U.S. patent application number 11/893430 was filed with the patent office on 2008-02-07 for automated bioculture and bioculture experiments system.
Invention is credited to Thomas F. Cannon, Laura K. Cohn, Peter D. Quinn.
Application Number | 20080032398 11/893430 |
Document ID | / |
Family ID | 27398907 |
Filed Date | 2008-02-07 |
United States Patent
Application |
20080032398 |
Kind Code |
A1 |
Cannon; Thomas F. ; et
al. |
February 7, 2008 |
Automated bioculture and bioculture experiments system
Abstract
The present invention provides a feedback controlled bioculture
platform for use as a precision cell biology research tool and for
clinical cell growth and maintenance applications. The system
provides individual closed-loop flowpath cartridges, with
integrated, aseptic sampling and routing to collection vials or
analysis systems. The system can operate in a standard laboratory
or other incubator for provision of requisite gas and thermal
environment. System cartridges are modular and can be operated
independently or under a unified system controlling architecture,
and provide for scale-up production of cell and cell products for
research and clinical applications. Multiple replicates of the
flowpath cartridges allow for individual, yet replicate cell
culture growth and multiples of the experiment models that can be
varied according to the experiment design, or modulated to desired
cell development of cell culture end-points. The integral flowpath
cartridge aseptic sampling system provides for dynamic analysis of
metabolic products or representative cells from the culture.
Inventors: |
Cannon; Thomas F.;
(Glenwood, MD) ; Cohn; Laura K.; (Alexandria,
VA) ; Quinn; Peter D.; (Washington, DC) |
Correspondence
Address: |
THELEN REID BROWN RAYSMAN & STEINER LLP
P. O. BOX 640640
SAN JOSE
CA
95164-0640
US
|
Family ID: |
27398907 |
Appl. No.: |
11/893430 |
Filed: |
August 15, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09967995 |
Oct 2, 2001 |
7270996 |
|
|
11893430 |
Aug 15, 2007 |
|
|
|
60236733 |
Oct 2, 2000 |
|
|
|
60236702 |
Oct 2, 2000 |
|
|
|
60236703 |
Oct 2, 2000 |
|
|
|
Current U.S.
Class: |
435/303.1 ;
435/283.1; 435/309.1; 435/317.1 |
Current CPC
Class: |
C12M 23/42 20130101;
Y10S 435/809 20130101; C12M 23/48 20130101; C12M 37/00 20130101;
C12M 29/10 20130101; C12M 41/00 20130101; C12M 29/04 20130101 |
Class at
Publication: |
435/303.1 ;
435/283.1; 435/309.1; 435/317.1 |
International
Class: |
C12M 3/00 20060101
C12M003/00; C12M 1/26 20060101 C12M001/26; C12N 5/02 20060101
C12N005/02 |
Claims
1. An incubator rack for supporting a plurality of cell culture
perfusion flowpath assembly cartridges, comprising: a horizontal
base having a surface with a plurality of grooves each adapted to
support a flowpath assembly cartridge; a plurality of data
interface connections for transmitting data between said rack and
said plurality of cartridges; and at least one control interface
for communication between said rack and an external computer.
2. The incubator rack of claim 1, further comprising a vertical
wall connectable to said horizontal base, wherein said vertical
wall is perpendicular to said plurality of grooves.
3. The incubator rack of claim 1, wherein said rack further
comprises a means for preventing a specific cartridge from being
inserted into more than one location on said rack.
4. The incubator rack of claim 1, wherein the rack further
comprises a fan.
5. The incubator rack of claim 1, wherein the rack further
comprises a vibration dampening system.
6. A biochamber convertible for use in static cell culture or in a
cell perfusion apparatus, said biochamber comprising: a first
chamber; a cover; a sealing means for rendering said first chamber
removably connectable to said cover and for sealing said cell
culture within said biochamber from biologic contaminants; at least
one insert positioned between the first chamber and the cover,
thereby forming a second chamber; and wherein said first chamber
and said second chamber are each in fluid communication with a
media stream during perfusion.
7. The biochamber of claim 6, wherein said seal means comprises a
means for verifying seal integrity.
8. The biochamber of claim 6, wherein said insert comprises a
material selected from the group consisting of: a hollow fiber
bundle; a plastic surface; a three dimensional matrix; and an
optically reflective surface.
9. The apparatus of claim 8, wherein said seal means comprises two
or more sealing interfaces.
10. The apparatus of claim 8, wherein said biochamber further
comprises at least one air gap between said two or more sealing
interfaces.
11. The apparatus of claim 8, wherein said two or more sealing
interfaces are capable of indicating seating of said interfaces by
a color change.
12. The biochamber of claim 6, further comprising at least one
means for diffusing media flow within said biochamber.
13. The biochamber of claim 6, further comprising a sampling
port.
14. The biochamber of claim 6, further comprising a n injection
port.
15. The biochamber of claim 6, further comprising a sampling
interface.
16. A sampling device for use with a cell culture perfusion loop,
said sampling device comprising: a fluidic pump for transporting a
routing fluid; a one way flow valve; a valve for diverting an
aliquot of sample from said perfusion loop to said one way flow
valve; wherein said fluidic pump and one way flow valve are
connected for transporting said routing fluid to said one way
valve; and a means for transporting said aliquot of sample and said
routing fluid from said one way flow valve to a collection device
or analysis instrument.
17. The automated sampling apparatus of claim 16, wherein said
routing fluid is air.
18. The automated sampling apparatus of claim 16, further comprises
at least one filter.
19. A sampling device for obtaining a sample from a cell culture
perfusion loop having: a pump means for causing a routing fluid to
be transported through tubing; a means for releasing an aliquot of
sample from said perfusion loop; a valve means, wherein said pump
means and said valve means are connected for transporting said
routing fluid; and a means for transporting said sample within said
routing fluid from said valve means to a sample collection device
or analysis instrument.
20. A method of culturing cells comprising: providing a biochamber
convertible for use in static cell culture or in a cell perfusion
apparatus, said biochamber comprising: a first chamber; a cover; a
seal rendering said first chamber removably connectable to said
cover; and at least one insert positioned between the first chamber
and the disposable cover, thereby forming a second chamber;
providing a biological cell; introducing said biological cell into
said biochamber; placing said biochamber in an incubator under gas
and temperature conditions permitting said cell to propagate to
achieve a desired cell density within said biochamber; inserting
said biochamber containing said desired density of cells into a
cell perfusion apparatus, such that said first chamber and said
second chamber are in fluid communication with medium flowing
through said cell perfusion apparatus; and operating said cell
perfusion apparatus under conditions permitting said cell to
propagate.
21. The method of claim 20, further comprising: removing said
biochamber from said cell perfusion apparatus; disconnecting said
first chamber from said cover; and harvesting said propagated cells
or a cell product from said biochamber.
22. The method of claim 21, wherein said cell product is removably
anchored to said insert.
23. The method of claim 21, wherein said cell product is in
suspension.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 09/967,995, filed Oct. 2, 2001 which claims the benefit of U.S.
Provisional Application Nos. 60/236,733, 60/236,702 and 60/236,703,
each filed Oct. 2, 2000.
FIELD OF THE INVENTION
[0002] The field of the invention is automated cell culture
systems, cell culture growth chambers and automated sampling
systems.
BACKGROUND OF THE INVENTION
[0003] Cell culture has been utilized for many years in life
science research in an effort to better understand and manipulate
the cellular component of living systems. Cells are typically grown
in a static environment on petri dishes or flasks. These cell
culture methods are very labor-intensive especially when a large
number of studies need to be performed.
[0004] Traditional cell culture systems depend on controlled
environments for cell maintenance, growth, expansion, and testing.
Typical cell culture laboratories include laminar flow hoods,
water-jacketed incubators, controlled access by gowned personnel,
and periodic sterilization procedures to decontaminate laboratory
surfaces. Personnel require extensive training in sterile
techniques to avoid contamination of containers and cell transfer
devices through contact with non-sterile materials. Despite these
measures, outbreaks of contamination in traditional cell culture
laboratories, e.g., fungus or bacterial contamination, commonly
occur, often with the impact of compromising weeks of research and
halting operations for days or weeks.
[0005] Trained technicians under a sterile, laminar flow hood
typically perform cell culture. Cells are grown in flasks or
bioreactors and maintained in incubators that provide the requisite
thermal and gas environment. Cultures are removed from incubators
and transported to a sterile hood for processing. Cells can be
harmed when removed from their thermal and gas environment. The
constant transport and manipulation of the culture represents an
opportunity for contamination that can cause weeks of work to be
wasted from a single bacterium. Traditional cell culture is very
labor intensive and uses a steady stream of sterile, disposable
products for each experiment. The nutrient cell culture medium
includes a color indicator that is visually inspected by the
technician on a daily basis, at a minimum. When the color is deemed
to indicate that the pH is falling out of healthy range the cells
are removed from the incubator, the old media is manually removed
and fresh media is injected. This process is adequate at best.
[0006] Perfusion systems provide a three-dimensional cell culture
environment that reproduces critical aspects of the dynamic in vivo
environment. In vitro perfusion systems allow tissue-engineered
cells to develop and organize as if inside the body. Biotechnology
companies, universities, and research institutes are attempting to
develop complex tissue replacements including liver, pancreas, and
blood vessels, among others. These complicated tissue products
require advanced biochamber perfusion systems that are capable of
mimicking in vivo development dependent stimulation. A perfusion
cell culture system's primary purpose is to provide a pump that
will continuously re-circulate medium. Standard experiment
manipulations, such as media replacement (when it is no longer at
the proper pH), cell and media sampling, and fluid injections, are
performed by a laboratory technician in a sterile hood. In an age
where genetically engineered products will be FDA approved and drug
compound costs are hundreds of millions of dollars, the traditional
way of performing cell culture is no longer acceptable.
[0007] One critical issue to be addressed in any cell culture
application involves precision reproducibility and the elimination
of site-to-site differences so that cell products and experiments
will be consistent in different biochambers or different physical
locations. This is particularly difficult to accomplish when
culture viability is determined solely on visual cues, i.e., medium
color and visualization under a microscope.
[0008] In a purely manual environment, quality control is
accomplished by selecting qualified personnel, providing them with
extensive training, and developing a system of standard operating
procedures and documentation. In an automated environment, the
principles of process validation are used to demonstrate that the
process is precise, reliably consistent, and capable of meeting
specifications. The principles of statistical process control are
then implemented to monitor the process to assure consistent
conformance to specifications.
[0009] The particular physical and biological requirements for the
growth and modification of cells and tissues of interest vary.
However, two key components are necessary in order to grow any of
these cells and tissues: cells that are capable of replicating and
differentiating, as needed, and an in vitro system containing
biocompatible materials that provide for the physiological
requirements for the cells to grow, such as surface attachment,
medium exchange, and oxygenation. These systems should be automated
and amenable for routine use by the thousands of research
laboratories, universities, tissue engineering companies,
hospitals, and clinics that perform research requiring consistent
and reliable results and also those that serve patients intended to
benefit from transplantation cells and tissues in native or
genetically altered form without adversely affecting product
quality and, particularly, product sterility.
[0010] Cell and organ transplantation therapy to date has typically
relied on the clinical facility to handle and process cells or
tissues through the use of laboratory products and processes
governed to varying degrees by standard operating procedures and
with varying regulatory authority involvement. The procedures to
date, however, generally have not required extensive manipulation
of the cells or tissue beyond providing short term storage or
containment, or in some cases, cryopreservation. With the addition
of steps that require the actual growth and production of cells or
tissues for transplantation, medium replacement, sampling,
injections of drug/compound dosing, physiologic and set-point
monitoring, and quality assurance data collection, there are many
considerations that need to be addressed in order to achieve a
reliable and clinically safe process. This issue is the same
regardless of whether the cell production is occurring at the
patient care location, as might be the case for the production of
cells for a stem cell transplant, or at a distant manufacturing
site, as might be the case for organ and tissue engineering
applications.
[0011] Platform-operated culture systems, typically referred to as
bioreactors, have been commercially available. Of the different
bioreactors used for mammalian cell culture, most have been
designed to allow for the production of high density cultures of a
single cell type. Typical application of these high density systems
is to produce a conditioned medium produced by the cells. This is
the case, for example, with hybridoma production of monoclonal
antibodies and with packaging cell lines for viral vector
production. These applications differ, however, from applications
in which the end-product is the harvested tissue or cells
themselves. While traditional bioreactors can provide some
economies of labor and minimization of the potential for
mid-process contamination, the set-up and harvest procedures
involve labor requirements and open processing steps, which require
laminar flow hood operation (such as manual media sampling to
monitor cell growth). Some bioreactors are sold as large benchtop
environmental containment chambers to house the various individual
components that must be manually assembled and primed.
Additionally, many bioreactor designs impede the successful
recovery of expanded cells and tissues and also can limit
mid-procedure access to cells for purposes of process monitoring.
Many require the destruction of the bioreactor during the
harvesting process.
[0012] It should therefore be appreciated that within tissue
engineering companies, cellular therapeutic companies, research
institutions, and pharmaceutical discovery companies there is a
need for an automated cell and tissue culture system that can
maintain and grow selected biological cells and tissues without
being subject to many of the foregoing deficiencies. There also is
a need for a lower cost, smaller, automated research and
development culture system which will improve the quality of
research and cell production and provide a more exact model for
drug screening.
SUMMARY OF THE INVENTION
[0013] The present invention provides a precision bioculture
support system, including a cell culture apparatus for use within
an incubator. The apparatus preferably includes at least one media
flowpath assembly cartridge having an outer shell or housing and
affixed thereto, a pump, at least one valve adapted to prevent or
divert media flow, a control interface, and a disposable sterile
media perfusion flowpath loop. The media perfusion loop is
removably attachable to the outer shell without breaching flowpath
sterility, and contains, in fluid communication, at least one
biochamber, a tubing in contact with the pump, at least one tubing
in contact with the valve, a gas permeable membrane exposed to
ambient air, and a media reservoir. In a preferred embodiment, each
cartridge has a control interface and battery pack or other power
source for stand alone operation. In another preferred embodiment,
the apparatus further includes an incubator rack that is removably
integratable with a plurality of flowpath assembly cartridges
without breaching flowpath sterility.
[0014] Another embodiment of the invention provides an incubator
rack for supporting a plurality of flowpath assembly cartridges.
The rack includes, in one embodiment, a plurality of grooves each
adapted to support a flowpath cartridge, a plurality of data
interface connections for transmitting data between the rack and
the cartridges, and a control interface for communication with an
external computer.
[0015] The invention further provides an automated sampling device
having a fluidic pump for transporting a carrier fluid, a valve for
diverting an aliquot of sample from a perfusion loop, a means for
sterilizing the carrier fluid, and a check valve. The pump, filter,
and check valve are connected in series by tubing for transporting
the carrier fluid and the diverted sample from the check valve to a
sample collection device or analysis instrument.
[0016] The invention further provides a biochamber which is
convertible for use in static cell culture or in a perfusion
apparatus. The biochamber includes a first chamber, a cover, a seal
rendering the first chamber removably connectable to the cover and
preventing contamination of the cell culture within the biochamber,
and at least one insert positioned between the first chamber and
the cover, thereby forming a second chamber.
[0017] Additional features and advantages of the invention will be
set forth in the description which follows and will be apparent
from the description or may be learned by practice of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 depicts a media flowpath assembly cartridge and
incubator rack in accordance with the invention.
[0019] FIG. 2 depicts a media flowpath assembly cartridge in
accordance with the invention.
[0020] FIG. 3 shows the outer shell of an exemplary cartridge and
its fixed components.
[0021] FIG. 4 shows an incubator rack in accordance with the
invention.
[0022] FIG. 5 shows a unitized, disposable flowpath perfusion loop
in accordance with the invention.
[0023] FIG. 6 is a schematic illustrating a cartridge and flowpath
assembly, including an integrated automated sampling apparatus.
[0024] FIG. 7 is a schematic illustrating an alternate embodiment
of a cartridge and flowpath assembly.
[0025] FIG. 8 is a schematic illustrating a further alternate
embodiment of a cartridge and flowpath assembly.
[0026] FIG. 9 depicts a drip chamber and noninvasive sensor in
accordance with the invention.
[0027] FIG. 10A shows an external cartridge controller
interface.
[0028] FIG. 10B shows a manual interface located on an individual
cartridge.
[0029] FIG. 11 shows an exploded view of a biochamber in accordance
with the invention.
[0030] FIG. 12 illustrates separate components of an alternate
biochamber embodiment.
[0031] FIG. 13 is a schematic illustrating an automated sampling
apparatus connected to a flowpath assembly cartridge perfusion loop
in accordance with the invention.
[0032] FIG. 14 depicts a pump and related structures in accordance
with the present invention.
[0033] FIGS. 15A and 15B illustrate alternate embodiments of a
valve for diverting media flow.
[0034] FIG. 16 illustrates the front face of a cartridge
embodiment.
[0035] FIG. 17 illustrates a biochamber dual o-ring and air gap
seal.
DETAILED DESCRIPTION OF THE INVENTION
[0036] Reference will now be made in detail to the presently
preferred embodiments of the invention which serve to explain the
principles of the invention. It is to be understood that the
application of the teachings of the present invention to a specific
problem or environment will be within the capabilities of one
having ordinary skill in the art in light of the teachings
contained herein.
[0037] The present invention provides an automated precision cell
culture system which includes one or a plurality of perfusion loop
flowpath cartridges that can be placed in an optional rack or
docking station which fits into an incubator. The incubator
provides the appropriate gas and thermal environment for culturing
the cells as each perfusion loop contains a means for passive
diffusion of air from the incubator environment. The system
provides for parallel processing and optimization through
continuous set point maintenance of individual cell culture
parameters as well as automated sampling and injection. The
invention further provides a biochamber which is convertible for
use as a static cell culture device or in a perfusion loop flowpath
cartridge.
[0038] As used herein, "cell culture" means growth, maintenance,
transfection, or propagation of cells, tissues, or their
products.
[0039] As used herein, "integratable" means parts or components
which are capable of being joined together for operation as a unit
for one or more data transfer or other functions.
[0040] As used herein, "without breaching flowpath sterility"
refers to the closed nature of the perfusion loop which remains
intact during various manipulations or movements such that each
flowpath assembly perfusion loop can be connected to a cartridge
housing, which in turn can be connected to a rack or docking
station, disconnected and then reconnected without exposing the
internal surfaces of the flowpath to environmental contaminants and
without the components of the perfusion loop flowpath losing fluid
communication with one another. Thus, the loop itself is preferably
a disposable, unitized system that can be removed from the
cartridge's outer shell without its components losing fluid
communication with one another. Moreover, an individual perfusion
loop can be moved or carried throughout a laboratory or other
facility, or to a separate lab or facility, as desired for separate
testing or analyses while its contents remain sterile.
[0041] Referring now to FIG. 1, the present invention provides one
or a plurality of media flowpath assembly cartridges, 1, which each
can be placed into docking station or rack, 2, which can then be
placed into a laboratory incubator. The incubator may be any
incubating device, and may be located in a laboratory, a
manufacturing facility, or any clinical or other setting in which
cell culture via incubation is desired. The incubator preferably
maintains a controlled environment of about 5% CO.sub.2 and about
20% O.sub.2 and controlled temperature, although any environment
may be used and selected by one of ordinary skill depending on the
particular end use application, given the teachings herein. The
incubator environment is typically separately controlled, while the
automated culture system of the invention is preferably controlled
by an external PC for integration of individual flowpath assembly
cartridges and system control through a docking station interface,
as described in detail below.
[0042] The illustrated embodiment of FIG. 1 includes an optional
lever 3 for facilitating the cartridge's integration and removal
from the rack. In alternate embodiments, a latch or other capture
device may be used. The illustrated embodiment also includes
optional access ports 4 to accommodate injection or sampling of
fluid. One or more connections 5 may also be included for
connecting power sources and computer control and data transfer
cables.
[0043] FIG. 2 illustrates an embodiment of the invention in which
an individual flowpath assembly cartridge is not attached to a
rack. In this illustrated embodiment, an optional cover 6 encloses
the cartridge's inner components. The cover may be removable and
may be connected to the cartridge's outer shell by a hinge. With
reference now to FIG. 3, a cartridge outer shell or housing 7
provides physical support for the internal components. The
embodiment shown in FIG. 3 illustrates internal hardware components
of a preferred cartridge outer shell or housing, including a pump
8, an optional oxygenator bracket 9, a biochamber 10, valves for
diverting media flow 11, a flow cell or drip chamber 12, a
noninvasive sensor 13, a series of access ports 4, an optional air
pump for sample routing 0.1 micron filtered air (not shown in FIG.
3), and an interface 15 for interfacing with a connection located
on the rack or with a separate power source. The flow cell or drip
chamber may be combined with a noninvasive sensor, for example a pH
sensor, to form a single component. In an alternate embodiment,
interface 15 may provide a connection for a computer cable for
control and data transfer. In another alternate embodiment,
interface 15 may provide fluid connection downstream to an inline
analyzer. The in-line analyzer may provide data on, for example,
cell metabolic activity. Many such in-line analyzers are suitable
for use in the present invention.
[0044] The incubator rack operates as a docking station for one or
preferably multiple cartridges when they are positioned within an
incubator during operation of the cell culture system. The rack or
docking station is preferably fabricated from a plastic material
and may be manufactured by, for example, injection molding.
Referring to FIG. 4, in one embodiment the rack has a horizontal
base 16 and a series of vertical dividers 17 forming grooves, or
tracks 18 for guiding the insertion of and supporting each flowpath
assembly cartridge. In one embodiment, the vertical dividers
provide a small space between each docked cartridge and its
adjacent or neighboring cartridge. The rack may also provide
orthogonal support via a vertical wall 19 preferably in the rear of
the rack. The rack may also have at least one connector 20 and 21,
preferably in the rear and affixed to the vertical wall, for
conveying power from a power source and for communication with an
external computer. Connectors may also be present for attaching a
power or communication cable to a single cartridge or to multiple
cartridges operating within the same rack. A series of connectors
may optionally be attached to a circuit board laid into a groove in
the rear plane of the rack (not shown). The rack may also include a
fan for circulating air within the incubator or a vibration
isolation and damping system.
[0045] To increase portability of a fully loaded rack, an open box
structure can be employed which further protects the front section
and secures the cartridges for transporting within the rack as a
unit. In the embodiment shown in FIG. 4, the rack accommodates
eight cartridges. An incubating device suitable for use in
accordance with the present invention can accommodate a rack
adapted to hold any desired number of cartridges. The rack may thus
be manufactured to include as many slots or tracks as can fit into
a standard laboratory incubator or other suitable incubating
device. A common laboratory incubator will readily support up to
ten cartridges or more on one shelf.
[0046] A lever action removal mechanism may be included to overcome
resistance of the electrical connectors to disengagement and thus
facilitate removal of each cartridge from the rack. In another
embodiment, an indicator illuminates when a cartridge is properly
connected to the electrical connectors or when a cartridge is not
receiving power. The indicator is preferably an LED. In a still
further embodiment, a battery power source is included on or in the
cartridge to provide back up power and power for when the cartridge
is transported or otherwise removed from the rack. A handle may be
located on each cartridge housing to facilitate its removal from
the rack. Such handles can include an indentation for grasping,
which may be located in various locations, preferably the top,
right-hand side, a foldaway handle, or any other mechanism for
facilitating manual transfer and portability of each cartridge. The
cartridge's outer shell is preferably made of plastic and may be
formed by injection molding. The cartridge may also include a
display or control panel. The cartridge may also include a circuit
board in one of numerous locations. A preferable location is on or
embedded in the back plane of the cartridge's outer shell. In an
alternate embodiment, a fold out stand on the bottom plane of the
cartridge outer shell may be included. The stand would allow the
user to place the cartridge on a desktop once the flow path is
inserted and is an aid to keep the cartridge in a vertical position
during some phases of sterile processing in the sterile hood. Prior
to inserting the cartridge into the rack, the stand can be rotated
90 degrees into a tucked away position. Any other stand or suitable
mechanism capable of providing support on a table or bench, or
other horizontal surface for an individual cartridge can be used,
if desired.
[0047] In one embodiment, cartridges may be integrated such that
two or more flowpaths are in fluid connection with each other for
conducting experiments. This embodiment is advantageous when, for
example, increased fluid volume, increased cell volume, or cell
co-culture is desired. Cell co-culture includes culturing a
different cell type in each cartridge. In an alternate embodiment,
larger cartridges with increased biochamber and media supply are
accommodated for scaled up cell culture.
[0048] The invention further provides a unitized, disposable
sterile media perfusion loop flowpath, which is removably
attachable to the outer shell or housing of the cartridge without
breaching flowpath sterility. The perfusion loop is preferably a
continuous flow perfusion loop, but can also function as a single
pass perfusion loop. In an alternate embodiment, the perfusion loop
is a single pass perfusion loop. The loop is preferably removable
from the cartridge housing as a single disposable unit. FIG. 5
illustrates one embodiment of a unitized perfusion loop according
to the invention. In FIG. 5, the loop is not connected to a
cartridge. As shown in the embodiment of FIG. 5, the media
perfusion loop or flowpath includes a media reservoir 22, tubing
23, an oxygenator 24, a biochamber or bioreactor 10, an interface
to accommodate an air supply 26 for sample removal, a filter 27 for
sterilizing air from the air supply, a sampling interface 28, and a
waste reservoir 29 (injection and sample reservoirs not shown in
this Fig.). In an alternate embodiment, the flowpath includes an
interface for connection with an analyzer. The oxygenator 24 is
preferably a passive diffusion oxygenator. The oxygenator may
comprise any gas permeable surface. In an alternate embodiment, the
oxygenator is a diffusion membrane positioned, for example, over a
valve manifold. In another alternate embodiment, the oxygenator is
a diffusion membrane positioned over the biochamber. Alternatively,
the oxygenator may be a hollow fiber for accommodating forced
gas.
[0049] In an alternate embodiment, more than one biochamber or
bioreactor is included in a single flowpath for increasing cell
volume or to provide co-culturing. The biochambers may be connected
in series or in parallel. Waste contained in the waste reservoir 29
may include spent media, cellular byproducts, discarded cells, or
any other component that enters the waste reservoir 29 through the
media perfusion loop. Sampling interface 28 may be any suitable
connection or surface forming a boundary through which a sample may
be extracted from the perfusion loop while eliminating or
minimizing any potential breach in flowpath sterility. Extraction
may be manual or automated. The sampling interface may, for
example, be a silicon injection site or a luer connection.
[0050] FIGS. 6, 7, and 8 illustrate alternative embodiments of a
media perfusion loop or flowpath arranged within a cartridge
housing in accordance with the present invention. Referring to FIG.
7, during operation, media contained in the media reservoir 22
travels via tubing through a tubing section in contact with a first
valve 30 which diverts a portion of the media to oxygenator 24. In
the illustrated embodiment, the diverted media then travels through
a flow cell 12 which is removably attachable to a noninvasive
sensor 13. In a preferred embodiment, the flow cell comprises a
drip chamber. As used herein, "noninvasive" means that the sensor
operates without invading or interfering with the sterility of the
perfusion loop. Noninvasive sensor 13 is preferably a pH sensor or
combination pH sensor/drip chamber. The flow cell provides a
selective barrier membrane which prevents proteins and other
substances in the media from interfering with the detection signal.
The membrane allows for easy transfer of hydrogen ions across the
membrane to the detection path of the sensor. The pH sensor
preferably includes LEDs and photodetectors for measuring light
transmission through cell culture media. The noninvasive sensor may
be an oxygen sensor or any other analyzer suitable for use in the
present invention.
[0051] In FIG. 7, the media then travels through a tubing section
in contact with a pump 8, then through a tubing section in contact
with a second valve 31, which diverts a portion of the flow either
directly to biochamber 10, or first through tubing which subjects
the circulating media to a first noninvasive oxygen sensor 32 then
to biochamber 10. In the illustrated embodiment, the media then
flows from the biochamber 10 past a second noninvasive oxygen
sensor 33 and through a tubing section in contact with a third
valve 34. In the illustrated embodiment, valve 34 may divert the
flow to a tubing section in contact with a fourth valve 35, which
in turn diverts the flow either back through tubing in contact with
first valve 30 for recirculation or to waste reservoir 29. The
first, second, third, or fourth valves may be pinch valves.
Alternatively, the first second, third, or fourth valves may be a
diverter valve routing manifold including means for flow reversal.
As illustrated, flow may also be diverted from biochamber 10,
through valve 34, and through first check valve 37 integrated with
a sampling apparatus for sampling the contents of the biochamber.
Alternatively, flow may be diverted from biochamber 10 through side
sampling port 36 and through a second check valve 38 integrated
with a sampling apparatus. The tubing section in contact with the
pump or valves may form a diaphragm. In alternate embodiments, the
perfusion loop can include additional diverter valves and Y
selector flowpath routings for cell sampling, intra-chamber media
sampling, reverse flow, and numerous other applications for which
diversion of flow is desired.
[0052] The sampling apparatus illustrated in FIG. 7 includes first
attachment point 39 for introducing air into the sampling tubing.
The air travels through a gas valve 40 to a filter 27 for
sterilizing the air, then through check valve 38, where it captures
a quantity of fluid from the perfusion loop and transports the
fluid as a unitized sample through second attachment point 41,
which may include a luer activated valve 42 as shown. The sampling
apparatus is preferably automated or may be operated manually. In a
preferred embodiment, samples may be diverted to a sample reservoir
and maintained in a fluid between samples. The fluid between
samples may be an anti-fungal fluid. In another embodiment, the
automated sampling system may flush the sample line before the
sample is taken, the flush being diverted to the waste reservoir
for insuring a fresh sample.
[0053] In another embodiment, the fluid may be automatically
diverted through a length of tubing to the cartridge front or to an
analyzer located outside the incubator. Samples may be diverted
from the recirculating flowpath fluid or from fluid residing in
direct contact with the cells. Fluid may be automatically routed by
a computer program, or a manual interface button. In another
embodiment, fluid may be removed via a syringe from the manual
sampling port.
[0054] In one embodiment of a biochamber, cells are grown in a
space outside fibers carrying fluid through the biochamber. This
space, which is sealed from the general fluid path other than
across the fiber wall, is referred to as the extra-cellular space
(ECS). In another biochamber embodiment, cells are grown in
suspension in the absence of fibers. Samples collected through
sampling port 36 may include samples from the ECS of biochamber 10.
Samples collected through sampling port 36 may include a suspension
of cells. Samples may also include circulating fluid from various
points in the perfusion loop.
[0055] FIG. 7 also illustrates an attachment point 43 through which
an injection into media reservoir 22 may be made, and an optional
stir bar 44 within the media reservoir. Fluid may be automatically
injected at intervals preprogrammed into the system. Programming
may occur via a manual interface or via an external computer.
[0056] FIGS. 6 and 8 show alternative embodiments including
alternate arrangements of several of the components illustrated in
FIG. 7. FIG. 6 also includes an optional handle 45, a noninvasive
LED sensor array 46 for, e.g., pH, glucose, or O.sub.2 level
detection and a display and control module 47, located on the
cartridge outer shell. FIG. 6 further illustrates an optional
cutaway 48 adjacent to the biochamber 10 for optical viewing or
video monitoring of the operating biochamber.
[0057] FIG. 8 includes an internal controller 49 with a user
interface, a pH sensor 51, and an internal air pump 50 for
integration with the sampling apparatus. In the illustrated
embodiment, pH sensor 51 may be invasive or noninvasive. In one
embodiment, pH sensor 51 is a pH probe.
[0058] Oxygenator 24 may be formed by coiling a length of gas
permeable silicon or similar tubing. The oxygenator may alternately
be a membrane positioned over a biochamber, valve, or another
component of the flowpath. In an alternate embodiment, the
oxygenator may be a hollow fiber membrane oxygenator. The
oxygenator is preferably exposed to ambient air within the
incubator during operation. The oxygenator brackets, if used, can
be any mechanical, magnetic, or other device suitable for affixing
a structure to the cartridge's outer shell.
[0059] The disposable portion of the pump, i.e., the pump tubing,
may be made from silicon tubing or other biocompatible or compliant
tubing which includes a one way check valve on either end. In one
embodiment, it is an integral portion of the unitized disposable
flow path and can be sterilized as such during manufacture of the
flowpath. The pump may also include a lid for holding the pump
tubing in place. Such a pump may operate by using a plate to
squeeze the diaphragm and displace the fluid through the one way
check valves. The fluid displacement can be modulated and a varied
pressure wave produced through variable electronic signals to the
direct drive motor. The pump itself may be affixed to the cartridge
housing. The pump may be removable from the flowpath and housing
for servicing or other purposes. In one embodiment, the pump is
capable of providing a fluid flow rate of about 4 mL/min to about
40 mL/min. The pump is regulated by a feedback control process in
concert with flow meters.
[0060] FIG. 14 illustrates one embodiment of a pump and related
structures according to the invention. In FIG. 14, fluid flows
through flowpath tubing 69 through a first one way flow valve 70 or
check valve, into pump tubing 71. Pump actuator 73 compresses pump
tubing 71 against pump lid or rigid backing 72, thereby forcing
fluid from the pump tubing through a second one way flow valve 74
or check valve, into flowpath tubing 75. Flowpath tubing 69 and
pump tubing 71 may be made of the same material or different
materials.
[0061] FIG. 15A illustrates an embodiment of a diverter valve
suitable for use in the present invention. In the illustrated
embodiment, fluid enters tubing 77 and is diverted to path 79 when
actuator 78 occludes path 80 by compressing its tubing against a
rigid surface 76. In FIG. 15B, fluid enters tubing 77 and is
diverted to path 79 when actuator 78 occludes path 80 by
compressing its tubing against surface 76. These figures provide a
top view and a cross-sectional view of such valves.
[0062] The valve tubing may be flow path tubing routed through a
slot in the valve. The valve tubing may be a diaphragm. The valve
may be used as a diverter valve by running a flow path tube into a
Y connector, then routing the two tubes through two slots on the
valve. Such a mechanism only pinches one path at a time, thus
allowing the user to select which path is active. Various valves
and tubing or diaphragm structures may be selected by one of
ordinary skill in the art given the teachings herein. The valve
actuator is preferably capable of being held in position without
external power. Suitable structures for attaching the unitized
perfusion flow path components to the corresponding fixed
structures of the cartridge housing include clips or any other
fastener which sufficiently secures the path without impeding its
operation.
[0063] In alternative embodiments, one or more noninvasive sensors
are spectroscopy sensor arrays containing a group of LED emitters
and detectors oriented such that absorption of light through the
media can be examined. Such a sensor can detect frequency spectrum
of the media, and provide, for example, pH level, glucose content,
or O.sub.2 content determinations using NIR wavelengths. The sensor
can be mounted to the cartridge. In a preferred embodiment, the
flow cell is a transparent tube. In another embodiment, the flow
cell is positioned in a groove within a block or other body affixed
to the inner surface of the cartridge outer shell. In an alternate
embodiment, the sensor and flow cell are incorporated into a single
unit.
[0064] The media and waste reservoirs may have a capacity of about
100 mL to about 150 mL each. However, any other size can be used
and the cell culture system of the present invention can
accommodate reservoirs of various fluid capacities. Fluid volumes
may be selected to accommodate a variety of different cell types.
Some cell types have metabolic needs in which fluid volume greater
than 150 mL is preferable. Some experimental protocols suitable for
use with the present invention use small volume injection of a test
compound, which can be provided from a reservoir within the
cartridge or injected by various other means as discussed herein.
The reservoirs may include a sealable, removable lid to allow fluid
to be placed into the reservoir. The lid may also include a drop
tube for drawing media or other material from the reservoir and a
filtered vent of about 0.2-micron or other suitable porosity to
maintain sterility. The reservoirs may be made of autoclavable
plastic or glass, or any suitable substance for use in holding
fluid in accordance with the present invention. The vented lid is
preferably made of sterilizable plastic.
[0065] Any sterile biocompatible tubing is suitable for use in the
present invention. Tubing is preferably silicone. Tubing may also
be a commercially available tubing such as Pharmed, Viton, Teflon,
or Eagle Elastomer. In one embodiment the tubing has an inside
diameter of about 3/32'' and an outside diameter of about 5/32'';
however, any other suitable dimensions may be used. Such tubing may
be utilized for, e.g., diaphragms, or tubing in connection with
valves, the oxygenator, and between components of the perfusion
loop.
[0066] FIG. 9 depicts one embodiment of a drip chamber and
noninvasive sensor for use in the sterile media perfusion loop.
During use, fluid flows through feed tube 52 and is released in
discrete droplets through drip aperture 53 into partially filled,
preferably transparent flow chamber 54 before exiting through
tubing at the bottom of the drip chamber. As the droplets fall from
the aperture, they pass through a noninvasive sensor which includes
housing 55 having an emitter array 56, a photodetector array 57,
and a computational chip 58. The emitter array and photodetector
count the droplets and, with the computational chip, determine
droplet frequency to calculate a flow rate or a volume of fluid
passing during an event. The drip chamber may be positioned between
the pump and the oxygenator (which precedes the cell biochamber) or
located at various positions within the perfusion loop. A
preferable location is downstream from the cell biochamber. Another
preferred location is upstream from the pump. The sensor may be
linked to a pump for providing precise injection of fluids to the
recirculating media stream. Injected fluids may include media,
drugs, or other additives.
[0067] Efficient collection of the tissue or cells at the
completion of the culture process is an important feature of an
effective cell culture system. One approach is to culture cells in
a defined space without unnecessary physical barriers to recovery,
so that simple elution of product results in a manageable,
concentrated volume of cells amenable to final washing in a
commercial, closed system or any suitable cell washer designed for
the purpose. An ideal system would allow for the efficient and
complete removal of all cells produced, including both adherent and
non-adherent cells. Thus, various different biochambers can be used
in accordance with the present invention. As used herein, a
biochamber includes any bioreactor suitable for use in accordance
with the invention and can include any such device for growing,
maintaining, transfecting, or expanding cells or tissues. The
biochamber may be, for example, a hollow fiber biochamber or
bioreactor having luer fittings for attachment to the flowpath.
Various biochambers and bioreactors are adaptable for use with the
media flowpath assembly cartridge of the present invention given
the teachings herein.
[0068] A particularly preferred biochamber is a biochamber
convertible for use in static cell culture or in a cell perfusion
apparatus and includes a first chamber, a cover, a seal rendering
the first chamber removably connectable to the disposable cover,
and at least one insert positioned between the first chamber and
the disposable cover, thereby forming a second chamber. The
preferred biochamber operates in two modes, open or closed. In the
presealed phase or mode, the biochamber acts as a petri dish and
allows for manual cell seeding and growth prior to sealing the
biochamber and attachment to a flow system. In a preferred
embodiment, the biochamber has a lip that acts as a sterile barrier
which allows for gas diffusion but keeps bacteria out of the cell
space. Cells can be grown in the ECS, which is sealed from the
general fluid path other than across the membrane wall. Once
sealed, the biochamber can be seeded with cells above and below the
membrane insert. Ports may also be used to collect extra membrane
samples throughout an ongoing experiment. In preferred embodiments,
the biochamber remains horizontal in orientation and cell retrieval
is carried out manually.
[0069] Referring now to FIG. 11, the illustrated biochamber
embodiment includes a bottom chamber 59, a cover 60, a brace 61 for
holding at least one insert 62 between the bottom chamber 59 and
the cover 60. The biochamber preferably includes diffusers on each
end 63 for modifying pressure characteristics of incoming fluid to
provide an evenly distributed flow. FIG. 12 shows components of an
alternate embodiment of a biochamber according to the invention,
including cover 60, braces 61, and insert 62 between two braces 61.
A membrane insert is shown 62. The biochamber may accommodate a
variety of selectable barrier inserts, such as hollow fibers and
membranes, for cell growth. Inserts suitable for use in the present
invention include semipermeable membranes. Additional inserts
suitable for use in the present invention include optically
reflective surfaces for enhanced contrast video microscope
observation, and a variety of three-dimensional growth matrixes
such as gels, elastin conduits, bio-absorbable materials, and
scaffolds for improved growth and cell orientation. The biochamber
can also accommodate inserts and diffusion patterns that allow
active laminar flow and passive flow techniques. Inserts are
preferably from about 0.001 inch to 0.1 inch thick. A grooved shelf
may be provided to align the membrane assembly and provide
structural support. FIG. 12 also includes connections 64 for
flowpath tubing from the biochamber to the perfusion loop.
[0070] Referring to FIG. 17, in one embodiment a biochamber
includes a seal utilizing an o-ring with dual sealing interfaces
and an integral air gap to prevent contamination of the biochamber.
The biochamber and o-ring sealing surfaces form an environmental
seal 88, an air gap 87, and a fluid seal 89. The combination seal
and air gap ensures that environmental contaminants cannot come
into contact with the fluid o-ring seal 90. Fluid o-ring seal 90
can provide microscopic fluid interface channels, which might
otherwise be transversed by biologic contaminants such as viruses,
mycobacterium, and bacteria. The o-ring air gap is formed when the
two halves 91 and 92 of the biochamber are mated and air, which has
been HEPA filtered or made sterile through any suitable method, is
trapped between the two o-ring interfaces. The environmental seal
88 prevents contaminants from reaching the air gap 87, which
provides an area void of fluids and fluid micro channels which, if
present could permit contamination or breaching of the fluid seat
90. The sealing o-ring and biochamber halves preferably form a
continuous color change to signal the appropriate mating and
seating of the sealing surfaces.
[0071] In alternate embodiments, the cover and base may have a
color verifiable sealing surface that is established and maintained
via threaded twist end caps or pressure maintenance solution. Such
a sealing surface may reveal one color when the cover and base are
sealed and a different color when the seal is broken. The sealing
surface can include ridges for securing mid chamber inserts, the
seal and inserts preferably being reversible and removable. In
particularly preferred embodiments, multiple chamber ports allow
access and flow to the central media chamber and to medium and cell
products captive on either side of the insert barrier. The chamber
ports also preferably provide fluid interfaces for automated
perfusion manipulations such as sampling and injections.
[0072] FIG. 13 is a schematic diagram of one embodiment of an
automated sampling apparatus according to the present invention.
The illustrated embodiment shows the sampling apparatus having an
air pump 50 connected to a plurality of flowpath assembly
cartridges, 1, housed within an incubator 67. Alternatively, each
cartridge can have its own air pump. A sample is collected by first
diverting a sample from the flowpath using a diverter valve 11. The
diverter valve may be a pinch valve. The sample travels to a one
way or check valve 37. Valve 40 (optional, for use with another
routing or carrier fluid source; otherwise air pump 50 is used) is
then opened. Air from air pump 50 passes through sterilizing filter
27 and through check valve 37, thus capturing the sample and
forcing it to a collection receptacle 68. The sterilizing filter
may be, for example, a 0.1 or 0.2 micron filter or a series of
filters, or any other method or structure suitable to render the
routing air or other carrier fluid free of biologic contaminants.
The valve 40 is only required if the routing fluid is other than
incubator air. A single air pump can be used with an external air
source and manifold off of the air source to a plurality of
cartridges. The preferred approach, however, is for each cartridge
to contain the necessary hardware to perform its own sampling. The
sampling apparatus may be automatically operated by pressing a
button located on the cartridge. The button preferably is marked to
indicate that it is for sampling. The button may be located on the
front of the cartridge. In another embodiment, the sampling
apparatus is operated through programmed control by an external
computer. The sample may be diverted to a collection container. In
one embodiment, the collection container is a tube. In another
embodiment, the collection container is positioned on the front of
the cartridge. The sample tubing may be flushed into the waste
stream before the sample is collected for ensuring a fresh sample.
In an alternate embodiment, the sample may be diverted to a sample
reservoir located on a stepper motor for collection of multiple
samples without operator intervention. Each sample may remain in a
sample reservoir until collected for analysis, allowing for sample
collection during periods of time when an operator is
unavailable.
[0073] The automated sampling apparatus eliminates potential
breaches of the sterile barrier and thus minimizes the risk of
contamination without the use of bactericides or fungicides, which
may interfere with the integrity of the sample. Potential problems
associated with traditional sterile barrier culture manipulations
and perturbations, such as removal of the cultures from their
temperature and gas environment to room temperature and room air
for processing under a sterile hood facility, are eliminated. A
computer controlled sterile air pump allows integration with
analysis instruments that require fixed timing by controlling
sample duration and pump speed. Residual medium may be removed via
a purge cycle of the collection device. In-line residual may be
minimized at the point of sterile media or cell diverter and
through the use of hydrophobic routing materials and surface
modification. Use of periodic sterile air purge through the sample
routing tube can be utilized to prevent aerosols and endotoxins
from migrating back through the sample routing tube. The routing
tube end when not interfaced with the collection device is
preferably maintained in an anti-microbial bath. The apparatus
provides a small sample (typically 0.5 to 5 mL), which is extracted
from the flow path or ECS of the cell biochamber and routed via a
bubble of sterilized air within the collection tube to the final
collection point. For certain samples and applications any suitable
alternative fluid carrier, liquid or gas, may be used to allow
transport of the sample within the system and to a collection
receptacle or analysis instrument.
[0074] In addition to automated sampling, the invention also
permits manual cell or tissue harvest, and manual cell seeding and
manipulation, under a sterile hood, with manual dual port syringe
flush cell seeding. In one embodiment, a manual access port is
provided for injection of cells. Injection may occur through the
manual access port via a syringe or needle.
[0075] In terms of growth condition optimization and process
control, the present invention provides for continuous set point
maintenance of various cell culture growth parameters through
sensor monitoring and feedback control of pump, valves, and other
equipment suitable for a given cell culture or tissue engineering
application. Data, pertaining to, for example, pH, temperature,
flow rate, pump pressure, waveform, and oxygen saturation can be
displayed and stored. The incubator is typically separately
controllable for temperature and gas conditions. System program and
status parameters, such as media flow and flow dynamics through low
drip flow chamber, inline pressure sensor(s), and pump motor
control, can be controlled via a computer interface allowing
operator control on a PC directly or allowing protected remote
communication and program modification via a modem or internet
connection. Sampling increments and drug dosing can also be
preprogrammed or entered directly on a separate computer or can be
entered via a touch pad or other interface located on the docking
station or in each cartridge.
[0076] The computer interface preferably provides a display for
real-time or logged data of parameters from each cartridge
including, for example, temperature, pH, flow rate, pump pulse
waveform, and various scheduled events, including, for example,
injection of fresh media and other fluids, and automated sampling.
The pH, flow rate, pump pulse waveform, and other parameters are
preferably feedback regulated from a set point selected and entered
by the operator. Temperature is preferably regulated by the
incubative environment. In one embodiment, the cartridge logs data
without need for a separate computer. In another embodiment, a
cartridge may include a digital identification when connected to
the rack, for the purpose of identifying the particular experiment
being run in the particular cartridge or the status of the
experiment upon disconnection. Each cartridge may be keyed to a
particular rack slot once operation begins, which prevents its
continued operation if disconnected and replaced into an incorrect
or different slot. Each cartridge preferably includes a manual
interface which includes LED's to indicate the cartridge's state of
operation, and which provides the operator an interface for
entering set points. The interfaces also may operate while the
cartridge is not in the rack.
[0077] Each cartridge preferably includes a local controller such
that each noninvasive sensor generates and transmits information in
the form of an electrical signal to the local controller. The
signal may be transmitted by an electrical connection either
directly to the local controller or first to an amplifier or
transmitter and then to the controller via a communication path or
bus. The communication may be transmitted serially or in
parallel.
[0078] FIG. 16 shows one embodiment of the front face of a
cartridge 81 of the present invention, including a display 82, LEDs
83, operator interface 84, sample collection tubes 85, and sites 86
for injection or sampling.
[0079] The controller includes information corresponding to a
measured value with a set point which is either preprogrammed
within it (such as in a chip) or can be entered using a touch pad
or interface located on the cartridge or as part of a PC or other
central computer system connected to the local controller. When the
controller receives the signal from the sensor, it determines
whether to move the process value closer to the programmed set
point (i.e., change the flow rate, divert media flow, etc.) and
transmits the information to the pump, altering its flow rate if
necessary, or to the valve, diverting media flow if necessary or
desired. This feedback control is preferably continuous throughout
operation of the system. Automatic warning alarms may be utilized
to alert the operator via, for example, telephone or internet
connection and are preferably audible.
[0080] The local controller may be connected by a communication
path to the connector located on the cartridge which in turn is
connected to the connector located on the rack when the cartridge
is docked. The rack can then be connected via a communication path
to a central computer or controller. The communication path
connected from the rack to the central computer can transmit
separate information from each of a plurality of cartridges docked
in the rack to the central computer. The central computer can also
transmit information to each cartridge or all cartridges via a
communication path from the computer to the rack and the rack to
each individual cartridge. The central computer can also store and
analyze information received from the cartridges.
[0081] Growth condition optimization is preferably achieved through
noninvasive monitoring and precision control of numerous
parameters, including flow rate, physiologic pressure and pulse
wave, media addition, oxygenation and pH. In addition, sampling,
fresh media addition, and drug dosing, etc., can be automated by
programming a valve to divert media flow at a desired time or in
accordance with a desired schedule. The process control parameters
can be modified as desired to provide additional features, such as
drug injection and biological function monitoring, to achieve the
desired optimal results in various research and clinical contexts
depending on the particular end use application.
[0082] Consistent with this growth condition optimization, each
cartridge can provide a separate experiment in which any
combination of configurations and events in a timed or threshold
triggered fashion can be maintained, including, for example, medium
re-circulation at a specified flow rate, pressure wave and shear.
Once programmed, each cartridge can be operated with only a power
source, such as through the attachment of a power cable or with an
on board battery pack, to facilitate individual cartridge
processing, analyses, or manipulation under sterile laminar flow
hoods or various external analytical devices.
[0083] The cell culture system can operate in several modes. A
recirculation mode keeps the media flowing through the closed
perfusion loop. Alternatively, a feed/sump mode can be used in
which valves divert the flowpath to supply fresh media from the
media reservoir and drain waste from the perfusion loop to the
waste reservoir. Switching modes may be achieved, for example, by
preprogramming a predetermined volume of fresh media to be injected
at predetermined intervals. Switching modes may also be achieved
through the feedback control loop connected to the pH sensor. For
example, the operator may input into the computer a desired pH set
point. When the pH sensor detects a pH level below the set point,
the system automatically injects a predetermined volume of media
into the recirculating flowpath. The pH is then continually
monitored and fresh media again injected as needed.
[0084] Drugs or other substances can be injected into the perfusion
loop or into the biochamber for testing their effects on the
growing cells and tissues. The invention further provides for
automated injection of drugs or other substances directly into the
media reservoir or the fluidic path leading to the desired area.
Alternatively, manual injections can be performed by using a
syringe and a septum attached to the media reservoir or through the
manual injection site on the cartridge front face. Such manual
injections may be performed with the cartridge remaining in the
incubator, or at another suitable location, such as, for example,
under a sterile hood during cartridge processing. Alternatively,
drop by drop additions may be added and allowed to enter the media
reservoir or fluidics stream.
[0085] Numerous end use applications can be achieved with the
apparatus of the present invention. Numerous kinds of cells,
including anchorage dependent and non anchorage dependent cells
(i.e., those capable of growth in suspension) and various tissues
can be grown, harvested, inoculated, and monitored through use of
the present invention. More complex cell models may be achieved by
using various inserts in the biochamber or through optimization of
growth parameters. The system may also be used in numerous genetic
and metabolic engineering applications.
[0086] Samples of fluid circulating in the loop can be extracted,
as can cells or tissues growing or being maintained in the
biochamber. Cells can be used in the apparatus to produce a final
product of interest, such as through hybridoma production of
monoclonal antibodies or other products, or cells themselves can be
cultured as the final product.
[0087] When a plurality of flowpaths are in operation together in a
rack, the system permits parallel optimization and scale up. An
operator can make one or more adjustments to one of the flowpath
loops, and quickly obtain information and assess its impact on the
cells or tissues being cultured. The apparatus also permits high
through put and quality assurance by providing the ability to
conduct parallel experiments or processes under identical
conditions. Multiple racks may also be removably connected and
operated together for multiple experiments or to scale up cell
production. The present invention also permits optimization of, for
example, any or all of the following: cell selection, growth and
viability, cell growth conditions, cell metabolism or bioproduct
production, development of medium for a particular cell type for
limited cell populations, processing of metabolic products, and
expansion to several cell products and cell co-cultivation.
[0088] The above description and examples are only illustrative of
preferred embodiments which achieve the features and advantages of
the present invention, and it is not intended that the present
invention be limited thereto.
* * * * *