Abstract:
The invention provides a bioreactor (10) for the culture of animal, vegetable, microbial of algal cells of their co-cultures, of the type including a body (12) which delimits an internal volume (14) capable of holding a culture liquid (16) and a gas volume (17) above the culture liquid (16), and which includes means for introducing (18) and/or extracting (20) elements respectively into and/or out of the internal volume (14) of the body (12), and of the type which includes means (22) for driving the body (12) in an oscillating movement so as to obtain agitation of the culture liquid (16), characterised in that the body (12) is a rigid vessel.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This document claims priority to French Application No. 02 06146, filed May 21, 2002 and U.S. Provisional Application No. 60/398,567, filed Jul. 26, 2002, the entire content of which is hereby incorporated by reference. 
    
    
     FIELD OF THE INVENTION 
     The invention provides a bioreactor for a culture of animal, vegetable, microbial or algal cells, of the type including a body which delimits an internal volume capable of holding a culture liquid and a gas volume above the culture liquid, and which includes an arrangement for introducing and/or extracting elements respectively into and/or out of the internal volume of the body. The bioreactor further includes a drive to move the body for oscillating movement so as to agitate the culture liquid. 
     BACKGROUND OF THE INVENTION 
     DISCUSSION OF BACKGROUND 
     The technological advances in the field of biotechnology are leading to an increase in the demands for animal, vegetable, microbial or algal cells, so that it is necessary to increase the production capacities for these cells. 
     Production of the cells is carried out by cultivating them in a culture liquid which includes components necessary for their growth and which may be brought into contact with a gas which also contains components necessary for the growth of the cells. In particular, so-called “aerobic” cells are brought into contact with oxygen in air, which is a component necessary for their development, by an arrangement for injecting air into the culture liquid. The culture liquid can also be agitated by an agitation arrangement in order to optimize the contact between the cells and the components necessary for their growth which are contained in the liquid and/or in the gas. 
     For the culture of cells under such conditions, it has been proposed to use vessels which are made of stainless steel complying with food requirements, with the designation Z2 CND17.12 (standard NF A02-004) or 316L (AISI standard), and in which the culture liquid is agitated by an internal agitator, for example of the paddle type. However, the culture of certain categories of cells, referred to as “phototropic,” requires significant illumination of the culture liquid, and vessels made of stainless steel only make it possible to provide relatively weak illumination of the culture liquid. Further, the use of an internal agitator causes “shearing” of the culture liquid, which damages the cells and slows their development. 
     It has also been proposed to cultivate cells in a bioreactor composed of a plurality of flasks, or bottles, which have horizontal axes and are arranged in a rotor that can be moved in continuous rotation about a horizontal axis, as described and represented in U.S. Pat. No. 6,066,497. Each bottle includes an arrangement to allow injection or withdrawal of certain products, respectively into or out of their internal volume. The dimensions of the bottles are small so that a person can transport them without difficulty. However, the bottles therefore have a relatively restricted maximum capacity. In addition, the bottles can be used only with a minimum quantity of liquid, so that it is relatively difficult to modify the quantity of culture liquid during culture. However, when it is desired to inject a product into the culture liquid, it is necessary to carry out one injection for each bottle, which multiplies the contamination risk of the liquid by the number of bottles. 
     Finally, as described in Document FR-A-2,519,020, it has also been proposed to cultivate the cells in a bioreactor which includes a more or less translucent plastic bag fitted on a plate driven in a seesaw movement. The bag is partially filled with the culture liquid, and the volume of the bag is made up by injecting air so as to allow gas to exchange between the gas and the cells. However, the air which is introduced into the bioreactor needs to be sterile, such that the bioreactor requires complex air-sterilization system which can be relatively expensive. In addition, the pressure exerted by the culture liquid on the walls of the bag naturally tends to make it adopt a substantially spherical shape, which, as is known, is the geometrical shape which makes it possible to have a minimum external surface area for a maximum internal volume. However, this configuration reduces the free surface area of the culture liquid which is in contact with the air. 
     SUMMARY OF THE INVENTION 
     It is an object of the invention to provide a bioreactor which makes it possible to have a larger free contact surface area of the culture liquid with the air present in the bioreactor for a given volume of culture liquid. 
     In accordance with a preferred form, the invention provides a bioreactor in which the body is a rigid vessel. 
     The preferred form of the invention can also include the following additional advantageous features alone or in combination:
     the body can be made of a material which is permeable to light;   the body can include an arrangement for increasing the surface area of the culture liquid which is in contact with the gas volume present in the upper part of the body;   the bioreactor can include at least one container which is open at the top, which is arranged inside the body and driven to move with the body and which, for at least one orientation of the body, is capable of isolating a certain quantity of culture liquid from the rest of the culture liquid contained in the body;   the bioreactor can include at least one air filter allowing gas exchange between the internal volume of the body and the outside;   the upper face of the body is at least partially open, and the upper face of the body can be closed in a sterile manner by a closure element;   the closure element can be a film which is permeable to air so as to form an air filter, or the closure element can be a lid which carries the filter and the arrangement for introduction and/or extraction of elements;   the body can be driven in an oscillating movement about a substantially horizontal axis;   the body can be driven in an alternating translation motion parallel to a substantially horizontal direction.   

    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Other characteristics and advantages of the invention will become apparent from the following detailed description, particularly when considered in conjunction with the drawings in which: 
         FIG. 1  is a schematic perspective representation of a bioreactor according to the teachings of the invention; 
         FIG. 2  is a view similar to that in  FIG. 1 , in which the body is driven in an alternating horizontal translation movement; 
         FIG. 3  is a detailed view on a larger scale of an arrangement for locking the closure element of the body; 
         FIG. 4  is a side view of the body represented in  FIG. 1 , in which the body is represented in its resting position; 
         FIG. 5  is a view similar to that in  FIG. 4 , in which the body is represented during an oscillation; 
         FIG. 6  is a view similar to that in  FIG. 4 , representing an alternative embodiment of the invention in which the body includes containers, each of which extends substantially over the full length of the body; 
         FIG. 7  is a view similar to that in  FIG. 5 , according to the alternative embodiment of the invention. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     Referring now to the drawings, non-limiting examples of embodiments of the invention will now be described. For reference purposes in the drawings, the vertical, longitudinal and transverse orientations according to the coordinate system V, L, T are indicated in the figures. Identical, similar or analogous elements will be denoted by the same reference numerals in the description which follows. 
       FIG. 1  represents a bioreactor  10  for the culture of, for example, animal, vegetable, microbial or algal cells, which includes a body  12  preferably made of a rigid material capable of transmitting light and delimiting, inside the vessel, a volume  14  intended to hold a culture liquid  16 . In use, the culture liquid  16  constitutes the medium in which the cells develop, and it contains nutrient elements necessary for the growth of the cells. A volume of gas  17 , for example air in the event that the cells being cultivated are “aerobic cells,” is present above the culture liquid  16 . 
     One parameter which influences the performance of the bioreactor  10  is the concentration of the cells in the culture liquid  16 , which must lie within a given range of values. Since the purpose of the bioreactor  10  is to produce cells, it is necessary to replenish and/or top off the culture liquid with a fresh culture liquid as they multiply. To this end, the illustrated bioreactor  10  includes an arrangement such as a conduit or passage shown at  18  for introducing elements into the internal volume  14  of the body  12 , in particular fresh culture liquid, which are designed so that the introduction of the fresh culture liquid takes place without introducing polluting foreign elements into the internal volume  14  of the body. The bioreactor  10  also includes an arrangement such as a conduit or passage  20  for extracting elements from the internal volume  14  of the body  12 , which are used in particular for withdrawing a small quantity of culture liquid  16 , for example, to allow an analysis to make it possible to check that the culture of the cells is proceeding correctly. 
     In the illustrated embodiment, the body  12  is driven in an alternating movement making it possible to obtain continuous or periodic mixing of the culture liquid  16 . This mixing of the culture liquid  16  makes it possible to ensure the gas exchanges between the cells and the gas  17  present above the liquid, in particular with the oxygen contained in air when culturing so-called “aerobic cells.” In addition, the mixing of the culture liquid  16  makes it possible to optimize the contact between the cells and the nutrient elements contained in the culture liquid  16 , and such an external agitation system makes possible to avoid any shearing of the culture liquid. 
     When the body  12  is driven in an alternating movement, the presence of the gas volume above the culture liquid  16  makes it possible to form turbulence or movement, which leads to the formation of waves (not shown). The result of this turbulence is that the cells are driven in a stirring movement in the culture liquid, at least one component of which is vertical, and therefore, a continuous change of the cells which are at the surface  16   s  of the culture liquid  16 , hence increasing the gas exchanges or interaction with the gas in the internal volume. 
     In the embodiment represented in  FIG. 1 , the bioreactor includes a set of actuators  22 , here arranged below the body  12 , which drive it in an oscillating movement about a horizontal transverse axis. The oscillating movement of the body  12  may be obtained by any other means or expedients as would be recognized by persons skilled in the art. For example, Document WO-A-00,66706, describes a bioreactor that includes an oscillation plate on which the body is fitted. 
     According to a variant which is represented in  FIG. 2 , the body or vessel  12  is driven in an alternating translation motion parallel to a horizontal longitudinal direction. The actuators are then oriented parallel to the direction of the movement, that is to say parallel to the longitudinal direction, and they can act on a vertical side wall  23  of the body  12 . 
     In order to improve the yield of the bioreactor, the free upper surface area  16   s  of the liquid  16 , which is in contact with the gases  17  contained in the internal volume  14  of the body  12 , needs to be as large as possible so as to increase the volume of the natural gas exchanges or interaction between the culture liquid  16  and the gas volume  17 , and also to allow movement of the liquid permitting it to be stirred. To this end, and according to the invention, the body  12  is preferably a rigid vessel. In the illustrated embodiment, the body has a rectangular parallelepiped shape. With this arrangement, when the body is in a resting position, it includes a horizontal rigid bottom  24  of longitudinal overall orientation and rigid vertical side walls  23 . 
     According to an alternative embodiment (not shown), the body  12  includes a rigid frame. The bottom  24  and the side walls  23  are made of a flexible material, and they are held in shape by the frame. 
     Since the body  12  is rigid, the surface area  16   s  of the culture liquid  16  is substantially constant for a given position of the vessel, regardless of the volume of culture liquid  16  present inside the body  12 . By contrast, with bioreactors in which the body is a flexible bag, the walls deform under the pressure exerted by the fluid, so that the surface area of the culture liquid which is in contact with the gases is then reduced. 
     In order to promote the growth of so-called “phototropic” cells, which need a great deal of light in order to be able to develop, the body  12  can be advantageously made of a rigid material which is permeable to light. This material is preferably a transparent polymer such as polycarbonate. One advantage of polycarbonate is it that can withstand temperatures of up to about 135° C., so that the body  12  can be sterilized in an autoclave. Sterilization of the body  12  is then greatly simplified compared with the sterilization of bioreactors for which it is carried out with steam and in situ, with complex and expensive assembly. 
     The upper face  26  of the body  12  is open, and allows the introduction  18  and the extraction  20  arrangements to pass therethrough. However, the culture of the cells requires a rigorous absence of foreign cells, so that the upper face  26  needs to be closed off to guarantee sterility of the bioreactor  10 . To this end, the bioreactor  10  preferably includes a closure element  28  which covers the upper face  26 , so that the internal volume  14  of the body  12  is protected from any external contamination. 
     In order for the closure of the upper face  26  to be leaktight and therefore sterile, the bioreactor  10  can include a seal. An example of a seal arrangement is shown in  FIG. 3  in which a seal is interposed between the closure element  28  and the body  12 , with the seal compressed by a locking arrangement  32 . The locking or clamping arrangement  32 , which here includes a screw-nut system, makes it possible to clamp or compress the seal  30  and to lock the closure element  28  in position. Preferably, the locking arrangement is arranged outside of the body  12  so that operation of the lock or clamp does not lead to contamination. Although a screw-nut locking/clamping arrangement is illustrated, it is to be understood that other locking/clamping arrangements can be used in accordance with the invention. 
     Due to the gas exchange between the cells and the air, it is necessary to constantly or regularly replenish the air which is present in the internal volume  14  of the body  12 . Replenishment of the air can be achieved by way of air filters  34  including, for example, a micropore membrane which lets through only molecules or atoms contained in the air, and which prevents the passage of any other cell which could contaminate the culture liquid  16 . Such filters permit so-called “passive” aeration, which does not perturb the gas equilibrium inside the bioreactor  10 , in contrast to the air-injection systems used in traditional bioreactors. 
     According to a first embodiment represented schematically in the figures, the closure element  28  is a rigid lid which carries the introduction  18  and the extraction  20  arrangements, and which carries a plurality of air filters  34 . 
     According to a second embodiment (not shown), the closure element  28  includes a film or a membrane which fully covers the upper face  26  of the body, and which is formed to have the same characteristics as the air filters  34 , that is to say letting through only the molecules and atoms contained in the gas, while preventing the passage of elements which may contaminate the culture liquid  16 . 
     The area of the surface  16   s  of the culture liquid  16  is limited by the dimensions of the body, in other words, by its length “L” and its width “l”. 
     In order to increase the surface area of culture liquid  16  which is in contact with the air, and according to an alternative embodiment or optional aspect of the invention, the body  12  can include a plurality of containers  36  which, in the illustrated embodiments, include concave elements open at the top in the general shape of bowls or dishes. Each container illustrated includes a horizontal transverse plate  38  which joins together the two vertical longitudinal walls  40  of the body  12  and the transverse end edges or walls  42  which are inclined upwards. 
     The containers  36  are arranged to extend above the culture liquid  16  when the body  12  is in its resting position represented in  FIG. 4 . They are thus arranged so that at least some of the containers  36  are immersed in the culture liquid  16  in at least one position of the body  12  other than its resting position, in particular during the oscillating movement of the body, as represented in  FIG. 5 . 
     In the illustrated embodiment, a first movement of the body represented in  FIG. 5 , immerses first containers  36   a  in the liquid. When the body  12  pivots about its oscillation axis in order to return to its resting position, these first containers  36   a  have each taken up a certain quantity of culture liquid  44 , and they then isolate it from the rest of the culture liquid  16 . Gas exchanges can thereby take place at the level of the surface  16   s  of the culture liquid, and at the level of the surface  44   s of the quantity of withdrawn liquid  44  temporarily stored in each container  36 . The total exchange surface area is therefore increased. 
     When the body  12  tilts to the opposite position from that represented in  FIG. 5 , it is inclined with respect to its resting position, the first containers  36   a  extend above the culture liquid  16 , and some (e.g., as represented at  46 ) of the quantity of withdrawn liquid  44  pours out of the first containers  36   a  into the rest of the culture liquid  16 . The quantity  46  which pours out of the containers makes it possible to increase the total surface area of the culture liquid owing to its own surface area. 
     When the body  12  returns to the position in which the first containers  36   a  are immersed, the quantity of withdrawn culture liquid  44  is re-introduced and mixes in with the rest of the culture liquid  16 . Combined with the stirring or agitating of the culture liquid  16 , the action of the containers  36  makes it possible to increase the gas exchange surface area of the culture liquid  16 . 
     According to a variant (not shown) of the invention, the body  12  can include a plurality of series or rows of containers  36  which are arranged at different distances from the bottom  24 , so that at least some of the containers  36  are effective regardless of the depth of the culture liquid  16  contained in the body  12 . Thus, plural containers can be provided at different horizontal and vertical positions within the body  12 . 
     According to an alternative embodiment of the invention represented in  FIGS. 6 and 7 , the body  12  includes a plurality of containers  36  distributed at different distances from the bottom  24 , and each container  36  is arranged at a different distance from the bottom  24  than the other containers  36 . The length of each container  36  is preferably sufficiently less than the length “L” of the body  12 , so that the surface area of the withdrawn quantity of culture liquid  44  is as large as possible, while leaving a space “e” between the container  36  and the front  48  and rear  50  transverse walls. Preferably, for the culture of “phototropic” cells, the containers  36  are made of the same transparent material as the body, so that they do not reduce the illumination of all the cells. 
     The bioreactor  10  makes it possible to add fresh culture liquid to the culture liquid  16 , without needing to interrupt the culture of the cells. In this way, the overall level of the culture liquid  16  inside the body  12  can be increased with each addition of fresh culture liquid. 
     As should be apparent, simple mechanical rearrangements or modifications are possible to provide alternative embodiments of the invention. For example, the introduction  18  and extraction  20  arrangements may be provided on a vertical wall of the body  12 . 
     A bioreactor according to the invention may also have a small quantity of cells at the start of the culture, for example 1 liter, which is transferred into a suitable volume of medium, for example 10 liters. Fresh culture liquid can be subsequently added as the cells grow, until reaching the maximum capacity of the bioreactor, for example 100 liters, without having to relocate the culture liquid from one bioreactor to another, hence limiting the contamination risk. 
     Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.