U.S. patent application number 12/460265 was filed with the patent office on 2016-05-19 for layered flask cell culture system.
This patent application is currently assigned to EMD Millipore Corporation. The applicant listed for this patent is Phillip Clark, Marc Emerick, Kurt Greenizen, Christopher A. Scott. Invention is credited to Phillip Clark, Marc Emerick, Kurt Greenizen, Christopher A. Scott.
Application Number | 20160137961 12/460265 |
Document ID | / |
Family ID | 41550918 |
Filed Date | 2016-05-19 |
United States Patent
Application |
20160137961 |
Kind Code |
A2 |
Clark; Phillip ; et
al. |
May 19, 2016 |
Layered Flask Cell Culture System
Abstract
The present invention is a cell cultivating vessel or device,
such as a single or multitier flask, including a cover having a top
plate, a side wall and a resealable port; an intermediate tray for
receiving cells and cell culture media, having a bottom plate, a
side wall, and a gap region formed between an interior upwardly
angled lip located on an interior portion of the intermediate tray
bottom plate and an adjacent outwardly angled side wall portion of
the intermediate tray bottom plate, wherein the lip has a outwardly
swooping curvilinear edge feature; and a base tray for receiving
the cells and cell culture media, including a bottom plate and a
side wall. The intermediate tray is positioned between the cover
and the base tray, such that the gap region of the intermediate
tray bottom plate is in alignment with the port located on the
cover, resulting in the port, the intermediate tray and the base
tray in fluid communication with one another which provides direct
access, such as by a user to remove and/or add cells, cell media,
and nutrients located on each of the intermediate and/or the base
trays. Alternatively, the cell cultivating flask includes a
plurality of intermediate trays stacked on top of one another and
the gap regions of each intermediate tray are in alignment with
each other and with the port on the cover.
Inventors: |
Clark; Phillip; (Wakefield,
MA) ; Greenizen; Kurt; (Bradford, MA) ; Scott;
Christopher A.; (Westford, MA) ; Emerick; Marc;
(Amesbury, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Clark; Phillip
Greenizen; Kurt
Scott; Christopher A.
Emerick; Marc |
Billerica
Billerica
Billerica
Billerica |
MA
MA
MA
MA |
US
US
US
US |
|
|
Assignee: |
EMD Millipore Corporation
Billerica
MA
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20100129900 A1 |
May 27, 2010 |
|
|
Family ID: |
41550918 |
Appl. No.: |
12/460265 |
Filed: |
July 16, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61207683 |
Feb 14, 2009 |
|
|
|
61134985 |
Jul 16, 2008 |
|
|
|
Current U.S.
Class: |
435/304.2 ;
435/294.1 |
Current CPC
Class: |
C12M 23/04 20130101;
C12M 23/58 20130101; C12M 23/08 20130101; C12M 23/34 20130101; C12M
23/22 20130101; C12M 37/02 20130101; C12M 25/06 20130101; C12M
23/38 20130101; C12M 21/08 20130101 |
International
Class: |
C12M 1/24 20060101
C12M001/24; C12M 3/00 20060101 C12M003/00; C12M 1/12 20060101
C12M001/12 |
Claims
1. A cell cultivating flask comprising: a cover including a top
plate, a side wall and a resealable port; an intermediate tray for
receiving cells and cell media having a bottom plate, an underside,
an upperside, a side wall, and a gap region formed between an
interior upwardly angled lip having a outwardly curvilinear edge
located on an interior portion of the bottom plate and an adjacent
outwardly angled side wall portion of the bottom plate; and a base
tray for receiving cells and cell media including a bottom plate,
an upperside and a side wall, wherein the intermediate tray is
positioned between the cover and the base tray, and the gap region
of the intermediate tray is in alignment with the port on the
cover, resulting in the port, the intermediate tray and the base
tray in fluid communication with one another providing direct
access to cells and cell media when located on each of the
intermediate and base trays.
2. The flask of claim 1, further comprising an additional one or
more intermediate trays stacked on top of one another, and the gap
regions of each intermediate tray are in alignment with each other,
and with the port on the cover, resulting in the port, the stacked
intermediate trays and the base tray in fluid communication with
one another providing direct access to cells and cell media when
located on each of the intermediate and base trays.
3. The flask of claim 2, wherein the side wall on each intermediate
tray stacked on top of one another is fused liquid tight to the
underside of the next adjoining intermediate tray.
4. The flask of claim 2, wherein the side wall on each intermediate
tray stacked on top of one another is ultrasonically welded to the
underside of the next adjoining intermediate tray, the side wall of
the intermediate tray adjacent the cover is ultrasonically welded
to the underside of the cover, and the side wall of the base tray
is ultrasonically welded to the underside of the adjoining
intermediate tray.
5. The flask of claim 1, wherein the resealable port consists of a
threaded cap.
6. The flask of claim 5, wherein the threaded cap includes an air
permeable fabric in communication with the interior space.
7. The flask of claim 1, wherein a pipette like device or syringe
is used to add or remove cells and cell cultivating media from the
intermediate or base trays.
8. The flask of claim 7, wherein the flask is made from a polymer,
glass or combination thereof.
9. A cell cultivating flask comprising: a cover including a top
plate, a side wall and a resealable port; a plurality of
intermediate trays stacked on top of one another for receiving a
cells and cell media, each tray having, a bottom plate, an
underside, an upperside, a side wall, and a gap region formed
between an interior upwardly angled lip having a curvilinear edge
located on an interior portion of the bottom plate and an adjacent
outwardly angled side wall portion of the bottom plate; and a base
tray for receiving cells and cell media including, a bottom plate,
and upperside, and a side wall, wherein the plurality of
intermediate trays are positioned between the cover and the base
tray, and the gap regions of each intermediate tray are in
alignment with each other, and with the port on the cover,
resulting in the port, the stacked plurality of intermediate trays
and the base tray in fluid communication with one another providing
direct access to cells and cell media when located on the
intermediate and base trays.
10. The flask of claim 9, wherein the side wall on each
intermediate tray stacked on top of one another is fused by a
liquid tight seal or bond to the underside of the next adjoining
intermediate tray; the side wall of the intermediate tray adjacent
the cover is fused by a liquid tight seal or bond to the underside
of the cover; and the side wall of the base tray is fused by a
liquid tight seal or bond to the underside of the adjoining
intermediate tray.
11. The flask of claim 9, wherein the resealable port consists of a
threaded cap.
12. The flask of claim 11, wherein the threaded cap includes an air
permeable fabric in communication with the interior space.
13. The flask of claim 9, wherein a pipette like device or syringe
is used to add or remove cells and cell cultivating media from the
intermediate or base trays.
14. The flask of claim 9, wherein the flask is made from a material
selected from the group consisting of a polymer, glass or
combination thereof.
15. The flask of claim 14, wherein the material is transparent,
translucent, opaque, a single color, multicolored or combinations
thereof.
16. A method of using a multitier cell culture flask comprising: a)
providing a multitier cell culture flask including, i) a cover
having a top plate, a side wall and a resealable port; ii) an
intermediate tray for receiving a cell culture media having, a
bottom plate having an underside, an upperside, a side wall, and a
gap region formed between an interior upwardly angled lip having a
outwardly swooping curvilinear edge located on an interior portion
of the bottom plate and an adjacent outwardly angled side wall
portion of the bottom plate; and iii) a base tray for receiving a
cell culture media including, a bottom plate and a side wall,
wherein the intermediate tray is positioned between the cover and
the base tray, and the gap region of the intermediate tray is in
alignment with the port on the cover, resulting in the port, the
intermediate tray and the base tray in fluid communication with one
another providing direct access to the cell media located on each
of the intermediate and base trays; b) providing cells and cell
media; c) adding the cells and cell media to the flask through the
port by a pipette-like device or a syringe; d) growing the cells in
the flask for a desired amount of time and under desired
conditions, and e) removing the cells and/or media from the flask
through the port via a pipette-like device or a syringe.
Description
CROSS-REFERENCED TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 61/207,683, filed on Feb. 14, 2009 and of
U.S. Provisional Patent Application No. 61/134,985, filed on Jul.
16, 2008, the entire contents of which are incorporated by
reference herein.
[0002] The present invention relates to a cell culture system
having a device or vessel with one or more tiers of cell growth
surfaces in fluid communication with one another. More
particularly, it relates to a flask system having multiple trays of
cell growth surfaces wherein cell growth media on each tray is
directly accessible through the same resealable port.
BACKGROUND OF THE INVENTION
[0003] Small-scale cell culture devices have existed in the form of
shaker flasks and roller bottles for years. More recently, single
layer and now multilayered flask bottles have been developed. The
current trend in research has demanded more cells to fulfill the
high throughput screen campaigns in drug discovery companies.
Additionally, the use of cell-based assays are rapidly increasing
because of the push to challenge potential drug candidates earlier
in the development process. A typical cell based assay or HTS
screening run can require from about 10.sup.9 to about 10.sup.10
cells. A standard single layered flask can deliver about 10.sup.7
cells. The current cell need requires researchers to maintain and
feed 10 to 100 standard flasks in order to reach a requisite number
of cells to run a given cell based assay or HTS screening, so new
multilayered formats are needed. Thus, it would be desirable to
have a cell culture system that provides a high number of cells
without a substantial increase in the number of standard flasks to
be maintained and fed.
[0004] One such product has two trays attached to each other and
inserted within a flask to form a three culture layered flask. The
flask is generally rectangular with a typical threaded bottle
opening at its forward most converging sidewall which is in the
form of bottle neck (See U.S. Pat. No. 5,310,676). Each tray has a
sidewall around its entire periphery and the sidewalls are sealed
to each other. In order to provide cell and liquid access to the
middle and lower tray one needs to build a tunnel along the end
wall of the trays or flask that liquidly interconnect the trays so
that liquid and cells can be flowed into and out of the trays.
[0005] Another product uses up to ten trays having each upper
surface covered by a gas permeable film. Each tray has a sidewall
extending upwardly from the surface. The trays are stacked together
and the sidewalls fused to form an integral mass. A manifold and
bottle neck sidewall with a typical threaded bottle opening is then
bonded to the front end of the trays to complete the flask. (See WO
2008/069902 A3). The manifold provides access to the opened end of
the trays.
[0006] The systems taught in U.S. Pat. No. 5,310,676 and WO
2008/069902 A3) each have drawbacks. Neither is easily accessible
with a pipette or syringe for the addition of fluid or cells,
sampling or removal of the cells upon completion of the growth
cycle. In fact, the use of a pipette or syringe is restricted
because the bottle opening is positioned on the sidewall and the
trays are blocking pipette access to the media and the cells. It
would be desirable to be able to pipette the media and cells
directly from a culture device, using standard pipetting tools and
standard cell culture techniques.
[0007] Application of cells and liquid is difficult and often
incomplete. U.S. Pat. No. 5,310,676 relies upon the molded in
tunnel to aid in its distribution which can be difficult and time
consuming. Liquid must be added to the flask in an upright position
which can lead to foaming and damage to critical proteins in the
media. Then the flask is rotated in various directions to ensure
that all trays receive an adequate amount of fluid. Likewise,
removal of the liquid and/or cells require passage through the
tunnel in order to be recovered. Lastly, the number of trays is
insufficient to effectively increase the high yields needed by
today's scientists.
[0008] WO 2008/069902 uses a manifold to distribute liquid and
cells into a flask by adding the liquid (containing cells and/or
growth media) to the neck portion of the flask. The neck is closed
and the flask is shaken tapped, or otherwise moved to dislodge any
air that would become trapped within the layers of the system.
Lastly as the space between the trays is very small and the gas
permeable film forms the upper limit of media on any given tray,
the thickness of the media is fixed at about 0.32 mls per cm.sup.2.
Depending on the cell types being cultured the media needs are
different. For example, a human or mouse stem cell is a highly
metabolically active cell and demands a high level of media,
typically in the range of 0.4 mls per cm.sup.2, whereas a slow
metabolizing cell line such as CHO, MDCK or fibroblast may only
require 0.2 mls per cm.sup.2. Since WO 2008/069902 has a fixed
media volume to fill the system, the researcher needs to adjust the
cell feeding, and maintain a schedule for each cell type being
cultured, or else waste expensive media and additives.
Additionally, scientists prefer to be able to vary the amount of
media they can use in order to optimize the growing conditions, a
parameter that WO 2008/069902 does not enable a scientist to
optimize. It would be desirable to provide a culture device having
layers that easily fill with media without the restriction of
tunnels, or the need to dislodge air entrapped within the
layers.
[0009] As the cells grow, the researcher routinely needs to access
the status of growth of the culture so that appropriate steps can
be taken, such as when the cells are near confluent the researcher
will detach them from the culture tray for use in assays, screens
or the like, or to reseed new culture systems to continue to expand
the cell line. It is important to recover the cells prior to 100%
confluency. These systems typically require the researcher to move
the culture system to an expensive microscope that may or may not
be in the culture area, to investigate the cell growth status. It
would desirable to be able to fill the culture device with the
desired amount of media and additives to satisfy the needs of the
cell type, as well as the researcher's work schedule. Additionally,
it would be desirable to provide a cell culture system that enables
visualization of the cells without having to transport the flask or
the like housing the cells to an expensive microscope.
[0010] Typically, laboratories that are working with high numbers
of cells as described, use a variety of automated equipment such as
multiwell plate handlers and liquid dispensing systems to increase
throughput and reduce data variability due to the operator error.
These culture systems can be automated, but are limited to systems
equipped with articulating arms that can grasp the system and pour
the liquid out into a receiver vessel. Systems with these complex
articulating arms are expensive and not available to most
laboratories. Therefore, most uses for the above culture systems
are limited to a manual operator manipulating the systems, which
runs counter to the work practices of high throughput laboratories.
Thus, it would be desirable to provide a cell culture system
compatible with the automated systems currently in most
laboratories, and not require special articulating arms to
automate.
SUMMARY OF THE INVENTION
[0011] The present invention provides a single or multitier cell
culture vessel which is easy to fill, easy to empty, easily
accessible by pipette or syringe, and provides high throughput
capabilities desired by the industry.
[0012] The present invention is based, at least in part, on a
multitier cell culture system including a device or vessel such as
a flask or the like, including a cover having a top plate, a side
wall, typically three or more, and a resealable port; an
intermediate tray for receiving a cell culture media, wherein the
intermediate tray includes a bottom plate, a side wall, typically
three or more, and a gap region formed between an interior upwardly
angled lip located on an interior portion of the intermediate tray
bottom plate, and an adjacent outwardly angled side wall portion of
the intermediate tray bottom plate, wherein the lip has a outwardly
swooping curvilinear interior edge feature; and a base tray for
receiving the cell culture media, including a bottom plate and a
side wall, typically three or more. The intermediate tray is
positioned between the cover and the base tray, such that the gap
region of the intermediate tray bottom plate is in alignment with
the port located on the cover, resulting in the port, the
intermediate tray and the base tray in fluid communication with one
another, which provides access, such as by a user, directly to the
cell media and cells located on each of the intermediate tray and
the base trays.
[0013] According to another embodiment, the present invention is
based, at least in part, on a multitier cell cultivating vessel or
device, such as a flask including a cover having a top plate, a
side wall, typically three or more, and a resealable port; a
plurality of intermediate trays stacked on top of one another for
receiving a cell culture media, each tray having a bottom plate
having an underside, an upperside, a side wall, typically three or
more, and a gap region formed between an interior upwardly angled
lip having a swooping curvilinear edge located on an interior
portion of the bottom plate and an adjacent outwardly angled side
wall portion of the bottom plate; and a base tray for receiving a
cell culture media having a bottom plate and a side wall, typically
three or more. The plurality of intermediate trays are positioned
between the cover and the base tray, and the gap regions of each
intermediate tray are in alignment with each other, and with the
port on the cover, resulting in the port, the stacked plurality of
intermediate trays and the base tray in fluid communication with
one another providing direct access to the cells and cell media
located on each of the intermediate and base trays.
[0014] According to another embodiment, the present invention is
based, at least in part, on a cell cultivating vessel or device,
such as a flask having a bottom and a top wall joined together by
one or more sidewalls, typically three or more, which are
substantially perpendicular to the top and bottom walls. The top
has a threaded opening on to which a vented cap is threadably
mated, wherein the top wall is preferably substantially planar. An
interior space is defined by the walls. The bottom wall has at
least two portions, preferably three; a first portion in one plane,
a second portion in a second plane above, and preferably parallel,
to the plane of the first portion and an interconnecting portion
which is at an angle to interconnect the first portion to the
second portion. In some embodiments, the interconnecting portion is
incorporated into the second portion such that the area of the
second portion adjacent the first portion is in the plane of the
first portion and the area of the second portion beyond that is at
an upward angle away from the first plane to a desired point or
second plane. The bottom walls extend out from the outside bottom
planar surface to form a perimeter skirt. The perimeter skirt forms
a linear transition to the end wall. The linear skirt transition
creates an angle such that when the cell culture system is in
position, the linear transitional skirt lies flat onto a work
surface such that all the internal plane portions (one, two, three
and four) are positioned at a positive angle such that liquid on
those surfaces will drain towards the access port end of the
culture system. This feature enables full recovery of spent media
during media changes and complete recovery of the cells post
culture. Trays are placed in the interior space, preferably only in
the area defined by the first portion of the bottom wall and a
portion of the interconnecting portion if used.
[0015] One object of the present invention is to provide a
multitier cell culture device that permits a user to pipette the
cell media and cells directly from the device, using standard
pipette tools and standard cell culture techniques.
[0016] It is another object of the present invention to provide a
cell culture system that provides a high number of cells without an
increase in the number of standard flasks to be maintained and
fed.
[0017] Another object of the present invention to provide a cell
culture system compatible with the automated systems currently used
in most laboratories, and not require special articulating arms to
automate.
[0018] A further object of the present invention to provide a cell
culture system that provides a multitier cell culture device
wherein each tier or tray easily fills with media without the
restriction of tunnels, or the need to dislodge air entrapped
within the separate levels.
[0019] Another object of the present invention to provide a
multitier cell culture system that provides a cell culture device
that can be easily filled or emptied with the desired amount of
media, additives and cells to satisfy the needs of the cell type,
as well as a researcher's work schedule.
[0020] It is another object of the present invention to provide a
cell culture system that provides visualization of the cells within
the culture device without having to transport the flask or device
housing the cells to an expensive microscope or other type of
optical viewing instrument.
[0021] Additional features and advantages of the invention will be
set forth in the detailed description which follows. Many
modifications and variations of this invention can be made without
departing from its spirit and scope, as will be apparent to those
skilled in the art. It is to be understood that both the foregoing
general description and the following detailed description, the
claims, as well as the appended drawings are exemplary and
explanatory only, and are intended to provide an explanation of
various embodiments of the present teachings. The specific
embodiments described herein are offered by way of example only and
are not meant to be limiting in any way.
IN THE DRAWINGS
[0022] FIG. 1 shows a cross-sectional view of a first embodiment of
the present invention.
[0023] FIGS. 2a and 2b show cross-sectional views of an additional
embodiment of the present invention.
[0024] FIG. 3 shows a cross-sectional view of first embodiment of
FIG. 1 in the unloading position of the present invention.
[0025] FIG. 4 shows a cross-sectional view of a series of first
embodiments of the present invention in stacked formation.
[0026] FIGS. 5A-E shows a top down cross-sectional view of various
sidewall embodiments according to the present invention.
[0027] FIGS. 6A-6B show perspective views of an additional
embodiment of the present invention.
[0028] FIG. 7 shows a perspective view of an additional embodiment
of the present invention.
[0029] FIG. 8 shows another perspective view of an additional
embodiment of the present invention.
[0030] FIG. 9 shows a perspective view of an additional embodiment
of the present invention.
[0031] FIG. 10 shows a perspective view of the individual
components of the embodiment in FIG. 10.
[0032] FIG. 11 shows an exploded view of an additional embodiment
of the present invention depicted in FIG. 10.
[0033] FIG. 12a shows a side view of the embodiment of the present
invention depicted in FIG. 11.
[0034] FIG. 12b shows a cross-sectional view of the embodiment of
the present invention depicted in FIG. 11.
[0035] FIG. 13 shows a perspective view of an additional embodiment
of the present invention.
[0036] FIG. 14 shows a perspective view of an additional embodiment
of the present invention.
[0037] FIG. 15 shows a perspective view of an additional embodiment
of the present invention filled with cell culture media.
[0038] FIG. 16a shows a perspective view of an additional
embodiment of the present invention containing cell culture media
equally filling the trays.
[0039] FIG. 16b shows a perspective view of the present invention
depicted in FIG. 15 in a tipped position in order to spread the
cell culture media throughout the trays.
DETAILED DESCRIPTION
[0040] For the purposes of this specification and appended claims,
unless otherwise indicated, all numbers expressing quantities of
ingredients, percentages or proportions of materials, reaction
conditions, and other numerical values used in the specification
and claims, are to be understood as being modified in all instances
by the term "about".
[0041] Accordingly, unless indicated to the contrary, the numerical
parameters set forth in the following specification and attached
claims are approximations that may vary depending upon the desired
properties sought to be obtained by the present invention. At the
very least, and not as an attempt to limit the application of the
doctrine of equivalents to the scope of the claims, each numerical
parameter should at least be construed in light of the number of
reported significant digits and by applying ordinary rounding
techniques.
[0042] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. Any numerical value, however,
inherently contains certain errors necessarily resulting from the
standard deviation found in their respective testing measurements.
Moreover, all ranges disclosed herein are to be understood to
encompass any and all subranges subsumed therein. For example, a
range of "1 to 10" includes any and all subranges between (and
including) the minimum value of 1 and the maximum value of 10, that
is, any and all subranges having a minimum value of equal to or
greater than 1 and a maximum value of equal to or less than 10,
e.g., 5.5 to 10.
[0043] As used herein, the singular forms "a," "an," and "the"
include plural referents unless the context clearly dictates
otherwise.
[0044] FIG. 1 shows an embodiment of the present invention, wherein
flask 2 preferably comprises a transparent, translucent or
nontransparent glass or plastic material. The flask 2 has a top
wall 4, a bottom wall 6 and three or more sidewalls 8 which extend
substantially perpendicular to and between the top wall 4 and the
bottom wall 6 to define an interior space 10. As shown the bottom
wall 6 and sidewalls 8 are formed as one piece. They may if desired
be formed of individual pieces if desired. Top wall 4 is liquid
tightly sealed to the upper edge 12 of the sidewalls 8 such as by
heat bonding, melt bonding, sonic vibration welding or adhesives.
Optionally as in this embodiment, the upper edge 12 of the
sidewalls has a feature, in this case a ridge 14 that mates with a
corresponding feature in this case a trough 16 in the bottom or
inner surface 18 of the top wall 4 so that the walls are properly
aligned and sealed together. If the top wall is desired to be
removal the top wall 4 can be secured to the sidewalls 8 by other
well-known means such as screws, clamps, sliding dovetails and the
like (not shown).
[0045] The top wall 4 has a port 20 that can be selectively opened
and closed. One such means for selectively opening and closing the
port 20 is a cap 22 which can and preferably has a vent 24 that
allows for the transfer of gases into and out of the interior 10
without contamination. This can be accomplished by using a frit,
metal such as stainless steel or plastic such as a POREX.RTM. frit
or a hydrophobic membrane or filter, all of which have a pore size
designed to keep out bacteria, dust and other such contaminants. A
typical pore size used in such a frit or filter or membrane is less
than about 0.65 micron, preferably less than about 0.4 micron and
more preferably about 0.22 micron.
[0046] As shown the bottom wall 6 in this embodiment has a first
substantially flat portion 24 and a second substantially portion 26
connected to each other by a substantially planar interconnecting
portion 28 that is on an angle so as to connect the two portions 24
and 26 together. The first portion 24 is at one desired horizontal
plane that is below that second horizontal plane of the second
portion 26. This means that when in its use position as shown in
FIG. 1, the first portion 24 is the lowermost portion of the
interior 10 of the device 2.
[0047] A portion of the interior 10 contains one or more cell
growing trays 30. These trays have a substantially flat bottom 32
and sidewalls 34 that run around the periphery of the tray 30. The
number of sidewalls 34 of the tray(s) 30 is equal in number to the
number of sidewalls of the flask 2. The front sidewall 36 of the
tray(s) is different than the rest of the sidewalls 34 of the
tray(s) 30 in that it is at upward angle away from the
substantially flat bottom 32 of each tray 30. The front sidewall 36
or lip provides open access to each tray for cells, liquids and
gases when they 30 are arranged within the flask 2. Also shown on
each tray 30 is an optional feature 38 which is a foot that extends
outwardly from the front sidewall 36 of each tray 30.
[0048] The bottom walls extend a distance from the outside bottom
planar surface to form a perimeter skirt. The perimeter skirt forms
a linear transition to the end wall. The linear skirt transition
creates an angle that when the culture system is in position the
transitional skirt flat onto a work surface all the internal plane
portions (one, two, three and four) are positioned at a positive
angle so that liquid on those surfaces will drain toward the access
port end of the culture system. The feature enables full recovery
of spent media during media changes and complete recovery of the
cells post culture.
[0049] The tray(s) 30 are spaced about the bottom wall 6 of the
flask 2 so that the inner surface 44 of the bottom wall acts as a
tray. In this embodiment the tray(s) are spaced from the bottom
surface 44 by detents formed on the opposite sidewalls 8. These
detents or rests extend outwardly into the interior 10 to an extent
sufficient to support the tray(s) in the interior 10. Typically
they can extend outwardly from the sidewalls into the space by a
distance of from about 3 mm to about 7 mm. Alternatively, the
bottommost tray 30 may have feet (not shown) formed on its bottom
surface 32 to provide the necessary spacing with the inner bottom
surface 44 of the interior 10. In another embodiment if the bottom
surface 44 is not desired as a cell growth layer, no detent or feet
are needed and the tray(s) bottom 32 may contact the bottom surface
44.
[0050] Preferably, the tray(s) 30 are simply stacked onto of each
other and retained in the flask by the spacing considerations,
making them narrow enough so that the tray(s) 30 once inserted
cannot disassociate from each other. Optionally, they may be sealed
to each other such as by the use of adhesives or heat bonding and
the like, or they may have a strap such as one or more tie wraps or
cable wraps (not shown) placed around them, or they may contain
sliding dovetails or snap fits (not shown) between their adjacent
surfaces to hold them together.
[0051] The angle of the interconnecting portion 28 is equal to or
greater than the angle of the front sidewall 36 of each tray 30 so
that when moved to its loading/unloading position as described in
FIG. 2 the liquid and cells can flow to their appropriate
locations. Typically, the angle of the interconnecting portion 28
is from about 10 degrees to about 60 degrees as measured by the
angle alpha 46 between the first plane of the first portion 24 and
the outer bottom surface 48 of the interconnecting portion 28.
Preferably the angle alpha 46 is between about 10 degrees and about
45 degrees, more preferably from about 15 degrees to about 30
degrees and most preferably about 22 degrees. The angle of the
front sidewall 36 as described above is equal the angle alpha 46 or
less than the angle alpha 46 but preferably is never greater than
the angle alpha 46. Additionally, top wall 4 may optionally have a
similar third portion 29 (as shown) that corresponds to the angle
alpha in a position directly above the interface between the first
portion 24 and the interconnecting portion 28 of the bottom wall 6.
This is a preferred option so that the space between the top tray
and the inner surface of the top wall 4 is the same as the space
between any other tray (if more than one is used) and the adjacent
surface above it (the bottom surface of the try above it for
example). Alternatively the top wall 4 may be substantially planar
across its length and the space between the top tray and the top
wall can be suitably enlarged if necessary to provide suitable flow
of liquids and gases into that tray (not shown).
[0052] The tray(s) 30 are contained within the interior 10 to the
area circumscribed by the first portion 24 and preferably at least
a portion of the interconnecting portion 28 of the bottom wall 6.
This leaves an open area 41 in the interior 10 around and adjacent
to the port 20 for the entrance and exit of liquids, cells and
gases. In the embodiment where the tray(s) 30 are not bonded or
otherwise secured to the bottom or sidewalls 6,8 of the flask 2,
foot 38 acts as a means for preventing the tray(s) from moving into
the open area 41 when the flask is tilted for unloading or if
desired for loading as well.
[0053] FIG. 2 shows the flask 2 when in its unloading position.
This may also be the loading position is desired. As shown, the
flask 2 is lifted at its rear 50 and tilted forward on its foot 42
that extends downwardly from the sidewall 8 adjacent the port 20. A
pipette or syringe or funnel (pipette 52 is shown) can be inserted
into the port 20 and have access to the open area 41 of the
interior 10. The second portion 26 when in the unloading optionally
loading position becomes the low point of the flask 2. This allows
one to easily recover cells or exchange media or add new media or
cells into the open area 41 without disturbing the tray(s) or
requiring one to tip the flask vertically as is required by the
prior art.
[0054] FIG. 3 shows an alternative embodiment of the design of
FIGS. 1 and 2. In this embodiment, the second portion becomes a
part of the interconnecting portion 28 such that there is a flat
first portion of the bottom 6 and only an angled interconnecting
portion 28 that meets and terminates in the front sidewall 54. The
second portion 26 in effect becomes the point at which the
interconnecting portion 28 meets the front sidewall 54. The device
2 is used the same way as described as in FIG. 2. All elements
common to FIGS. 1 and 3 retain the same reference numbers, perform
the same functions, and have the same attributes as described in
reference to the embodiment of FIG. 1.
[0055] FIG. 4 shows a series of flasks 2 according to the present
invention. Due to their design with the relatively flat bottom
portions 24 and top wall 4 they can be alternately stacked on top
of each other to save floor or hood space during incubation and
growth.
[0056] As previously described the flask may have three or more
sidewalls 8. FIGS. 5A-D show three different sidewall embodiments
of the flask 2 to illustrate this feature. FIG. 5A shows a
triangular-shaped flask 2 having three sidewalls 8A. FIG. 5B shows
a polygonal sidewall configuration of four more sidewalls with
sidewalls 8B and 8D being linear or straight sidewalls and
sidewalls 8C being angle or tapered or curved sidewalls. Sidewalls
8c may be an extension of sidewalls 8B in which case there are 3
sidewalls or they may be separate sidewall portions in which the
flask has 5 sidewalls in this embodiment. FIG. 5C shows a
rectangular-shaped design with four sidewalls 8D-8G. FIG. 5D shows
another design with five sidewalls 81-8M. FIG. 5E shows a single
circular sidewall 8N.
[0057] The culture system of the invention, when in use, includes a
flask filled and/or emptied by a pipette, syringe or similar
device, having the culture system positioned on a work surface and
having the threaded opening in an upward position. The researcher
dispenses the media and cells into the system. The media amount can
vary depending on the cell type being cultured, i.e., more media
for highly metabolic cells such as stem cells. The researcher seals
the system with a gas permeable closure, such as a threaded cap
with a hydrophobic bacterial retentive microporous matrix enabling
free exchange of gas from outside of the system to the inside. The
culture system is tipped to its side, so that the media freely
fills the layers insuring a uniform amount between each tray. The
system is tipped on to the side wall opposite the closure then
tipped forward, resulting in the culture system seated with the
first and third portion surfaces substantially planar to the work
surface. In this position the media and cells spread uniformly
across each culture layer and the first portion surface. This
readied culture system is typically placed into an environmental
controlled chamber, incubation, for the cell growth phase.
[0058] The researcher needs to periodically investigate the status
of the culture during the growth phase. This activity typically
consists of the culture system being removed from the incubator and
transported to a microscope that may or may not be in close
proximity. In one embodiment, as shown in FIGS. 6A and 6B, flask 2
additionally includes a lens 54 that be may be molded in the top
wall or the bottom walls (not shown) with the focal surface being
the first adjacent culture layer. Ideally, the magnification from
this lens is 10.times. to 40.times. for low level viewing of the
cell culture. Preferably, the magnification is 25.times. to
40.times. so that visualization of cell growth status and cell
detachment during cell recovery is visible with the naked eye.
Additionally, a camera system 56 such as the Dino-Lite system
(BigC.com Corp., Torrance, Calif.) can be secured via a coupling
mechanism or mounting system 52 to the top wall 4 or the bottom
wall (not shown) to provide image capture. Ideally, this
configuration is maintained within the incubator such that
monitoring can occur without a researcher having to enter the
incubator and disrupt the growth phase of the cells. Additionally,
the use of a wireless connection permits the remote monitoring of
the cell culture. (not shown)
[0059] In another embodiment, as shown in FIG. 7, flask 2 may also
include one or more of the following features a) an overmolded seal
plastic seal 57 around the perimeter of the flask; b) a non-skid,
preferably plastic, button 55 to secure the flask 2 from slipping
during the recovery of spent media, cell post cultures or the like
when the flask is tipped; c) overmolded plastic ribs or other such
feature that provides a user with a non-slip gripping or handling
surface. In a preferred embodiment, the plastic used to make these
features comprises a thermoplastic elastomer (TPE) with a hard
durometer greater than 49 shore A in order to provide the user an
appropriate tactile feel for gripping or otherwise handling the
flask.
[0060] In another alternative embodiment, as shown in FIG. 8, flask
2 may also include an easy to open tear-away element 60 or the like
in one of the sidewalls 8, wherein the easy to open tear-away
element 60 is preferably a component of the overmolded seal 57.
[0061] In another alternative embodiment, as shown in FIGS. 9 and
11, a multitier cell cultivating multitier flask 100 comprising a
cover 94 including a top plate 98, side walls 96 and a resealable
port 20; an intermediate tray 92 for receiving cells and a cell
culture media 105, the intermediate tray having a bottom plate 95,
side walls 97, and a gap region 89 formed between an interior
upwardly angled lip 78 having a swooping curvilinear interior edge
99 located on an interior portion of the bottom plate and an
adjacent outwardly angled side wall 87 of the bottom plate; and a
base tray 90 for receiving cells and a cell culture media,
including a bottom plate 93 and side walls 91, wherein the tray is
positioned between the cover and the base tray, such that the gap
region of the bottom plate of the tray is in alignment with the
port located on the cover, resulting in the cover, the tray and the
base tray in fluid communication with one another which enables a
user to directly access each of the cell media located on the tray
and the base tray.
[0062] In another alternative embodiment as depicted in FIGS. 9 to
16, a cell cultivating flask 100 includes a cover 94 having a top
plate 98, one or more side walls 96 and a resealable port 20; a
plurality of intermediate trays 92 for receiving cells and a cell
culture media 105, each intermediate tray 92 having a bottom plate
95, one or more side walls 97, and a gap region 89 formed between
an interior upwardly angled lip 78 having a swooping curvilinear
interior edge 99 located on an interior portion of the bottom plate
and an adjacent outwardly angled side wall 87 of the bottom plate;
and a base tray 90 for receiving cells and a cell culture media,
having a bottom plate 93 and one or more side walls 91. The
plurality of intermediate trays 92 are positioned between the cover
94 and the base tray 90, such that the plurality of intermediate
trays are stacked on top of one another, and the gap regions of the
bottom plates 95 of each intermediate tray are in alignment with
each other and with the port located on the cover, resulting in the
port, the plurality of intermediate trays and the base tray in
fluid communication with one another which enables a user to
directly access each of the cell media located on the plurality of
intermediate trays and base tray, with a standard pipetting device
and the like.
[0063] As shown in FIG. 13, the side walls 97 on each intermediate
tray 92 stacked on top of one another are preferably fused to form
a liquid tight seal 135, to the underside of the side walls 97 on
next adjoining intermediate tray. In one embodiment, the side walls
on each intermediate tray stacked on top of one another are
ultrasonically welded 135 to the underside of the side walls of the
next adjoining intermediate tray 92; the side wall of the
intermediate tray 92 adjacent the cover 94 is ultrasonically welded
145 to the underside of the side wall of the cover 94, and the side
wall of the base tray 90 is ultrasonically welded 155 to the
underside of the side wall of the adjoining intermediate tray
92.
[0064] The resealable port 20 can be selectively opened and closed.
One such means for selectively opening and closing the port 20 is a
cap 22, which can be threaded and preferably has a vent 24 that
allows for the transfer of gases into and out of the interior 10
without contamination. This can be accomplished by using a frit,
metal such as stainless steel or plastic such as a POREX.RTM. frit
or a hydrophobic membrane or filter, all of which have a pore size
designed to keep out bacteria, dust and other such contaminants. A
typical pore size used in such a frit or filter or membrane is less
than about 0.65 micron, preferably less than about 0.4 micron and
more preferably about 0.22 micron.
[0065] In another alternative embodiment, as shown in FIGS. 9 to
16, a multitier cell cultivating flask 100, 110, 115 includes a
bottom wall 88 of a base plate 90 having a first substantially flat
portion 86 and a second substantially flat portion 84 connected to
each other by a substantially planar interconnecting portion 81
that is on an angle so as to connect the portions 84 and 86
together.
[0066] As depicted in FIGS. 12a and 12b, base plate 90 includes
first portion 86 configured as a substantially horizontal plane
that is below the second substantially horizontal plane of the
second portion 84 such that media, cells and the like 105 will
evenly disperse along the upper surface of base plate 90 because
angled portion 81 prevents cells and media 105 from pooling on
portion 84. When the flask 110 is in use as depicted in FIGS. 12a,
12b and 15, first portion 86 of base plate 90 is the lowermost
portion of the interior 120 of the device 110.
[0067] As depicted in FIGS. 16a and 16b, when flask 110 is placed
on one side as shown in FIG. 16a each tray equally fills with media
105. Alternatively, when flask 110 is placed in an upright position
as shown in FIG. 16b, each tray equally fills with media 105
again.
[0068] As shown in FIGS. 10, 12a and 12b, a portion of the interior
120 contains one or more cell growing trays 92. These trays have a
substantially flat bottom portion 76, a substantially planar lip
portion 78 that is on an upward angle away from the substantially
flat bottom 76 of each tray 92, and sidewalls 97 that run around
the periphery of the tray 92. The lip portion 78 has an interior
edge portion 99. The number of sidewalls 97 of the tray(s) 92 is
equal in number to the number of sidewalls of the flask 110. The
front sidewall 87 of the tray(s) is different than the rest of the
sidewalls 97 of the tray(s) 92 in that it is angled outward and
away from the interior swooping curvilinear edge 99 of the lip 78
of the bottom portion of the trays. The front sidewall 87 and the
interior curvilinear edge 99 of the lip portion 78 provides open
access or a gap region 89 to each tray for cells, liquids and gases
105 when arranged within the flask 110.
[0069] The disclosure set forth above may encompass multiple
distinct inventions with independent utility. Although each of
these inventions has been disclosed in its preferred form(s), the
specific embodiments thereof as disclosed and illustrated herein
are not to be considered in a limiting sense, because numerous
variations are possible. The subject matter of the inventions
includes all novel and nonobvious combinations and subcombinations
of the various elements, features, functions, and/or properties
disclosed herein. The following claims particularly point out
certain combinations and subcombinations regarded as novel and
nonobvious. Inventions embodied in other combinations and
subcombinations of features, functions, elements, and/or properties
may be claimed in applications claiming priority from this or a
related application. Such claims, whether directed to a different
invention or to the same invention, and whether broader, narrower,
equal, or different in scope to the original claims, also are
regarded as included within the subject matter of the inventions of
the present disclosure.
* * * * *