Abstract:
The present invention relates to cell culture in bioreactors, such as flexible cellbag bioreactors. More closely the invention relates to a method and system for determining the cell density in a bioreactor culture and for controlling the perfusion rate of a suspension culture of cells in a bioreactor, comprising measuring the oxygen uptake of primary mononuclear cells in a non-static bioreactor,

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to cell culture in bioreactors, such as flexible cellbag bioreactors. More closely the invention relates to a method and system for determining the cell density in a bioreactor culture and for controlling the perfusion rate of a suspension culture of cells in a bioreactor, comprising measuring the oxygen uptake of primary mononuclear cells in a non-static bioreactor, 
       BACKGROUND OF THE INVENTION 
       [0002]    Flexible cell culture bags, are currently used to culture and expand primary peripheral blood mononuclear cells (particularly T cells) for transplantation into patients. Cells are grown within a contained cellbag and high cell densities are achieved by using media perfusion, where fresh media is added to the culture and spent media is removed. The rate of media perfusion is dependent upon the concentration of cells within the cellbag, the perfusion rate increasing with increased cell concentration. Monitoring of the growth rate and concentration of the cultured cells requires sampling from the cellbag. When opened, the sampling port exposes the culture to the external environment which carries the risk of contamination of the culture. 
         [0003]    There are a variety of inline (in situ) cell counters that have been developed for monitoring of cell growth of cells grown in suspension cultures. These include optical probes (for example focused beam reflectance measurement (FBRM) probes and particle vision measurement (PVM) system), and in situ microscopes. However these all require probe immersion into the culture from an external source, which compromises sterility. These solutions are also large, expensive and would be difficult to integrate into a cellbag. They are designed to be used with stainless steel bioreactors rather than flexible cellbags. 
         [0004]    Optical probes which measure the pH and level of dissolved oxygen (DO) in a bioreactor culture are available. Dissolved oxygen readings can be used to determine the oxygen uptake rate (OUR) of the bioreactor culture. It is understood that that oxygen uptake rates correlate with cell concentration, such as described in for example Eur. J Appl Microbiol Biotechnol (1981) 12:193-197 which shows that the greater the number of cells in the culture, the higher the OUR reading. This method relates to culturing of immortalised cell lines for protein production where maintaining a constant high level of dissolved oxygen is a priority. Dissolved oxygen readings are used to determine whether the agitation rate of the bioreactor should be increased or whether oxygen sparging is required to keep the DO levels above a nominal critical level. 
         [0005]    A method to monitor cell growth and regulate perfusion rate of sensitive cells that does not compromise the cells or the closed culture environment would be desired. 
       SUMMARY OF THE INVENTION 
       [0006]    The present inventors have found that an inverse linear relationship exists between dissolved oxygen concentrations and cell density for primary cells cultured in suspension culture. As the number of cell increases, the concentration of dissolved oxygen decreases. The relationship allows cell concentration to be predicted based on the dissolved oxygen reading, thereby negating the requirement to use a sampling port. In the present invention readings of oxygen uptake are used to set the media perfusion rate. 
         [0007]    Thus, in a first aspect the invention relates to a method for regulating perfusion rate in a suspension culture of cells in a non-static bioreactor, comprising registering the oxygen uptake of primary mononuclear cells in the bioreactor; predicting the cell number of said primary mononuclear cells by a pre-determined correlation of the registered oxygen uptake with the cell number; and controlling the perfusion rate in response to the registered oxygen uptake. The method is applicable to suspension cultures in any type of rocking, stirred or agitated bioreactor. The bioreactor is preferably kept at a constant rocking rate, preferably at least during the registration of the oxygen uptake. 
         [0008]    The registered oxygen uptake is dissolved oxygen (DO) uptake or oxygen uptake rate (OUR), which measures the change in dissolved oxygen over time. In a preferred embodiment of the method, the DO uptake is measured. 
         [0009]    Preferably the oxygen uptake is measured by a sensor integrated in the bioreactor. The sensor is preferably an optical sensor which is embedded into the bioreactor, such as in the bottom of a cell bag. A cable may be attached from the outside which leads to a controlling computer. Measurements will be taken continuously and recorded. No manipulation of the bioreactor/cellbag is required. 
         [0010]    Preferably the rocking rate of the bioreactor is 1-25 rpm, more preferably 10-15 rpm. The method of the invention is especially suitable for suspension culture of T-cells, or other sensitive cells. 
         [0011]    In a preferred embodiment the registered oxygen uptake is dissolved oxygen (DO), the rocking rate is 10-15 rpm and the bioreactor is a flexible cell bag. 
         [0012]    In a second aspect, the invention relates to a bioreactor system, comprising a computer terminal ( 1 ), a bioreactor control unit ( 2 ), a gas outlet line ( 3 ), a sensor cable ( 4 ), a cell expansion system ( 5 ), a rocking platform ( 6 ), a bioreactor ( 7 ), a gas inlet port ( 8 ), a sensor ( 9 ), such as an optical probe, for measuring oxygen uptake, a media waste line ( 10 ), a media feed line ( 11 ), and pump unit ( 12 ), a media feed reservoir ( 13 ) and a media waste reservoir ( 14 ), wherein the pump unit, the cell expansion system and bioreactor control unit are connected to the computer terminal ( 1 ) which controls the rate that the pump unit operates which controls the rate of media exchange in response to oxygen uptake readings. 
         [0013]    The sensor ( 9 ) may be an optical probe or an electrochemical probe. 
         [0014]    In a preferred embodiment of the bioreactor system the sensor ( 9 ) measures dissolved oxygen (DO) or oxygen uptake rate (OUR). 
         [0015]    Preferably sensor is an optical probe which measures DO and is embedded in the bottom of a flexible cell bag. 
         [0016]    The invention presents the ability to use dissolved oxygen (DO) readings or oxygen uptake rate (OUR) to guide a perfusion strategy. In embodiments of the invention the perfusion may be automated (by writing a software program) based on DO or OUR readings. Using dissolved oxygen readings to set perfusion rates instead of cell count also enables automation of this process and negates the requirement to manually change perfusion settings, which reduces the chance of human error compromising the culture. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0017]      FIG. 1  shows data of an inverse linear relationship between dissolved oxygen (DO) concentration and concentration of primary human T cells in a 1 L culture on a Xuri™ bioreactor set at an angle of 6° and a rocking rate of 15 rpm of the bioreactor platform. 
           [0018]      FIG. 2  shows the relationship between DO and cell concentration when the Xuri™ bioreactor is set at a rock rate of 10 rpm and at a 6° angle. 
           [0019]      FIG. 3  is a schematic view of a bioreactor system according to the invention and shows the main features of the invention. 
           [0020]      FIG. 4  is a schematic view of a cellbag used in the bioreactor system of  FIG. 3 . 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0021]    The invention will now be described more closely in association with the accompanying drawings and some non-limiting Examples. 
         [0022]    The invention is a method to predict the density of primary lymphocytes grown in a bioreactor in situ. The invention uses a linear relationship between dissolved oxygen concentration and cell concentration to predict cell densities in the bioreactor culture. The invention allows perfusion rates to be set based on dissolved oxygen concentrations. A program which would allow perfusion rates to be adjusted based on dissolved oxygen readings can be written into the software that runs any selected bioreactor. 
         [0023]    The linear relationship between DO/OUR and cell concentration has never been shown for primary mononuclear cells before. The relationship was surprisingly tight and it is because of this tightness that it may be incorporated into the software control of a bioreactor, such as a WAVE or Xuri™ bioreactor. This means that cell number can be predicted and perfusion rates can be changed without having to take a sample from the cellbags. 
         [0024]    A preferred example of the bioreactor system of the invention is shown in  FIG. 3  wherein ( 1 ) is a computer terminal, ( 2 ) a cellbag bioreactor control unit, ( 3 ) a gas outlet line, ( 4 ) a DO optical cable, ( 5 ) a Xuri™ Cell Expansion System, ( 6 ) a rocking platform, ( 7 ) a cellbag bioreactor, ( 8 ) a gas inlet port, ( 9 ) an optical probe for measuring DO, ( 10 ) a media waste line, ( 11 ) a media feed line, ( 12 ) and pump unit, ( 13 ) a media feed reservoir and ( 14 ) a media waste reservoir. The pump unit, the Xuri™ Cell Expansion System and cellbag Bioreactor Control Unit are connected to the computer terminal. Software in the computer terminal controls the rate that the pump unit operates which controls the rate of media exchange. 
         [0025]      FIG. 4  shows a cellbag for use in the bioreactor system of  FIG. 3  comprising of ( 15 ) the fastening rods for attachment to the rocking platform, ( 16 ) a feed line, ( 17 ) a waste line, ( 18 ) a gas exhaust tube, ( 19 ) a gas inlet tube, ( 20 ) a perfusion filter, ( 21 ) an optical probe to measure dissolved oxygen, ( 22 ) a harvest port and ( 23 ) a sampling port. The perfusion filter is internal to the cellbag. The DO probe is embedded in the bottom of the cellbag and is in direct contact with the culture environment. 
       EXAMPLE 1 
     Defining the Relationship Between Dissolved Oxygen Levels and Cell Concentration 
       [0026]    Method: Peripheral Blood Mononuclear cells were cultured in static culture with anti-CD3/28 activation beads and 20 ng/ml of IL-2 for 5 days. During static culture the cell concentration was maintained at 5×10E05/ml though the addition of media. At day 5 of culture the cells were transferred to a 2 L cellbag for culture on the Xuri W25 bioreactor for a further 5-9 days. Cells were maintained at 5×10E05/ml through the addition of media until the final volume of 1 L had been reached, after which the cell concentration was allowed to increase as the cells continued to proliferate. The rocking platform was set at an angle of 6° and a rock rate of 15 rpm and kept constant throughout the course of the culture. Media perfusion was enabled once the cell concentration had reached 2×10E06/ml. The cellbags contained optical sensors for dissolved oxygen (DO) and measurement of DO levels were recorded every 24 hrs simultaneously with a cell count being taken. These data were compiled from 7 independent experiments, each using a different donor. 
         [0027]    A plot of DO verses cell concentration was drawn and a linear trendline determined ( FIG. 1 ). The formula of the trendline in this example was 
         [0000]        DO=− 1.58 E -06*Cell density+90.01 
         [0028]    Using this formula the cell density of a given culture could be predicted from the DO readings at any point during the culture period provided the rocking rate was maintained at 15 rpm and the rocking angle was maintained at 6°. 
         [0029]    The same data was generated for cultures where the rocking platform was set at 10 rpm and 6° ( FIG. 2 ). The data was compiled from two separate experiments using 2 donors. A plot of DO versus cell concentration was drawn and a trendline with formula generated. The formula of the trendline in this example was 
         [0000]        DO =−2.88 E -06*Cell density+89.81
 
         [0030]    These data show that the slope of the line is dependent on the rocking rate of the bioreactor. Therefor to use these formulas to predict cell density the rocking conditions must be kept constant throughout the culture period. 
       EXAMPLE 2 
     Predicting Perfusion Rates Based on Dissolved Oxygen Readings 
       [0031]    The ability to use DO to predict cell density can be further exploited to include an automated method of setting perfusion rates in T cell cultures. Perfusion rates are dictated by cell density, as the higher the number of cells, the higher the metabolic load and the greater the rate of media exchange that is required. 
         [0032]    The standard perfusion rates used in the above experiments were as follows 
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 Cell concentration 
                 Perfusion rate 
               
               
                   
                   
               
             
             
               
                   
                  2 × 10 6 /ml 
                 500 ml/day 
               
               
                   
                 10 × 10 6 /ml 
                 750 ml/day 
               
               
                   
                 15 × 10 6 /ml 
                 1000 ml/day  
               
               
                   
                   
               
             
          
         
       
     
         [0033]    Based on the trendlines of DO vs cell concentrations, the perfusion rates can be set using DO readings instead of cell concentration 
         [0000]    
       
         
               
               
               
             
           
               
                   
               
               
                 DO reading at 15 rpm 
                 Do reading at 10 rpm 
                 Perfusion rate 
               
               
                   
               
             
             
               
                 &gt;88 
                 &gt;84 
                 none 
               
               
                 75-88 
                 61-84 
                 500 ml/day 
               
               
                 67-75 
                 47-61 
                 750 ml/day 
               
               
                 &lt;67 
                 &lt;47 
                 1000 ml/day  
               
               
                   
               
             
          
         
       
     
         [0034]    An automated set-up can be configured ( FIG. 3 ) whereby the DO readings, which are collected continuously throughout the culture via the DO optical sensor embedded in the Cellbag bioreactor ( FIG. 4 ) are recorded by the software and the software then adjusts the pump rates that control the amount of media to be perfused in the Cellbag bioreactor. This would eliminate the need for manual changing of the perfusion rates by the operator during the culture period. At the beginning of the bioreactor culture the operator would be required to instruct the software which perfusion rate to use based on the DO readings, as exemplified in the above table, but thereafter no further input from the operator would be required.