The results of the FACS analysis are shown in Table 1 below which indicates
that MSCs grown on the embossed carriers in bioreactor conditions showed a marker
profile consistent with what is expected of these cells (also shown by the control cells
grown under static conditions in T-flasks). The top row of the table indicates the
specific markers assayed by FACS. Plus (+) symbol indicates that marker was
present (>90%) and the minus (-) sign indicates a marker that was not detected (<5%).
The expression of the 6 positive markers listed above are designated positive if
expression was observed in >90% of the cells while the 5 negative markers were
designated as such if expression was observed for <5%of the cells.
Table 1[0085] 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 scope of the invention.Claims:
1. A carrier for growing adherent cells, comprising:
one or more outer surfaces; and
one or more structured indentations on one or more of the outer surfaces,
wherein the carrier has a length at least about 0.2 mm, a width at least about 0.2
mm, and a height in a range from about 0.05 mm to 1.2 mm and each of the
structured indentations has a major axis in a range from about 0.1 mm to 0.5 mm,
a minor axis in a range from about 0 .1mm to 0.5 mm and a depth in a range from
about 0.025 mm to about 0.5 mm.
2 . The carrier of claim 1, wherein the carrier comprises two outer surfaces for
growing adherent cells.
3 . A cell culture system comprising one or more of the carriers of claim 1,
wherein the cell culture system is a bioreactor.
4 . A kit for culturing cells, comprising a disposable housing pre-loaded with
the carrier of claim 1.
5 . A carrier for growing adherent cells, comprising:
one or more outer surfaces; and
a single structured indentation on at least one surface, wherein the single
structured indentation has a length of at least about 1mm, a width of at least about
lmm, a height in a range from about 1mm to 10mm, and a wall-thickness of the
carrier in a range from about 0.05 mm to 2 mm.
6 . A method of culturing adherent cells, comprising:
providing a carrier for growing the cells, comprising one or more outer
surfaces; and one or more structured indentations on one or more of the outer
surfaces, wherein the carrier has a length at least about 0.2 mm, a width at leastabout 0.2 mm, and a height in a range from about 0.05 mm to 1.2mm and each of
the structured indentations has a major axis in a range from about 0.1 mm to 0.5
mm, minor axis in a range from about 0.1 mm to 0.5 mm and depth in a range
from about 0.025 mm to about 0.5 mm, and
growing the cells on the carrier.
7 . A method of making a carrier for growing cells, comprising:
a) providing a flat polymer film;
b) forming on the flat polymer film, on one or more sides, one or more
structured indentations;
c) imparting a surface treatment to the film comprising one or more of
corona discharge treatment, gas plasma treatment, chemical
functionalization or coating; and
d) cutting the treated polymer film into a plurality of portions.
8 . A method of making a carrier for growing cells, comprising:
a) providing one or more flat polymer films;
b) forming on the flat polymer film, on one or more sides, one or more
structured indentations;
c) cutting the polymer films into a plurality of portions; and
d) imparting a treatment to the portions comprising one or more of corona
discharge treatment, gas plasma treatment, chemical functionalization or
coating.
9 . The method of claim 8 wherein the carrier is cytophilic.
10. A method of making a carrier for growing cells, comprising:
a) providing two flat polymer films;
b) forming on the two flat polymer films, a plurality of structured
indentations on at least one surface of each of the two films;c) laminating the two polymer films together so that at least two
outwardly facing surfaces comprise a plurality of the structured
indentations;
d) cutting the laminated polymer film into a plurality of portions; and
e) imparting a treatment to the portions comprising one or more of corona
discharge treatment, gas plasma treatment, chemical functionalization or
coating.