Patent Abstract:
A bioreactor system for growing and conditioning tissue for research and implantation in a human or animal body is disclosed which includes one or more tissue growth chambers for growing and conditioning tissue, each chamber being defined by a housing and providing a fluid culture media cavity which can act as a reservoir. A construct for growing three-dimensional tissues is housed in each tissue growth chamber. Each chamber is connected to a source of pressurized air for applying a controlled pressure to the chamber media cavity. The tissue growth chambers can be mounted on an agitation device such as a shaker system which enhances mass transport within the chamber media cavity. A control system is provided to control the pressure and temperature of the pressurized gas delivered to the chamber media cavity and subsequently to the tissue construct.

Full Description:
CROSS-REFERENCED TO RELATED APPLICATIONS 
     This application is a non-provisional application of Application No. 61/000,485, filed Oct. 26, 2007 and claims priority from that application which is also deemed incorporated by reference in its entirety in this application. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     Not applicable 
     BACKGROUND OF THE INVENTION 
     I. Field of the Invention 
     The present invention relates to a method and apparatus for growing and conditioning tissue engineered medical products and in particular to a method and apparatus for a servocontrolled bioreactor with a dynamic pressurization system and a chamber with an integrated nutrient reservoir for conditioning tissue engineered medical products (TEMPs) in the orthopedic, secretory organ and vascular areas. TEMPs can include, without limitation, various types of tissue in several areas. Within the orthopedic area TEMPS can include: cartilage, bone, meniscus, ligaments, tendons, muscle, and other musculo-skeletal devices. Within the secretory organ area TEMPs can include liver, kidney, skin and other organs. Within the vascular area TEMPs can include, but are not limited to, heart valves, blood vessels, and cardiac patches. Other TEMPs can include: cornea, bladder, urethra, small intestine or any other tissue that might be replaced in the body. 
     Tissue engineering is a rapidly growing area that seeks to create, repair and/or replace tissues and organs by using combinations of cells, biomaterials, and/or biologically active molecules. It is an interdisciplinary field that integrates aspects of engineering, and other quantitative sciences, with biology and medicine. Research and technology development in tissue engineering promises to revolutionize current methods of health care treatment and significantly improve the quality of life for millions of patients. As one indication of the scope of the problem that tissue engineering addresses, worldwide organ replacement therapies utilizing standard organo-metallic devices consume 8 percent of medical spending, or approximately $350 billion per year. Organ transplantation is another option for replacing damaged or diseased tissue, but one that is severely limited by donor availability. Tissue-engineered products hold the promise for true functional replacement. However, despite early laboratory successes, few functional tissue engineered products are currently available for clinical use. 
     Researchers have sought to develop living alternatives to traditional “man-made” medical devices. These TEMPs use the patient&#39;s own cells to create replacement devices that can be cultured and grown once they are implanted. Through design and fabrication of biomaterials and specification of cells or biomolecules, it is hoped that TEMPs will play a major role in many future regenerative medicine therapies. In the orthopedic area considerable energy is being expended on the development of tissue engineered cartilage, meniscus, bone, ligaments or tendon replacements. Likewise, similar efforts are being made to develop new replacements for heart valves, arteries, heart muscle tissue and venous valves. Tissue engineered replacements for secretory organs such as the liver, kidney and skin also hold great promise for future therapies. Tissue engineered skin replacements are already available and are dramatically improving the outcomes for patients with diabetic ulcers, as well as burn victims and those undergoing certain cosmetic therapies. 
     The field of tissue engineering in recent years has included the development of bioreactors which provide a means of conditioning a developing tissue by applying mechanical stresses to a construct (cells or cell seeded substrate while circulating nutrient media around and or through the construct. Cells and tissues grown in bioreactors able to mimic physiological conditions including mechanical forces have enhanced tissue development, mechanical properties and function. These bioreactor systems typically include a bioreactor chamber coupled to a device which generates motion and applies forces, pressures and or deformations to the tissue construct via a mechanical feed-through (push or pull rod). Additionally, these systems typically include a separate reservoir that contains a nutrient media for sustaining the cells within the tissue construct. The reservoir is often connected to the bioreactor chamber via tubing and a mechanical pumping system. The biochemistry of the nutrient media can be maintained by exposing it to the environment that is created within an incubator system. The incubator maintains the temperature, as well as gas concentrations (CO 2 , O 2 , etc.). The exposure of the media to the environment is often accomplished by placing a vented reservoir directly in the incubator or by circulating the media through gas permeable tubing located inside the incubator. Gas concentrations can also be bubbled through the media to maintain appropriate culture conditions. 
     II. Related Art 
     Other bioreactor systems have addressed aspects of the need for an apparatus dedicated to growing and conditioning tissue engineered medical products. As will become apparent, the present device described in this patent application surpasses those systems in several respects. 
     Spaulding et al. (U.S. Pat. No. 5,330,908), (U.S. Pat. No. 6,001,643) and Schwartz (U.S. Pat. No. 5,437,998), (U.S. Pat. No. 5,665,594) disclose bioreactor systems that operate at ambient pressure and in which mechanical stresses to the cells are applied by rolling the chamber about its cylindrical axis. Conversely, the bioreactor system of the present invention applies a static or varying pressure to the chamber and subsequently the tissue construct within while the chamber is agitated via a multi-axis motion device. 
     Dunkleman et al. (U.S. Pat. No. 5,792,603), Peterson et al (U.S. Pat. No. 5,846,828) and Vilendrer et al (U.S. Pat. No. 7,348,175) disclose bioreactor systems for vascular grafts in which an alternating or varying flow or backpressure from an external media storage device is provided via a tube and pumping system. Control of the flow and/or back pressure is provided by an external flow pump. Whereas, in the present system, the sealed chamber and media reservoir are combined thus eliminating the need for an interconnecting tube to create flow. Additionally, pressurization in the present system may be provided directly via a high pressure air source and servovalve controlled by a microprocessor servocontrol system which supplies specific air pressures to one side of a deformable membrane. As the membrane deforms the pressure is transferred to the interior of the chamber. Applegate et al. (U.S. Pat. No. 5,843,766) discloses a bioreactor system for grafts with inlet and outlet ports for evenly distributing media flow across and generally parallel to the tissue substrate that also requires a separate external nutrient fluid reservoir. In the present system, the agitation of the chamber and deformation of the membrane aid in convective mass transport. 
     Amrani et al. (U.S. Pat. No. 5,902,937) discloses an in vitro testing system for testing blood/tissue interaction that utilizes twin chambers with top and bottom tissue membranes and an internal conduit for conducting blood between the chambers. Blood flow actuation is provided by pistons that alternately depress the membranes on each chamber to displace blood from one chamber to the other. Naughton et al. (U.S. Pat. No. 6,008,049) discloses a diffusion gradient bioreactor system for conditioning tissue engineered liver whereby the tissue is exposed to two nutrient flows. The primary purpose of their device is to move solutes through the device via flow. With the present system only one sealed chamber is needed and it is not interconnected with another chamber to create flow. Also, as indicated above, pressurization in the present invention may be provided directly through a deformable membrane. The high pressure air source is controlled via a microprocessor-controlled servovalve. 
     Flatt et al (U.S. Pat. No. 6,060,306) discloses a bioreactor chamber with a substrate that is sealed to the sides of the chamber. Using various mechanical actuation means, a pressure differential is created across the substrate to create a fluid flow through it. In the present system, the substrate is not sealed to the sides of the chamber so any applied pressure does not create a differential across the substrate to create a fluid flow through it. 
     Peterson et al (U.S. Pat. No. 6,121,042) and Carpentier et al (U.S. Pat. No. 6,210,957 B1) disclose bioreactor systems that provide flow about a substrate that is attached to a structure. These systems pump fluid through the chamber using inlet and outlet ports, or actuate the structure to control media flow characteristics around the substrate. In contrast, the substrate of the present system is not attached to a structure but is free to move within the chamber nor is fluid pumped through the chamber to create controlled media flow characteristics. Mechanical agitation of the chamber to enhance fluid mass transport is provided via the shaking motion of a mechanical shaking system and a hydrostatic stress may be provided by pressurization of the membrane as indicated above. 
     Smith et al (U.S. Pat. No. 6,171,812 B1) and Vilendrer (U.S. Pat. No. 7,410,792 B2) further disclose a bioreactor system that has means for perfusion and application of forces to the substrate within the bioreactor chamber. In the present system, neither of these forces within the chamber is explicitly provided. 
     Takagi et al (U.S. Pat. No. 6,432,713 B2), (U.S. Pat. No. 6,599,734 B2), (U.S. Pat. No. 6,607,917 B2), (U.S. Pat. No. 6,921,662 B2) describes bioreactor systems which utilize a sealed chamber that is interconnected to a medium reservoir via a tube or circuit. Pressurization of the chambers is provided via actuator driven pistons and control of the system is accomplished using regulators and timers or a microcomputer that turns various valves on at the appropriate times. Tagaki et al (U.S. Pat. No. 7,144,726) also describes a bioreactor chamber with fluid port and a magnetically driven pressing plate for creating pressurization within the chamber. These references do not suggest the use of a sealed combined chamber. 
     While some of the above described systems have worked in certain applications, there remains a need in the art to simplify the method and apparatus for growing and conditioning tissue while maintaining environmental control. 
     SUMMARY OF THE INVENTION 
     The present invention addresses a need in the art for a method and apparatus for growing and conditioning tissue and other needs which will be appreciated by those skilled in the art upon reading and understanding the teachings of the present invention. 
     The subject matter of the present development relates to a bioreactor system for growing and conditioning tissue in various embodiments including a bioreactor system for three-dimensional tissue growth and stimulation. This system features a bioreactor tissue growth chamber capable of dynamic pressurization and an integrated fluid culture media reservoir which does not require a flow loop. In accordance with the present invention, it has been discovered that beneficial mechanical stress can be applied directly to a construct by deforming a membrane on one or more surfaces of the tissue growth chamber without the need for mechanical feed-throughs. Additionally, integrating the reservoir within the bioreactor chamber can eliminate the culture media flow loop and associated tubing. Consequently, the present invention features a bioreactor or tissue growth chamber that couples the stress application with the nutrient media exchange mechanism in one unit. 
     One embodiment includes a bioreactor system for growing and conditioning tissue for research or future implantation in a patient. The system includes one or more tissue growth chambers for growing and conditioning the tissue and each tissue growth chamber includes a housing which is a fluid culture media reservoir for growing and conditioning at least one tissue construct that may include one or more substrates for growing three-dimensional tissues. The tissue growth chamber includes separate ports for introducing pressurized air or other gas and fluid culture media. The bioreactor system further includes at least one pressurized gas space proximal to or contained within the chamber and a control system to selectively control the pressure and temperature of the gas (normally air) from the source of pressurized gas which is transmitted to the tissue growth chamber via a non gas-permeable membrane to create a dynamic hydrostatic state of stress to developing tissues or cells. In this manner, a programmable static or dynamic pressure profile can be delivered. A substrate on which the three-dimensional tissue construct may be grown may include bio-compatible three-dimensional framework having interstitial spaces bridgeable by cells. The one or more tissue growth chambers may be mounted on a shaker to agitate the culture media to enhance convective mass transport. At least one device for facilitating gas exchange within the chamber is provided to maintain the appropriate gas concentration (atmosphere). This may be a gas-permeable membrane between the chamber and a desired gas environment. 
     The embodiment may include a system for distributing pressurized air and mechanically agitating tissue growth chambers which are designed to be contained in an incubator and interface with a computer-controlled system and a source of dried, heated air, normally external to the incubator. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       In the drawings: 
         FIG. 1A  shows a bioreactor chamber according to one embodiment of the present invention; 
         FIG. 1B  is a cross-sectional view of the bioreactor chamber shown in  FIG. 1A ; 
         FIG. 2  shows a schematic functional diagram of a microprocessor servocontrolled bioreactor system according to one embodiment of the present invention; 
         FIG. 3  illustrates one embodiment of a pressure manifold assembly shown schematically in  FIG. 2  including various components that are mounted to the manifold; 
         FIGS. 4A and 4B  depict assemblies that fit within the incubator according to one embodiment of the present invention,  FIG. 4A  showing a manifold assembly and  FIG. 4B  showing bioreactor chambers, shaker plate and shaker; 
         FIG. 5  depicts the assemblies of  FIGS. 4A and 4B  as assembled in an incubator; and 
         FIG. 6  depicts an entire system as it may be operated in a laboratory. 
     
    
    
     DETAILED DESCRIPTION 
     In the following detailed description, reference is made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration specific embodiments in which the invention may be practiced. The embodiments shown and described are meant as examples only and are not intended to limit the scope of the concepts of the invention in any manner. Illustrative embodiments are described in sufficient detail to show a full grasp of the invention by the inventors and to enable those skilled in the art to practice the invention. It will be understood that the embodiments may be combined, or that other embodiments may be utilized and that structural, logical and electrical changes may be made without departing from the spirit and scope of the present invention. The following detailed description provides examples, the scope of the present invention being defined by the appended claims and their equivalents. 
     The present disclosure relates to a method and apparatus for a bioreactor system for three-dimensional tissue stimulation featuring dynamic pressurization and an integrated reservoir which does not require a flow loop. This will be described with reference to  FIGS. 1A, 1B, 2, 3 and 4 .  FIG. 1A  shows a bioreactor or tissue growth chamber, generally at  100  and  FIG. 1B  depicts a cross-sectional view of the tissue growth chamber  100  of  FIG. 1A . One or more of these chambers may be used with the tissue growing and conditioning system of the invention. In one current embodiment, for example, up to 12 chambers can be included. 
     As seen in  FIG. 1B , a tissue construct  102  has been created and placed into a main hollow chamber or media cavity  104  defined by a main housing  106  which is closed by upper and lower endcaps  108  and  110 , respectively. The term “construct” may refer to the tissue itself or a tissue/substrate combination. The chamber media cavity  104  is further sealed on both upper and lower ends with membranes as at  112 ,  114 , respectively. In this embodiment, membrane  112  is a non gas-permeable material and membrane  114  is gas-permeable and communicates with vent holes for gas exchange shown at  119  to facilitate gas exchange between the chamber media cavity  104  and a desired external gas environment. Other chamber embodiments may include different membrane configurations utilizing as few membranes as none or as many as necessary to achieve the desired media cavity pressurization and or media gas concentration. 
     The tissue construct is housed in the tissue growth chamber but is not attached to the chamber. The substrate may be permeable and the culture media can circulate through the substrate to contact all surfaces of the construct. The substrate may be fabricated from a material designed to facilitate three dimensional tissue growth and include a biocompatible three dimensional framework  103  that includes interstitial spaces  105  that are bridgeable by cells (not shown). That substrate material may be biological in nature and be comprised of extracellular matrix, decellularized tissue, native tissue explants, polymer matrices, woven fiber meshes, porous ceramic lattices, porous metal structures, or any other type of material that supports cell growth and differentiation. The tissue construct may also be substrate free, and be comprised of cells alone. The tissue construct is three dimensional in nature and may be tissue-engineered cartilage, skin, bone, liver, lung or some other tissue-engineered graft or medical device. It will be appreciated that the tissue can be grown without using a substrate as by using a cell suspension or aggregate of cells. 
     The tissue growth chamber membranes may be either gas permeable or non gas permeable and are made out of a material that permits elastic deformation. The membranes may either be flat or curved or fabricated with a specific geometry to cover and seal the media cavity appropriately. The membranes  112 ,  114  are held in place by a pair of opposed endcaps/support structures at  108 ,  110 , respectively, that sandwich/clamp the membranes between the endcaps and the main housing sidewall  106 . Optionally, and particularly, if the upper membrane is relatively stiff, spaced opposed o-ring members as at  116  and  118  or some other equivalent sealing mechanism, such as glue, soft deformable sealant, tape, caulk, silicone grease, or the like, may be used to seal the membrane  112  between the chamber main housing sidewall  106  and the endcap  108 . The membranes  112 ,  114  may also be fixed, bonded, or a permanent component of either the endcaps  108 , 110  or the chamber main housing sidewall  106 . Sealing mechanisms may also be provided for the membrane  114  between housing sidewall  106  and endcap  110 . 
     The endcaps themselves are held in place by the compressive action of a plurality of fasteners that may be bolts  120  ( FIG. 1A ) that are threaded through washers  122  and endcap  110  and are threaded into corresponding openings in opposite endcap  108  at  124 . It will be appreciated that the bolts may be replaced by other suitable fastening elements such as, for example, threaded tie rods provided with nuts on either end. Alternatively, the endcaps  108 ,  110  may also be held and sealed against the membranes  112 ,  114  and chamber main housing sidewall  106  via threaded connections or some other clamping means. 
     Access is provided to the chamber after it has been sealed, such that media may be deposited/injected into the chamber media cavity  104  via a media port  130  (inlet/outlet access port) which includes an access opening into the chamber media cavity  104 . The media port  130  includes a media fitting  132 , o-ring seal  134  and plug fitting  136 . 
     The o-ring seal  134  in some embodiments may be replaced by any of several other sealing techniques and devices including Teflon® (polytetrafluoroethylene) tape, silicone grease, interference thread fit, glue, epoxy, caulk, or any other satisfactory sealing mechanism. The media port may also be machined, molded or directly fabricated as an integral part of the chamber main housing. Depending on the application, the chamber may be constructed without an access port or provided with as many media ports as is required for injecting media and bleeding out air. For some applications, it may be desirable for air to remain in the chamber. Once the chamber cavity  104  is filled or partially filled with media, the media fitting  132  is closed as with plug fitting  136 . An optional additional port  138  may be provided in the chamber main housing to accommodate a pressure transducer to verify and monitor the internal chamber pressure. 
     The application of a hydrostatic state of stress is an important aspect of the operation of the bioreactive chamber. Accordingly, a pressurized gas fitting  138  is provided to supply controlled pressurized gas (or air) into a pressurized gas space  140  in endcap  108 . In the embodiment of  FIGS. 1A and 1B , the pressurized gas fitting  138  is a quick-disconnect fitting, but any type of fitting that allows the user to connect a pressurized air/gas supply to the chamber could be used. The pressurized gas space is open to membrane  112  such that a change in gas pressure in space  140  causes Membrane A  112  to deform and increase the pressure in the chamber media cavity  104 . 
     In the illustrative embodiment, membrane  112  is non-permeable and membrane  114  is permeable. Therefore, gas exchange can occur through membrane  114  and vent holes  142  provided in endcap  110 . Of course, in the event that no gas exchange is required, as is the case in certain applications, both membranes could be non gas-permeable. In certain other applications, the addition of pressurized gas directly to the inside of the chamber may be desired and, in such embodiments, membrane  112  is selected from gas permeable materials and membrane  114  is of a non gas-permeable material. In that arrangement, the pressurized gas may contain the appropriate concentrations of gas species needed for successful cell culture. 
       FIG. 2  shows an embodiment of a dynamic pressurization and control system, generally  200 , suitable to be used to create a dynamic hydrostatic stress state in the chamber  104  of  FIGS. 1A and 1B  for the tissue construct  102 . The pressurization system, as will be described below, includes a Pressure Pack shown in dashed lines at  204  for creating a pressurized supply of air or other system gas which may be generally in the 80-150 psig range and to provide a vacuum in the 5 psia range, however, any pressure and vacuum range that would be required is acceptable. 
     Air to be pressurized starts out as ambient air that is drawn into the system through a filter  206  and then warmed by an associated heater  208 , which may be a tubular air heater as in the illustrative embodiment. The warmed air travels to a proportional valve  210  attached to a manifold assembly indicated by a dotted line at  212  located inside the incubator  202 . The proportional valve  210  controls the amount of air that is drawn past a vacuum gauge  214  into a vacuum tank  216 , through a compressor  218  and into a pressure tank  220 . The compressor  218 , in turn, is coupled to an AC motor  222  which is controlled by an AC motor inverter drive  224  which controls the motor at a constant rpm. 
     Pressure tank  220  is used as an accumulator for storing pressurized air supplied by compressor  218 . A pressure relief valve is provided at  226  with integrated pressure gauge  228 . The pressure relief valve may be set to any desired pressure and is normally set to about 100 psig (or any other appropriate pressure limit) as a safeguard to prevent over pressurizing the system. A pressure transducer  230  is connected to the pressure tank and to a controller  232  which is also connected to control proportional valve  210  to complete a feedback loop with control software to maintain a desired pressure in the tank  220 . In this manner, if the pressure in the tank  220  falls below the desired pressure level, the controller  232  sends a signal to proportional valve  210  to open incrementally to increase air flow to the compressor and thereby increase the tank pressure. Conversely, if the pressure in the tank is above the desired pressure level, the controller signals the proportional valve to begin to close, which decreases the available air that can be compressed and the air pressure in the tank decreases. 
     Prior to being supplied to the incubator bioreactor chamber, the pressurized air is passed through a filter  234  (or series of filters) to remove any particles and then through an air dryer  236  to prevent moisture from accumulating in the line. Moisture is expelled out of the dryer into the ambient air via a purge line  238 . 
     Upon entering the incubator, the pressurized air travels through the manifold assembly  212 . A three-way ported servovalve  240  when commanded in one direction ports the pressurized air supply to a gas distribution manifold  244  which, in turn, subsequently supplies pressurized air to the chambers  100 . Pre-drying the air reduces the potential for corrosion of the servovalve  240 . Prior to encountering the servovalve  240 , the air is passed through an air heater  242  wherein it is heated to match the temperature inside the incubator. A pressure transducer  246  on gas distribution manifold  244  monitors the pressure inside the manifold  244 . If the servovalve  240  is in a completely closed position and the air supply to the gas distribution manifold  244  is blocked, as shown in  FIG. 2 , the air slowly escapes out of a bleed valve  248 . The servovalve  240  is controlled via an electric voltage signal output from the system controller  232 . The system controller  232  monitors the pressure in bioreactor chambers  100  via pressure transducer  246  or optional pressure transducer  249  through port  247  in chamber  100  and sends a control signal to the servovalve  240  to maintain the desired pressure waveform. Additionally, the controller  232  can be used to monitor the pressure output signal of an optional pressure transducer  250  which is connected directly to a chamber  100  and can be used for calibration purposes to correlate the pressure in chamber  100  with the pressure in the gas distribution manifold  244 . Optional pressure transducer  249  can also be used as an alternate feedback device to control the servovalve  240  mounted on the gas distribution manifold  244 . 
     When the servovalve  240  is commaned to provide pressure, high pressure gas enters the gas distribution manifold  244  and pressurizes the chambers  100 . When the servovalve is commanded to reduce pressure, it connects manifold  244  with the vacuum tank  216  as needed to reduce the chamber pressures to a lower value. The pressure command can take any of many forms to control the pressure supplied to the manifold on a steady or time-variable basis including a static setting, a sinusoidal, trapezoid, ramp or physiologic waveform. A personal computer  252  provides a user interface for communicating with the controller  232 . Additionally, controller  232  can be configured to communicate conditions within the bioreactor system to the outside world as via a data acquisition system (DAQ)  254  and alarm  256 . Advantageously, in this manner, using a non gas-permeable membrane  112 , the pressurized air supply can be kept separate from the gas mixture inside the incubator, yet produce the desired hydrostatic pressure effect on growing tissue. 
     It will also be appreciated that the configuration of  FIG. 2  could also be constructed with all (or some combination of) the components of the manifold assembly located outside the incubator. In embodiments for certain applications, it is acceptable for the air from the incubator to mix with the pressurized air supply. In such embodiments, inlet filter  206  and bleed-valve/silencer  248  can be moved inside the incubator  202  and the inlet air would not need to be heated between the filter  206  and the proportional valve  210 . 
       FIG. 3  illustrates one embodiment of a pressure manifold assembly including gas manifold components shown schematically in  FIG. 2 . The manifold assembly is mounted on an incubator shelf  300  designed to slide into a standard incubator shelf holder (not shown). High pressure air from the pressure pack is received in a servovalve manifold  302  through a high pressure inlet fitting  303  and is routed through servovalve  240  where it is either diverted to a high pressure outlet  304 , or directed to the gas distribution manifold  244 . In the manifold, pressure is set to the desired dynamic pressure using feedback from the pressure transducer  246  which is connected to the center of the gas distribution manifold  244 . The pressurized air/gas exiting the manifold is supplied to a plurality of tissue growth chambers  100  through a plurality of manifold distribution fittings  305  which may be connected by non gas-permeable chemically inert tubing (not shown) to each chamber. 
     As indicated, the pressure in the chambers  100  may be reduced as needed by commanding the servovalve  240  to connect to the vacuum tank  216  which rapidly reduces the air/gas pressure until the appropriate pressure has been reached. Air/gas exits the servovalve by traveling through a vacuum outlet fitting  305  connected to vacuum tank  216 . 
     It will be recognized that in a different embodiment, an additional air supply may be required to maintain an appropriate pressure, therefore a secondary air inlet (in addition to the proportional valve inlet  210 ) may be necessary. Accordingly, the servovalve manifold  302  in  FIG. 3  includes a secondary vacuum inlet that has been shown sealed with a plug at  306 , which can be used if larger airflow is necessary. 
       FIG. 4A  illustrates another view of a mounted manifold assembly  212  mounted on an incubator shelf  300 . The shelf is carried on a pair of support legs  310  and  312  with attachment feet  314  and  316 , respectively which are designed to mount in an incubator as shown in  FIG. 5 . 
       FIG. 4B  shows a plurality of bioreactor chambers  100  mounted atop a Belly Dancer Shaker System  400  designed to be contained in an incubator. Additional openings are provided at  402  to accommodate additional chambers. The shaker system is one built by Stovall Life Science, Inc. of Greensboro, N.C. but equivalent systems may be used. The shaker provides a means of selectively cyclically agitating the cell culture media to enhance nutrient mass transport through the construct and gas transfer across the gas permeable membrane  114  ( FIG. 2 ). The shaker system includes a shaker module  400  and a connected shaker plate  404  or platform on which are mounted the plurality of chambers  100 . 
     It will be appreciated that the agitating movement of the bioreactor chamber creates varying motion in the nutrients containing fluid culture medium which may aid cell seeding on the substrate or help facilitate cell aggregation and adhesion as well as the transfer of nutrients within the substrate. The movement of the chamber may further generate varying multi-axial stresses in the tissue construct which provides added stimulation which may enhance tissue development, mechanical properties and function. 
       FIG. 5  depicts the assemblies of  FIGS. 4A and 4B  as assembled in an incubator  202  where the actual tissue growth is carried on. Such incubators are available commercially and, of course, include their own temperature control systems to create the proper temperature environment for carrying on the tissue growth. 
       FIG. 6  depicts a typical view of an entire system, including all the auxiliary devices as would be typically set up in a tissue growth laboratory. Note that the pressurized air supply or pressure pack  204 , together with heaters  208 ,  242 , filter  206  and air drier  236  are situated outside the incubator  202 , but are connected to the incubator through tubing connections as shown. Auxiliary gas tanks  600 ,  602  are also provided and are generally connected to the incubator gas inlet ports, but may be connected to media inlet ports of tissue growth chambers inside the incubator  202 . Electrical control is supplied by controller  232  in combination with a personal computer. Both components may be programmed in any manner desired to control the tissue growth environmental conditions. Other embodiments may use alternative control platforms including but not limited to:
     Programmable Logic Controllers, Personal Digital Assistants, mini computer, super computer, etc.   

     The embodiments provided herein are intended to demonstrate representative embodiments of the present subject matter. Variations in structure and design are possible without departing from the scope of the present invention, which is defined by the appended claims and their equivalents. Other embodiments for bioreactor configurations may include the combination of various elements and configurations as provided herein. 
     This invention has been described herein in considerable detail in order to comply with the patent statutes and to provide those skilled in the art with the information needed to apply the novel principles and to construct and use embodiments of the example as required. However, it is to be understood that the invention can be carried out by specifically different devices and that various modifications can be accomplished without departing from the scope of the invention itself.

Technology Classification (CPC): 2