METHOD FOR CULTURING CELLS

Abstract

The invention refers to a method for culturing cells. The cells are cultured in a substrate, wherein in the substrate a fluid channel system is formed, comprising a culturing channel having a culturing access to the outside, a storage channel having a storage access to the outside, and a transfer channel having a transfer access to the outside, wherein the culturing channel and the storage channel are fluidically connected to each other via the transfer channel. The method comprises supplying an initial cell suspension through the culturing access into the culturing channel or through the transfer access via the transfer channel into the culturing channel, growing a cell culture in the culturing channel, transferring cells of the cell culture from the culturing channel into the storage channel via the transfer channel, wherein the transfer is carried out by supplying a transfer liquid through the culturing access and transporting the transfer liquid through the culturing channel and the transfer channel into the storage channel, and storing and/or concentrating and/or homogenizing and/or diluting a cell suspension containing the transferred cells in the storage channel.

Claims

1. Method for culturing cells in a substrate (1), wherein in the substrate (1) a fluid channel system (1a) is formed, comprising a culturing channel (2a, 2b, 2c) having a culturing access (5a, 5b, 5c) to the outside, a storage channel (3a, 3b, 3c) having a storage access (6a, 6b, 6c) to the outside, and a transfer channel (4) having a transfer access (7a, 7b) to the outside, wherein the culturing channel (2a, 2b, 2c) and the storage channel (3a, 3b, 3c) are fluidically connected to each other via the transfer channel (4), and wherein the method comprises: supplying an initial cell suspension (12a′) through the culturing access (5a, 5b, 5c) into the culturing channel (2a, 2b, 2c) or through the transfer access (7a, 7b) via the transfer channel (4) into the culturing channel; growing a cell culture in the culturing channel; transferring cells of the cell culture from the culturing channel (2a, 2b, 2c) into the storage channel (3a, 3b, 3c) via the transfer channel (4), wherein transferring is carried out by supplying a transfer liquid through the culturing access (5a, 5b, 5c), and transporting the transfer liquid through the culturing channel (2a, 2b, 2c) and the transfer channel (4) into the storage channel (3a, 3b, 3c); and performing one or more selected from the group consisting of: storing, concentrating, homogenizing, and diluting a cell suspension containing the transferred cells in the storage channel (3a, 3b, 3c).

2. Method according to claim 1, wherein all method steps in which liquids are moved in the fluid channel system (1a) are carried out by means of active components (13a-13c, 15a-15c, 16a-16d, 17a, 17b, 18a-18c), particularly pumps and/or valves, arranged exclusively outside the substrate (1), particularly automatically by controlling the active components (13a-13c, 15a-15c, 16a-16d, 17a, 17b, 18a-18c).

3. Method according to claim 2, wherein by actuating the active components (13a-13c, 15a-15c, 16a-16d, 17a, 17b, 18a-18c), particularly automatically by controlling the active components (13a-13c, 15a-15c, 16a-16d, 17a, 17b, 18a-18c), one or more selected from the group consisting of the following is set: which liquid is supplied to the fluid channel system (1a), a sequence of liquids supplied to the fluid channel system (1a), through which access liquid is supplied to the fluid channel system (1a), through which access liquid is taken from the fluid channel system (1a), the transport path of liquid in the fluid channel system (1a), the flow rate of liquid in the fluid channel system (1a), and when liquid is one or more selected from the group consisting of: supplied, transported further, and discharged, wherein the setting is carried out particularly by one or more selected from the group consisting of: establishing and/or interrupting connections between a liquid container (12a-12d) in which the respective liquid is stored, and the fluid channel system (1a), for example by, particularly automatically, opening and/or closing valves and/or switching multiway valves, and applying a pressure suitable for transporting the respective liquid into the fluid channel system (1a) and/or in the fluid channel system (1a), for example by, particularly automatically, actuating pumps.

4. Method according to claim 1, wherein no active components (13a-13c, 15a-15c, 16a-16d, 17a, 17b, 18a-18c), no measurement device (22a-22d, 23), and no controller (20) are arranged in the substrate (1).

5. Method according to claim 1, wherein the cells of the cell culture are detached from the walls of the culturing channel (2a, 2b, 2c) by one or more selected from the group consisting of: enzymatically and by shear forces arising during moving of a liquid in the respective culturing channel (2a, 2b, 2c) before they are transferred via the transfer channel (4) into the storage channel (3a, 3b, 3c).

6. Method according to claim 1, comprising optical inspection of the cells during one or more selected from the group consisting of: growing, during detachment and while the cells are located in the storage channel (3a, 3b, 3c).

7. Method according to claim 1, wherein the culturing channel (2a, 2b, 2c) is a first culturing channel (2a, 2b, 2c) and the transfer liquid is a first transfer liquid, and wherein the fluid channel system (1a) comprises a second culturing channel (2a, 2b, 2c) fluidically connected to the transfer channel (4), and wherein the method comprises transferring the cells from the storage channel (3a, 3b, 3c) via the transfer channel (4) into the second culturing channel (2a, 2b, 2c) by means of a second transfer liquid and growing an additional cell culture by multiplying the cells in the second culturing channel (2a, 2b, 2c).

8. Substrate (1) for culturing cells by the method according to one of the preceding claims, wherein a fluid channel system (1a) is formed in the substrate (1), comprising: a culturing channel (2a, 2b, 2c) for growing a cell culture, comprising a culturing access (5a, 5b, 5c) to the outside; a storage channel (3a, 3b, 3c) for one or more selected from the group consisting of: storing, concentrating, homogenizing, and diluting a cell suspension containing the transferred cells, comprising a storage access (6a, 6b, 6c) to the outside; a transfer channel (4) with a transfer access (7a, 7b) to the outside, wherein the culturing channel (2a, 2b, 2c) and the storage channel (3a, 3b, 3c) are fluidically connected to each other via the transfer channel (4).

9. Substrate (1) according to claim 8, wherein the fluid channel system (1a) one or more selected from the group consisting of: a shape, arrangement, orientation, and connection of the channels, that exclusively by one or more selected from the group consisting of: selectively opening and closing the accesses and applying pressure to the fluid channel system (1a) liquids are moved through the fluid channel system (1a) on the transport paths designated for the respective method step or are moved through the fluid channel system (1a) at flow velocities required for the method step.

10. Substrate (1) according to claim 8, wherein the culturing channel (2a, 2b, 2c), the storage channel (3a, 3b, 3c) and the transfer channel (4) are arranged in one plane or wherein a mouth of the culturing channel (2a, 2b, 2c) into the transfer channel (4) is arranged along the transfer channel (4) offset from a mouth of the storage channel (3a, 3b, 3c) into the transfer channel (4).

11. Substrate (1) according to claim 8, wherein the longitudinal axis of the transfer channel (4) is arranged at an angle greater than or equal to 50°, particularly greater than or equal to 55°, particularly greater than or equal to 65°, particularly greater than or equal to 75°, particularly greater than or equal to 85° and particularly substantially perpendicular to the respective longitudinal axis of the culturing channel (2a, 2b, 2c).

12. Substrate (1) according to claim 8, wherein the culturing channel (2a, 2b, 2c) is narrowed towards the transfer channel (4) or wherein the storage channel (3a, 3b, 3c) is narrowed towards the transfer channel (4).

13. Substrate (1) according to claim 8, wherein at least one wall of the culturing channel (2a, 2b, 2c), particularly at least a partial area of the floor or the ceiling or the side walls has a surface property of the material, selected from the group consisting of: a material property, a structural property, and a coating (2a′) that promotes the adhesion of cells, particularly a coating selected from the group consisting of: a hydrophilic coating and a cell-adhering coating, or wherein at least one wall of the culturing channel (2a, 2b, 2c), particularly at least a partial area of the floor or of the ceiling or of the side walls, is provided with a surface modification which is switchable with respect to its physical or chemical properties in such a way that the switching causes detachment of the cells from the surface, particularly with a thermoresponsive surface modification, particularly with a coating with poly-N-isopropylacrylamide (PNIPAM)-based polymers, or wherein at least one wall of the culturing channel (2a, 2b, 2c), particularly at least a partial area of the floor, is provided with a regenerable surface modification which particularly is configured in such a way that the cell adhesion can be changed, activated and/or deactivated, particularly can be changed via competitive inhibition, or wherein at least one wall of the culturing channel (2a, 2b, 2c), particularly at least a partial area of the floor and/or of the side walls, has a structuring which is configured in such a way that the growth surface is increased, particularly has one or more selected from the group consisting of: a saw tooth profile, a wave profile, a lamellar structure, fibrous areas, porous areas, and sponge-like areas, respectively.

14. Substrate (1) according to claim 8, wherein at least one wall selected from the group consisting of: the transfer channel (4) and of the storage channel (3a, 3b, 3c) have a surface property of the material that prevents the growing-on of cells.

15. Substrate (1) according to claim 8, wherein the floor of the storage channel (3a, 3b, 3c) has a structuring configured in such a way that the movement of cells lying on the floor is impeded at least in one direction.

16. System comprising a substrate (1) according to claim 8 and at least three liquid containers (12a-12d) arranged outside the substrate (1), wherein the culturing access (5a, 5b, 5c), the storage access (6a, 6b, 6c) and the transfer access (7a, 7b) are each fluidically connected to at least one of the liquid containers (12a-12d), active components (13a-13c, 15a-15c, 16a-16d, 17a, 17b, 18a-18c), which are configured and arranged in such a way that, when actuated, they cause one or more selected from the group consisting of: a controlled supply of liquids from the liquid containers (12a-12d) into the fluid channel system (1a), a controlled transport of liquids in the fluid channel system (1a), and a controlled discharge of liquids from the fluid channel system (1a), wherein all active components (13a-13c, 15a-15c, 16a-16d, 17a, 17b, 18a-18c) are arranged outside the substrate (1).

17. System according to claim 16, wherein the at least three liquid containers (12a-12d) comprise two storage containers (12a, 12b, 12c) for process liquids, and one waste container (12d), and wherein: the culturing access (5a, 5b, 5c) is connected to a valve (15a, 15b, 15c) configured to establish fluid connection between the culturing channel (2a, 2b, 2c) and at least one of the storage containers (12a, 12b, 12c) and to establish fluid connection between the culturing channel (2a, 2b, 2c) and the waste container (12d), or wherein the culturing access (5a, 5b, 5c) is connected to two valves (15a, 15b, 15c) one of which is configured to establish a fluid connection between the culturing channel (2a, 2b, 2c) and at least one of the storage containers (12a, 12b, 12c) and the other of which is configured to establish a fluid connection between the culturing channel (2a, 2b, 2c) and the waste container (12d), or the storage access (6a, 6b, 6c) is connected to a valve (16a, 16b, 16c, 16d) configured to establish fluid connection between the storage channel (3a, 3b, 3c) and the waste container (12d), or the transfer access (7a, 7b) is connected to a valve (17a, 17b) which is configured to establish a fluid connection between the transfer channel (4) and the waste container (12d), and particularly wherein the transfer access is connected to the valve (17a, 17b) or an additional valve, wherein the valve (17a, 17b) or the additional valve is configured to establish a fluid connection between the transfer channel (4) and at least one of the storage containers (12a, 12b, 12c).

18. System according to claim 16, comprising one or more selected from the group consisting of: a measurement device (22a-22d) arranged outside the substrate (1) and configured to measure process measurands, and a measurement device (23) arranged outside the substrate (1) and configured to optically inspect the cells in the substrate (1).

19. System according to claim 16, comprising a controller configured to control the active components (13a-13c, 15a-15c, 16a-16d, 17a, 17b, 18a-18c) such that a method according to one of claims 1 to 7 is carried out, the controller particularly being configured to regulate values of parameters to be set on the active components (13a-13c, 15a-15c, 16a-16d, 17a, 17b, 18a-18c) on the basis of target values and measured actual values of process measurands which are detected by the measurement device (22a-22d) for measuring process measurands.

Description

[0113] Further features and advantages are explained below with reference to the exemplary drawings, wherein

[0114] FIG. 1 shows a schematic not to scale top view of a substrate of a first embodiment;

[0115] FIG. 2 shows a schematic not to scale top view of a substrate of a second embodiment;

[0116] FIG. 3 shows a schematic not to scale top view of a substrate of a third embodiment;

[0117] FIG. 4 shows a schematic not to scale top view of a substrate of a fourth embodiment;

[0118] FIG. 5 shows a schematic not to scale representation of a system of a fifth embodiment using the example of a substrate of the first embodiment;

[0119] FIG. 6 shows a schematic not to scale representation of a system of a sixth embodiment using the example of a substrate of the second embodiment;

[0120] FIG. 7 shows a schematic not to scale representation of a system of a seventh embodiment using the example of a substrate of the third embodiment;

[0121] FIG. 8 shows a schematic not to scale representation of a system of a seventh embodiment using a substrate of the fourth embodiment as an example;

[0122] FIG. 9 shows a schematic not to scale representation of a system of an eighth embodiment.

[0123] In the following and in the Figures the same reference numerals are used for the same or corresponding elements in the various embodiments unless otherwise specified.

[0124] FIGS. 1 to 4 show first to fourth embodiments of a substrate 1 according to the invention. In each of the embodiments shown herein a fluid channel system 1 a is formed in the substrate 1, comprising a first culturing channel 2a, a storage channel 3a, and a transfer channel 4. The first culturing channel has a culturing access 5a. The storage channel has a storage access 6a and the transfer channel has a transfer access 7a. The transfer channel may further optionally have a second transfer access 7b.

[0125] In FIG. 1 it is exemplarily indicated that the first culturing channel may comprise a coating 2a′. In particular, this can be a cell-adherent coating or a hydrophilic coating. Furthermore, it is exemplarily indicated that the transfer channel and the storage channel comprise a coating 4′ and 3a′, respectively. Each of the coatings may be a cell-repellent coating or a hydrophobic coating. The coatings are not shown in FIGS. 2 to 4 but may also be provided in the corresponding embodiments.

[0126] In the embodiments shown in FIGS. 2 to 4, the fluid channel system further comprises one or a plurality of additional culturing channels, for example a second culturing channel 2b and a third culturing channel 2c and one or a plurality of additional storage channels, for example a second storage channel 3b and a third storage channel 3c. The additional culturing channel(s) each have their own culturing access 5b and 5c, respectively. The additional storage channel(s) each have their own storage access 6b or 6c, respectively.

[0127] In FIG. 2, channels 2b, 2c and 3b are shown by dashed lines. This illustrates that any number of additional channels can be provided, wherein particularly the number of storage channels and the number of culturing channels do not have to be identical. In contrast, FIG. 3 shows an embodiment in which the number of storage channels is equal to the number of culturing channels.

[0128] As shown in FIGS. 1 to 4, each of the culturing channels is fluidically connected to the transfer channel. In this example, each of the culturing channels is narrowed towards the transfer channel, that is, the diameter of the mouth of the respective channel is smaller than the diameter in other areas of the channel, which, however, is optional. Furthermore, each of the storage channels is fluidically connected to the transfer channel. The longitudinal axes 8a to 8c of the culturing channels are arranged substantially perpendicular to the longitudinal axis 9 of the transfer channel, which, however, this is not mandatory.

[0129] In FIGS. 1 to 3, each of the storage channels is narrowed towards the transfer channel which, however, is optional. Here, in addition, the longitudinal axes 10a to 10c of the storage channels are arranged along the transfer channel offset from the longitudinal axes 8a to 8c of the culturing channels, here exemplarily also substantially perpendicular to the longitudinal axis of the transfer channel.

[0130] In FIG. 4 the longitudinal axis 10a of the storage channel 3a is substantially parallel to the longitudinal axis of the transfer channel. The substrate comprises a fluid connection 24 between the storage channel and the transfer channel. The fluid connection has a diameter smaller than the diameter of the storage channel, and its longitudinal axis in this example is arranged substantially perpendicular to the longitudinal axis of the transfer channel and offset from the longitudinal axes of the culturing channels along the transfer channel. The storage channel 3a in FIG. 4 may have an additional storage access 6d in addition to the storage access 6a.

[0131] By way of example, in the first to fourth embodiments all channels are arranged in one plane. In particular, when the substrate is used as intended their longitudinal axes are arranged horizontally.

[0132] The substrate can be transparent, particularly formed of a transparent plastic material. In particular, the substrate can be configured in such a way that the cells contained therein can be inspected microscopically.

[0133] FIGS. 5 to 8 schematically show not to scale fifth to eighth embodiments of a system 11a to 11d for culturing cells, here exemplarily comprising the substrates shown in FIGS. 1 to 4. However, this is optional and other substrates according to the invention may be used in any of the systems. In particular, the shape, the orientation, the number, the interconnection and/or the coating of the channels in the fluid channel system may be different from those described with reference to FIGS. 1 to 4.

[0134] FIGS. 5 to 8 each show by way of example three storage containers for process liquids, for example a first storage container 12a for initial cell suspension 12a′, a second storage container 12b for cell culture medium 12b′, and a third storage container 12c for a liquid 12c′ for detaching the cells. It is understood that additional storage containers may be provided for process liquids. Further, as shown in the Figures, the system may optionally comprise a waste container 12d. A pump 13a to 13c may be connected to each of the storage containers 12a to 12c, respectively. In each case, a filter 14a to 14c, for example a sterile filter, can optionally be arranged between the pump and the storage container. Depending on the process, a different number of storage containers may also be provided. If necessary, only one pump may also be provided for a plurality of storage containers. The system may then comprise, for example, a valve which is configured in such a way that it can be used to set to which of the storage containers pressure is applied.

[0135] At least one valve may be connected to each of the accesses. In the Figures, the culturing access 5a is connected to a first valve 15a, the storage access 6a is connected to a second valve 16a, and the transfer access 7a is connected to a third valve 17a. If provided, the additional culturing accesses 5b and 5c may each be connected to a fourth valve 15b and 15c, the additional transfer access 7b may be connected to a fifth valve 17b, the additional storage accesses 6b and 6c may each be connected to a sixth valve 16b and 16c or the port 6d may be connected to an additional valve 16d.

[0136] As shown in FIGS. 5 to 8, the storage containers 12a, 12b and 12c may each be connected to a conduit 19 via a valve 18a, 18b, 18c. The conduit may be connected to the culturing access 5a of the first culturing channel via valve 15a, to the transfer access 7a of the transfer channel via valve 17a and optionally to the storage access 6a of the storage channel via valve 16a. At least one of the remaining accesses 5b, 5c, 6b, 6c, 6d, and/or 7b may also each be connected to the conduit via a valve 15b, 15c, 16b, 16c, 16d, and 17b, respectively. Each of the accesses connected to the conduit can be supplied with one of the liquids in the liquid containers 12a to 12c by suitable valve positioning of the valves 18a to 18c and of the valve associated with the respective access.

[0137] Alternatively or additionally to being connected to the conduit, each of the accesses may be connected to the waste container 12d via the corresponding valve, optionally via a flow meter 18d. The measured values from the flow meter can be used to clearly determine the flow rate in the system if, as shown herein, the system is a closed system. The measure values of the flow meter 18d can then be used as a control and/or regulating variable, particularly for actuating the valves.

[0138] In the embodiment shown in FIG. 8, the storage channel 3a has two accesses 6a and 6d. Here, both accesses are connected to the conduit and to the waste container via valves. Alternatively, one of the accesses, for example access 6a, may be connected exclusively to the conduit 19 via valve 16a, and the other access, for example access 6d, may be connected exclusively to the waste container 12d via valve 16d.

[0139] FIG. 9 shows an alternative system 11e. The system also comprises four liquid containers 12a to 12d, for example the storage containers and waste containers described above, three pumps 13a to 13c, and filters 14a to 14c. Further, it shows valves 15a, 16a, 17a and 17b connected to accesses 5a, 6a, 7a, 7b, respectively. As an example, the system is shown with the substrate of the first embodiment, but any substrate according to the invention may be used instead. Here, the three liquid containers are not connected to a common conduit 19. Each of the liquid containers has its own conduit 19a to 19c, and the liquid can be supplied to each of the accesses via a valve 20a to 20c through the valve connected to the respective access.

[0140] In the embodiment shown here, initial cell suspension from the container 12a can be supplied to the first culturing channel 2a via the valve 20a and the valve 15a. Liquid from the container 12b can be supplied to the accesses 15a, 16a or 17a via the multiway valve 20b. Liquid from the container 12c, for example liquid for detaching cells, can be supplied to the first culturing channel via the valve 20c. In addition, liquid from the first culturing channel 2a can be supplied to the waste container 12d through the valve 15a, liquid from the transfer channel 4 can be supplied to the waste container through the valve 17b, and liquid from the storage channel 3a can be supplied to the waste container through the valve 16a.

[0141] In each of FIGS. 5 to 9 an optionally provided control means 21 is shown.

[0142] FIG. 5 shows optionally provided sensors 22a to 22d. For example, sensor 22a is configured and arranged to measure the humidity in the area of the substrate. Sensor 22b is configured and arranged to measure the temperature in the area of the liquid container. Sensor 22c is configured and arranged to measure the temperature in the area of the substrate. Sensor 22d may be a sensor for measuring the oxygen or CO.sub.2 content of the air in the system. In particular, one or a plurality of the sensors may be arranged within a housing 25 along with the substrate and/or the liquid containers.

[0143] Furthermore, an optionally provided means for optical inspection 23 is indicated schematically. This can be a microscopy means. The substrate may be in the form of a microscopy carrier. The means for optical inspection may be arranged at least partially within the housing, which enables monitoring without intervention in the system. Alternatively, the optical inspection means may be arranged outside the housing and the housing may be removable.

[0144] The sensors, the optical inspection means and the housing are not shown in FIGS. 6 to 9, but the systems shown there may also comprise one or a plurality of these sensors, an optical inspection means and/or a housing.

[0145] In the following, a method for culturing cells is illustrated, here exemplarily using the system of the sixth embodiment, as shown in FIG. 6. Said method can also be carried out analogously with other systems, particularly those described above. In the following, possible modifications suitable for a differently configured system are illustrated at the appropriate point by way of example.

[0146] In this example the liquid container 12a contains an initial cell suspension, a liquid in which the cells to be seeded are suspended. The liquid container 12b contains a cell culture medium. The liquid container 12c contains a liquid for detaching cells, for example trypsin. If such a liquid is not required for the corresponding application, it is understood that this storage container can be renounced.

[0147] In the first step liquid is transported from the storage container 12a into the first culturing channel using the pump 13a. For this purpose, the valves 15a, 17a and 18a can be actuated. All other valves are closed in this example. Opening the valve 18a enables initial cell suspension from the storage container 12a to enter the conduit 19.

[0148] Depending on whether the valve 15a or 17a is opened to the conduit, the initial cell suspension is transported into the first culturing channel via access 5a or 7a, respectively, provided the other valve is opened to the waste container 12d. The initial cell suspension remains in the culturing channel or flows slowly through the first culturing channel. The cells settle in the first culturing channel and may adhere to the walls of the channel. The liquid of the initial cell suspension can then optionally be transported out of the first culturing channel into the waste container or remain in the culturing channel for the time being.

[0149] In the first culturing channel, the cells begin to multiply. Optionally, a nutrient liquid can be transported into the first culturing channel, for example via the same path as the initial cell suspension, and then be discharged from the same when the nutrients have been used up.

[0150] In an optional second step, when the cells have multiplied and formed a first cell culture, the liquid for detaching the cells is transported from the storage container 12c to the first culturing chamber using the pump 13c. For this purpose, valves 15a, 17a and 18c are actuated. All other valves are closed in this example. Opening the valve 18c allows the liquid from the storage container 12c to enter the conduit 19. From there, the liquid enters the first culturing chamber in the same way as the initial cell suspension did before. The second step may be renounced if the cells do not or not very strongly grow on the walls of the culturing chamber. In particular, if the growing-on is not strong the detachment may be purely mechanical, for example by shear stress.

[0151] In a third step, the cells are transferred from the first culturing channel via the transfer channel into the first storage channel. For this purpose, the valve 18b is opened so that cell culture medium from the liquid container 12b enters the conduit 19. The cell culture medium is introduced into the first culturing channel through the culturing access 5a using the pump 13b and is transported into the storage channel through the transfer channel. In the process the cells are transferred into the storage channel. In this step, the valve 15a is opened to the conduit. Moreover the valve 16a is opened for example towards a waste container.

[0152] The method for culturing cells according to the invention may already end after the cells have been transferred to the storage channel. The cells can be temporarily stored and/or inspected in the storage channel.

[0153] Optionally, the method may further comprise one or a plurality of the following steps.

[0154] For example, in an optional fourth step concentration of the cell suspension can be carried out for example in the storage channel. For this purpose, the property of the cells that they sink to the floor is used. By setting a sufficiently low flow velocity and optionally supported by structuring the floor of the storage channel, the cells remain in the storage channel while part of the cell culture medium is transported further out of the storage channel so that the cell concentration is increased.

[0155] Alternatively or additionally, in an optional fifth step the cell suspension in the storage channel can be homogenized for example by mixing it. Optionally, after homogenization an optical inspection of the cells can be carried out, which are now arranged more uniformly in the storage channel. If a homogeneous cell distribution is present due to the homogenization, an optical inspection of the cells at a few positions in the substrate is sufficient to make representative statements about the entire cell culture.

[0156] Alternatively or additionally, in an optional sixth step dilution of the cell suspension located in the storage channel or the transfer channel can be carried out. For this purpose, the valve 18c can be opened so that cell culture medium enters the conduit and is transported into the storage channel 3a by means of the pump 13c, particularly through the access 6a via the valve 16a. At the same time another valve for example valve 15a or, if present, valve 15b, 17a or 17b, is opened for example towards the waste container.

[0157] If no further culturing of the cells in the substrate has to be carried out following these steps, a substrate without additional culturing channels can be used, for example as shown in FIGS. 1 and 5.

[0158] If the method comprises further culturing steps, a seventh step is a transfer of the cells through the transfer channel into the second culturing channel 2b. For this purpose, the valve 18b can be opened so that cell culture medium enters the conduit and is transported into the storage channel 6a by means of the pump 13b, particularly as shown here through the access 6 via the valve 16a. In addition, the valve 15b is opened for example towards the waste container. The cell culture medium is then transported together with the cells from the storage channel via the transfer channel into the second culturing channel. There, the cells can multiply and thus a second cell culture can be grown.

[0159] The steps described above can be repeated for additional culturing chambers.

[0160] In this case, the cells of each of the cell cultures can each be temporarily stored in the first storage channel or can each be transported to a separate storage channel associated with the respective culturing channel, for example storage channels 3b or 3c, and stored there and optionally further processed and/or inspected.

[0161] It is also conceivable that a plurality of culturing channels are provided in a substrate to which initial cell suspension is supplied. In other words, a plurality of first culturing channels may be provided. Thus, a plurality of first cell cultures can already be grown in the first culturing step. In other words, a plurality of first cell cultures can be grown in parallel with each other. Thus, many cells can be grown within a short time. If instead the same number of culturing channels is used sequentially, the method can be automated over a longer period of time without using channels twice.

[0162] If additional culturing steps each take place in additional culturing channels, a consistent surface quality can easily be ensured for each of the culturing steps. The method can then be carried out without preparation or cleaning of the first culturing channel. However, a second culturing step in the first culturing channel instead of or in addition to a culturing step in another culturing channel is also conceivable. In this case, a return from the storage channel to the first culturing channel would be carried out through the transfer channel, wherein particularly a cleaning step for cleaning the culturing channel can be carried out between the culturing steps. This is advantageous in that a more compact substrate can be used.

[0163] A detailed method is described below by way of example using a system with at least two culturing channels, for example a system as shown in FIG. 6, 7 or 8. All valves that are not described as opened are closed in the respective step. The method may comprise the following steps.

[0164] Cell seeding in the first culturing channel 2a: valve 18a is opened to the conduit, valve 15a is opened to the waste container, valve 17a is opened to the conduit. The applied pressure can for example be 20 mbar. The initial cell suspension flows from the storage container 12a through the conduit and the transfer channel into the culturing channel 2a. There, the cells sink to the floor and adhere. Cell division begins. Alternatively, valve 15a may be opened to the conduit and valve 17a may be opened to the waste container. The cells are then supplied directly (i.e. not via the transfer channel) to the culturing channel.

[0165] Washing away non-adherent and dead cells, for example 5 min to 60 min, particularly 20 min after cell seeding: valve 18b is opened towards the conduit, valve 15a is opened towards the waste container, valve 17a is opened towards the conduit. The applied pressure may be for example 50 mbar. Cell suspension is flushed through the culturing channel via the transfer channel to flush away non-adherent and dead cells. Alternatively, valve 15a can be opened to the conduit and valve 17a can be opened to the waste container. The cells then are directly (i.e. not via the transfer channel) transported away from the culturing channel from the substrate.

[0166] Trypsinization, for example when the cell culture has a predetermined cell density, for example after 48 hours: valve 18c is opened to the conduit, valve 15a is opened to the waste container, valve 17a is opened to the conduit. The applied pressure may be for example 20 mbar. Trypsin-containing solution flows from the storage container 12c through the transfer channel into the culturing channel 2a, remains there and detaches cells. The solution can have effect until a predetermined amount of cells is detached from the channel surface, for example about 5 minutes.

[0167] Cell transfer and concentrating: valve 18b is opened to the conduit, valve 15a is opened to the conduit, valve 16a is opened to the waste container. The applied pressure may be for example 100 mbar. Cell culture medium from the storage container 12b is pumped through the first culturing channel, the transfer channel, and the storage channel. For example, the volume of liquid supplied may be approximately 1.2 times the volume of the culturing channel. In the process, cells are transported through the transfer channel into the storage channel. Cells that are still attached to the surface of the culturing channel can be detached by the shear force and transferred as well. In the storage channel the cells can be retained for example by barriers, which may be formed in the form of a structuring of the floor of the storage channel or by a membrane at the access of the storage channel. Cell culture medium is transported away and cells remain in the storage channel.

[0168] Dilution: valve 18b is opened to the conduit, valve 16a is opened to the conduit, valve 17b is opened to the waste container. The applied pressure may be for example 20 mbar. Cell culture medium from the storage container 12b is transported out of the substrate through the storage channel and the transfer channel. In the process cells are transported out of the storage channel in a controlled manner. Depending on the duration or amount of liquid and flow velocity, the amount of cells remaining can be adjusted. For example, the volume of liquid supplied can be about two times the volume of the storage channel.

[0169] Optional mixing of liquid with the cells in the storage channel to obtain a more homogeneous cell distribution: for this purpose the liquid can be moved back and forth by applying positive and negative pressure. This step can be carried out before and/or after dilution.

[0170] Transfer to the and cell seeding in the second culturing channel 2b: valve 18b is opened to the conduit, valve 15b is opened to the waste container, valve 16a is opened to the conduit. The applied pressure may be for example 20 mbar. Cell culture medium from the storage container 12b transfers cells from the storage channel 6 via the transfer channel into the second culturing channel 2b. There, the cells sink to the floor and adhere. Cell division begins. Optionally, valve 17a may additionally be opened to the conduit and cell culture medium may be introduced into the substrate through transfer access 7a and combined with cells transferred from the storage channel during transfer of cells in the transfer channel, for example, to carry out dilution (alternatively or in addition to the aforementioned dilution in the storage channel).

[0171] Alternative to cell seeding in the second culturing channel: removal of the cells from the substrate.

[0172] The above sequence of steps can be repeated using another or the same storage channel for each repetition.

[0173] It is understood that features mentioned in the previously described embodiments are not limited to these particular combinations and are also possible in any other combinations.