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

Abstract

A device for the supply or discharge of medium into or out of a culture vessel comprises a housing on which a first aperture and a plurality of second apertures are arranged. The first aperture and the second apertures have fluid-conducting connection to one another in a manner to supply a medium from the first aperture via the second apertures into the interior of the culture vessel or to discharge same in reversed direction therefrom, when the device is attached on the culture vessel. The plurality of second apertures are configured to generate a plurality of medium sub-streams arranged in parallel to one another of the medium that is to be supplied or that is to be discharged. Further disclosed are a culture vessel and a method of cultivating microbiological systems.

Claims

1. A device configured to supply or discharge a medium into or out of a culture vessel, 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.

2. The device as claimed in claim 1, wherein 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.

3. The device as claimed in claim 2, wherein at least one of the plurality of fluid conduits is configured at least partially as nozzle.

4. The device as claimed in claim 2, wherein at least one of the plurality of fluid conduits is configured at least partially as laminar nozzle configured to generate a laminar medium sub-stream.

5. The device as claimed in claim 4, wherein the nozzle has an internal cross section which at least sectionally narrows in the direction of the second aperture associated with said nozzle.

6. The device as claimed in claim 2, wherein at least one of the plurality of fluid conduits has a tubular terminal section which is curved toward that second aperture of the plurality of second apertures which is associated with the at least one fluid conduit.

7. The device as claimed in claim 2, wherein a shared intermediate chamber is arranged in the housing for fluid-conducting connection between the first aperture and the plurality of fluid conduits.

8. The device as claimed in claim 1, wherein the first aperture is arranged centrally on a surface of the housing.

9. The device as claimed in claim 1, wherein the first aperture is arranged terminally on a tube section projecting beyond a surface of the housing.

10. The device as claimed in claim 1, wherein the housing comprises a base body and a cover configured to releasably closing the base body.

11. A culture vessel, 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.

12. The culture vessel as claimed in claim 11, further comprising one or more plug-in apertures arranged on the upper side of the vessel body, wherein the plug-in apertures are configured to plug-in the at least one device.

13. The culture vessel as claimed in claim 11, wherein 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, wherein at least one of the plurality of fluid conduits is directed, at least in sections thereof, perpendicularly to a bottom side of the vessel body.

14. The culture vessel as claimed in 11, wherein 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.

15. The culture vessel as claimed in claim 14, wherein the first and the second device are arranged at two mutually opposite edges of the vessel body.

16. The culture vessel as claimed in claim 11, wherein the plurality of second apertures are respectively arranged with an adjustable distance from a bottom side of the vessel body.

17. The culture vessel as claimed in claim 11, wherein the vessel body comprises a connection for at least one of electrical current and voltage arranged for applying the at least one of electrical current and voltage on the vessel body (12).

18. The culture vessel as claimed in claim 11, configured as a bioreactor for the cultivation of microbiological systems

19. A method of cultivating microbiological systems, comprising using a culture vessel, the culture vessel 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.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Exemplary embodiments of the invention are shown in the drawings, and are described hereinbelow with reference to said drawings, in which:

[0050] FIG. 1 shows a perspective view of a bioreactor for cultivating microbiological systems, comprising devices for the supply and discharge of medium;

[0051] FIG. 2 shows another perspective view of the bioreactor from FIG. 1;

[0052] FIG. 3 shows a perspective view of another embodiment of a bioreactor;

[0053] FIG. 4 shows a perspective view of a vessel body of a bioreactor in isolation;

[0054] FIG. 5 shows a perspective view of a device for the supply or discharge of medium in isolation;

[0055] 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;

[0056] 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

[0057] FIG. 8A-D shows microscope images of a cell culture obtained by means of the bioreactor from FIG. 7.

EMBODIMENTS

[0058] FIG. 1 shows a bioreactor 10A for the cultivation of microbiological systems, for example of cells and/or microorganisms. The bioreactor 10A comprises a vessel body 12 to receive a medium, for example a nutrient medium, and/or a gas such as oxygen. The bioreactor 10A 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.

[0059] As can be seen from FIG. 1, the vessel body 12 comprises a first section 12a 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 12b 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 12a having the shape of a rectangular parallelepiped.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] FIG. 2 shows another perspective view of the bioreactor 10A 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.

[0064] 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 12a in the direction of the bottom side 20, and respectively terminate in a second aperture 36i-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.

[0065] 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 34a, b, c, d. A first nozzle section 34a, 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 34a can be fixed on the cover 28. Alternatively, the first nozzle section 34a of the nozzle 34 can be arranged as a bore within, and running through, the vessel body 30.

[0066] A second nozzle section 34b, which is likewise configured as cylinder, extends from the first nozzle section 34a in the direction of the bottom side 20 of the vessel body 12. As can be seen in FIG. 2, the two sections 34a, b have the same internal diameter. The second section 34b is preferably arranged as longitudinal extension of the first section 34a fixed on the cover 28. Alternatively, the second nozzle section 34b can be fixed on the base body 30.

[0067] A third nozzle section 34c extends from the second nozzle section 34b in the direction of the bottom side 20 of the vessel body 12. As can be seen in FIG. 2, the third nozzle section 34c 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.

[0068] The plurality of nozzles 34 provide a fluid conduit between the first aperture 32 and each of the second apertures 36i-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.

[0069] 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.

[0070] Arranged at the end of the respective third nozzle section 34c is a tubular fourth nozzle section 34d. The fourth nozzle section 34d extends from the third nozzle section 34c to the second aperture 36i-vi, and has a curvature in the direction toward the second aperture 36i-vi. The aperture direction of the second aperture 36i-vi therefore differs, by an angle that is preferably 90°, from the direction in which the other nozzle sections 34a, 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.

[0071] 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.

[0072] 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 35i-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 34a′, 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.

[0073] As can be seen in FIG. 2, the second apertures 36i-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 36i-iv) from the bottom side 20 is preferably variable. The second apertures 35i-iv of the second device 16 are also arranged with a preferably variable distance h′ from the bottom side 20.

[0074] FIG. 3 shows a perspective view of another bioreactor 10B with configuration similar to that of the bioreactor 10A from FIGS. 1 to 3, the only difference being that in the case of the bioreactor 10B 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 34a′, b′, c′, a tubular fourth nozzle section 34d′ with a curvature. The second apertures 36i-iv of the first device 14 on the one hand and the second apertures 36′iiv 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 10B are shown in transparent view.

[0075] 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.

[0076] 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 47a, b, there being a narrowing section 49 configured in the transition from the first to the second nozzle section 47b.

[0077] FIG. 6 shows a perspective view of another bioreactor 10C, 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.

[0078] The respective bioreactor 10A, 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.

[0079] 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 10A or 10B 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′.

[0080] 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.

[0081] The cultivation system 60 shown in FIG. 7 is used to carry out laboratory experiments whose results are shown in FIG. 8A-D.

[0082] CnT-02 (obtainable by way of example from CELLnTEC advanced cell systems AG, Berne, Switzerland) supplemented+1 mM CaCl.sub.2 is used as stratification medium for the conduct of experiments.

[0083] Urothelium cells from urethers are used as cell culture.

[0084] The cells are cultivated to confluence at 37° C. with 5% CO.sub.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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.