Patent Publication Number: US-2021189316-A1

Title: Device for supplying and discharging a medium; culture vessel having such a device and method of cultivating microbiological systems by using such a culture vessel

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of international patent application PCT/EP2019/073866, filed on Sep. 6, 2019 designating the U.S., which international patent application claims priority from German patent application 10 2018 122 745.0, filed on Sep. 17, 2018. The entire contents of these priority applications are incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to a device for supplying or discharging a medium into or out of a culture vessel. The invention further relates to a culture vessel comprising such a device, and to a method of cultivating microbiological systems, in particular cell cultures, using such a culture vessel. 
     BACKGROUND 
     A device of the type mentioned above is used for growing or cultivating microbiological systems, in particular cells or microorganisms, in a culture vessel, for example in a bioreactor. 
     It is thus possible to keep alive microbiological systems, such as cells, tissue and/or microorganisms, outside of an organism, in order to permit study of their development in greater detail. During the cultivating of cells (cell culture) and during what is known as “tissue engineering” it is necessary to provide, to the cells located in the culture vessel, in particular adherent cells or cells in suspension, a medium that comprises the substances needed for the development of the cells, for example nutrients or oxygen. 
     A device of the type mentioned at the outset serves for the supply of the medium; this medium, which by way of example is taken from a medium reservoir, is introduced through a first aperture into the housing of the device attached on the culture vessel. In this case, the device serves as supply device. Since the first aperture has fluid-conducting connection to a second aperture, the incoming medium can reach the second aperture and emerge through same. Finally, the emerging medium reaches the interior of the culture vessel, and can be received by the cells located there. 
     The device mentioned above can also serve as discharge device for the discharge of the medium, in particular of the used-up nutrient medium, from the culture vessel. In this case, the nutrient medium is removed by suction from the interior of the culture vessel through the second aperture into the housing of the discharge device attached on the culture vessel, and the nutrient medium is thus discharged from the housing by way of the first aperture. The used-up medium can thus be removed from the culture vessel. 
     The principle of operation described above for supply and discharge of a medium is known by way of example from DE 102 01 259 A1. Provided in the device disclosed there are an inlet connection bore and a return connection bore, intended for introduction of nutrient medium and optionally also oxygen into the cell culture space within the container by way of appropriate conduit connections or flexible-tube connections. 
     However, the known device has the disadvantage of only limited controllability of the flow of the nutrient medium or of the oxygen in the cell culture space. Thereby, the nutrient medium entering through the inlet connection bore into the cell culture space cannot be conducted at a defined flow rate in the direction of the return connection bore. Large variations of the quantity of nutrients or oxygen present in the cell culture space therefore occur, so that the cells to be cultivated can be subject to temporal and/or spatial over- or undersupply; this, however, can adversely affect cell development. 
     SUMMARY 
     It is an object of the present invention to provide a device of the type mentioned in the introduction with the aim of permitting better control of the flow of the medium in the interior of the culture vessel, in order to reduce, or entirely avoid, variations of the quantity of the medium present in the culture vessel. 
     According to an aspect of the invention, a device configured to supply or discharge a medium into or out of a culture vessel is provided, comprising a housing, a first aperture and a plurality of second apertures arranged on the housing, the plurality of second apertures being connected with the first aperture in fluid-conducting manner to supply a medium from the first aperture via the second apertures into an interior of the culture vessel or to discharge a medium from the interior of the culture vessel in reversed direction, when the device is attached on the culture vessel, such that a plurality of medium sub-streams, arranged in parallel to one another, of the medium to be supplied or to be discharged are generated. 
     Because each of the plurality of second apertures is connected in fluid-conducting manner with the first aperture, the medium entering through the first aperture into the housing of the device, in particular of the supply device, can reach each of the plurality of second apertures. The incoming medium can in turn emerge through these and finally reach the interior of the culture vessel. A plurality of medium strings or medium sub-streams are thus produced from the incoming medium stream, these being arranged in parallel to one another. An incoming stream is thus advantageously distributed into a plurality of outgoing streams, thus achieving particularly uniform distribution of the overall flow of the medium in the interior of the culture vessel. 
     The arrangement of the plurality of second apertures is moreover particularly advantageous for reducing, or entirely avoiding, swirling effects. Because of a plurality of medium sub-streams arranged in parallel to one another are generated, mixing of different medium sub-streams is greatly reduced, and this has a favorable effect on minimization of swirling or turbulence effects in the flow of the medium. The flow behavior of the medium in the interior of the culture vessel can thus be better controlled. 
     The abovementioned advantages also apply to the case in which the device serves as discharging device for discharging the used-up medium by way of the plurality of second apertures into the housing of the device and finally outward (for example into a return conduit). Here again, the arrangement of the plurality of second apertures promotes a uniform flow distribution of the medium in the interior of the culture vessel. Here again, turbulence effects in the medium stream are moreover reduced or entirely avoided. 
     The particular advantage for cell development, alongside the reduced variation of the quantity of ingredients in the interior of the culture vessel, is minimization of swirling or turbulence effects, and in particular laminar flow of the medium. If swirling effects are present, the cells located in the culture vessel, in particular adherent cell lawns, fixed cell assemblages, or biopsy samples and tissue samples, can be subjected to a swirling motion that can damage, or entirely destroy, the interior structure of the cells. This risk is advantageously reduced by the provision of the plurality of medium sub-streams, arranged in parallel to one another, of the medium that is to be introduced or to be discharged. 
     Increased laminarity of flow of the medium moreover permits achievement of an almost constant flow rate, thus permitting mechanical stimulation of the adherent cells or cell lawns and/or tissue during growth of the cells. Growth conditions provided during cell development and cell differentiation can thus simulate natural conditions. 
     Continuous and dynamic cultivation of cells and tissues under controllable, in particular laminar, flow conditions is advantageously permitted. The present invention is therefore particularly suitable for the cultivation of adherent urothelium cells intended to expand in multiple layers. The present invention can moreover also be used for short-, medium- and/or long-term studies of other adherent cell types. 
     In a preferred embodiment, the first aperture is connected with each of the plurality of second apertures respectively via one of a plurality of fluid conduits arranged in parallel to one another. 
     The medium stream entering into the first aperture is thus, upstream of the plurality of second apertures, already divided into a plurality of sub-streams. This facilitates the generation of the plurality of medium sub-streams of the medium that is to be introduced or that is to be discharged, and this promotes reduction of swirling effects in the interior of the culture vessel. 
     In another preferred embodiment, at least one of the plurality of fluid conduits is configured at least partially as nozzle, in particular as laminar nozzle for generating a laminar medium sub-stream of the plurality of medium sub-streams. 
     A nozzle is a flow duct with a cross section that changes in flow direction, and has the advantage that medium supply and medium discharge take place with no, or only with slight, loss of flow velocity. The nozzle advantageously promotes a defined flow rate of the medium in the interior of the culture vessel. 
     The nozzle preferably comprises a laminar nozzle for generating a laminar medium sub-stream. It is thus easily and advantageously possible to generate at least one of the plurality of medium sub-streams as laminar medium sub-stream. The laminar nozzle can have a tube diameter that, for flow velocities of the medium that are appropriate for the development of the cells, gives a Reynolds number that is below 2300, preferably below 2000. 
     In another preferred embodiment, the nozzle has an external and/or internal cross section which at least sectionally narrows in the direction of the second aperture associated with said nozzle. 
     This type of nozzle is particularly advantageous for achieving a particularly uniform flow rate. The controllability of the medium flow is thus further improved. 
     In another preferred embodiment, at least one of the plurality of fluid conduits has a tubular terminal section which is curved toward the second aperture associated with the at least one fluid conduit. 
     The curvature results in an advantageously uniform change of the flow direction of the medium before exit from the second aperture and/or before entry into the second aperture, for example from a direction perpendicular to the bottom of the culture vessel to a direction parallel to the bottom. It is thus possible to mitigate, or entirely avoid, any abrupt change of direction of the medium stream, thus further reducing swirling effects. 
     In another preferred embodiment, at least one of the plurality of fluid conduits is directed at least sectionally perpendicularly to a bottom side of a vessel body of the culture vessel. 
     This has the advantage that the force exerted by the intrinsic weight of the medium can be utilized for the supply of medium when the device is arranged on the culture vessel, where the bottom side of the vessel body is oriented horizontally. The power needed for the pump for introducing the medium into the device is thus advantageously reduced. 
     In another preferred embodiment, a shared intermediate chamber is configured in the housing for the fluid-conducting connection between the first aperture on the one hand and the plurality of fluid conduits on the other hand. 
     The shared intermediate chamber is connected not only to the first aperture but also to the plurality of second apertures via the plurality of fluid conduits. The shared intermediate chamber advantageously permits a particularly uniform flow transition between the first aperture and each of the plurality of second apertures. 
     In another preferred embodiment, the first aperture is arranged centrally on a surface of the housing and/or is arranged terminally on a tube section projecting beyond the surface of the housing. 
     The first aperture thus arranged promotes a uniform distribution of the medium that is to be introduced and/or that is to be discharged. The tube section projecting beyond the surface of the housing permits easy connection for flexible tubes which by way of example are connected to a medium reservoir and/or a pump for the supply or discharge of medium. 
     In another preferred embodiment, the housing comprises a base body and a cover for the releasable closure of the base body. 
     In this embodiment, the device of the invention is configured in two parts. Easier handling of the device of the invention is thus achieved, alongside greater ease of replacement of the cover and of the base body. 
     According to another aspect of the invention, a culture vessel, in particular a bioreactor for the cultivation of microbiological systems, for example of cells and/or microorganisms, is provided, comprising a vessel body configured to accommodate a medium and extending from an upper side to a bottom side, and at least one device configured to supply or discharge a medium into or out of the vessel body and arranged on an upper side of the vessel body, the at least one device comprising: a housing, a first aperture and a plurality of second apertures arranged on the housing, the plurality of second apertures being connected with the first aperture in fluid-conducting manner to supply a medium from the first aperture via the second apertures into an interior of the vessel body or to discharge a medium from the interior of the vessel body in reversed direction, such that a plurality of medium sub-streams, arranged in parallel to one another, of the medium to be supplied or to be discharged are generated. 
     The culture vessel, in particular the bioreactor vessel, is preferably treated by a sterilization method conventionally used in tissue engineering, so that it can be used in good manufacturing practice (GMP) production processes. 
     In a preferred embodiment, at least one plug-in aperture arranged to plug-in the at least one device is arranged on the upper side of the vessel body. 
     This permits accommodation of the device on the vessel body of the culture vessel in a manner that is secure and particularly easy to implement. 
     In another preferred embodiment, the at least one device comprises, on the vessel body, a first device for the supply of the medium, and a second device for the discharge of the medium. 
     With this measure, it is possible to utilize the abovementioned advantages of the device of the invention not only for supplying the medium but also for discharging the medium. The controllability of the flow rate in the culture vessel is advantageously further increased. 
     Preferably, the first and the second devices are arranged at two mutually opposite edges of the vessel body. 
     This can provide particularly uniform flow of the medium between the two edges of the vessel body. This advantageously promotes cell development of the adherent cells in the entire region between the edges of the vessel body. 
     In another preferred embodiment, the plurality of second apertures are respectively arranged with an adjustable distance from the bottom side of the vessel body. 
     It is thus advantageously possible to avoid damage to the device caused by impacts involving the bottom side of the vessel body. It is moreover possible to achieve particularly precise control of the flow behavior of the medium in the vessel body by adjusting the distance of the second apertures from the bottom side. 
     In another preferred embodiment, the vessel body comprises a connection for electrical current and/or voltage arranged for the application of an electrical current and/or an electrical voltage on the vessel body. 
     With this measure it is possible to establish an internal membrane potential of the individual cells located in the vessel body, in order to promote growth and cell proliferation. The typical electrical voltage to be applied on the culture vessel, in particular bioreactor vessel, is by way of example in the range of about 0 mV to 100 mV. 
     According to another aspect of the invention, a method of cultivating microbiological systems, for example cells and/or microorganisms, comprises using a culture vessel according to one of the above embodiments. 
     Further advantages and features will be apparent from the description below and from the attached drawing. 
     It is self-evident that the abovementioned features and the features that will be explained below can be used not only in the respective stated combination but also in other combinations, or alone, without leaving the scope of the present invention. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Exemplary embodiments of the invention are shown in the drawings, and are described hereinbelow with reference to said drawings, in which: 
         FIG. 1  shows a perspective view of a bioreactor for cultivating microbiological systems, comprising devices for the supply and discharge of medium; 
         FIG. 2  shows another perspective view of the bioreactor from  FIG. 1 ; 
         FIG. 3  shows a perspective view of another embodiment of a bioreactor; 
         FIG. 4  shows a perspective view of a vessel body of a bioreactor in isolation; 
         FIG. 5  shows a perspective view of a device for the supply or discharge of medium in isolation; 
         FIG. 6  shows another perspective view of the vessel body from  FIG. 4 , where the device from  FIG. 5  is attached on the vessel body in order to form a bioreactor; 
         FIG. 7  shows a diagrammatic view of a system for the cultivation of microbiological systems, wherein the system comprises the bioreactor from  FIG. 6 , a medium reservoir and a pump, connected to one another by a plurality of flexible tubes, and 
         FIG. 8A-D  shows microscope images of a cell culture obtained by means of the bioreactor from  FIG. 7 . 
     
    
    
     EMBODIMENTS 
       FIG. 1  shows a bioreactor  10 A for the cultivation of microbiological systems, for example of cells and/or microorganisms. The bioreactor  10 A comprises a vessel body  12  to receive a medium, for example a nutrient medium, and/or a gas such as oxygen. The bioreactor  10 A moreover comprises a first device  14  for the supply of medium into the vessel body  12 , and a second device  16  for the discharge of medium from the vessel body  12 . The vessel body  12  extends from upper side  18  to a bottom side  20  not shown here (see  FIG. 2 ), there being, arranged between the upper side  18  and the bottom side  20 , an internal space  19  (see  FIG. 2 ) of the vessel body  12 . The vessel body  12  moreover comprises a lateral face  26 , on which a vessel neck  22  which can be closed by a cap  23  is arranged. 
     As can be seen from  FIG. 1 , the vessel body  12  comprises a first section  12   a  which has the shape of a rectangular parallelepiped and in which the upper side  18  runs in essence parallel to the bottom side  20 , and a second section  12   b  which has the shape of a trapezoid and in which, at the side having the vessel neck, the upper side  18  is inclined in the direction of the bottom side  20 . The first and the second device  14 ,  16  are attached at two mutually opposite edges  13 ,  15  of the first section  12   a  having the shape of a rectangular parallelepiped. 
     The first device  14  comprises a base body  30  and a cover  28  for releasable closure of the main body  30 . Arranged centrally on a surface  29  of the cover  28  is a first aperture  32 , arranged at the end of a tube section  32  projecting beyond the surface  29  of the cover  28 . The cover  28  and the base body  30  form a housing of the first device  14 . 
     The second device  16  is similar to the first device  14  in likewise comprising a cover  28 ′ for releasable closure of a base body  30 ′, wherein, centrally arranged on a surface  29 ′ of the cover  28 ′, is a tube section  33 ′ which has, at its end, a first aperture  32 ′ of the second device  16 . 
     Flexible tubes for the feed or return of a medium, for example a nutrient medium comprising a plurality of ingredients, or a gas such as oxygen, can be attached to the first aperture  32 ,  32 ′, and the flexible tubes can be attached to a pump and/or a medium reservoir. 
       FIG. 2  shows another perspective view of the bioreactor  10 A from  FIG. 1 , wherein the first and second devices  14 ,  16  are shown in more detail. For illustrative purposes, the vessel body  12  and the first and second devices  14 ,  16  are shown in transparent view. 
     The first device  14  is by way of example a supply device, and comprises a plurality of nozzles  34  which are connected in fluid-conducting manner with the tube section  33  via a shared intermediate chamber  38  extending in a longitudinal direction of the first device  14 . The nozzles  34  extend from the shared intermediate chamber  38  perpendicularly to the upper side  18  of the rectangular parallelepiped-shaped first section  12   a  in the direction of the bottom side  20 , and respectively terminate in a second aperture  36   i - vi . The plurality of nozzles  34  here are respectively inserted into the internal space  19  of the vessel body  12  via a plug-in aperture  40 , these being arranged on the upper side  18  of the vessel body  12 . 
     The plurality of nozzles  34  are distributed over the entire length of the shared intermediate chamber  38 , and arranged at a distance from one another. Each nozzle  34  comprises a plurality of successive nozzle sections  34   a, b, c, d . A first nozzle section  34   a , which is configured as cylinder, extends within the base body  30  of the first device  14 , preferably from the shared intermediate chamber  38  to the level corresponding to the associated plug-in aperture  40  of the vessel body  12 . The first nozzle section  34   a  can be fixed on the cover  28 . Alternatively, the first nozzle section  34   a  of the nozzle  34  can be arranged as a bore within, and running through, the vessel body  30 . 
     A second nozzle section  34   b , which is likewise configured as cylinder, extends from the first nozzle section  34   a  in the direction of the bottom side  20  of the vessel body  12 . As can be seen in  FIG. 2 , the two sections  34   a, b  have the same internal diameter. The second section  34   b  is preferably arranged as longitudinal extension of the first section  34   a  fixed on the cover  28 . Alternatively, the second nozzle section  34   b  can be fixed on the base body  30 . 
     A third nozzle section  34   c  extends from the second nozzle section  34   b  in the direction of the bottom side  20  of the vessel body  12 . As can be seen in  FIG. 2 , the third nozzle section  34   c  has a conical shape, where the external cross section and the internal cross section of the conical shape narrows in the direction toward the bottom side  20 . 
     The plurality of nozzles  34  provide a fluid conduit between the first aperture  32  and each of the second apertures  36   i - iv . The nozzles  34  arranged in parallel to one another advantageously produce a plurality of medium sub-streams (medium strings) of the incoming medium, arranged in parallel to one another. The fluid conduits are moreover distributed over the length of the first device  14 , and therefore also over the length of the edge  13 , and the incoming medium stream is therefore particularly uniformly distributed over the area of the basal side  20 . This greatly reduces, or entirely avoids, temporal or spatial over- or undersupply of the cells in the vessel body  12 . 
     The spatial separation of the nozzles  34  moreover greatly reduces, or entirely avoids, disadvantageous mixing of the plurality of medium sub-streams of the medium that is to be introduced. This promotes laminar flow, or flow with little swirling and little turbulence, of the medium in the internal space  29  of the vessel body  12 , with defined flow rate, and allows a laminar flow configuration. The flow behavior of the medium is advantageously easier to control, thus providing a further reduction of temporal or spatial over- or undersupply of the cells in the vessel body  12 . Cultivation of the microbiological systems located in the vessel body  12 , in particular of the adherent cell types/cell lawns and/or microorganisms, is thus further promoted. 
     Arranged at the end of the respective third nozzle section  34   c  is a tubular fourth nozzle section  34   d . The fourth nozzle section  34   d  extends from the third nozzle section  34   c  to the second aperture  36   i - vi , and has a curvature in the direction toward the second aperture  36   i - vi . The aperture direction of the second aperture  36   i - vi  therefore differs, by an angle that is preferably 90°, from the direction in which the other nozzle sections  34   a, b, c  extend. The curvature prevents sudden change of the flow direction during emergence into the internal space  19 , thus further reducing swirling/turbulence effects and further increasing the laminarity of flow of the medium. 
     At least one of the nozzles  34  can moreover be configured as laminar nozzle suitable for producing a laminar medium sub-stream of the medium that is to be supplied. It is preferable that the laminar nozzle has a Reynolds number that is below 2300, more preferably below 2000, for a flow velocity, and also a viscosity of the medium to be supplied, that is conventional for the cell culture. 
     The second device  16  is by way of example a discharge device, and likewise comprises a plurality of fluid conduits in the form of nozzles  34 ′, each of which extends between a shared intermediate chamber  38 ′ and a second aperture  35   i - vi . The only difference from the nozzles  34  of the first device  14  is that the nozzles  34 ′ of the second device  16  respectively have only the first, the second and the third nozzle section  34   a ′, b′, c′. As can be seen in  FIG. 2 , the first and the second device  14 ,  16  are oriented in relation to one another in a transverse direction. Advantageously, turbulence effects are further reduced, and a defined flow rate of the medium in the internal space  19  is further promoted. 
     As can be seen in  FIG. 2 , the second apertures  36   i - vi  of the first device  14  are arranged spaced apart from the bottom side  20  of the vessel body  12 . The distance h (measured from the center of the respective second aperture  36   i - iv ) from the bottom side  20  is preferably variable. The second apertures  35   i - iv  of the second device  16  are also arranged with a preferably variable distance h′ from the bottom side  20 . 
       FIG. 3  shows a perspective view of another bioreactor  10 B with configuration similar to that of the bioreactor  10 A from  FIGS. 1 to 3 , the only difference being that in the case of the bioreactor  10 B the first and second device  14 ,  16  are identical. Each nozzle  34 ′ of the second device  16  therefore comprises, alongside the first, second and third nozzle section  34   a ′, b′, c′, a tubular fourth nozzle section  34   d ′ with a curvature. The second apertures  36   i - iv  of the first device  14  on the one hand and the second apertures  36 ′ i iv of the second device  16  on the other hand are arranged to face toward one another. Advantageously, swirling/turbulence effects are further reduced, and laminarity of flow of the medium is further increased. Here again, for illustrative purposes, the various components of the bioreactor  10 B are shown in transparent view. 
       FIG. 4  shows another perspective view of the vessel body  12  from  FIGS. 1 to 3 . As can be seen in  FIG. 4 , the plug-in apertures  40  are configured as cutouts in the upper side  18  which are arranged in a row along the first edge  13  of the vessel body  12 , at a distance from one another. In the same way, a plurality of plug-in apertures  40 ′ are configured as cutouts which are arranged in a row along the second edge  15  of the vessel body  12 , at a distance from one another. 
       FIG. 5  shows a perspective view of another device  42 ,  42 ′ for the supply or discharge of medium which is similar in its design to the first and the second device  14 ,  16  from  FIGS. 1 to 3 . Here again, the first aperture  44  is configured at the end of the tube section  41  projecting beyond the surface of the cover  43 . The cover  43  is of flat design, and is placed on the base body  45  to provide releasable closure of same. In addition, the base body  45  comprises a plurality of cutouts through which the nozzles  47  can pass, and a sealant  48  in the form of a gel is provided here to seal the intervening space between the respective nozzle  47  and the cutout associated therewith. As can be seen in  FIG. 5 , the nozzle  47  comprises a first and a second nozzle section  47   a, b , there being a narrowing section  49  configured in the transition from the first to the second nozzle section  47   b.    
       FIG. 6  shows a perspective view of another bioreactor  10 C, which comprises the base body  45  from  FIG. 4 , and also two devices  42 ,  42 ′ from  FIG. 5 . In one of the two devices  42 ,  42 ′, the cover  43  is separated from the base body  45 , and therefore only the base body  45  is attached on the vessel body  12 . The bioreactor  100  is therefore in a partially built-up condition. The arrangement of the base body  45  is such that each of its plurality of cutouts respectively vertically overlaps a plug-in aperture  40 ′ associated therewith of the vessel body  12 . It is therefore possible, on closure of the base body  45  by the cover  43 , to introduce the nozzles  47  of the device  42 ′ through the cutouts and the plug-in apertures  40 ′ into the vessel body  12 . 
     The respective bioreactor  10 A, B, C can be produced from glass and/or plastic. By way of example, the vessel body is produced primarily from glass, while the supply device, and the discharge device, is produced primarily from at least partially transparent plastic. The visibility of the medium flowing within the vessel body or through the supply device or discharge device is thus advantageously improved. 
     Finally,  FIG. 7  shows a greatly simplified diagrammatic depiction of a cultivation system  60 . The cultivation system  60  comprises, alongside the bioreactor  100 , a medium reservoir  54  and a pump  55 , the various components here being connected to one another by way of a plurality of flexible tubes  52 ,  52 ′ for the conduct of the medium through the system. A first flexible tube  56  between the medium reservoir  54  and the pump  55 , and also a second flexible tube  52  between the pump  55  and the bioreactor  100 , serve as input conduit, the directions of flow of the medium here being indicated by the arrows  53 ,  57 . A third flexible tube  56 ′ between the medium reservoir  54  and the pump  55 , and also a fourth flexible tube  52 ′ between the pump  55  and the bioreactor  100 , serves as return conduit, the directions of the medium here being indicated by the arrows  53 ′,  57 ′. It is also possible to use the bioreactor  10 A or  10 B instead of the bioreactor  100 , the technical advantages thus achievable being the same. The flexible tubes  52 ,  52 ′ are respectively connected by way of an adapter  50 ,  50 ′ to the respective device  42 ,  42 ′. 
     The quantity of the medium  58  present in the vessel body  12  can be selected in a manner dependent on the shape and size of the vessel such that the medium  58  covers the cells located in the vessel body  12 . The quantity present is preferably such that the level of the medium  58  is in the range of 0.001 mm to 20 mm above the bottom side  20 . The flow rate of the medium is preferably in the range of 0.001 mL/min and 100 mL/min. With the aid of the devices  42 ,  42 ′ it is possible to establish a flow configuration in which the supplied medium and the discharged medium has the same constant flow rate. 
     The cultivation system  60  shown in  FIG. 7  is used to carry out laboratory experiments whose results are shown in  FIG. 8A-D . 
     CnT-02 (obtainable by way of example from CELLnTEC advanced cell systems AG, Berne, Switzerland) supplemented+1 mM CaCl 2  is used as stratification medium for the conduct of experiments. 
     Urothelium cells from urethers are used as cell culture. 
     The cells are cultivated to confluence at 37° C. with 5% CO 2  in the bioreactor  100  shown in  FIG. 7 . During the duration of the experiment, the used-up medium is replaced by freshly constituted medium every two days. When confluence is complete, stratification of the cells is induced with the aid of the abovementioned stratifying medium. From that moment onward, the stratifying medium is replaced daily, the volume thereof present being 5 ml. 
     The flexible tubes  52 ,  52 ′, and also the pump  55 , are then attached to the bioreactor  100 , the pump  55  here being adjusted to achieve and maintain a flow rate of 100 to 150 ml per hour. 
     The stratification of the culture is maintained until the fourteenth day of the experiment. During the course of the experiment, a pellicle forms in the bioreactor  100 ; this separates from the bottom of the bioreactor  100  after a period of ten days.  FIG. 8A  shows a micrograph of the cell culture on the fourth day of the experiment (i.e. before separation of the pellicle) under 5× magnification.  FIG. 8B  shows another micrograph of the cell culture on the tenth day of the experiment (i.e. on the day on which the pellicle separated) under 10× magnification.  FIG. 8C  shows a micrograph of the cell culture on the twenty-fourth day of the experiment (i.e. after separation of the pellicle) under 5× magnification.  FIG. 8D  shows another micrograph of the cell structure from  FIG. 8C  under 10× magnification. 
     The separation of the multilayer cell pellicle is aided by a dispase (25 U/ml). As soon as the cell pellicle has separated from the bottom of the bioreactor  100 , the dispase is removed by suction and 5 ml of PBS is pipetted into the system. The cell pellicle is transferred to a Petri dish and stored on ice. 
     The laboratory results show that in the bioreactor  100  it is possible to achieve appropriate cell propagation, which is progressed to confluence ( FIG. 8A ). Stratification in the experiment produces a urothelium in the form of a multilayer pellicle ( FIGS. 8C and 8D ). The bioreactor  100  can therefore be used to produce a biological tissue.