Abstract:
This invention relates to a cell expansion system and to a method of determining when to harvest cells from the cell expansion system.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit of U.S. Provisional Application No. 61/088,223 filed Aug. 12, 2008 and U.S. Provisional Application No. 61/160,082 filed Mar. 13, 2009. 
     
    
     BACKGROUND 
       [0002]    Human stem cells, which have been expanded in culture from a small amount of donor cells, can be used to repair or replace damaged or defective tissues and have broad clinical applications for treatment of a wide range of diseases. Recent advances in the area of regenerative medicine demonstrate that stem cells have unique properties such as self-renewal capacity, the ability to maintain the unspecialized state, and the ability to differentiate into specialized cells under particular conditions. 
         [0003]    As an important component of regenerative medicine, the bioreactor or cell expansion system plays a role in providing optimized environments for cell growth and expansion. The bioreactor provides nutrients to the cells and removal of metabolites, as well as furnishing a physiochemical environment conducive to cell growth in a closed, sterile system. Cell expansion systems can be used to grow other types of cells as well as stem cells. 
         [0004]    Many types of bioreactors are currently available. Two of the most common include flat plate bioreactors and hollow fiber bioreactors. Flat plate bioreactors enable cells to grow on large flat surfaces, while hollow fiber bioreactors enable cells to grow either on the inside or outside of the hollow fibers. 
         [0005]    It is not current practice to look inside a bioreactor to determine when to harvest the expanded cells without destroying the sterility of the closed system. A way to determine when to harvest the cells while still maintaining sterility is necessary. It is to such methods that the present invention is directed. 
       SUMMARY OF THE INVENTION 
       [0006]    The invention relates to a method of determining when to harvest cells from a cell growth chamber of a cell expansion system. The steps include measuring the number of cells initially loaded into the cell growth chamber; measuring the concentration of lactate generated at various times throughout a cell expansion cycle; and using the measurements to determine when to harvest the expanded cells from the cell growth chamber at the end of a cell expansion cycle. 
         [0007]    The invention also relates to a cell expansion system which includes a cell growth chamber containing cells; at least one pump and a digital computer. The digital computer further includes at least one processor; a memory; a user interface; and a controller interface. The user interface is configured to allow an operator to enter the number of cells initially loaded into the cell growth chamber and the concentration of lactate measured at various times during a cell expansion cycle. The memory takes the number of cells initially loaded into the cell growth chamber and the concentration of lactate measured at various times throughout the cell expansion cycle to determine when the cells in the cell growth chamber should be harvested. The memory then sends a signal through the processor to the controller interface to instruct the at least one pump to pump fluid through the cell growth chamber to remove the cells from the cell growth chamber. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0008]      FIG. 1  is a front side elevation view of an embodiment of a cell growth chamber. 
           [0009]      FIG. 2  is a schematic of an embodiment of a cell expansion system. 
           [0010]      FIG. 3  is a graph depicting when to initiate a harvest procedure. 
           [0011]      FIG. 4  is a flow chart of the steps of an embodiment of a method for determining when to initiate a harvest procedure. 
           [0012]      FIG. 5  is a block diagram illustrating a digital computer aspect of the cell expansion system. 
       
    
    
     DETAILED DESCRIPTION 
       [0013]    As discussed above, a number of bioreactor or cell growth chamber configurations exist for culturing cells. No particular configuration is required for this invention. However, as but one example, not meant to be limiting, is a hollow fiber bioreactor shown in  FIG. 1 . 
         [0014]    With reference now to  FIG. 1 , an example of a cell growth chamber  100  is shown in front side elevation view. Cell growth chamber  100  has a longitudinal axis LA-LA and includes cell growth chamber housing  104 . In at least one embodiment, cell growth chamber housing  104  includes four openings or ports: IC inlet port  108 , IC outlet port  120 , EC inlet port  128 , and EC outlet port  132 . 
         [0015]    Fluid in a first circulation path enters cell growth chamber  100  through IC inlet port  108  at a first longitudinal end  112  of the cell growth chamber  100 , passes into and through the intracapillary side (referred to in various embodiments as the intracapillary (“IC”) side or “IC space” of a hollow fiber membrane) of a plurality of hollow fibers  116  (the hollow fibers are also generally referred to as a membrane), and out of cell growth chamber  100  through IC outlet port  120  located at a second longitudinal end  124  of the cell growth chamber  100 . The fluid path between the IC inlet port  108  and the IC outlet port  120  defines the IC portion  126  of the cell growth chamber  100 . Fluid in a second circulation path flows in the cell growth chamber  100  through EC inlet port  128 , comes in contact with the extracapillary side or outside (referred to as the “EC side” or “EC space” of the membrane) of the hollow fibers  116 , and exits cell growth chamber  100  via EC outlet port  132 . The fluid path between the EC inlet port  128  and the EC outlet port  132  comprises the EC portion  136  of the cell growth chamber  100 . Fluid entering cell growth chamber via the EC inlet port  128  is in contact with the outside of the hollow fibers  116 . Small molecules (e.g., water, oxygen, lactate, etc.) can diffuse through the hollow fibers from the interior or IC space of the hollow fibers to the EC space, or from the EC space to the IC space. Large molecular weight molecules such as growth factors are typically too large to pass through the hollow fiber membrane, and remain in the IC space of the hollow fibers. The media may be replaced as needed. Media may also be circulated through an oxygenator to exchange gasses as needed. Cells can be contained within the first circulation path and/or second circulation path, and can be on either the IC side and/or EC side of the membrane. 
         [0016]    Although cell growth chamber housing  104  is depicted as cylindrical in shape, it could have a variety of shapes, such as a rectangular cube. Cell growth chamber housing  104  can be made of any type of biocompatible polymeric material. 
         [0017]    Referring now to  FIG. 2 , a schematic of one possible embodiment of a cell expansion system (CES) is shown. CES  200  includes first fluid circulation path  202  (also referred to as the “intracapillary loop” or “IC loop”) and second fluid circulation path  204  (also referred to as the “extracapillary loop” or “EC loop”). First fluid flow path  206  is fluidly associated with cell growth chamber  100  to form first fluid circulation path  202 . Fluid flows into cell growth chamber  100  through IC inlet port  108 , through hollow fibers in cell growth chamber  100 , and exits via IC outlet port  120 . Pressure gauge  210  measures the pressure of media leaving cell growth chamber  100 . Media flows through IC circulation pump  212  which can be used to control the rate of media flow. Media then flows through valve  214 . As those skilled in the art will appreciate, additional valves and/or other devices can be placed at various locations to isolate and/or measure characteristics of the media along portions of the fluid paths. Accordingly, it is to be understood that the schematic shown represents one possible configuration for various elements of the CES and modifications to the schematic shown are within the scope of the one or more present inventions. 
         [0018]    With regard to the IC loop, samples of media can be obtained from sample port  216  or sample coil  218  during operation. Pressure/temperature gauge  220  disposed in first fluid circulation path  202  allows detection of media pressure and temperature during operation. A biosensor (not shown) may also be fluidly disposed in first  202  fluid circulation path to allow for the measurement of metabolite levels within the fluid flow paths. Media then returns to IC inlet port  108  to complete fluid circulation path  202 . Cells grown/expanded in cell growth chamber  100  can be flushed out of cell growth chamber  100  via line  296  and valve  298  or redistributed within hollow fibers for further growth. 
         [0019]    Second fluid circulation path  204  includes second fluid flow path  222  that is fluidly associated with cell growth chamber  100  in a loop. Fluid in second fluid circulation path  204  enters cell growth chamber  100  via EC inlet port  128 , and leaves cell growth chamber  100  via EC outlet port  132 . Media is in contact with the outside of the hollow fibers in the cell growth chamber  100 , thereby allowing diffusion of small molecules into and out of the hollow fibers. 
         [0020]    Pressure/temperature gauge  224  disposed in the second fluid circulation path  204  allows the pressure and temperature of media to be measured before the media enters the EC space of the cell growth chamber  100 . Pressure gauge  226  allows the pressure of media in the second fluid circulation path  204  to be measured after it leaves the cell growth chamber  100 . With regard to the EC loop, samples of media can be obtained from sample port  230  during operation. A sample coil  218  such as that found in the IC loop may also be found in the EC loop to remove samples of EC media. 
         [0021]    After leaving EC outlet port  132  of cell growth chamber  100 , fluid in second fluid circulation path  204  passes through EC circulation pump  228  to oxygenator  232 . Second fluid flow path  222  is fluidly associated with oxygenator  232  via oxygenator inlet port  234  and oxygenator outlet port  236 . In operation, fluid media flows into oxygenator  232  via oxygenator inlet port  234 , and exits oxygenator  232  via oxygenator outlet port  236 . Oxygenator  232  adds oxygen to and removes bubbles from media in the CES. In various embodiments, media in second fluid circulation path  204  is in equilibrium with gas entering oxygenator  232 . The oxygenator  232  can be any appropriately sized oxygenator known in the art. Air or gas flows into oxygenator  232  via filter  238  and out of oxygenator  232  through filter  240 . Filters  238  and  240  reduce or prevent contamination of oxygenator  232  and associated media. 
         [0022]    In the configuration depicted for CES  200 , fluid media in first fluid circulation path  202  and second fluid circulation path  204  flows through cell growth chamber  100  in the same direction (a co-current configuration). Those of skill in the art will recognize that CES  200  can also be configured to flow fluid in a counter-current conformation. Those of skill in the art will also recognize that the respective inlet and outlet ports can be disposed in the cell growth chamber at any location. 
         [0023]    In accordance with at least one embodiment, cells and fluid media can be introduced to fluid circulation path  202  via first fluid inlet path  242 . Fluid container  244  (e.g., Reagent) and fluid container  246  (e.g., IC Media) are fluidly associated with first fluid inlet path  242  via valves  248  and  250 , respectively. For purposes of priming the various inlet paths, first and second sterile sealable input priming paths  208  and  209  are provided. Cells and fluid proceed through heat exchanger  252  (if used), IC inlet pump  254 , and into air removal chamber  256 . Air removal chamber  256  is fluidly associated with first circulation path  202 . The air removal chamber  256  may include one or more ultrasonic sensors to detect air or the lack of fluid at certain measuring positions within the air removal chamber  256 . For example, ultrasonic sensors may be used near the bottom and/or near the top of the air removal chamber  256  to detect air or fluid at these locations. Air or gas purged from the CES  200  during portions of the priming sequence can vent to the atmosphere out air valve  260  via line  258  that is fluidly associated with air removal chamber  256 . 
         [0024]    Fluid container  262  (e.g., Cell Inlet Bag (or Saline Priming Fluid)) is fluidly associated with the first fluid circulation path  202  via valve  264 . Additional fluid can be added to first or second fluid circulation paths  202  and  204  from fluid container  266  (e.g., Wash Solution) and fluid container  268  (e.g., EC Media). Fluid container  266  is fluidly associated with valve  270  that is fluidly associated with first fluid circulation path  202  via distribution valve  272  and first fluid inlet path  242 . Alternatively, fluid container  266  can be fluidly associated with second fluid inlet path  274  by opening valve  270  and closing distribution valve  272 . Likewise, fluid container  268  is fluidly associated with valve  276  that is fluidly associated with first fluid circulation path  202  via first fluid inlet path  242 . Alternatively, fluid container  268  is fluidly associated with second fluid inlet path  274  by opening valve  276  and closing valve distribution  272 . 
         [0025]    In the IC loop, fluid is initially advanced by the IC inlet pump  254 . In the EC loop, fluid is initially advanced by the EC inlet pump  278 . An air detector  280 , such as an ultrasonic sensor, may be associated with the EC inlet path  284 . 
         [0026]    In at least one embodiment, first and second fluid circulation paths  202  and  204  are connected to waste line  288 . When valve  290  is opened, IC media can flow through waste line  288  that leads to the heat exchanger  252  and then to waste bag  286 . Likewise, when valve  292  is opened, EC media can flow through waste line  288  that leads to the heat exchanger  252  and then to waste bag  286 . The heat exchanger  252  serves to recover heat from the waste line  288  and make such heat available for heating fluids entering via the first or second fluid inlet paths  242  and  274 , respectively. 
         [0027]    Cells can be harvested via cell harvest path  296 . Here, cells from cell growth chamber  100  can be harvested by pumping media containing the cells through cell harvest path  296  and valve  298  to cell harvest bag  299 . When harvesting cells, or at other times as may be desired, distribution pump  294  can pump media through a connector path  282  located between the first and second fluid circulation paths  202  and  204 . 
         [0028]    Various components of the CES can be contained or housed within an incubator (not shown), wherein the incubator maintains cells and media at a desirable temperature. 
         [0029]    As will be recognized by those of skill in the art, any number of fluid containers (e.g., media bags) can be fluidly associated with the CES in any combination. It will further be noted that the location of the air removal chamber, or sensors independent of the air removal chamber, can be at any location in the CES before IC inlet port  108 . 
         [0030]    As shown in  FIG. 5 , a digital computer  1600  is operatively associated with the CES  200 . The digital computer  1600  includes memory  1604 , at least one processor  1608 , and a user interface  1612  for receiving instructions from a user via a user input device  1616  (mouse, keyboard, keypad, touch screen, optical sensor or verbal command). In addition, the digital computer  1600  comprises a CES controller interface  1620  for relaying information to and from CES elements  1624  such as sensors pressure, temperature, and biosensor) and for instructing various mechanical systems of the CES  200  such as the pumps and valves. The digital computer  1600  may be in communication with additional sensors for monitoring other aspects of the CES  200 , such as whether one or more fluid bags are low and/or empty. Programming utilized by the digital computer  1600  may comprise, by way of example and not limitation, software or firmware. 
         [0031]    During an expansion cycle, some amount of fluid is typically removed from the fluid circulation paths (the IC and/or EC circuit) at various times throughout the cell expansion cycle and analyzed for the amount of metabolites and other by-products of cell growth in the fluids. The fluid removed from the fluid flow circuits may be run through any commercially available blood gas analyzer (the blood gas analyzer used in this instance was a Siemens 800 series) to measure the amounts of metabolites contained in the fluid. Using a blood gas analyzer, the concentration of lactate (or glucose) is measured in mM/L. Other methods of measurement such as direct chemistry may also be used. 
         [0032]    Metabolites may also be measured using a biosensor. Any commercially available biosensor may be used. If the biosensor is sterile, or is made of a material which may be sterilized with ethylene oxide or gamma irradiation, it may be fluidly connected directly into the fluid lines (in-line). If the biosensor is not able to be sterilized, it may be indirectly connected into the fluid lines via a sterile barrier filter. 
         [0033]    Glucose and lactate molecules are small enough that they diffuse equally across the membrane, and are in equilibrium. Therefore, accurate measurements can be taken by any means on either the IC  202  or EC  204  side, or in waste line  288 . Fluid may be removed from the IC loop  202  through sampling port  216  or sample coil  218  and/or from the EC loop  204  through sample port  230 . 
         [0034]    Aerobically growing cells consume glucose and oxygen and produce lactate. The more cells that are present in a cell growth chamber, the more glucose and oxygen are consumed and lactate generated. When cells are at a high density, particularly adherent cells, cell expansion slows due to increased cell-cell interaction between colonies. Cell clumping or aggregation also occurs at high cell density. It is currently not routine practice to look directly inside a cell growth chamber to see if cells are growing into each other without destroying the sterility of the system. Therefore, it would be advantageous if metabolic products of cell growth such as lactate, or products consumed during cell growth such as glucose and oxygen could be used as an indirect measurement to determine if cells were reaching confluence and should be harvested. 
         [0035]    An algorithm may be generated to determine the number of cell doublings, which, in turn, determines the best time to reseed or harvest the cells before cell growth slows. The number of doublings can be determined using lactate mass generated and the number of cells initially loaded into the cell expansion system. 
         [0000]    Reseeding or harvesting should occur when the number of doublings=d′ 
         [0036]    N=number of cells in the system 
         [0037]    N 0 =starting number of cells loaded into the cell growth chamber 
         [0000]    Rate of change of mass of lactate m L  is proportional to N 
         [0000]    
       
         
           
             
               Doubling 
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               rate 
             
             = 
             
                
               
                  
                 t 
               
             
           
         
       
     
         [0000]    The algorithm is used to determine the number of doubles, which, in turn, determines the best time to reseed or harvest. The number of doubles can be determined from lactate mass m L  and the initial number of cells N in the system. 
         [0000]    
       
         
           
             
               m 
               L 
             
             = 
             
               
                  
                 
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                   t 
                 
               
               ∝ 
               N 
             
           
         
       
     
         [0038]    m L =g L N=g L N 0 (2) d  where g L  is generation rate of lactate (m L (g))/t(hr)/N 
         [0039]    N=N 0 (2) d  This is the starting number N 0  of cells times the doubling rate (2) d    
         [0000]      ln  m   L =ln  g   L +ln  N   0   +d ′ ln 2 
         [0000]    for d=d′ 
         [0000]      ln  m   L   =c +ln  N   0  where  c =ln  g   L   +d ′ ln 2 
         [0000]    The log of the lactate mass m L  equals a constant (c)+the log of the starting number (N 0 ) of cells 
         [0040]    The rate of lactate produced is proportional to the number of cells in the system at any point in time. As increased cell numbers cause decreased cell growth rate, it is expected that lactate generation rate would also decrease. It should also be noted that the numbers that are initially put into the algorithm will change both c and the point at which the line begins to flatten out. 
         [0041]    Using the number of cells initially loaded in the cell growth chamber, the number of cells harvested, and the lactate generation rate at harvest, an algorithm can be generated which can be used to determine when to harvest the cells before cell growth slows and the cells begin to aggregate. This is shown in  FIG. 4 . 
         [0042]    The algorithm can be depicted graphically as shown in  FIG. 3 . As would be expected, and as shown from the graph, the curve ceases to be exponential and starts to flatten out when the colonies have gotten large enough to grow into each other. 
         [0043]    As but one example, not meant to be limiting, if 10 million cells were initially loaded into a cell growth chamber, and the operator wanted 140 million cells at harvest, the cells should be harvested when the lactate generation rate is around 2.2 mM/day. 
         [0044]    The physical manifestation of the algorithm ( FIG. 3 ) can be used by an operator to determine when to harvest cells, or the algorithm can be embedded into a computer program, for use with digital computer  1600 . 
         [0045]    In at least one embodiment, and as shown in  FIG. 4 , the digital computer  1600  is connected to a processor having a memory containing the algorithm. During the course of a cell expansion cycle, when a certain amount of lactate has been generated by the expanding cells, the processor will notify the operator that it is time to harvest the cells. In another embodiment, the processor might initiate harvesting protocols when a certain amount of lactate has been generated. 
         [0046]    This algorithm can be used to determine when the optimal time to harvest adherent cells is for any type of bioreactor. 
         [0047]    It will be apparent to those skilled in the art that various modifications and variations can be made to the structure and methodology of the present invention without departing from the scope or spirit of the invention. Rather, the invention is intended to cover modifications and variations provided they come within the scope of the following claims and their equivalents.