BIOREACTOR AND METHOD FOR CULTIVATING BIOLOGICAL CELLS ON SUBSTRATE FILAMENTS

Abstract

The invention relates to a bioreactor (100) which is designed for cultivating biological cells (1), comprising a container (10) configured to receive a cultivation medium (2), and a plurality of substrate filaments (20) which are arranged in the container (10) and are configured for a temporary adherent coupling of the biological cells (1) to the substrate filaments (20). The substrate filaments (20) are provided with a surface layer (21) which is switchable between an adherence state, in which the biological cells (1) can be QI coupled adherently to the surface layer (21), and a release state, in which the adherent coupling of the biological cells (1) to the surface layer (21) is reduced in comparison to the binding state. The invention also relates to a method for processing biological cells (1) in the bioreactor (100).

Claims

1. A bioreactor which is configured for culturing biological cells, comprising a container which is configured for receiving a culture medium, and a plurality of substrate filaments which are arranged in the container and are configured for temporary adherent coupling of the biological cells to the substrate filaments, wherein the substrate filaments are provided with a surface layer which can be switched between an adherence state, in which the biological cells can be coupled to the surface layer in an adherent manner, and a release state, in which the adherent coupling of the biological cells to the surface layer is reduced compared to the adherence state.

2. The bioreactor according to claim 1, wherein the surface layer is configured to switch between the adherence state and the release state in response to light.

3. The bioreactor according to claim 2, wherein the substrate filaments comprise light guides, which are each configured for connection to a switching light source device and for switchable illumination of the surface layer out of the light guide.

4. The bioreactor according to claim 3, wherein the switching light source device comprises a plurality of separately switchable light sources, and the substrate filaments are each configured for connection to one of the light sources, wherein the surface layer of each substrate filament is individually switchable between the adherence state and the release state by activating the associated light source.

5. The bioreactor according to 3, wherein the switching light source device part of the bioreactor and is securely connected to the light guides.

6. The bioreactor according to claim 3, wherein the light guides are configured for a switchable illumination of the surface layer with evanescent waves, each of which penetrates from inside the light guides into the surface layer.

7. The bioreactor according to claim 2, wherein the substrate filaments are configured with at least one of liquid-crystal switchable light elements, light-emitting diodes and chemiluminescent light elements, which are each configured for switchable illumination of the surface layer.

8. The bioreactor according to claim 2, wherein the surface layer comprises at least one of a light-responsive hydrogel layer, and a functionalization layer on the surface of the substrate filaments.

9. The bioreactor according to claim 1, wherein the surface layer is configured to switch between the adherence state and the release state in response to heat.

10. The bioreactor according to claim 1, wherein the substrate filaments comprise hollow fibers, which are each configured for connection to a temperature-control device and for switchable temperature control of the surface layer with a temperature-control medium flowing through the hollow fibers.

11. The bioreactor according to claim 10, wherein the temperature-control device comprises a plurality of separately switchable temperature-control elements, and the hollow fibers are each configured for connection to one of the temperature-control elements, wherein the surface layer of each hollow fiber is individually switchable between the adherence state and the release state by activating the associated temperature-control element.

12. The bioreactor according to claim 9, wherein the surface layer formed from a temperature-responsive hydrogel.

13. The bioreactor according to claim 1, wherein the substrate filaments comprise flexible fibers which extend in the container.

14. The bioreactor according to claim 1, wherein the container has an elongate form, and the substrate filaments extend in a longitudinal direction of the container.

15. The bioreactor according to claim 1, wherein the container has at least two fluid connectors and is configured to be flowed through by a liquid flushing medium, wherein in the release state, the adherent coupling of the biological cells to the surface layer is reduced such that the biological cells can be separated from the substrate filaments by flow forces of the flushing medium.

16. The bioreactor according to claim 1, wherein the container has at least one sensor connector which is configured for integrating at least one sensor into the bioreactor.

17. A method for processing biological cells in the bioreactor according to claim 1, comprising the steps of: setting the adherence state of the substrate filaments, adherent coupling of the biological cells to the substrate filaments, culturing the adherently coupled biological cells, setting the release state of the substrate filaments, and detaching the biological cells from the substrate filaments.

Description

[0031] Further details and advantages of the invention are described below with reference to the appended drawings. The drawings show:

[0032] FIG. 1: a schematic depiction of the bioreactor according to the first embodiment of the invention;

[0033] FIG. 2: further details of the bioreactor according to the first embodiment of the invention;

[0034] FIG. 3: a schematic illustration of the release of biological cells from the surface of a substrate filament;

[0035] FIG. 4: a schematic depiction of the second embodiment of the bioreactor according to the invention; and

[0036] FIG. 5: a schematic depiction of the bioreactor with further components.

[0037] Preferred embodiments of the invention are described below with exemplary reference to bioreactors which are provided with light-responsive or heat-responsive substrate filaments. The implementation of the invention is not limited to these embodiments.

[0038] Alternatively, light-responsive and heat-responsive substrate filaments can be combined in the bioreactor. Furthermore, the invention is not limited to the tubular form of the bioreactor shown by way of example. Depending on the application of the invention, other forms of the bioreactor, for instance box or sphere forms, can be selected. Furthermore, in a deviation from the examples shown, active light sources can be provided in the container of the bioreactor, on the substrate filaments and/or on an inner wall of the container. Details of the invention are described in particular with reference to the design and arrangement of the substrate filaments and the operation of the bioreactor. Details of the culturing of biological cells, in particular the differentiation of adherently-growing stem cells, are not described here, since they are known per se from conventional culturing processes.

[0039] According to the schematic partial view in FIG. 1, the first embodiment of the bioreactor 100 according to the invention comprises a container 10, in which a plurality of substrate filaments 20 are arranged. According to FIG. 1A, the container 10 has the form of a hollow cylinder. The container 10 is shown open here, but in operation has a container wall which is closed on all sides, optionally with fluid and sensor connectors (see FIG. 2), windows and/or further access openings.

[0040] The substrate filaments 20 extend in the axial direction of the container 10. The axial length of the container 10 is for example 20 cm, and the diameter of the container 10 is for example 5 cm.

10000 substrate filaments 20 are arranged in the container 10, for example. The container 10 is filled with a culture medium 2, which flushes around the substrate filaments 20. The culture medium 2 preferably flows through the container 10 (see FIGS. 2 and 5). The culture medium 2 comprises for example mTeSR 1 medium (mTeSR is a product name).

[0041] Each substrate filament 20 comprises a light guide 22 with a switchable surface layer 21, as illustrated in the schematic partial sectional view in FIGS. 1B and 1n the schematic partial perspective view in FIG. 1C. The light guide 22 is a compact fiber, for example made of glass. The diameter of the light guide 22 is for example 200 μm.

[0042] The surface layer 21, comprising for example a light-responsive alginate with PNIPAAm coupled thereto, is arranged on the surface of the light guide 22. The thickness of the surface layer 21 is for example 0.01 μm. The surface layer 21 is switchable between an adherence state, in which the biological cells 1 are coupled to the surface layer 21 in an adherent manner (see FIGS. 1B and 1C), and a release state, in which the biological cells 1 can be detached from the surface layer 21 (see FIG. 3B).

[0043] FIG. 2 shows further details of the first embodiment of the bioreactor 100 according to the invention. At the axial ends of the container 10, the substrate filaments 20 emerge from the container wall as a common bundle 23. A switching light source device 30 comprises two light sources 31, for instance two laser sources, which are adapted for irradiating the free ends of the substrate filaments 20. The irradiation is switchable by actuating the light sources 31 or by shutters (not shown), such that the substrate filaments 20 can be varied between an illuminated state and an unilluminated state. Preferably, the surface layer 21 (see FIG. 1B) is designed such that in the unilluminated state the adherence state of the substrate filaments 20 is set and in the illuminated state the release state of same is set.

[0044] FIG. 2 additionally shows two fluid connectors 11 which are arranged for permanent or temporary connection to a fluid system and which enable supply and discharge of a liquid medium to and from the container 10. The fluid connectors 11 are for example connected to a pump and a medium reservoir, as described below with reference to FIG. 5. The fluid connectors 11 are directly connected to the interior of the container 10 for receiving the culture medium, and thereby enable the automated exchange of media during culturing, in particular expansion, but also the inoculation/removal of cells in suspension. Furthermore, the container 10 is provided with two sensor connectors 12. A temperature sensor, a glucose sensor, a lactate sensor and/or an optical sensor, in particular a camera, are for example arranged in the sensor connectors 12.

[0045] FIG. 3 schematically illustrates details of the method for cell processing, in particular the switching of the substrate filaments 20 from the adherence state (FIG. 3A) into the release state (FIG. 3B). The adhesion and expansion of the cells 1 occurs on the substrate filaments 20 which run in the inside of the container 10. The substrate filaments 20 are bundled at the ends and exit the container 10 (see FIG. 2) as a compact light guide bundle. The core of the substrate filaments 20 is the light-guiding material of the light guides 22. The light guided therethrough is emitted in a diffuse manner over the entire light guide 22 at a specific wavelength. The emitted light excites the surface layer 21 located on the light guide 22 and chemically or physically switches properties. For example, a polymer of arginylglycylaspartic acid (RGD) can be switched from adhesive to non-adhesive (and vice-versa) via the APABA linker (4-[(4-aminophenyl)azo]benzocarbonyl), to which the RGD peptide has been conjugated, by light at a wavelength of 366 or 450 nm. If light-switchable calcium scavengers (diazo-2) are introduced into ionotropic hydrogels, the crosslinking from the hydrogel can be bound by the irradiation of light and thus degrade the hydrogel. In this case, adherent cells on such a layer detach from the culturing surface and pass, as suspended cells, into the interior of the container filled with culture medium, from which they can be transported out of the bioreactor via the connected fluid system.

[0046] The light guide 22 is unilluminated in the adherence state (no irradiation of the substrate filaments, see FIG. 2). The biological cells 1 are adherently coupled to the surface layer 21 in the adherence state. In this state, the biological cells 1 are cultured with the culture medium 2 (see FIG. 1A), for example for an expansion of the cell culture on the substrate filaments 20 and/or for a differentiation of the cells 1. If a predetermined culture result has been achieved, the light guides 22 are illuminated with the light sources 31 (see FIG. 2). The surface layer 21 is switched into the release state, such that the cells 1 detach from the surface layer 21. The detachment can be promoted by a movement of the culture medium in the surroundings of the substrate filaments.

[0047] FIG. 4 schematically shows the second embodiment of the bioreactor 100 according to the invention. In this embodiment, the substrate filaments 20 comprise hollow fibers 25 which extend in the axial direction of the cylindrical container 10. The hollow fibers 25 are connected to a pump and a temperature-control medium reservoir (not shown). The temperature of the hollow fibers 25 can be adjusted by a temperature-control medium which flows through the hollow fibers 25. For example, switching between the adherence state at a lower temperature and the release state at a higher temperature (or vice-versa) can be provided.

[0048] The bioreactor 100 according to FIG. 4 can be dimensioned as described above with reference to FIG. 1. For example, 5000 hollow fibers 25, with an internal diameter of 500 μm and surface layer made of PNIPAAm with a thickness of 0.05 μm, are provided.

[0049] FIG. 5 shows a schematic overview of the bioreactor 100, for example according to the first or second embodiment of the invention, with the container 10, in which the substrate filaments (not shown) are arranged, the switching light source device 30 or the temperature-control device 40 and a control device 50. In the illustrated example, the container 10 is provided with four fluid connectors 11, which are coupled to a first fluid system comprising a first pump and a culture medium reservoir 14, to a second fluid system comprising a second pump 15 and a flushing medium reservoir 16. The control device 50 is connected to the components 30, 40 and the pumps 13, 15, and also to the sensors at the sensor connectors 12. In addition, the fluid system can be provided with blocking elements, for example switchable valves, in order to be selectively coupled to the container 10. The blocking elements can also be controlled with the control device 50. Alternatively, only one fluid system can be provided, if the cells are flushed out of the container with the culture medium.

[0050] The provision of the control device 50, which is for example formed by a computer circuit, advantageously affords the possibility of automating operation of the bioreactor 100. A control loop can be created, wherein a culture state of the cells is detected as a function of signals from the sensors at the sensor connectors 12 or as a function of a predetermined culture protocol. Depending on the culture state, the pump 13 can be controlled to supply the culture medium, or the switching of the substrate filaments from the adherence state into the release state can be triggered. Furthermore, in the release state, the control device 50 can be used to control the pump 15 for flushing the cultured cells out of the container 10 and for collecting the detached cells in the flushing medium reservoir 16 and for discharging the cells for further cell processing (see arrow).

[0051] The invention is not limited to the above-described preferred embodiments. Rather, a plurality of variants and modifications is possible, which also form part of the inventive concept and are therefore within the scope of protection. The invention particularly also claims protection for the subject matter and the features of the dependent claims and the combinations thereof, regardless of the claims referred to in each case.