U.S. patent application number 14/814267 was filed with the patent office on 2015-11-26 for multilayered cell culture apparatus.
The applicant listed for this patent is Corning Incorporated. Invention is credited to Gregory Roger MARTIN, Allison Jean TANNER.
Application Number | 20150337252 14/814267 |
Document ID | / |
Family ID | 37243347 |
Filed Date | 2015-11-26 |
United States Patent
Application |
20150337252 |
Kind Code |
A1 |
MARTIN; Gregory Roger ; et
al. |
November 26, 2015 |
MULTILAYERED CELL CULTURE APPARATUS
Abstract
A multilayered cell culture apparatus for the culturing of cells
is disclosed. The cell culture apparatus is defined as an integral
structure having a plurality of cell culture chambers in
combination with tracheal space(s). The body of the apparatus has
imparted therein gas permeable membranes in combination with
tracheal spaces that will allow the free flow of gases between the
cell culture chambers and the external environment. The flask body
also includes an aperture that will allow access to the cell growth
chambers by means of a needle or cannula. The size of the
apparatus, and location of an optional neck and cap section, allows
for its manipulation by standard automated assay equipment, further
making the apparatus ideal for high throughput applications.
Inventors: |
MARTIN; Gregory Roger;
(Acton, ME) ; TANNER; Allison Jean; (Portsmouth,
NH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Corning Incorporated |
Corning |
NY |
US |
|
|
Family ID: |
37243347 |
Appl. No.: |
14/814267 |
Filed: |
July 30, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14254280 |
Apr 16, 2014 |
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14814267 |
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13904171 |
May 29, 2013 |
8846399 |
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14254280 |
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13591566 |
Aug 22, 2012 |
8470589 |
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13904171 |
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12783217 |
May 19, 2010 |
8273572 |
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13591566 |
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11433859 |
May 11, 2006 |
7745209 |
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12783217 |
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60702896 |
Jul 26, 2005 |
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Current U.S.
Class: |
435/325 |
Current CPC
Class: |
C12N 5/0602 20130101;
C12M 25/06 20130101; C12M 23/58 20130101; C12M 23/04 20130101; C12M
23/08 20130101; C12M 23/34 20130101; C12M 29/04 20130101; C12M
41/00 20130101; C12M 25/04 20130101; C12M 23/24 20130101; C12M
23/38 20130101; C12M 25/02 20130101 |
International
Class: |
C12M 1/04 20060101
C12M001/04; C12M 1/12 20060101 C12M001/12 |
Claims
1. (canceled)
2. A method of culturing cells in a gas permeable multi-shelf cell
culture apparatus, the method comprising: adding cells and media
into a gas permeable multi-shelf apparatus comprising two or more
culture compartments, each compartment including a shelf comprised
of gas permeable, liquid impermeable material for cells to reside
upon, each shelf connected to an opposing surface, a fluid pathway
shared by said culture compartments, and each said shelf is in
contact with a gas space, whereby said apparatus is incubated in
the presence of ambient gas suitable for cell culture, oriented in
a position such that said culture compartments are located one
above the other, each said shelf is in a horizontal position with
said gas space located below it, cells reside upon at least a
portion of each said shelf, said culture compartments include media
in contact with said shelf and said opposing surface, and ambient
gas resides within each said gas space and is in contact with each
shelf.
3. The method of claim 2, wherein a culture compartment support is
in contact with a portion of each said shelf.
4. The method of claim 3, wherein said culture compartment support
includes projections that make contact with said shelf.
5. The method of claim 4, wherein said projections are dispersed
throughout said gas space.
6. The method of claim 2, wherein each said shelf is parallel to
its said opposing surface.
7. The method of claim 2, wherein the ratio of the volume of media
within said apparatus to the surface area of said gas permeable,
liquid impermeable walls is in the range of 0.25 ml/cm.sup.2 to 0.4
ml/cm.sup.2.
8. A method of culturing cells, the method comprising: adding cells
and media into a gas permeable multi-shelf apparatus comprising two
or more culture compartments, each having a gas permeable, liquid
impermeable wall for cells to reside upon and an opposing wall, a
portion of each said gas permeable, liquid impermeable wall in
contact with a culture compartment support, a manifold that creates
a fluid pathway between said culture compartments, at least a
portion of each said gas permeable, liquid impermeable wall is in
contact with a gas space, and culturing said cells while said
apparatus resides in the presence of ambient gas suitable for cell
culture and in a position wherein said culture compartments reside
at differing elevations, said culture compartments are full of
media, each said gas permeable, liquid impermeable wall is held in
a horizontal position by said culture compartment support, cells
reside upon at least a portion of each gas permeable, liquid
impermeable wall, said ambient gas suitable for cell culture
resides in each said gas space and makes contact with each said gas
permeable, liquid impermeable wall.
9. The method of claim 8, wherein said culture compartment support
includes projections that make contact with said gas permeable,
liquid impermeable wall.
10. The method of claim 9, wherein said projections are dispersed
throughout said gas space.
11. The method of claim 8, wherein each said gas permeable, liquid
impermeable wall is parallel to its said opposing wall.
12. The method of claim 8, wherein the ratio of the volume of media
within said apparatus to the surface area of said gas permeable,
liquid impermeable material is in the range of 0.25 ml/cm.sup.2 to
0.4 ml/cm.sup.2.
13. A static method of culturing cells, the method comprising:
adding cells and media into a gas permeable multi-shelf cell
culture apparatus comprised of more than one culture space, each
culture space including a shelf comprised of gas permeable, liquid
impermeable material for cells to reside upon, each shelf connected
to an opposing surface that is oriented in parallel to the shelf,
each culture space is connected to a common fluid pathway, a gas
space in contact with at least a portion of said gas permeable,
liquid impermeable material of each shelf, a shelf support
structure in contact with a portion of each said shelf and acting
to maintain the shelf in a planar state, and culturing said cells
when said cell culture apparatus is in a location that includes
ambient gas suitable for cell culture and said cell culture
apparatus is in a position wherein each said shelf is in a
horizontal position and a gas space is located below each said
shelf, said shelves oriented one above the other, the space between
each shelf and its opposing surface includes media in contact with
the shelf and its opposing surface, and ambient gas is in the gas
space located below each shelf.
14. The method of claim 13, wherein said shelf support structure
includes projections that make contact with said shelf.
15. The method of claim 14, wherein said projections are dispersed
throughout said gas space.
16. The method of claim 13, wherein the ratio of the volume of
media within said apparatus to the surface area of said shelves is
in the range of 0.25 ml/cm.sup.2 to 0.4 ml/cm.sup.2.
17. A method of culturing cells, the method comprising: adding
cells and media into a gas permeable multi-shelf apparatus
comprising more than one culture compartment, each having a gas
permeable, liquid impermeable shelf and an opposing wall, a gas
space in communication with each said gas permeable, liquid
impermeable shelf, and a manifold providing said culture
compartments a shared fluid pathway, and placing said apparatus in
contact with ambient gas suitable for cell culture in a position
such that said culture compartments are oriented one above the
other, media is in a static state, each said gas permeable, liquid
impermeable shelf resides in a horizontal position with its
opposing wall residing above it and ambient gas makes contact with
at least a portion of the underside of each said gas permeable,
liquid impermeable shelf.
18. The method of claim 17, wherein a portion of each said gas
permeable, liquid impermeable shelf is in contact with a culture
compartment support.
19. The method of claim 18, wherein said culture compartment
support includes projections that make contact with said gas
permeable, liquid impermeable shelf.
20. The method of claim 19, wherein said projections are dispersed
throughout said gas space.
21. The method of claim 17, wherein each said gas permeable, liquid
impermeable shelf is parallel to its said opposing wall.
22. The method of claim 21, wherein the ratio of the volume of
media within said apparatus to the surface area of said gas
permeable, liquid impermeable shelves is in the range of 0.25
ml/cm.sup.2 to 0.4 ml/cm.sup.2.
23. A method of culturing cells, the method comprising: adding
cells and media into a gas permeable multi-shelf apparatus
comprising more than one cell compartment, each cell compartment
having a shelf for cells to reside upon comprised of a gas
permeable, liquid impermeable membrane connected to an opposing
wall, a manifold connecting said cell compartments, each said shelf
in contact with projections that hold the shelf a planar position,
said projections allowing ambient gas to make contact with each
said shelf, whereby the cells are cultured within said apparatus
when said apparatus is in contact with ambient gas suitable for
cell culture and in a position wherein each said shelf is
horizontal, said shelves at differing elevations, ambient gas is in
contact with the underside of each said shelf, each said cell
compartment is full of media, and the cells reside upon each said
shelf.
24. The method of claim 23, wherein each said shelf is parallel to
its said opposing wall.
25. The method of claim 23, wherein the ratio of the volume of
media within said apparatus to the surface area of said shelves is
in the range of 0.25 ml/cm.sup.2 to 0.4 ml/cm.sup.2.
26. A method of culturing cells, the method comprising: adding
cells and media into a gas permeable multi-shelf apparatus
comprising two or more culture spaces, each space having a gas
permeable, liquid impermeable surface for cells to reside upon and
an opposing surface, projections acting to maintain said gas
permeable, liquid impermeable surface in a planar position so that
it can be oriented in a horizontal position with said projections
acting to allow ambient gas to make contact with the underside said
gas permeable, liquid impermeable surface, a fluid pathway in
communication with each said culture space, and culturing said
cells while said apparatus is in contact with ambient gas suitable
for cell culture, the orientation of said apparatus being such that
each said gas permeable, liquid impermeable surface resides at a
different elevation than the other said gas permeable, liquid
impermeable surfaces, said culture spaces are full of media that is
in a static state, each said gas permeable, liquid impermeable
surface is in a horizontal position, and ambient gas is in contact
with at least a portion of the underside of each said gas
permeable, liquid impermeable surface.
27. The method of claim 26, wherein each said gas, permeable liquid
impermeable surface is parallel to its said opposing surface.
28. The method of claim 26 wherein the ratio of the volume of media
within said apparatus to the surface area of said gas permeable,
liquid impermeable surfaces is in the range of 0.25 ml/cm.sup.2 to
0.4 ml/cm.sup.2.
29. A method of culturing cells comprising: incubating cells in the
presence of ambient gas suitable for cell culture, said cells
residing in a gas permeable multi-shelf cell culture apparatus
wherein said cells reside upon more than one shelf, each shelf
comprised of gas permeable, liquid impermeable material, each shelf
connected by a fluid pathway that is integral to said apparatus, a
portion of the bottom of each shelf is in contact with a shelf
support that maintains the shelf in a horizontal position, said
shelves located at differing elevations, said ambient gas is in
contact with the bottom of each said shelf, each said shelf has an
opposing surface connected to it and residing above it, and media
is in static contact with each shelf and its opposing surface.
30. The method of claim 29, wherein each said shelf support
includes projections that are in contact with said gas permeable
shelf.
31. The method of claim 30, wherein each said gas permeable shelf
is parallel to its said opposing surface.
32. The method of claim 30, wherein each said gas permeable shelf
is rectangular.
33. The method of claim 29, wherein the ratio of the volume of
media within said apparatus to the surface area of said shelves is
in the range of 0.25 ml/cm.sup.2 to 0.4 ml/cm.sup.2.
34. A method of culturing cells consisting essentially of: adding
cells and media into a gas permeable, liquid impermeable cell
culture apparatus; and incubating said apparatus in contact with
ambient gas suitable for cell culture, wherein said cells reside
upon more than one shelf, each shelf comprised of gas permeable,
liquid impermeable material, a portion of the bottom of each said
shelf is in contact with a shelf support that maintains the shelf
in a horizontal position and at a different elevation relative to
other shelves, said ambient gas is in contact with the bottom of
each said shelf, each said shelf is connected to an opposing
surface residing above it, the space between each said shelf and
its opposing surface is full of media, and the space between each
said shelf and its opposing surface is in communication with a
common fluid pathway.
35. The method of claim 34, wherein each said shelf support
includes projections that make contact with said shelf.
36. The method of claim 34, wherein said common fluid pathway is
full of said media.
37. The method of claim 34, wherein said ambient gas suitable for
cell culture is located within a cell culture incubator that
includes carbon dioxide.
38. The method of claim 34, wherein said shelf is parallel to its
opposing surface.
39. The method of claim 34, wherein the ratio of the volume of
media within said apparatus to the surface area of said shelves is
in the range of 0.25 ml/cm.sup.2 to 0.4 ml/cm.sup.2.
40. A method of culturing cells, the method comprising: adding
cells and media into a gas permeable multi-shelf apparatus
comprising more than one culture compartment comprised at least in
part of gas permeable material, each culture compartment having a
gas permeable, liquid impermeable planar shelf for cells to reside
upon connected to an opposing surface, a shared fluid pathway
connecting said culture compartments, a gas space in contact with
at least a portion of each gas permeable, liquid impermeable shelf,
each gas permeable, liquid impermeable shelf in contact with a
culture compartment support, and placing said apparatus within a
cell culture incubator that includes ambient gas suitable for cell
culture, said apparatus oriented in a culture position within said
cell culture incubator such that said culture compartments reside
one above the other, at least a portion of said ambient gas enters
each said culture compartment by way of its said shelf, at least a
portion of said cells are located upon said shelves, said culture
compartments are full of media, and said shelves are
horizontal.
41. A method of culturing cells, the method comprising: a first
step of adding cells and media into a gas permeable multi-shelf
apparatus comprising two or more culture spaces, each having a gas
permeable, liquid impermeable shelf for cells to reside upon and an
opposing surface, a portion of each said gas permeable, liquid
impermeable shelf in contact with a culture compartment support
that exposes at least a portion of each said gas permeable, liquid
impermeable shelf to ambient gas and maintains said gas permeable,
liquid impermeable shelf in a flat position when said gas
permeable, liquid impermeable shelf is oriented in a horizontal
position with media residing upon it, and a manifold that creates a
fluid pathway between said culture compartments; and a second step
consisting essentially of placing said gas permeable multi-shelf
apparatus in a cell culture incubator wherein said apparatus is in
a position such that said culture compartments reside one above the
other, each said gas permeable, liquid impermeable shelf is in a
horizontal position, cells reside upon at least a portion of each
gas permeable, liquid impermeable shelf and said culture
compartments include media.
42. The method of claim 41, wherein said cell culture incubator
includes carbon dioxide.
43. The method of claim 41, wherein the ratio of the volume of
media within said apparatus to the surface area of said gas
permeable, liquid impermeable shelves is in a range of 0.25
ml/cm.sup.2 to 0.4 ml/cm.sup.2.
44. The method of claim 41, wherein each said gas permeable, liquid
impermeable shelf for cells to reside upon is parallel to its
opposing surface.
45. A method of culturing cell comprising: incubating cells in the
presence of ambient gas suitable for cell culture, said cells
residing in a gas permeable multi-shelf cell culture apparatus
wherein said cells reside upon more than one shelf, each shelf
comprised of gas permeable, liquid impermeable material, each shelf
open to only one shared fluid pathway, said shared fluid pathway is
located within said apparatus, a portion of the bottom of each
shelf is in contact with a shelf support that maintains the shelf
in a horizontal position, said ambient gas is in contact with the
bottom of each said shelf, each said shelf has an opposing wall
connected to it and residing above it, and said apparatus contains
media in a static state.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/699,157, filed on Apr. 29, 2015, which is a continuation of
U.S. application Ser. No. 13/904,171, filed on May 29, 2013, which
is a continuation of U.S. application Ser. No. 13/591,566, filed on
Aug. 22, 2012, which is a continuation of U.S. application Ser. No.
12/783,217, filed on May 19, 2010, which is a divisional of U.S.
application Ser. No. 11/433,859, filed on May 11, 2006, which
claims the benefit of priority to U.S. Provisional Application Ser.
No. 60/702,896 filed on Jul. 26, 2005 and entitled "Multilayered
Cell Culture Apparatus" which is incorporated by reference herein
in.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the cellular
biological field and, in particular, to a cell cultivating
flask.
BACKGROUND OF THE INVENTION
[0003] In vitro culturing of cells provides material necessary for
research in pharmacology, physiology, and toxicology. The
environmental conditions created for cultured cells should resemble
as closely as possible the conditions experienced by the cells in
vivo. One example of a suitable environment for culturing cells is
a common laboratory flask such as demonstrated in U.S. Pat. No.
4,770,854 to Lyman. The cells attach to and grow on the bottom wall
of the flask, immersed in a suitable sustaining media. The flask is
kept in an incubator to maintain it at the proper temperature and
atmosphere.
[0004] Although most cells will tolerate a hydrogen ion
concentration (pH) range of 6.8 to 7.8, the optimal pH for growth
of mammalian cells is 7.2 to 7.4. For the optimal pH to be
maintained during cell cultivation, the cell culture medium must
contain a buffering system.
[0005] Frequently, pH is maintained by using a bicarbonate
buffering system in the medium, in conjunction with an incubator
atmosphere of approximately 5 to 7 percent carbon dioxide by
volume. The carbon dioxide reacts with the water to form carbonic
acid which in turn interacts with bicarbonate ions in the medium to
form a buffering system which maintains the pH near physiological
levels. Entry of carbon dioxide from the incubator into the cell
culture flask is generally achieved by using a loosely fitting or
vented cap or cover so that the small opening remains for the
exchange of gas between flask and incubator. Further, flasks have
been sold that are made from impact resistant polystyrene plastic
which is permeable to water vapor, oxygen and carbon dioxide.
However, relying only on the gas exchange through the polystyrene
is generally ineffective since the vessel wall thickness greatly
decreases the permeability rate. Further still, flasks have been
made having a cell growth surface that is itself an extremely thin
(approximately 0.004 inches thick) flexible, gas permeable
membrane. While this type of construction allows for gas exchange,
the flexibility and thinness of the growth surface makes the growth
of a uniform surface difficult and contributes to problems
associated with the durability of the flask.
[0006] Gas exchange, particularly the utilization of oxygen by the
cells, is a factor that limits the area for cell growth within a
cell culture flask. Since flasks for cell culture typically grow
attachment dependent cells in a monolayer roughly equal in size to
the footprint of the flask, media volume is therefore restricted to
an area within the flask permissive to the diffusion of oxygen.
Oxygen and carbon dioxide are of particular importance to the
culturing of cells. The supply of oxygen for cellular respiration
and metabolic function in conventional cell culture containers
occupies the head space of the container, e.g., the void space in
the container that is above the surface of the cell culture medium.
Thus, the volume of the container and the surfaces within
conventional cell culture containers are inefficiently used. This
results in limiting the rate of gas exchange and/or restricting the
equilibration of gases. There is a need for a cell culture flask
that can provide an increased surface area for cell growth while
still permitting sufficient gas exchange for the multitude of
attachment dependent cells.
[0007] Desirably, many flasks are stacked together in the incubator
and a number of cultures are simultaneously grown. Small variations
in the growth medium, temperature, and cell variability have a
pronounced effect on the progress of the cultures. Consequently,
repeated microscopic visual inspections are needed to monitor the
growth of the cells. As such, cell culture flasks are typically
constructed of optically clear material that will allow such visual
inspection.
[0008] With the advent of cell-based high throughput applications,
fully automated cell culture systems have been the subject of
serious development work (see e.g. A Review of Cell Culture
Automation, M. E. Kempner, R. A. Felder, JALA Volume 7, No. 2,
April/May 2002, pp. 56-62.) These automated systems employ
traditional cell culture vessels (i.e. common flasks, roller
bottles, and cell culture dishes) and invariably require
articulated arms to uncap flasks and manipulate them much like the
manual operator.
[0009] There is a need for a cell culture apparatus having a rigid
structure that is capable of providing an increased surface area
for cell growth while also providing necessary gas exchange. Even
further, it is desirable to produce a greater cell yield within
commonly known flask volumes while permitting gas exchange at a
surface of cell attachment.
[0010] Additionally, the desired cell culture apparatus will be
suitable for use in the performance of high throughput assay
applications that commonly employ robotic manipulation.
SUMMARY OF THE INVENTION
[0011] According to an illustrative embodiment of the present
invention, a cell growth apparatus for efficient culturing of cells
is disclosed. The illustrative apparatus includes a unitary body
including a bottom tray defining a cell growth area and a top
plate, connected by side walls and end walls. At least one aperture
located along any periphery of the apparatus permits access to the
internal volume. At least one gas permeable substrate/membrane is
affixed to a support internal to the body of the apparatus. A
tracheal space/chamber permits gases from an external atmosphere to
be exchanged across the gas permeable, liquid impermeable membrane,
into and out of the cell culture chamber(s). Further, the tracheal
space is an air chamber confined by an outer vessel body.
Communication between a tracheal chamber and a cell growth chamber
provides a uniformity of conditions for cellular growth.
Furthermore, a uniform gaseous distribution can be beneficial in
providing consistency in the culturing environment.
[0012] One embodiment of a cell growth apparatus of the present
invention includes a plurality of cell growth chambers, each having
a gas permeable, liquid impermeable surface and an opposing
surface. At least one tracheal chamber is in communication with at
least one gas permeable, liquid impermeable surface of a cell
growth chamber so that cells can exchange gases (e.g. oxygen,
carbon dioxide, etc.) with an external environment. The cell growth
apparatus of the present invention has at least one tracheal
chamber incorporated with a plurality of cell growth chambers
combined into one integral unit. The integral unit thus has
multiple growth surfaces in any assembled arrangement. A preferred
embodiment of a cell growth apparatus of the present invention
alternates each cell growth chamber with a tracheal chamber in a
vertical successive orientation whereby each cell growth chamber
includes a substantially planar horizontal surface supporting the
growth of attachment-dependent cells. The cell growth surface,
however, may be planar and/or nonplanar to accommodate the surface
area for growth. A modified or enhanced surface area in combination
with one or more tracheal spaces enables a diversified area for
growing cells. Subsequently, another embodiment of the present
invention may include an arrangement of surfaces intermediary to
cell growth surfaces and tracheal spaces. As such, cell growth
chambers may be adjoined and configured so that they still have
communication with a tracheal chamber.
[0013] When a plurality of cell culture chambers are arranged with
tracheal chambers formed there-between, the tracheal chambers
permit gaseous exchange between the gas permeable, liquid
impermeable surface of a cell culture chamber and the external
atmosphere. In a preferred embodiment of the present invention,
each cell culture chamber alternates with a tracheal chamber
allowing the cells greater access to external gaseous exchange.
[0014] One embodiment of the apparatus of the present invention
utilizes a gas permeable, liquid impermeable membrane as the
opposing surface of a cell culture chamber. In such an embodiment,
a plurality of gas permeable substrates (internal to the body of
the apparatus) can be incorporated to increase surface area for
cellular growth. Preferably then, the apparatus is capable of being
rotated to facilitate the growth of attachment-dependent cells on
an alternate surface. Each gas permeable substrate may have a
tracheal space above and/or below it. One such embodiment is
capable of incorporating one or more tracheal spaces between each
stacked gas permeable substrate/layer. Additionally, the gas
permeable membrane(s) may be treated or coated to promote cell
growth.
[0015] Another embodiment of the present invention includes one or
more supports to form a shelf internal to the apparatus. As such,
each shelf would have at least one gas permeable substrate affixed.
An alternative embodiment may incorporate lateral ribs traversing
the flask body such that an internal gas permeable membrane would
be further capable of supporting cellular growth. When such
supports or lateral ribs are utilized, a plurality of gas permeable
membranes can be arranged or housed within the support itself or
affixed to one or more surfaces of the supports. It would therefore
be important then, when stacking the layers or gas permeable
substrates, to include a tracheal space between each layer of cell
growth. Preferably, the tracheal space(s) provide uniform gaseous
distribution within the cell culture chamber of the internal
apparatus. Completely filling the apparatus with media would allow
for optimal cellular nutrient exchange. Consequently, the
uniformity of conditions for cellular growth may include a
determined media volume per unit surface area. In another aspect,
an integral unit of the cell culture apparatus comprises a
plurality of modules, each having a cell growth chamber and a
tracheal chamber. The plurality of modular gas permeable substrates
are utilized to permit a plurality of cell chambers and tracheal
chambers to be arranged to form one unitary apparatus of the
present invention. The plurality of layers of gas permeable
substrates are further capable of being interconnected or adjoined
to provide a multiplicity of areas for cellular growth. The
plurality of modules may be interconnected in series or staggered
to permit continuous flow. For easy assembly and disassembly,
individual units having snap-like features could be securely and
easily adjoined.
[0016] In another embodiment of the present invention, the
apparatus comprises a manifold to access the cell growth chambers
of an integral unit. The manifold may further be capable of
directing the flow of air, liquid, media and/or cellular material
within the cell culture chamber.
[0017] While many embodiments of the present invention are suitable
for static cultures, another embodiment of the present invention
staggers the gas permeable substrates within the flask to permit
continuous flow through the cell culture chamber. The staggered
layers allow media to continuously flow or perfuse through the
apparatus.
[0018] One embodiment of the present invention provides compliance
with conventionally sized and shaped containers currently used such
that the apparatus, device or flask of the present invention may be
utilized with various equipment and instrumentation. Thus, the
apparatus of the present invention may have a substantially
rectangular footprint and a substantially uniform height. The
rectangular footprint would have dimensions that are substantially
identical to an industry standard footprint dimension for
microplates. One embodiment of the configuration of the apparatus
then may include a neck and/or cap located within the substantially
rectangular footprint and that does not exceed the height of the
integral unit.
[0019] Another embodiment of the apparatus of the present invention
may comprise stand-offs either rising from an exterior surface of
the top plate or descending from an exterior surface of the bottom
tray.
[0020] For the addition and removal of media, the cell growth
apparatus has at least one access port to access multiple growth
chambers. Each cell growth chamber, however, may have individual
access ports. Supplementary, the apparatus is capable of being
equipped with a septum seal accessible opening or aperture either
integrated within the body of the apparatus itself, or as a part of
a cap. When a cap is utilized, one embodiment of the apparatus of
the present invention, having a height as measured by the distance
between an outermost plane of the bottom tray and an outermost
plane of the top plate, has a cap, cover, and/or septum covering
the aperture. The cap may have a diameter that does not exceed the
height of the apparatus/flask so as to prevent interference when
the flasks are stacked. Additionally, the cap may be integrally
included in a top surface, side, and/or corner region of the
apparatus. The apparatus of the present invention may have an
aperture which defines an entry portal and another which may define
an exit portal. When gas permeable substrates are stacked, the
entry and exit portals may be positioned in a parallel or staggered
assembly so as to permit flow or perfusion through cell culture
chambers within the body of the apparatus.
[0021] Convenience then dictates the utilization of one or more
optical components, such as microscopic lenses, in communication
with individual cell growth chambers. These lenses would allow
observation of one or more layers of cell growth. Also, and
advantageously so, the apparatus is shaped and configured to enable
robotic access to the interior of the apparatus without requiring
cumbersome robotic arm manipulation.
[0022] The present invention also includes a method of culturing
cells in the apparatus of the present invention. The method
initially involves providing a apparatus for the growth of cells as
previously described. Gas permeable substrates are first assembled
into the desired configuration of the apparatus followed by
introduction of cells and/or media into the cell culture chamber of
the apparatus. Thereafter, the flask can then be incubated to meet
the desirable conditions for the growth of cells. Rotation of the
apparatus further permits the culturing of cells on an alternate
surface of the gas permeable substrate.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The invention is best understood from the following detailed
description when read with the accompanying drawing figures. It is
emphasized that the various features are not necessarily drawn to
scale. In fact, the dimensions may be arbitrarily increased or
decreased for clarity of discussion.
[0024] FIG. 1A is a perspective external view of an illustrative
embodiment of the apparatus of the present invention.
[0025] FIG. 1B is a cross-sectional perspective side view of an
illustrative embodiment of the present invention.
[0026] FIG. 1C is a partial internal side view of intermediary
supports and gas permeable growth surfaces of FIG. 1A.
[0027] FIG. 2 is a top view of supports utilized in another
embodiment of the present invention.
[0028] FIG. 3 is an external top view of a frame supporting a gas
permeable membrane in another embodiment of the present
invention.
[0029] FIG. 4 is a cross-sectional side view of another
illustrative embodiment of the present invention.
[0030] FIG. 5 is an internal side view of the interconnected
chambers of one embodiment of the present invention.
[0031] FIG. 5A is a side view of the external frame/body of the
embodiment of FIG. 5.
[0032] FIG. 6 is an individual unit of one embodiment of the
present invention.
[0033] FIG. 7 is another individual unit or tray of an embodiment
of the present invention.
[0034] FIG. 8 is an alternative embodiment of the present
invention.
[0035] FIG. 9 is another embodiment of the present invention.
DETAILED DESCRIPTION
[0036] In the following detailed description, for purposes of
explanation and not limitation, exemplary embodiments disclosing
specific details are set forth in order to provide a thorough
understanding of the present invention. However, it will be
apparent to one having ordinary skill in the art that the present
invention may be practiced in other embodiments that depart from
the specific details disclosed herein. In other instances, detailed
descriptions of well-known devices and methods may be omitted so as
not to obscure the description of the present invention.
[0037] An external view of a apparatus in accordance with one
embodiment of the present invention is shown in FIG. 1. The
apparatus 100 of this embodiment takes the form of a flask 100; the
flask 100 comprises an outer vessel body 101 (see FIG. 1A) defined
by a top plate 110, a bottom tray 120, sidewalls 112, and end walls
114. Disposed within the flask 100 are individual cell growth
chambers 111 as can be seen more clearly in a cross-sectional
illustration in FIGS. 1B and 1C. The individual cell growth
chambers 111 are each defined by a generally transparent bottom
surface 113 and a generally transparent top surface 115. The
surfaces 113 and 115 are attached to the flask body 101 along the
sidewalls 112 and end walls 114. Preferably, at least one bottom
surface 113 within each chamber 111 is gas permeable, liquid
impermeable material and capable for the growth of cells 117. Each
top surface 115 is preferably a rigid, generally gas impermeable
material (preferably transparent) that will provide support to the
cell growth chamber 111. In this embodiment, supports 119 allow a
gas permeable membrane 113 to be securely adhered thereto in a
leak-proof sealing to the flask body 101. Tracheal spaces 118 are
created between each cell growth chamber 111. The opposing top
surface 115 of the chamber 111 defines an upper wall to the cell
growth chamber 111 as well as a bottom portion of a tracheal
chamber 118. The tracheal chamber 118 is therefore inclusive of a
gas permeable, liquid impermeable surface 113 of a first cell
growth chamber and an opposing surface 115 to a second growth
chamber 111. Supports 119 further provide structural arrangements
to integrally incorporate the surfaces 113 and 115 in forming
growth chambers 111 in alternation with tracheal air spaces 118
within the unitary flask 101. Each cell growth chamber 111
therefore alternates with a tracheal chamber 118 in vertical
successive orientation. Accessibility to the cellular growth
chambers 111 is achieved via an aperture 120 within the flask body
101. The aperture 120 having a necked opening 121 is connected to
the cell growth chambers 111 via a manifold 104. The manifold 104
is a portal for manipulation of flask contents. In this embodiment,
the necked opening 121 is covered by a cap 122 allowing the flask
to be completely filled with media 127 without leakage.
[0038] In one embodiment of the present invention, the chambers 111
permit cellular growth on gas permeable membranes 113 such that
multiple cell growth chambers 111 are integral with the body 101 of
the apparatus 100 and are capable of being completely filled with
nutrient media for the growth of cells. The series of tracheal air
spaces 118 through the apparatus 100 provide gaseous communication
between the cells 117 of the internal volume of the apparatus and
the external environment. The tracheal spaces 118 allow oxygenation
of media located within cell growth chambers 111 through the gas
permeable surfaces 113. Further, the tracheal chambers 118 may take
the form of any air gap or space, and do not allow entrance of
liquid. As a result, a rigid cell culture apparatus 100 having
multiple growth chambers 111, alternating with tracheal spaces 118,
is cooperatively constructed to afford the benefit of equivalent
gaseous distribution to a large volume of cells 117. Supplementary,
the aperture 120 of the flask is resealable by way of a septum
and/or cap 122 to prevent contents of the flask from spilling.
[0039] The apparatus 100 of the present invention may be made by
any number of acceptable manufacturing methods well known to those
of skill in the art. In a preferred method, the apparatus 100 is
assembled from a collection of separately injection molded parts.
Though any polymer suitable for molding and commonly utilized in
the manufacture of laboratory ware may be used, polystyrene is
preferred. Although not required, for optical clarity, it is
advantageous to maintain a thickness of no greater than 2 mm.
[0040] The bottom tray 120 and top plate 110 are preferably
injection molded. Various sizes and shapes of the supports 119 may
be incorporated to facilitate positioning of the membranous layers
113 for cell culture 117 within the internal flask body 101. A top
view of another embodiment of the present invention (FIG. 2) has
supports 219 as elevated stand-offs 219 along a frame or edge 202
of the flask 100. The supports 219 are rigid structures to support
a sheet of gas permeable membrane 213 adhered to the frame 202, as
well as provide a structural framework to allow multiple layers
(rigid or membranous 213) to be formed within the flask 200.
Alternatively, FIG. 3 illustrates an inner surface 313, whereby
only a portion of each cell growth chamber 300 is gas permeable.
For instance, a rigid frame 302 may support a permeable membrane
313.
[0041] Gas permeable, liquid impermeable substrates 113 may be
comprised of one or more membranes known in the art. Membranes
typically comprise suitable materials that may include for example:
polystyrene, polyethylene, polycarbonate, polyolefin, ethylene
vinyl acetate, polypropylene, polysulfone, polytetrafluoroethylene
(PTFE) or compatible fluoropolymer, a silicone rubber or copolymer,
poly(styrene-butadiene-styrene) or combinations of these materials.
As manufacturing and compatibility for the growth of cells permits,
various polymeric materials may be utilized. For its known
competency, then, polystyrene may be a preferred material for the
membrane (of about 0.003 inches in thickness, though various
thicknesses are also permissive of cell growth). As such, the
membrane may be of any thickness, preferably between about 25 and
250 microns, but ideally between approximately 25 and 125 microns.
The membrane 113 allows for the free exchange of gases between the
interior of the flask and the external environment and may take any
size or shape, so long as the membrane is supportive of cellular
growth. A preferred embodiment would include a membrane 113 that is
additionally durable for manufacture, handling, and manipulation of
the apparatus.
[0042] The gas permeable membrane 113 is properly affixed to the
supports 119 by any number of methods including but not limited to
adhesive or solvent bonding, heat sealing or welding, compression,
ultrasonic welding, laser welding and/or any other method commonly
used for generating seals between parts. Laser welding around the
circumference of the membrane 130 is preferred to establish a
hermetic seal around the membrane region such that the membrane is
flush with and fused to the face of the supports 132 such it
becomes an integral portion of the interior surface of the
apparatus. Once the gas permeable membrane 130 is adhered, then the
top plate 110 and bottom tray 120 may be joined. The parts are held
together and are adhesive bonded along the seam, ultrasonically
welded, or laser welded. Preferably, laser welding equipment is
utilized in a partially or fully automated assembly system. The top
plate and tray are properly aligned while a laser weld is made
along the outer periphery of the joint.
[0043] Advantageously and in order to enhance cell attachment and
growth, the surfaces internal to the apparatus 100 are treated to
enable cell growth. Treatment may be accomplished by any number of
methods known in the art which include plasma discharge, corona
discharge, gas plasma discharge, ion bombardment, ionizing
radiation, and high intensity UV light.
[0044] Finally, when a cap 122 is provided, it may be a screw cap,
snap-fit cap, cap with septum, cap with air holes, or any cap known
in the art. Preferably, a cap 122 is utilized in which a septum is
integral with the cap 122. This will allow a cannula, tip or needle
to access the contents of the apparatus 100 without the need for
unscrewing. The septum is leak proof, puncturable and capable of
resealing once the needle, tip or cannula is removed from the
apparatus, even after multiple punctures. In one embodiment, the
cap 122 is positioned to access the contents of the apparatus 100
via an end wall 114. As well, the cap 122 may be positioned on a
top surface 110. Additionally, the cap arrangement can also be
located such that the cap 122 does not protrude from the
rectangular footprint as determined by the periphery of the
apparatus 100. Other accessibility options may include a neck and
cap arrangement within a corner region of the apparatus 100, such
that the cap 122 would not protrude from the periphery of the
apparatus body 101.
[0045] In use, the apparatus 100 of the current invention is
employed according to accepted cell growth methods. Cells are
introduced to the apparatus 100 though the aperture via the neck
(or through a septum in the aperture). Along with the cells 117,
media 127 is introduced such that the cells are immersed in the
media. The apparatus is arranged such that the cell containing
media covers the cell growth surfaces 113. Advantageously, the
apparatus 100 is capable of being completely filled with media
since the gas permeable membranes 113 in combination with the
tracheal spaces 118 provide uniform gas distribution to the cell
growth surfaces 113. This will further ensure the flow and exchange
of gases between flask interior and the external environment. The
apparatus is then placed within an incubator and may be stacked
together with similar vessels such that a number of cell cultures
are simultaneously grown. The apparatus is situated such that the
bottom tray 120 assumes a horizontal position (or vertical position
depending on the cell culture application). Another advantage of
the apparatus 101 of the present invention is its enhanced capacity
to grow cells on an opposing surface 115 when the apparatus is
rotated 180.degree.. Thus, when the apparatus is rotated, cells can
be cultured on an alternate surface 115. As such, it would be
beneficial to have the surface 115 composed of a gas permeable
material. Where only gas permeable membranes are layered
intermediary to the apparatus, cell growth is therefore enabled on
both of its gas permeable surfaces 113/115.
[0046] Cell growth is monitored from time to time by microscopic
inspection through the generally transparent interior and exterior
surfaces of the apparatus 100. Easier accessibility and greater
visibility of cellular growth can be visualized when optical lenses
having varying magnifications are employed in the external body
101. Additionally, optical lenses may be integrated within other
internal surfaces of the apparatus 100.
[0047] Additionally, during the cell growth process, it may become
necessary to extract the exhausted media and insert fresh media. As
previously described, media replacement may be achieved through
insertion of a canula, for example, through the septum.
Alternatively, the media may be replaced by removing the cap 122,
in embodiments that offer this option. Once the cells are ready for
harvesting, a chemical additive such as trypsin is added to the
apparatus through the septum. The trypsin has the effect of
releasing the cells from the surfaces of the apparatus. The cells
can then be harvested from the flask.
[0048] A cap and neck arrangement is not necessary, however, for an
apparatus 400 of the present invention (FIG. 4). As illustrated in
this embodiment, supports 432 separate a series of tracheal spaces
440 between each growth layer 450. The tracheal air spaces provide
uniform gas distribution within the flask 400 to each cell culture
layer 450. In this embodiment, the media in the individual cell
growth chambers does not mix as these chambers 450 can be
considered separate, and possibly, modular units 450 for easy
assembly of the apparatus 400. The chambers 450, however, may be
interconnected via hollow supports 432. In one embodiment, access
to the interior of the apparatus 400 may be accomplished directly,
through plugged ports or apertures 460 that are on an end wall 414
to allow accessibility to each cell culture/media layer 450.
Another easy means of access may employ septa as coverings for the
apertures 460.
[0049] Septa are capable of being integrally affixed to the body of
the apparatus 400 by any of the aforementioned methods for affixing
a membrane to the wall of the apparatus. The septa may take any
form well known to those of skill in the art including a slit
arrangement useful for blunt needles and as generally described in
WO 02/066595, the contents of which are incorporated herein by
reference. Possible materials that may be employed in making the
septa include natural and synthetic elastomeric materials
including, but not limited to silicone rubber, fluoro-carbon
rubber, butyl rubber, polychloroprene rubber, a silicone elastomer
composite material, thermoplastic elastomer, medical grades of
silicone rubber, polyisoprene, a synthetic isoprene, silicone,
santoprene and fluoropolymer laminate and combinations thereof. In
a preferred embodiment, the elastomeric material is substantially
nontoxic to cultured cells. Moreover, a universal septum may cover
each aperture 460 while still allowing access to each individual
layer of cell growth 450. This embodiment of the flask 400 may be
preferred when stacking of the apparatus 400 is required, or when
significant robotic manipulation is encountered since it eliminates
the need for cap displacement.
[0050] FIGS. 5 and 5A illustrate another embodiment of the present
invention. As illustrated in partial internal and external
cross-sectional views, respectively, a multilayered culture vessel
501 of the present invention is a perfusion system 500. Multiple
gas permeable substrates 530 are adhered to supports/frames 532 and
stacked in a parallel configuration permitting an airway or
tracheal space 540 to separate each cellular growth layer 550. As
in previous embodiments, the gas permeable substrates/membranes 530
in combination with the tracheal chambers 540 define the cell
culture system 500. The tracheal spaces 540, alternating with
layers of transparent gas permeable membrane(s) 530 and supports
532, provide air/gas exchange with media and cell cultures 550 on
an alternate or opposing surface of the gas permeable substrate
530. As such, liquid media inside the apparatus 501 is capable of
being contained within a layer 550. In addition, the tracheal air
chambers 540 under each cell growth surface 550 have gaseous
communication between the cells/media layers 550 and external
environment via the series of openings 541 formed between the
supports 532 in the external apparatus body 501. The necked opening
560 comprises one aperture which defines an entry portal 562 and
one aperture which defines an exit portal 564. The entry portal 566
and exit portal 568 in conjunction with the necked opening 560
allows access to the internal volume/layers 550 of the apparatus
500. Furthermore, in this embodiment of the apparatus/vessel 501, a
raised rim 580 serving as a standoff 580 is located on the surfaces
of both the top plate 510 and bottom plate 520. The standoff rim
580 is intended to contact the bottom tray 520 of an identical
vessel that is stacked on top the apparatus 501. Stacking makes
efficient use of incubator space. Another attribute of having a
standoff rim 580 is the allowance of an air gap between stacked
flasks; the air gap is important for allowing gas exchange through
any vent that may be incorporated into an upper or underside
surface of the apparatus 501, and further prevents damage to the
gas permeable membrane 530. Other alternatives for standoffs 580
include raised corners, posts, ledges, or any other feature that
will allow spacing between successively stacked flasks. Preferably,
the bottom plate 520 is molded with a rim 580 around the periphery
that can engage with a standoff rim 580 from an immediately
adjacent apparatus to ensure lateral stability of the stacked
vessels.
[0051] For exemplary purposes and not limitation, cell seedlings,
media exchange, and/or cell harvesting can be accessed via the
entry portal(s) 566 and exit portal(s) 568. In combination with the
portals 566/568, linear fluid flow restrictors 564 can act as
manifolds to evenly direct flow during cell harvesting.
Additionally, for exemplary purposes only and not limitation, an
embodiment of the present invention incorporates a staggered
configuration of gas permeable substrates 530 in conjunction with
the supports 532 so as to allow continuous flow or perfusion
through the vessel 501. Various arrangements of the layers 550 and
stacked substrates 530, however, would permit utilization of the
vessel 501 for static cell culture or cell culture in a perfusion
system as discussed, including parallel, symmetrical, or
asymmetrical arrangements.
[0052] For easier accessibility and manufacturing of the
multilayered apparatus 501, the arrangement of cell growth layers
550 and stacked substrates 530 into individual modular units may be
preferred. As such, a modular unit of one embodiment of the present
invention is illustrated in FIG. 6. An individual modular unit 600
comprises a support network 632 in combination with gas permeable
membranes/substrates 630. A plurality of modular units 600 are
capable of being interconnected and/or interlocked or adhered
together to provide a multiplicity of growth surfaces 630/631 that
can be easily assembled or disassembled into a unitary multilayered
vessel for cellular culture. Vertical stacking of the modular units
600 would be analogous to interconnecting building blocks. Any
number of cell growth layers 600 could be assembled or disassembled
to provide a wide range of accessibility options to each modular
cell growth unit 600. One embodiment of the present invention
utilizes supports 632 forming a shelf/frame 633 along a periphery
of the individual unit 600 in addition to lateral ribs 635 spanning
or bisecting the distance internal to the frame 633. The
transparent gas permeable substrate(s) 630 are adhered to supports
632 such that air gaps or tracheal spaces 640 are formed between
each cell layer of gas permeable substrate 630 to allow gas
distribution throughout the unitary apparatus when multiple trays
are assembled into one vessel body. The tracheal spaces 640 have
gaseous exchange with the external atmosphere via the tracheal
openings/ports 641 in the external frame 633. Further, the tracheal
spaces 640 provide air/gas exchange with media and cell cultures on
a [primary] surface 630 and an alternate or secondary surface 631,
both surfaces 630/631 capable of cell growth. As seen in this
embodiment, peripheral ridges or elevations 637 of the support
system 632 are utilized to facilitate stacking of the modular units
600. The gas permeable membrane 630, however, may be adhered to any
of the surfaces of the support system 632 or peripheral edge 637 so
as to provide a leak-proof gas permeable substrate 630 in
combination with the modular unit 600 and further permitting
multiple areas for cell growth on the gas permeable surfaces
630/631. Additionally, an open end 633 of the frame 632 is a
feature to permit fluid flow when multiple modular units 600 are
stacked and adhered together into a unitary body so as to be
utilized in perfusion devices. Furthermore, one embodiment of the
apparatus of the present invention encompasses one gas permeable
substrate 630 providing a primary growth surface 630, as well as an
[optional] gas permeable substrate 631 providing a secondary growth
surface 631 adhered to an underside of the frame network 632.
[0053] As seen in FIG. 7, another embodiment of the present
invention utilizes a modular unit 700 inclusive of a cap 762
covering an aperture 760. A manifold 764 permits access to the
internal cell culture layer 750. A unitary cell culture chamber is
capable of being constructed when individual units 600 and/or 700
are stacked. Further, when combined, the internal cell culture
layers 650 and/or 750 would be accessible via the aperture 760 to
the unitary cell culture chamber.
[0054] In utilizing the vessels of the current invention, various
methods in the industry may be employed in accordance with accepted
cell growth culturing. As discussed in a previous embodiment, cells
are introduced to the flask though the neck or through the septum.
Along with the cells, media is introduced such that the cells are
immersed in the media. The apparatus is arranged such that the
cell-containing media covers the cell growth surfaces.
Advantageously, the apparatus is capable of being completely filled
with media since the gas permeable membranes in combination with
the tracheal spaces provide uniform gas distribution to the cell
growth surfaces. This will furthermore ensure the flow and exchange
of gases between flask interior and the external environment. The
apparatus is then placed within an incubator and may be stacked
together with similar flasks such that a number of cell cultures
are simultaneously grown. The flask is situated such that the
bottom tray assumes a horizontal position (or vertical position
depending on the cell culture application). The flask can then be
rotated to permit the culturing of cells on an alternate surface.
Where only gas permeable membranes are layered intermediary to the
apparatus, cell growth is enabled on upper and under sides of the
membrane (opposing gas permeable surfaces).
[0055] Cell growth can be monitored from time to time by
microscopic inspection through the generally transparent surfaces.
If more detailed visual inspection of the cell growth layers is
required, optical lenses can be integrated into the body or frame
of the apparatus. As such, varying magnifications of the optical
lenses would permit viewing within individual layers without
disassembly of the apparatus. Optical lenses may be incorporated
into any surface or modular unit, as well, preferably when the
units are capable of being disassembled for observational
analysis.
[0056] During the cell growth process, it may become necessary to
extract the exhausted media and insert fresh media. As previously
described, media replacement may be achieved through insertion of a
canula, for example, through the septum. Alternatively, the media
may be replaced by removing the cap, in embodiments that offer this
option. Once the cells are ready for harvesting, a chemical
additive such as trypsin is added to the flask through the septum.
The trypsin has the effect of releasing the cells from the vessel
surfaces. The cells are then harvested from the apparatus.
[0057] As discussed, the embodiments of the present invention are
for exemplary purposes only and not limitation. Supplementary,
tracheal spaces are capable of being formed above and/or below the
support network when the trays are stacked upon one another where
peripheral ridges of individual modular units permit gaps of air to
flow through gas permeable substrates to cell growth areas when the
units are interconnected. The tracheal spaces formed within the
individual units are further capable of including a diversified
network of supports, intersecting and/or alternating gas permeable
membrane with supports and air/tracheal spaces.
[0058] The gas permeable substrates utilized in the embodiments of
the present invention are capable of cell growth and gas exchange
with the external environment, achieving uniform gaseous
distribution throughout the cell culture vessel. Furthermore, the
apparatus of the present invention may utilize horizontal or
vertical designs having surfaces arranged for uniform gaseous
distribution to cell growth areas. As seen in one embodiment of the
present invention in FIG. 8, vertical growth surfaces or gas
permeable substrates 830 are separated by tracheal spaces 840. The
tracheal spaces 840 allow for the exchange of oxygen, carbon
dioxide, and other various gases between the respiratory/gas
permeable surfaces 830 that the cells grow on and the incubator or
external atmosphere where the apparatus 800 is stored while the
cells are given time to grow. The apparatus 800 of the present
invention may include a cap 862 and/or a manifold 864, as well,
which is unitary with the vessel body 800.
[0059] Another embodiment of the present invention (FIG. 9) is an
apparatus 900 that includes an external manifold 965 allowing
access to individual cell growth layers 930 via a septum as
discussed previously. The units 930 are modular and joined together
to handle as one. Furthermore, tracheal spaces 940 allow uniform
gaseous distribution to cell growth areas 930 throughout the flask
900. The uniformity of conditions for cellular growth may include a
determined media volume per unit surface area. Though the
determined ratio of volume per unit surface area has previously
been known within a confined range of about 0.5-1.0 ml/cm.sup.2,
the ratio is no longer limiting due to the direct access of the
cells to gaseous exchange via the gas permeable membrane upon which
the cells grow. While efficient use of media is still preferable,
any volume of media may be utilized in an apparatus of this
invention, the apparatus of which may be any size and/or take any
shape. Further, the enhanced capabilities of the present invention
may incorporate tracheal spaces in combination with cell growth
chambers into standardized or conventionally-sized containers. One
embodiment of the apparatus of the present invention includes an
increased surface area for cellular growth preferably with a ratio
of media volume per unit surface area in the range of about
0.25-0.50 ml/cm.sup.2; however, the dimensions and confines for
cellular growth are unlimited. One such embodiment would include a
height of about 2.8 mm. As stated previously, however, the height
is unrestricted so long as it permits area for the growth of cells.
Furthermore, by completely filling the cell growth chambers with
media, the cells have access to optimal nutrient exchange.
[0060] The embodiments of the present invention may be modified to
take the shape of any device, container, apparatus, vessel, or
flask currently used in industry. Specifically, cylindrical or
alternative vessels may utilize gas permeable substrates (internal
to the vessel) in combination with tracheal chambers or spaces to
provide an improved culturing environment for the growth of cells.
A spiral or alternative approach inclusive of a tracheal chamber
would therefore be possible. Further, although tracheal chambers
may take many forms and be of any size, the passageway-like
chambers are: a) confined air spaces, b) in communication with a
gas permeable membrane that is permissive to cell growth, and c)
communicative with the external environment via open direct access
and/or additional gas permeable membranes.
[0061] As presented, the multiple embodiments of the present
invention offer several improvements over standard vessels
currently used in industry. The improved cell culture devices
remarkably enhance the volume of cells that are capable of being
cultured in the volume enclosed by traditional cell culture
vessels. The various benefits are attributable to the multi-layered
arrangement of gas permeable membranes assembled into a unitary
vessel. Successive layering of individual growth chambers and
tracheal chambers inclusive of the gas permeable membranes makes
oxygen and other gases from the external environment available to
the internal contents of the apparatus. Specifically, gaseous
exchange with the nutrient media is conducive to an even
distribution of cell growth when gas permeable membranes are
utilized on at least one potential growth surface. The cell growth
apparatus is capable of fully utilizing its capacity by allowing
cells access to optimal volumes of nutrient media and direct
oxygenation via the tracheal spaces. Additional benefits are
afforded to the cell culturing apparatus in which the exterior
framework is rigidly constructed, conveniently offering easy
handling, storage, and accessibility.
[0062] In one embodiment, the present invention has a footprint
conforming to industry standard for microplates (5.030+/-0.010
inches by 3.365+/-0.010 inches). For this reason, the neck portion
is preferably recessed within the overall rectangular footprint.
The advantage of providing an apparatus with such a footprint is
that automated equipment designed specifically for the manipulation
of microplates may be utilized with this apparatus with very little
customized modification. Similarly the height, or the distance
between the outer most portion of the bottom tray and the outer
portion of the top plate, is approximately 0.685+/-0.010 inches. At
any rate, the present invention is not intended to be limited in
any way by the aforementioned preferred dimensions and in fact may
be constructed to any dimension.
[0063] As exemplified, the apparatus may include any unitary
structure, vessel, device or flask with the capacity to integrally
incorporate substrates in successive orientation. The invention
being thus described, it would be obvious that the same may be
varied in many ways by one of ordinary skill in the art having had
the benefit of the present disclosure. Such variations are not
regarded as a departure from the spirit and scope of the invention,
and such modifications as would be obvious to one skilled in the
art are intended to be included within the scope of the following
claims and their legal equivalents.
* * * * *