Bioreactor design and process for engineering tissue from cells

Abstract

A scaled-up multi-coaxial fiber bioreactor, and variations of this bioreactor. The device is characterized by a hollow housing and an array of from about 20 to about 400 modules of hollow fibers, where each module includes at least three coaxial semipermeable hollow fibers. The innermost fiber provides a boundary for an innermost compartment which is connected to inlet and outlet ports. Arranged coaxially around the central hollow fiber are several other hollow fibers with their respective compartments, each compartment defined by a respective annular space between adjacent fibers and each including inlet and outlet ports. An outermost compartment for permitting integral aeration is the space between the outer side of the outermost fibers and the inner side of the housing, and has inlet and outlet ports. The hollow housing has inlet and outlet manifolds and flow distributors for each of the compartments. In a preferred embodiment the bioreactor is used as an extracorporeal liver. Liver cells, are introduced into one or more annular compartments and media and aeration are provided in others. Plasma from an ailing patient is introduced into another compartment for biotransformation of blood-borne toxins and biosynthesis of proteins, lipids, and other metabolic products.

1 . A bioreactor, comprising: 
 (a) a housing having an inner side comprising: a gas introduction means integral to the housing; and a gas expiration means integral to the housing;    (b) an array of a plurality of modules of hollow fibers, residing within the housing, each module comprising: 
 (i) a plurality of coaxial hollow fibers, each having an inner side and an outer side, including an innermost hollow fiber and an outermost hollow fiber;  
 (ii) a plurality of compartments, comprising: a first compartment defined by the inner side of the innermost hollow fiber; and  
 (iii) at least one additional compartment defined by a respective annular space between adjacent fibers of the plurality of coaxial hollow fibers; and  
   (c) an outermost compartment defined by a space within the inner side of the housing which is not occupied by the plurality of modules.    
   
   
       2 . The bioreactor of  claim 1 , where the hollow fibers are semipermeable.  
   
   
       3 . The bioreactor of  claim 2 , where the hollow fibers comprise a material selected from the group consisting of polysulfone, polypropylene, nylon, polyester, polytetrafluoroethylene, cellulose acetate, and mixed esters of cellulose.  
   
   
       4 . The bioreactor of  claim 1 , where the first compartment, the at least one additional compartment and the outermost compartment each further comprise at least one inlet port and at least one outlet port.  
   
   
       5 . The bioreactor of  claim 1 , where the bioreactor further comprises at least  10   9 cells.    
   
   
       6 . The bioreactor of  claim 5 , where the cells are liver cells.  
   
   
       7 . The bioreactor of  claim 6 , where the liver cells are selected from the group consisting of porcine liver cells and human liver cells.  
   
   
       8 . The bioreactor of  claim 4 , where the housing further comprises at least one inlet manifold and at least one outlet manifold for the first compartment and at least one inlet manifold and at least one outlet manifold for each additional compartment.  
   
   
       9 . The bioreactor of  claim 8 , where at least one manifold further comprises a flow distributor.  
   
   
       10 . The bioreactor of  claim 9 , where at least one compartment further comprises an extracellular matrix.  
   
   
       11 . The bioreactor of  claim 1 , where at least one annular space is about 0.2 millimeters to about 0.8 millimeters.  
   
   
       12 . The bioreactor of  claim 1 , where the bioreactor is sterilized by a means selected from the group consisting of autoclaving, ethylene oxide and gamma radiation.  
   
   
       13 . The bioreactor of  claim 1 , wherein the innermost hollow fiber has a length of about 2 centimeters to about 50 centimeters.  
   
   
       14 . The bioreactor of  claim 8 , where the housing has a first end and a second end, and where each inlet port and each exit port are at the first end of the housing.  
   
   
       15 . The bioreactor of  claim 8 , further comprising: microfibers substantially parallel to the modules of hollow fibers.  
   
   
       16 . The bioreactor of  claim 15 , where the microfibers further comprise at least one aeration inlet port and at least one aeration outlet port.  
   
   
       17 . The bioreactor of  claim 1 , where at least one coaxial hollow fiber is saturated with perfluorocarbon.  
   
   
       18 . The bioreactor of  claim 1 , where at least one coaxial hollow fiber has a pore size less than 1×10 −6  m.  
   
   
       19 . The bioreactor of  claim 1 , where at least one coaxial hollow fiber has a pore size less than 0.1×10 −6  m.  
   
   
       20 . The bioreactor of  claim 1 , where at least one coaxial hollow fiber has a pore size less than 0.05×10 −6  m.  
   
   
       21 . The bioreactor of  claim 1 , where at least one compartment further comprises cells mixed with an extracellular matrix.  
   
   
       22 . A method of supplying cell biosynthesis products to a patient in need thereof, comprising: pumping intravenous feeding solution through a compartment of the bioreactor of  claim 5;  collecting the output; and intravenously feeding the output to the patient.  
   
   
       23 . A serially-linked bioreactor, comprising a plurality of bioreactor subunits, each bioreactor subunit comprising: 
 (a) a housing having an inner side comprising: a gas introduction means integral to the housing; and a gas expiration means integral to the housing;    (b) an array of a plurality of modules of hollow fibers, residing within the housing, each module comprising; 
 (i) a plurality of coaxial hollow fibers, each having an inner side and an outer side, including an innermost hollow fiber and an outermost hollow fiber;  
 (ii) a plurality of compartments, comprising: a first compartment defined by the inner side of the innermost hollow fiber; and at least one additional compartment defined by a respective annular space between adjacent fibers of the plurality of coaxial hollow fibers; and  
   (c) an outermost compartment, defined by a space within the inner side of the housing which is not occupied by the plurality of modules; and    (d) at least one compartment of one bioreactor subunit linked serially to at least one compartment of at least one other bioreactor subunit.    
   
   
       24 . The bioreactor of  claim 23 , where each bioreactor subunit further comprises at least 10 9  cells.  
   
   
       25 . The bioreactor of  claim 24 , where the cells are liver cells.  
   
   
       26 . The bioreactor of  claim 25  where the cells are selected from the group consisting of human liver cells and porcine liver cells.  
   
   
       27 . The bioreactor of  claim 24 , where at least one compartment of each bioreactor subunit further comprises an extracellular matrix.  
   
   
       28 . A method of treating a patient in need thereof comprising: 
 (a) introducing plasma of a patient into a bioreactor subunit of the serially linked bioreactor of  claim 23 ,    (b) forcing at least a portion of the plasma to flow radially through a cell compartment of the bioreactor subunit to form a biotransformed effluent;    (c) introducing the biotransformed effluent into a second bioreactor subunit of the bioreactor of  claim 23;     (d) forcing at least a portion of the biotransformed effluent to flow radially through a cell compartment of the second bioreactor subunit to form supplemented plasma; and    (e) returning the supplemented plasma to the patient's circulatory system.    
   
   
       29 . A multi-coaxial hollow fiber bioreactor, comprising: 
 (a) a housing comprising an inner side; and    (b) a module of hollow fibers, comprising: at least three coaxial semipermeable hollow fibers, including an innermost fiber having an inner side, the inner side defining a first compartment which comprises at least one innermost inlet port and at least one innermost outlet port; a plurality of compartments, each compartment defined by a respective annular space between adjacent fibers of the at least three hollow fibers, including at least one outer inlet port and at least one outer outlet port,    where each compartment comprises a flow communication means between the respective annular space, the respective outer inlet port and the respective outer outlet port and where one of the annular spaces contains eucaryotic cells; and    (c) an outermost compartment defined by a space between the outer side of the outermost fiber of said at least three hollow fibers, and the inner side of the housing, and comprising at least one outermost inlet port, and at least one outermost outlet port;    the housing comprising at least one inlet manifold and at least one outlet manifold for each of the compartments; where at least one of the compartments contains eucaryotic cells.    
   
   
       30 . A method of cell culture, comprising: introducing viable cells into a compartment of the bioreactor of  claim 29 , and passing nutrient medium through coaxially adjacent hollow fibers.  
   
   
       31 . A method of manufacture of a coaxial bioreactor, comprising: 
 (a) bonding outer semipermeable hollow fibers to a manifold,    (b) inserting middle semipermeable hollow fibers into the outer semipermeable hollow fibers,    (c) bonding the middle semipermeable hollow fibers to a second manifold,    (d) inserting the inner semipermeable hollow fibers into the middle semipermeable hollow fibers, and    (e) bonding the inner semipermeable hollow fibers to a third manifold.    
   
   
       32 . The method of  claim 31  where inserting middle semipermeable hollow fibers into the outer semipermeable hollow fibers is vacuum-assisted.  
   
   
       33 . The method of  claim 31  where inserting inner semipermeable hollow fibers into the middle semipermeable hollow fibers is vacuum assisted.  
   
   
       34 . An apparatus for assembly of a bioreactor, comprising: 
 (a) a vacuum head, attached to a negative pressure source, the vacuum head comprising: a hollow housing; a mesh to retain hollow fibers, where the mesh is affixed to the housing; and a holder for a manifold;    (b) a vessel for holding polyurethane epoxy and ends of hollow fibers.    
   
   
       35 . A device for maintaining viable eucaryotic cells, comprising: 
 (a) an annular compartment, having an annular space,    (b) two compartments, adjacent and coaxial to said annular space, where each adjacent compartment contains a liquid, and    (c) an integral aeration supply for the annular space.    
   
   
       36 . The device of  claim 35 , in which the annular space is about 0.2 to about 0.8 mm.  
   
   
       37 . The device of  claim 35 , additionally comprising a second annular compartment having a second annular space.  
   
   
       38 . A method of treating a patient in need thereof, the device comprising: 
 (a) circulating plasma from a patient into a device, comprising: 
 (i) an annular compartment, having an annular space and a complement of eukaryotic cells therein,  
 (ii) at least two compartments, adjacent and coaxial to said annular space, where each adjacent compartment contains a liquid,  
 (iii) an integral aeration supply for the annular space; and  
   (b) allowing a portion of the plasma to traverse the annular compartment.    
   
   
       39 . The method of  claim 38 , in which the said traversing is facilitated by pressure.  
   
   
       40 . The method of  claim 38 , where said portion of the plasma is returned to the patient's circulation system.  
   
   
       41 . The method of  claim 38 , in which said device additionally comprises a second annular compartment having a second annular space and a complement of eukaryotic cells therein.  
   
   
       42 . The method of  claim 39 , in which said device additionally comprises a second annular compartment having a second annular space and a complement of eukaryotic cells therein.  
   
   
       43 . The method of  claim 40 , in which said device additionally comprises a second annular compartment having a second annular space and a complement of eukaryotic cells therein.  
   
   
       44 . The method of  claim 42 , in which said portion of the plasma is returned to the patient's circulation system.  
   
   
       45 . A method of selecting a radial flow rate to enhance cell viability in a bioreactor comprising semi-permeable fibers, comprising: measuring a first hydraulic pressure associated with a first semi-permeable fiber and a second hydraulic pressure associated with a second semi-permeable fiber to obtain a pressure differential and adjusting the first hydraulic pressure, the second hydraulic pressure, or a combination thereof to select one or more radial flow rates so as to improve cell viability.  
   
   
       46 . The method of  claim 45  in which the first fiber and the second fiber are coaxial.  
   
   
       47 . The method of  claim 46  in which the radial flow rate is selected based on the formula:  
     
       
         
           
             
               Δ 
               ⁢ 
               
                   
               
               ⁢ 
               P 
             
             = 
             
               
                 Q 
                 
                   2 
                   ⁢ 
                   π 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   L 
                 
               
               [ 
               
                 
                   
                     ln 
                     ⁡ 
                     
                       ( 
                       
                         
                           r 
                           b 
                         
                         
                           r 
                           a 
                         
                       
                       ) 
                     
                   
                   
                     K 
                     1 
                   
                 
                 - 
                 
                   
                     ln 
                     ⁡ 
                     
                       ( 
                       
                         
                           r 
                           d 
                         
                         
                           r 
                           c 
                         
                       
                       ) 
                     
                   
                   
                     K 
                     2 
                   
                 
               
               ] 
             
           
         
       
     
     where ΔP is the pressure differential, Q is the radial flow rate, L is the length of the shorter of the first and second fiber lengths, r a  is the radial distance from the centerline of the bioreactor to the inner surface of the first fiber, r b  is the radial distance from the centerline of the bioreactor to the outer surface of the first fiber, r c  is the radial distance from the centerline of the bioreactor to the inner surface of the second fiber, r d  is the radial distance from the centerline of the bioreactor to the outer surface of the second fiber, K 1  is the hydraulic permeability of the first fiber, and K 2  is the hydraulic permeability of the second fiber.  
   
   
       48 . A computer readable medium including instructions therein for calculating a radial flow rate in a bioreactor comprising semi-permeable fibers, said instructions including the steps of: 
 (a) receiving measurements of hydraulic permeability for each of at least two coaxial semi-permeable fibers,    (b) receiving measurements of hydraulic pressure for at least two coaxial semi-permeable fibers, and    (c) estimating said radial flow rate between said coaxial semi-permeable fibers.