Automated bioculture and bioculture experiments system

Abstract

The present invention provides a feedback controlled bioculture platform for use as a precision cell biology research tool and for clinical cell growth and maintenance applications. The system provides individual closed-loop flowpath cartridges, with integrated, aseptic sampling and routing to collection vials or analysis systems. The system can operate in a standard laboratory or other incubator for provision of requisite gas and thermal environment. System cartridges are modular and can be operated independently or under a unified system controlling architecture, and provide for scale-up production of cell and cell products for research and clinical applications. Multiple replicates of the flowpath cartridges allow for individual, yet replicate cell culture growth and multiples of the experiment models that can be varied according to the experiment design, or modulated to desired cell development of cell culture end-points. The integral flowpath cartridge aseptic sampling system provides for dynamic analysis of metabolic products or representative cells from the culture.

What 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 transporting 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 cover;    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 diffuser.  
     
     
         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  6 mm 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 amino 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.