WO2003085101A1 - Automated bioculture and bioculture experiments system - Google Patents

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 (7), with integrated, aseptic sampling and routing (26, 27, 28) 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 (7) 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.

Description

AUTOMATED BIOCULTURE AND BIOCULTURE EXPERIMENTS SYSTEM

[0001] This application is a continuation-in-part of U.S. Patent

Application Serial Number 09/967,995, filed October 2, 2001.

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, in which each experiment uses a stream of sterile, disposable

products. 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 ttaining 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

frequent transport and manipulation of the culture represents an opportunity for

contamination from a single bacterium that can cause weeks of work to be

wasted.. 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 deviating from a 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 complex tissue products

require advanced biochamber perfusion systems that are capable of mimicking in

vivo development dependent stimulation for optimal tissue development. 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 traditionally 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 elirnination of site-to-site and batch-to-

batch 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 subjective 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 for cell and /or tissue culture: cells that are capable of

replicating and potentially differentiating and an in vitro system containing

biocompatible materials that provide for the physiological requirements for the

cells to remain viable, proliferate, differentiate, and /or organize into desired

tissue structures. These requirements include temperture, pH, surfaces

appropriate for cell attachment, nutrient exchange, waste removal, 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 and while

mamtaining flowpath indentity.

[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 or other data storage/user interface device. The rack

provides structural alignment and access to each of the resident cartridges and

biochambers contained therein. The access area and alignment structure allow

for a variety of interfaces through the access port including, for example, video

microscopy, mechanical stimulation, and growth and turbidity interrogations.

[0015] The invention further provides an automated sampling

capability having a fluidic pump for transporting a carrier fluid, a check valve for

diverting an aliquot of sample from a perfusion loop, and a means for

maintaining the sterility of the carrier fluid.. The pump, filter, and check valve

are connected in series by tubing or other means of sterile fluid routing for transporting the carrier fluid and the diverted sample from the check valve to a

sample collection device or analysis instrument. The sample collection device

connection may include a heat source or other means to re-sterilize the

connection. In a preferred embodiment, the fluid routing system is disposable

to limit opportunities for cross-contamination.

[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 providing for an 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] Figure 1 depicts a media flowpath assembly cartridge and

incubator rack in accordance with the invention.

[0019] Figure 2 depicts a media flowpath assembly cartridge in

accordance with the invention.

[0020] Figure 3 shows the outer shell of an exemplary cartridge and its

fixed components. [0021] Figure 4 shows an incubator rack in accordance with the

invention.

[0022] Figure 5 shows a unitized, disposable flowpath perfusion loop

in accordance with the invention.

[0023] Figure 6 is a schematic illustrating a cartridge and flowpath

assembly, including an integrated automated sampling apparatus.

[0024] Figure 7 is a schematic illustrating an alternate embodiment of

a cartridge and flowpath assembly.

[0025] Figure 8 is a schematic illustrating a further alternate

embodiment of a cartridge and flowpath assembly.

[0026] Figure 9 depicts a drip chamber and noninvasive sensor in

accordance with the invention.

[0027] Figure 10A shows an external cartridge controller interface.

Figure 10B shows a manual interface located on an individual cartridge.

[0028] Figure 11 shows an exploded view of a biochamber in

accordance with the invention.

[0029] Figure 12 illustrates separate components of an alternate

biochamber embodiment.

[0030] Figures 13A and B are schematics illustrating an automated

sampling apparatus connected to a flowpath assembly cartridge perfusion loop in

accordance with the invention. [0031] Figure 14 depicts a pump and related structures in accordance

with the present invention.

[0032] Figures 15A and 15B illustrate alternate embodiments of a

valve for diverting media flow.

[0033] Figure 16 illustrates the front face of a cartridge embodiment.

[0034] Figure 17 illustrates a biochamber dual o-ring and air gap seal.

[0035] Figures 18A and B illustrate a biochamber providing tensile

mechanical stimulation to a tissue specimen.

[0036] Figures 19A and B illustrate a biochamber providing

compressive mechanical stimulation to a tissue specimen.

[0037] Figure 20 illustrates an embodiment having an alternate

valving configuration in accordance with the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0038] 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.

[0039] 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.

[0040] As used herein, "cell culture" means growth, maintenance,

differentiation, transfection, or propagation of cells, tissues, or their products.

[0041] 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.

[0042] 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.

[0043] 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% C0₂ and about 20% 0₂ and controlled

temperature and relative humidity, 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 or control pod for integration of individual

flowpath assembly cartridges and system control through a docking station

interface, as described in detail below. The control pod is a user-interface with

embedded microprocessors and a graphical user interface, but is not necessarily a

PC.

[0044] The illustrated embodiment of Figure 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.

[0045] 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 in¬

line 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.

[0046] 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.

To allow access to the biochambers aligned in the docking station, an access

window may be provided, for example, below the horizontal plane of the biochamber(s). The window allows interface with video imaging systems,

mechanical stimulation systems or other devices.

[0047] 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.

[0048] 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.

[0049] 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 large scale cell and/or tissue culture.

[0050] 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. The valve manifold is potentially

a series of cam-like devices that rotate into a variety of positions. As they rotate,

they deform the membrane in various positions such that the position of the

overlay provides for fluid diffusion into predetermined directions through the

etched plastic below. This membrane also allows for the diffusion of gas and

may take the place of a separate oxygenator. In this embodiment, tubing is only

used at the biochamber interface and may or may not be utilized at the fluid reservoir interfaces. 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. Additionally,

in alternate embodiments, any or all of the preceding gas exchange interfaces

may use active mass transport of gas across the interface rather than passive

diffusional transport.

[0051] 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.

[0052] Figures 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 may allow 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.

[0053] 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.

[0054] The sampling apparatus illustrated in Figure 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 discretely with a

fluid "spacer" (air or liquid) 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 to insureg a fresh sample.

[0055] 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 and the

fluidic loop flow may be reversed to improve homogeneity of the sample. 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.

[0056] 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-membranous or 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. Samples may be collected via ultra filtration of the

perfusion loop media through a membrane into the extra-membranous space

and directed to the sample routing tube. [0057] Figure 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.

[0058] 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 0₂ 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. This configuration allows for visualization on a standard

laboratory or other microscope, viewed from below with an additional light

source available mounted on the cartridge, above the biochamber. An alternate

embodiment allows the cartridge to be used with a modified docking station.

The docking station may include a fixed CCD video camera that translates in

and out along the y axis (depth of field). Alternatively, a CCD video camera

may be mounted on an x-y-z translator that allows the camera to translate under

each biochamber and then translate along the y (depth of field) axis for real-time

visualization of the cultures. These images are sent to a computer or monitor

that is located outside the incubator environment. In an alternative

embodiment, this camera is outfitted to provide real-time fluorescent images when used in conjunction with fluorescent dyes, etc. and fluorescent light

activation.

[0059] 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.

[0060] 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.

[0061] The disposable portion of the pump, i.e., the pump tubing,

may be made from silicon tubing or other biocompatible or compliant tubing

which may include 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 one way check valves.

The fluid displacement can be modulated and a varied pressure wave produced through variable electronic signals to the fluid displacement actuator.. 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

4mL/min to about 40 mL/min. The pump is regulated by a feedback control

process in concert with flow meters. The pump motor may include a direct drive

with stepper or infinite position control. Position control can be modulated via

computer control to create a variety of pressure head and pulse wave signatures

including physiologic wave forms.

[0062] In an alternative embodiment, this pump is increased to a

larger volume, multi-head pump. The pump head may be changed depending

on the desired flowrate. The larger volume head allows the system to pump at

100-300 mL/min. The head design, in concert with a suitable valving

configuration, may be used to generate, for example, a cardiac-signature

pumping profile, with diastolic and systolic pressure. The larger flowrate pump

may be used in concert with two high flow sensors placed within the fluid loop.

These sensors allow for closed-loop feedback control of the pump to allow for

precision flowrates. This higher flowrate is important for several cell types,

especially for cardiovascular applications. An example is endothelial cells that

typically line blood vessels. The endothelial cells require a high shear stress to

organize appropriately in the lumen of the vessel. Another example of a cell

type that requires hydrodynamic mechanical stresses is mesenchymnal cells that require such stimuli to generate a cohesive, robust extracellular matrix similar to

those of native tissues. This embodiment may include a biochamber capable of

housing a tubular tissue and applying controlled hydrodynamic training regimes

to the tissue. In this embodiment, the tubular tissue may be based upon an

acellularized tubular matrix with appropriate mechanical and biochemical

properties to promote vascular tissue genesis.

[0063] 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.

Another alternative embodiment of the pump is to allow for mechanical forces to

be translated through the biochamber and directly to the cells and/or tissues.

This is especially important for cells that are typically exposed to large levels of

force in vivo and require such forces to maintain regular cellular and/or tissue

function, like bone, tendon, ligament, cartilage, or muscle cells and/or tissues.

[0064] In one embodiment for mechanically stimulating the cells

and/or tissues housed within the biochamber, the biochamber is fitted with an

elastic or similar membrane that allows for deformation and force translation

directly into the cell biochamber. This membrane interfaces with a cam or solenoid that is mounted underneath the biochamber, either on the cartridge or

within the docking station. This cam interfaces directly with the membrane and

deforms it to translate a force onto the cells. This technique also aids diffusion

into three dimensional cell scaffolds that are placed as inserts into the

biochamber and which can potentially impede flow through the normal flow

direction In an alternate embodiment, a cam or lever is placed at the end of the

biochamber instead of under it. The biochamber is mounted on the cartridge in

such a way that the cam provides a rocking motion, ensuring even distribution of

suspended cells within the biochamber. In an alternate embodiment, the

mechanical stimulation is provided by a hydrodynamic pressure head. Figs. 18A

and 18B illustrate an alternate embodiment useful for mechanical stimulation of

a tissue or cell specimen housed within the biochamber. In the illustrated

embodiment, a linear actuator 94 is positioned below the biochamber. The

linear actuator acts upon a lever arm 95 that passes through a flexible substrate

93 which is continuous with or embedded within the lower housing 97 of the

biochamber. The lever arm 95 interacts with tissue structure 96 housed within

the biochamber causing mechanical stimulation of the tissue specimen within the

tissue structure. In the illustrated embodiment, media flows through the

biochamber in the direction represented by arrows 98, but this direction may be

reversed. In this embodiment, as illustrated in Fig. 18B, the fulcrum 99 of lever

arm 95 is located within flexible substrate 93. In such a configuration, very

small displacements within the flexible substrate caused by action (represented by double-headed arrow 106) from linear actuator 94 result in comparatively larger

displacements within the biochamber.

[0065] Figs. 19A and 19B illustrate another alternate embodiment

useful for mechanical stimulation of a tissue or cell specimen housed within the

biochamber. Fig. 19A represents operation of the biochamber 104 in media

flow mode and Fig. 19B represents operation in compression mechanical

stimulation mode. The compression mode induces flow in the system with a

check valve. The biochamber's interior space is defined by two compliant

membranes 102. An actuator 101 is positioned above or below the biochamber

104 and a fixed surface 103 is positioned opposite the actuator, such that the

biochamber 104 is positioned between the actuator 101 and the fixed surface

103. The movement of the actuator against the biochamber can provide

compressive forces 107 to the cells and or tissues 105 housed within the

biochamber, as illustrated in Fig. 19B by compressing the biochamber 104

against the fixed surface 103. The embodiment illustrated in Fig. 19A and 19B

is appropriate for compressive connective tissues, including meniscus found in

load-bearing joints. In the illustrated embodiment, media flows through the

biochamber in the direction represented by arrows 98, but this direction may be

reversed.

[0066] 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 Figure 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 .

[0067] 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.

[0068] In an alternate embodiment, illustrated in Fig. 20, the valves

108 are located on a disposable flow-path 109 and are actuated by lifters 110

located within the non-disposable cartridge 113. The lifters may include valve

actuators 112 which contact the valves to occlude the flowpath and divert flow.

The lifters may be controlled by a cam-shaft 111 that is rotated to control the

position of the lifters, and thus the open/closed state of the valves. The disposable flow path integrates with the cartridge by swinging into place similar

to the manner in which an audio cassette swings into place in a front-loading

audio cassette player. In this way the flow path and thereby the valves are

precisely aligned with the valve actuators on the cam-shaft lifters. This design

allows for precise fluid routing in a mechanically robust system that is amenable

to a multitude of different fluid flowpath arrangements depending upon the

position of the cam shaft, which is computer-controlled and based upon the

user's input.

[0069] 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 0₂ 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.

[0070] The media and waste reservoirs may have a capacity of about

100 mL to about 500 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, either within the incubator or in large volume carboys placed outside the incubator (for volumes larger than 500 ml). 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-

rnicron 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.

[0071] 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. Tubing diameters may be used to control pressure heads

since flow rate through the system may be determined by the pressure head and the point of greatest resistance to flow in the system. In an alternate

embodiment, this concept is used to advantage to limit the flow or maximize the

flow to a specific section of the flowpath. In an alternative embodiment, the

tubing diameter and length is minimized to minimize the total residual volume

in the tubing and to maximize the effect of diluted substances in the media, such

as pharmaceuticals or growth factors.

[0072] 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. [0073] Figure 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 element or array 56, a photodetector element or

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.

[0074] 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 extra-membranous space,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 membranous samples throughout an

ongoing experiment. In preferred embodiments, the biochamber remains

horizontal in orientation and cell retrieval is carried out manually.

[0075] 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. Figure 12 also includes

connections 64 for flowpath tubing from the biochamber to the perfusion loop.

[0076] Referring to Fig. 17, in one embodiment a biochamber

includes a seal utilizing a gasket 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 gasket 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 gasket 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 gasket 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 seal 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.

[0077] 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. In an

alternative embodiment, the sealing method may include a modified tongue and

groove interface fabricated from compliant materials to lock into the groove such

that the internal pressure on the seal reinforces the tongue and groove. The two

surfaces may then seal in much the same manner as do those of a Tupperware™

container or Zip-Loc™ bag. More than one groove may be employed to provide

for trapped air, to prevent ambient air from reaching the liquid seal interface.

[0078] Figure 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. In a preferred embodiment, each sample collection reservoir may

be connected by a resterilizable connector where a resterilization technique, such

as the application of heat or steam, is applied to the connector to resterilize the

interface after the sample has been retrieved.

[0079] 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

extra-membranous space 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.

[0080] 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.

[0081] 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 or control pod 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 control pod

touch pad or other interface located on the docking station or in each cartridge.

[0082] 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 an unique 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. Alternatively, each

cartridge may operate independent of rack slot location via the cartridge's unique

digital identifier. 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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. [0088] 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.

[0089] 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. [0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] Tissue engineering of small diameter vascular graphs (SDVG) is one example of the numerous processes which can be advantageously conducted within automated bioculture and bioculture experiment systems according to the present invention. SDVG's are intended as a vascular replacement therapy for patients with cardiovascular disease in need of vessel replacement for small (<6mm) vessels. Cardiovascular disease is the leading cause of morbidity and mortality in the developed world, and artificial replacement therapies for SDVGs currently have 40% thrombosis rates after 6 months. Treatment by replacement with autologous grafts is not ideal in that it does not address those patients without appropriate tissues for grafting and leaves a functional vascular deficit at the donor site. The ideal replacement leaves no functional deficit in the individual, withstands the immediate mechanical demands of the implant sight, does not promote thrombogenesis, and can adapt to long-term requirements of the tissue. To be commercially and clinically feasible, xenosourced and/or minimally-invasively harvested autosourced cells or materials should be used. Previous attempts have used immunogenic xenografts, neonatal allogeneic cell sources, or have not been mechanically sufficient.

[0095 ] Systems according to the invention using precision automated feed back control can mimic the in vivo developmental environment for optimal tissue development. In addition, systems according to the invention provide biocompatible materials and physiological requirements for cells to maintain viability, proliferate, differentiate and organize into SDVGs and various complex tissue structures depending on the cells used, selection of biochamber, perfusion conditions, and media and other factors.

[0096] An improved SDVG product and method according to the present invention utilizes an acellularized conduit (e.g., non-immunogenic elastin) to provide a natural substrate with appropriate geometry and compliance. The conduit is placed in a biochamber according to the invention and seeded with autologous cells that can be harvested by minimally-invasive outpatient biopsies. An automated bioculture system according to the invention reproduces the dynamic in vivo environment and thus mechanically "trains" the seeded conduit in the presence of media additives that promote the development of tissue comprised of differentiated cells and extracellular matrix (ECM) capable of withstanding the mechanical loads placed on the tissue in vivo. The vessels, once they have achieved appropriate mechanical properties, are then seeded with liposuction or similarly non-invasevely harvested autologous vascular endothelial cells (VEC) and trained in the perfusion system of the invention prior to surgical implantation.

[0097] More specifically, in this embodiment an SDVG is manufactured, preferably utilizing a large volume, multi-head pump, in concert with a valving configuration to generate a cardiac signature pumping profile with diastolic and systolic pressures. The high flow rate pump, together with a biochamber capable of housing a tubular tissue conduit, is utilized to apply high shear stress and controlled hydrodynamic training of the tissue within the perfusion loop. The SDVG manufacture process utilizes as a tissue scaffold starting material a xenogeneic or synthetic conduit (potentially elastin) with appropriate dimensions. The conduit is seeded with appropriate cells, for example, (of autologous or heterologous source) fibroblasts (potentially from punch skin biopsy), smooth muscle cells (potentially from carotid artery biopsy), and myofibroblasts. The seeding may use orbital shaking or any suitable method, if desired, to enhance cell adhesion. The cells are then allowed to remodel the conduit while being perfused in the bioreactor and monitored through automated sampling, video microscopy, etc. During this stage (from a few weeks to several months in duration) the cells differentiate and develop an organized ECM and form with the ECM a coherent tissue. Media may be supplemental with various factors, including, for example, one or more of the following, to promote production of ECM: ascorbic acid, copper ion, and amino acids. Growth and differentiation optimization are readily achieved using automated feed back controlled monitoring and precision adjustment of flow rate, physiological pressure and pulse wave, media addition, oxygenation and pH.

[0098] VECs (from, e.g., autologous liposuction harvest) are then

introduced to the ECM, and perfusion is continued for about 2-7 days or other

time period sufficient to produce a functional SDVG. Such hydrodynamic

training available in the invention causes the VECs to differentiate, form a

functional neo-endothelium. The resulting tissue engineered product, now

rendered non-thrombogenic my virtue of the neo-endothelium, is then removed

from the biochamber and surgically implanted to a patient in need of an SDVG.

[0099] 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.

Claims

CLAIMSWhat is claimed as new and desired to be protected by Letters Patent:

1. A culture apparatus for use within an incubator, said apparatus comprising:

a rack for supporting at least one flowpath assembly cartridge;

at least one media flowpath assembly cartridge, said cartridge including:

a housing ;

a pump;

at least one valve adapted to prevent or divert media flow;

a control interface;

a sterile media perfusion flowpath loop removable from

said housing without breaching flowpath sterility, said media perfusion loop

containing:

at least one biochamber;

an oxygenator; and

a media reservoir.

2. The apparatus of claim 1, further comprising an access port between said rack and said at least one flowpath assembly cartridge.

3. The apparatus of claim 1, further comprising an access port between said rack and said at least one biochamber.

4. The apparatus of claim 1, wherein said cartridge further comprises at least one noninvasive sensor.

5. The apparatus of claim 1, further comprising a flow sensor.

6. The apparatus of claim 5, wherein said flow sensor comprises a drip chamber.

7. The apparatus of claim 4, further comprising a flow cell removably positionable within said noninvasive sensor.

8. The apparatus of claim 5, wherein said flow sensor is removably positionable within a noninvasive sensor.

9. The apparatus of claim 1, wherein said oxygenator comprises a gas permeable membrane.

10. The apparatus of claim 9, wherein said gas permeable membrane permits active mass transport of gas into the flowpath.

11. The apparatus of claim 9, wherein said gas permeable membrane permits diffusion of oxygen from an incubator environment into the flowpath.

12. The apparatus of claim 11, wherein said gas permeable membrane is positioned over a valve manifold.

13. The apparatus of claim 12, wherein said valve manifold comprises one or more rotatable cams with associated lifters.

14. The apparatus of claim 13, wherein said membrane is capable of being deformed by the lifters via rotation of said one or more cams.

15. The apparatus of claim 1, wherein said cartridge further comprises a data interface.

16. The apparatus of claim 15, wherein said rack further comprises a data interface for integration with the data interface of the cartridge.

17. The apparatus of claim 1, wherein said rack further comprises a data interface for integration with an external controller.

18. The apparatus of claim 1, wherein said rack further comprises a video camera positioned to obtain images from said at least one biochamber.

19. The apparatus of claim 18, wherein said video camera is a CCD video camera.

20. The apparatus of claim 4, wherein said noninvasive sensor is selected from the group consisting of a pH sensor, a glucose content sensor, an oxygen sensor and a spectroscopy sensor.

21. The apparatus of claim 4, wherein said pump is regulated by feedback control via data received from said noninvasive sensor.

22. The apparatus of claim 1, further comprising a means for providing mechanical forces through the biochamber to a cell or tissue within said biochamber.

23. The apparatus of claim 22, wherein said biochamber further comprises a membrane capable of deformation, wherein said membrane provides force translation into said biochamber for mechanically stimulating a cell or tissue within said biochamber.

24. The apparatus of claim 23, further comprising a cam connectable to said rack or said cartridge and positioned to interface with the membrane, and wherein said membrane deformation is provided by contact with said cam.

25. The apparatus of claim 21, wherein said pump is a multi-head pump.

26. The apparatus of claim 25, wherein said apparatus is capable of generating a cardiac-signature pumping profile.

27. The apparatus of claim 21, wherein said pump is capable of modulation via computer control.

28. The apparatus of claim 27, wherein said modulation creates a pressure head signature.

29. The apparatus of claim 27, wherein said modulation creates a pulse wave signature.

30. The apparatus of claim 29, wherein said pulse wave signature comprises a predetermined physiologic wave form.

31. The apparatus of claim 4, wherein said valve is regulated by feedback control via data received from said noninvasive sensor.

32. The apparatus of claim 1, further comprising a sampling interface.

33. The apparatus of claim 1, wherein said media perfusion loop further comprises a waste reservoir.

34. The apparatus of claim 32, wherein said sampling interface is in communication with an automated sampling device.

35. The apparatus of claim 1 further comprising an injection interface.

36. The apparatus of claim 1, wherein said biochamber further comprises a flexible substrate and said cartridge further comprises:

an actuator positioned adjacent to the biochamber; and

a lever arm that passes through said flexible substrate, wherein said actuator acts upon said lever arm to interact with a tissue within said biochamber.

37. The apparatus of claim 36, wherein said lever arm has a fulcrum located within the flexible substrate.

38. The apparatus of claim 1, wherein said cartridge further comprises:

a rotatable cam shaft connectable to at least one lifter, said lifter having at least one valve actuator for contacting said valve to prevent or divert media flow.

39. The apparatus of claim 1, further comprising a sampling device having:

a fluidic pump for transporting a routing fluid;

a means for sterilizing said 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, sterilizing means, and one way flow valve are connected in series by tubing for transporting routing fluid; and

a tube for ttansporting said aliquot of sample within said routing fluid from said one way flow valve to a collection device or analysis instrument.

40. The apparatus of claim 39, further comprising a connecting means between said tube for transporting the aliquot of sample and said collection device.

41. The apparatus of claim 40, wherein said connecting means further comprises a heat source for sterilizing the connection.

42. The apparatus of claim 39, wherein said one way flow valve is a check valve.

43. The apparatus of claim 39, wherein said routing fluid is air.

44. The apparatus of claim 39, wherein said sterilizing means comprises at least one filter.

45. A media flowpath assembly cartridge, comprising:

a housing;

a pump;

at least one valve adapted to prevent or divert media flow;

a control interface;

a sterile media perfusion flowpath loop removably attachable to

said housing without breaching flowpath sterility, said media perfusion loop

containing:

at least one biochamber;

an oxygenator; and

a media reservoir.

46. The cartridge of claim 45, further comprising a power source for stand alone operation.

47. The cartridge of claim 45, further comprising a control interface for stand alone operation.

48. The cartridge of claim 47, wherein said control interface provides for operation through an external computer.

49. The cartridge of claim 47, wherein said control interface provides for operation through a user interface.

50. The cartridge of claim 49, wherein said user interface comprises a control pod.

51. The cartridge of claim 50, wherein said control pod comprises at least one embedded microprocessor.

52. The cartridge of claim 50, wherein said control pod comprises a graphical user interface.

53. The cartridge of claim 45, further comprising a sensor and wherein said pump is regulated by feedback control via data received from said sensor.

54. The cartridge of claim 45, further comprising a sensor and wherein said valve is regulated by feedback control via data received from said sensor.

55. The cartridge of claim 45, wherein said oxygenator permits diffusion from an incubator environment into the flowpath.

56. The cartridge of claim 55, further comprising an automated sampling device having:

a fluidic pump for transporting a routing fluid;

a means for sterilizing said 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, sterilizing means, and one way flow valve are connected in series by tubing for transporting routing fluid to said one way flow valve; and a tube for transporting said aliquot of sample within said routing fluid from said one way flow valve to a collection device or analysis instrument.

57. The cartridge of claim 45, further comprising an injection interface.

58. The cartridge of claim 45, further comprising a noninvasive sensor selected from the group consisting of a pH sensor, a glucose sensor, an oxygen sensor and a spectroscopy sensor.

59. The cartridge of claim 45, wherein said at least one biochamber is convertible for use in static cell culture or in a cell perfusion apparatus and comprises:

a first chamber;

a seal rendering said first chamber removably connectable to said cover; and

at least one insert positioned between the first chamber and the cover, thereby forming a second chamber.

60. The cartridge of claim 59, wherein said biochamber further comprises a difϊuser.

61. The cartridge of claim 59, wherein said seal further comprises two or more sealing interfaces.

62. The cartridge of claim 61, wherein said biochamber further comprises at least one air gap between said two or more sealing interfaces.

63. The cartridge of claim 61, wherein said two or more sealing interfaces are capable of indicating seating of said interfaces by a color change.

64. The cartridge of claim 45, wherein said housing comprises a cutaway for monitoring the biochamber during operation.

65. The cartridge of claim 64, wherein said monitoring is selected from the group consisting of optical viewing, video monitoring, visualization via a microscope, and visualization via an inverted microscope.

66. The cartridge of claim 45, further comprising a light source.

67. A sampling device for use with a cell culture perfusion loop, said sampling device comprising:

a fluidic pump for transporting a routing fluid;

a means for sterilizing said 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, sterilizing means, and one way flow valve are connected in series by tubing for transporting said routing fluid to said one way valve; and

a tube for transporting said aliquot of sample and said routing fluid from said one way flow valve to a collection device or analysis instrument.

68. The sampling device of claim 67, further comprising a connecting means between said tube for transporting the aliquot of sample and said collection device.

69. The sampling device of claim 68, wherein said connecting means further comprises a heat or steam source for sterilizing the connection.

70. The sampling device of claim 67, wherein said tubing for transporting said routing fluid is disposable.

71. A method of manufacturing a vascular tissue, comprising:

providing a perfusion apparatus for use within an incubator, said apparatus comprising:

a rack for supporting at least one flowpath assembly cartridge;

at least one media flowpath assembly cartridge, said cartridge including:

a housing;

a pump;

at least one valve adapted to prevent or divert media flow;

a control interface;

a sterile media perfusion flowpath loop removable from said housing without breaching flowpath sterility, said media perfusion loop containing:

at least one biochamber;

a pump;

a valve;

an oxygenator;

a media reservoir;

introducing an acellularized conduit into said biochamber;

introducing a first biological cell into said biochamber; operating said perfusion apparatus under conditions and for a sufficient time sufficient for development of tissue comprising differentiated cells and extracellular matrix;

introducing a second biological cell onto said extracellular matrix; and

operating said perfusion apparatus under conditions and for a time period sufficient to produce a vascular tissue.

72. The method of claim 71, wherein said tissue is a vascular graft of less than about 6mm in diameter.

73. The method of claim 71, wherein said conduit is xenogeneic or allogeneic.

74. The method of claim 71, wherein said conduit is a synthetic conduit.

75. The method of claim 71, wherein said conduit comprises elastin.

76. The method of claim 71, wherein said first biological cell is from an autologous source.

77. The method of claim 71, wherein said first biological cell is a fibroblast.

78. The method of claim 71, wherein said first biological cell is a smooth muscle cell.

79. The method of claim 71, wherein said first biological cell is a myofibroblast.

80. The method of claim 71, wherein said introducing the first biological cell into said biochamber comprises rotation or orbital shaking.

81. The method of claim 71, wherein said medium reservoir comprises a factor or factors for promoting production of extra cellular matrix.

82. The method of claim 81, wherein said factor is selected from the group consisting of ascorbic acid, copper ion, and arnino acids.

83. The method of claim 71, wherein said second biological cell is a vascular endothelial cell.

84. The method of claim 83, wherein said vascular endothelial cell is from an autologous source.

85. The method of claim 71, wherein said first biological cell is an endothelial cell.

86. The method of claim 71, wherein said first biological cell is a mesenchymal cell.

87. The method of claim 71, wherein said operating said apparatus comprises applying hydrodynamic mechanical stress to cells with in said biochamber.

88. The method of claim 71, wherein said pump provides mechanical forces through the biochamber to cells within said biochamber.

89. The method of claim 71, wherein said biochamber comprises a membrane capable of deformation, and wherein said membrane provides force translation into said biochamber for mechanically stimulating cells within said biochamber.

90. The method of claim 89, wherein said perfusion apparatus further comprises a cam positioned to interface with the membrane, and wherein said membrane deformation is provided by contact with said cam.

91. A tissue manufactured by the method of claim 71.

92. The apparatus of claim 1, wherein said perfusion flowpath loop further comprises a unique digital identifier.

93. The method of claim 71, wherein said perfusion flowpath loop further comprises a unique digital identifier.