WO2021018480A1

WO2021018480A1 - Provision of cell cultures

Info

Publication number: WO2021018480A1
Application number: PCT/EP2020/067413
Authority: WO
Inventors: Konrad Mueller-Auffermann
Original assignee: Krones Ag
Priority date: 2019-07-29
Filing date: 2020-06-23
Publication date: 2021-02-04
Also published as: DE102019211243A1; EP4004182A1; CN114174488A

Abstract

The present invention relates to a method for providing a cell culture, comprising the steps of: filling a substrate into a propagator, inoculating the substrate in the propagator with a first cell culture, propagating the cell culture in the propagator in order to obtain a desired amount of a second cell culture in the propagator, wherein the second cell culture has a sedimented portion and a suspended portion, and removing, as desired, some of the sedimented portion of the desired amount of the second cell culture from a lower region of the propagator and/or some of the suspended portion of the desired amount of the second cell culture from an upper region of the propagator.

Description

Provision of cell cultures

Field of invention

The present invention relates to the provision of cell cultures including the propagation of the cell cultures in a propagator and the withdrawal of the propagated cell cultures from the propagator.

State of the art

Cell cultures, such as yeast, are generally propagated (propagated) in propagators, i.e. bioreactors in the form of tanks. Usually, the propagators are equipped with circulating devices, such as stirrers, or circulation lines, in order to thoroughly mix the inoculated substrate. In addition, the propagators are typically equipped with temperature control devices in order to sterilize the substrate and / or to set a desired temperature during the propagation. The temperature control devices can include heating and / or cooling surfaces and external heat exchangers.

A prior art propagation method is illustrated in FIG. A cleaned propagator 1 is filled via a substrate inlet 2 with a substrate, in the example shown in FIG. 1A from below, and filled with a pure cell culture previously grown in the laboratory, for example a yeast culture used for brewing beer, for example with the aid of a so-called Carlsberg Piston 3, inoculated. With the aid of a circulation pump 4, the pure culture is mixed with the substrate, and a gas, such as air, for cell reproduction can optionally be introduced via a gas inlet 5. Usually the dilution of the cells with substrate at this stage is 1:10 to 1: 100. As soon as the desired concentration of cells has been reached in the propagator 1 (in the case of yeasts about 80 to 120 million cells per ml), the substrate is again added (see FIG. 1B) for a renewed dilution of usually 1:10 to 1: 100 μm to prevent product inhibition (by alcohol and CO2) and to cause further growth. The gas supply and circulation can be kept constant or used at intervals. This process is repeated until the desired total amount of cells has been produced. Since the agitators or circulating devices used have to be specifically designed for the masses received in the propagators, a smaller pre-propagator 1 and a larger main propagator 6 are typically used, with the amount obtained from the pre-propagator being fed into the main propagator 6, in which the above be The process described is carried out in the same way (FIG. 1C). Finally, the cell culture obtained in this way (the so-called propagate) can be drawn off from below and, for example, fed to a fermenter for carrying out a fermentation.

However, the conventional multi-stage propagation described in several propagators / assimilators of different sizes is relatively complex. Above all, the propagated cell culture is withdrawn for further use, for example in a fermentation process, in an undifferentiated manner without any consideration of the specific properties of the different types of cells in the cell culture.

However, the sedimentation behavior and the flocculation behavior of the cells vary greatly according to the age and size distribution of the cell culture, its genetic variability, vitality and viability. However, these behaviors are important for subsequent processes that use the preserved cell culture, such as fermentation processes. For example, when fermenting wort, different sedimentation and flocculation behavior of the yeast cultures used are required depending on the desired type of beer or technology. This is not given sufficient consideration in the conventional process described for producing a cell culture.

The object of the present invention is to simplify the provision of a cell culture and to improve it with regard to the differentiation of the cell culture obtained and to be used further.

Description of the invention

The above-mentioned object is achieved by a method for providing a (target) cell culture. This procedure includes the steps:

Filling a substrate into a propagator (tank); Inoculating the substrate in the propagator with a first cell culture (for example with a pure culture produced in the laboratory; for example a yeast for beer production);

Propagating the first cell culture in the propagator in order to obtain in the propagator a desired amount of a second cell culture with a desired concentration (from cells to substrate), the second cell culture having a sedimented portion and a floating portion (of cells); and optionally withdrawing at least part of the sedimented portion of the desired amount of the second cell culture from a lower area of the propagator and / or at least part of the floating portion of the desired amount of the second cell culture from an upper area of the propagator, whereby the (target- ) Cell culture (as a fully propagated first cell culture) is provided.

Here and in the following, the term “propagator” also includes an assimilator, i.e. a tank for growing and multiplying cell cultures. The second cell culture represents the nth generation of the first cell culture, where n is greater than two. The (target) cell culture obtained by the process is ready for further use, for example in a fermentation process. In contrast to the prior art, this (target) cell culture, which is provided for further use, such as a fermentation process, can be produced in one and the same propagator. In principle, however, it can also be fed to a further propagator for further propagation.

The optional subtraction includes the selection of whether subtraction should be made from the upper area or the lower area of the propagator. The selection can be made via a corresponding distribution station with controllable / regulable valves, via which the supply and discharge of media into and out of the propagator is controlled / regulated (see also detailed description below). Such a selection is not provided in the prior art. Advantageously, the method according to the invention thus enables a (target) cell culture to be made available for further use, which is differentiated according to the sedimentation behavior and flocculation behavior of the cells of the (target) cell culture obtained.

In a further development, the propagation can comprise the following steps carried out one after the other in the order specified: 1) a first propagation of the first cell culture (of the inoculated substrate) in the propagator until a first concentration of cells to substrate is reached (and thus a first propagate is obtained);

2) adding more substrate to the propagator;

3) a second propagation in the propagator until a second concentration of cells to substrate (which may be equal to the first concentration) is reached (and thus a second propagate is obtained); and

4) Repeat steps 2) and 3) until the desired amount of the second cell culture (as a finished propagate) with the desired concentration of cells to substrate is obtained.

If the desired amount of the second cell culture with the desired concentration has already been obtained after the mentioned second propagation, there is no further repetition, i.e. step 4) can then be omitted.

The finished propagation, from which the (target) cell culture can then optionally be withdrawn for further processes, is thus produced iteratively from the start cell culture, preferably in one and the same propagator.

The propagator can have a first (lower) inlet / outlet with a first opening in one, in particular lower, end area of the propagator and a second (middle) inlet / outlet with a second opening in an area of the propagator that is closer than the first opening is arranged at the center of the propagator. The second opening is therefore further away from the end area than the first opening and is in particular above the cell culture sediment that forms in the propagator. The optional removal of the part of the sedimented portion of the desired amount of the second cell culture through the first opening of the first inlet / outlet and the optional removal of the part of the floating portion of the desired amount of the second cell culture can be done through the second opening of the second inlet. / Rejection.

In particular, the first and second inlet / outlet line can be routed parallel to the longitudinal axis of the propagator so that the second inlet / outlet line extends further into the propagator than the first inlet / outlet line. The first and second inlet / outlet lines can be passed through the floor of the propagator, in which case the second opening is higher than the first opening. For example, the first opening can be in the bottom of the propagator. Furthermore, the second inlet / outlet line can be guided in a tube-in-tube configuration within the first inlet / outlet line, for example concentrically. In the tube-in-tube configuration, one or more further inlet / outlet lines with different height levels of their respective opening can be routed and serve to remove part of the desired amount, in particular part of the floating portion, of the second cell culture.

The propagator can also have a third inlet / outlet line with a third opening, which is guided within the first inlet / outlet line and extends further into the propagator than the second inlet / outlet line, and in this case the optional removal of the Part of the floating portion of the desired amount of the second cell culture can be done optionally through the second opening of the second inlet / outlet and / or the third opening of the third inlet / outlet.

Before the propagation and / or during the propagation and / or after the propagation, the contents of the propagator can be circulated at least temporarily in at least one partial area of the propagator. For example, the method can comprise at least a temporary circulation of the inoculated substrate (before the actual propagation) and / or the propagating first cell culture during propagation in the propagator and / or the second cell culture in at least a partial area of the propagator.

The at least temporary circulation can take place via a circulation line connected to the propagator (which is located outside the propagator) and with the aid of a circulation pump connected to the circulation line. In particular, the at least temporary circulation can take place via at least one of the above-mentioned first inlet / outlet, second inlet / outlet and third inlet / outlet. Here, the at least temporary circulation can at least temporarily, in particular after sedimentation or for sedimentation of the propagating first cell culture in a lower area of the propagator, only in an area between the second opening of the second inlet / outlet line (22) and the third opening the third inlet / outlet line (23) take place, so that in particular the sediment created or created by the sedimentation is essentially not circulated.

In addition, the method can include the step of supplying zinc and / or copper to the substrate / propagate during and / or after propagation, in particular via a zinc anode and / or copper anode (or electrodes) which is arranged in said circulation line . In addition, nutrients and / or growth-promoting substances can be used before and / or during the propagation in a targeted manner, for example via one of the above-mentioned first to third inlet / outlet lines. Before the propagation and / or during the propagation, air, oxygen and / or another gas or gas mixture can also be passed into the propagator. This can happen via the above-mentioned circulation line connected to the propagator and an air supply device connected to it.

Furthermore, a method for fermenting wort is provided with the step of filling a fermentation tank with the wort and the step of delivering the (target) cell culture provided according to one of the methods described above into the fermentation tank.

A retrofit method is then provided for retrofitting a conventional propagator and / or previously otherwise used tanks. The method is a method for retrofitting a propagator and / or previously otherwise used tank, which has an inlet / outlet line at one end area, with a first additional and in particular additionally a second additional inlet / outlet line through the inlet / outlet line in the propagator and / or previously otherwise used tank and connecting the inlet / outlet line, the first additional inlet / outlet line and in particular the second additional inlet / outlet line via a distribution station to a circulation line into which a circulation pump is connected, with the help of which a content of the propagator and / or tanks previously used otherwise can be circulated via the inlet / outlet and / or the first further inlet / outlet and / or in particular the second further inlet / outlet. The distribution station has valves for opening and closing the various inlet and outlet lines.

A device for providing a cell culture, which comprises a propagator and a control / regulating unit, is also provided, the control / regulating unit being designed to carry out the method according to one of the examples described above.

Further features and exemplary embodiments and advantages of the present invention are explained in more detail below with reference to the drawings. It should be understood that the embodiments do not exhaust the scope of the present invention. It is further understood that some or all of the features described below can also be combined with one another in other ways.

FIG. 1 shows the production of a cell culture according to the prior art. Figure 2 shows a propagator as it can be used in an embodiment of the method according to the invention.

FIGS. 3A to 3G serve to illustrate an embodiment of the method according to the invention for providing a cell culture.

The present invention relates to a method for providing a (target) cell culture, in which a propagator / assimilator (tank) is used for propagating (multiplying, growing) a cell culture. An example of a device with such a propagator 10 is shown in FIG. The propagator / assimilator 10 has a conically tapered lower region

11, in which optionally a portion of a propagated cell culture can sediment. An inlet / outlet line 12 is passed through the tip of the cone and verbun with a circulation line 13, in which a circulation pump 14 for circulating the contents of the propagator 10 is connected. The circulation pump 14 can be designed to be adjustable in speed and, for example, frequency-controlled. Furthermore, an air supply device 15 is connected to the circulation line 13 in order to be able to direct air (or another gas) into the interior of the propagator 10. The air supply device 15 enables or accelerates the growth of the cells in the propagator 10. A zinc anode to increase the zinc content to improve cell growth and / or a copper anode for desulfurization can be arranged in the circulation line 13.

In addition, the propagator 10 has an inlet / outlet line 16 provided as an alternative to the circulation line 13, which is higher and in particular above the opening of the inlet / outlet line 12, which is guided through the tip of the cone and can be arranged in the tip itself is in the propagator forming cell culture sediment. In the example shown in Figure 2, the further inlet / outlet line 16 ends laterally in the tank. Alternatively, the further inlet / outlet line 16 can be within the inlet / outlet line passed through the tip of the cone

12 extend and further above them open into the propagator 10 (see also the description of FIGS. 3A to 3G below). The propagator 10 can also have a temperature control device, in particular for cooling.

According to the invention, a (target) cell culture is provided by selectively withdrawing the cell culture produced in the propagator 10 through the inlet / outlet line 12 or the inlet / outlet line 16. In this way, on the one hand, a portion of the cell culture that is characterized by deposited cells and, on the other hand, a portion of the cell culture that is characterized by floating cells can be obtained for further use, for example in a fermentation process. Of course, a mixture of these cells with different sedimentation and floating or flocculation behavior can also be obtained with a desired mixing ratio. Since the cells are often separated (filtered) before the end product is filled, or fermentation is greatly slowed down if it settles too quickly, the flocculation and sedimentation behavior is an important quality parameter of a cell culture used for fermentation.

In particular, the propagator 10 can be used for beer production. For certain types of beer and the technology tailored to them, for example, a “dusty character” (ie the highest possible proportion of cells in suspension) of the population is preferred in order to clarify a homogeneous suspension, for example with the help of centrifugation, and to prevent beer losses from the classic yeast harvest from below to reduce. With other types of beer, on the other hand, the aim is to sediment the culture as quickly and completely as possible after fermentation, for example to be able to harvest the yeast from the tank as completely as possible from below and thus also to simplify the diatomaceous earth or membrane filtration that follows after storage.

The weaning behavior of the cells is genetically influenced by the strains used, the substrate composition, the cell size, the age distribution as well as the vitality and viability of the culture, which is why the number of reuses and mixing ratios (propagation yeast to harvest yeast) should be coordinated. Furthermore, of course, as many of those yeast cells as possible should be used to make the wort that have the desired settling properties. These conditions mentioned above can be taken into account in the method according to the invention using, for example, the device shown in FIG. 2, as was previously not possible in the prior art.

One embodiment of the method according to the invention for providing a (target) cell culture, for example for a fermentation process such as the fermentation of wort, is described below with reference to FIGS. 3A to 3G.

In the method illustrated in FIGS. 3A to 3G, a device with a propagator 20 is used, which is characterized in that it has several, in this case three, inlets / outlets (inlets / outlets) 21, 22 and 23. The three inlet / outlet lines 21, 22 and 23 are provided in a tube-in-tube configuration in which the middle inlet / outlet line 22 and the upper inlet / outlet line 23 extend (for example, concentrically) through the lower inlet / Ausleitung 21 extend. The lower inlet / outlet line 21 has an opening at the bottom in the propa- Gator 20, for example in the cone tip, the middle inlet / outlet line 22 optionally has a displacement body of suitable geometry (attached to the middle inlet / outlet line 22, for example, in a double cone shape) and an opening above that of the lower inlet / outlet line 21 and the upper inlet / outlet line 23 has an opening above that of the central inlet / outlet line 22. The middle inlet / outlet line 22 extends further into the propagator 20 than the lower inlet / outlet line 21, and the upper inlet / outlet line 23 thus extends further into the propagator 20 than the middle inlet / outlet line 22. In other embodiments, inlet / outlet lines can reach into the propagator from above or from the side.

The inflow / outflow of media into the propagator 20 or out of the propagator 20 is regulated or controlled via a distribution station 24. Here, the inflow / outflow via the lower inflow / outflow line 21 via a valve system 24a of the distribution station 24, the inflow / outflow via the middle inflow / outflow line 22 via a valve system 24b of the distribution station 24 and the inflow / outflow via the upper inlet / outlet line 23 regulated or controlled via a valve system 24c of the distribution station 24. The propagator 20 can also have a temperature control device, in particular for cooling, wherein an external heat exchanger for heating and / or cooling in the circulation line 25 can also be provided additionally or alternatively.

The distribution station 24 connects the inlet / outlet lines 21, 22 and 23 to a circulation line 25 into which a circulation pump 26 is integrated. A zinc anode (electrode) for increasing the zinc content as required and / or a copper anode (electrode) for desulfurization as required can also be arranged in the circulation line 25. The circulation pump 26 can be designed to be adjustable in speed and, for example, frequency-controlled. Furthermore, sampling stations 27, a measuring technology station 28 and a regulation / control station 29 are provided in the circulation line 25 or are connected to it. The measurement technology station 28 can be used, for example, to determine an extract content (a concentration), a cell count (for example using a turbidity measurement or capacitive measurement), a pH value, a fill level, a temperature, a conductance, an oxygen / CO 2 measurement, etc. to serve.

A substrate is introduced into the propagator 20 via a process connection having a valve 30 and the lower inlet / outlet line 21.

The substrate can be a wort without foreign organisms, ideally it can be adequately equipped with nutrients and growth substances as well as usable sugars, one temperature of 10-28 ° C at inoculation and between 6-20 ° P original wort. If there is a lack of nutrients and growth substances, these can optionally be added to the substrate, in which case zinc enrichment and / or copper enrichment according to requirements can also be carried out by applying voltage to appropriate electrodes.

All the valves 24a, 24b and 24c of the distribution station 24 can be opened in order to keep the flow rate of the introduced substrate low (see FIG. 3A). Here and below, an open valve position is indicated by a filled valve symbol and a closed valve position by an empty valve symbol. Flow directions of the media are indicated here and below by arrows. In the example shown in FIG. 3A, the substrate is introduced up to a level which is above the height level of the opening of the central inlet / outlet line 22. In other examples the level can be lower.

Furthermore, an air supply device 31, an inoculation point 32 for supplying a starting cell culture and a valve 33, which can open a line, which can be connected to a gully, to discard a medium can be found in the circulation line 25 or connected to it.

To start a first propagation of the cell culture, as shown in Figure 3B, the substrate is inoculated with the culture organism (start cell culture), such as a start yeast cell culture, via the inoculation point 32 and the lower inlet / outlet 21 and the medium obtained in this way, at least at time intervals, is circulated with the aid of the circulating pump 26 and ventilated at least temporarily via the air supply device 31 to enable aerobic cell growth.

The optional displacement body serves as a flow breaker and flow guide to control the flow of the medium during the circulation process in the propagator 20. The inoculation can be done with a so-called Carlsberg flask 34 and / or with an alternatively treated culture (e.g. spray-dried, suction-filtered, solid or liquid-enriched and / or previously frozen culture). If desired, different valves 24a, 24b, 24c on the pressure side of the circulation pump and thus inlets / outlets 21, 22, 23 can be selected to ensure ideal mixing even when the middle inlet / outlet 22 and / or upper inlet / outlet is not covered 23 to ensure.

When a desired concentration of cells of the cell culture, about 80 to 120 million yeast cells per ml of substrate, has been reached in the course of the first propagation in the propagator 20, further substrate is fed into the propagator 20 via the lower inlet / outlet line 21 (see Fig He Figure 3C), for example, a 1:10 to 1: 100 dilution of the cell culture with substrate to achieve, so that a growth inhibition (for example due to an increased alcohol and CO2 content and a lack of growth substances) can be avoided. Again, circulation and ventilation takes place. The direction of flow can be selected by a suitable control / regulation of the valves 24a, 24b and 24c of the distribution station 24.

The process sequence shown in FIGS. 3B and 3C can be repeated until the desired amount of cell culture with the desired concentration of cells in the substrate has been produced in the propagator 20. In the process status shown in FIG. 3D, a circulation is shown as an example, in which the upper inlet / outlet line 23 is used for the inflow of the circulated medium and the entire container contents are thus homogenized.

From a certain point in time, at least the lower area of the propagator 20 should no longer be actively flowed through, i.e. there should be no circulation of the medium in this lower area, so that the cells can settle and accumulate and thus form a sediment S, as shown in FIG. 3E is. If desired, however, a circulation can still take place between the height levels of the openings of the central inlet / outlet line 22 and the upper inlet / outlet line 23 in order to further homogenize there, as is also shown in FIG. 3E. In the process status shown in FIG. 3E, a circulation is shown by way of example, in which the upper inlet / outlet line 23 is used for the inflow of the circulated medium.

When the desired amount of cell culture with the desired concentration of cells in the substrate has been produced in the propagator 20, a portion of this amount can optionally be withdrawn through one or more of the inlet / outlet lines 21, 22 and 23. So floating cells can be drawn off from an upper area of the propagator 20 via the upper inlet / outlet line 23 and / or the middle inlet / outlet line 23, as shown in FIG. 3F. The withdrawal takes place via the valve 30 of the process connection. Desirably, a small portion (about 1 to 10%) of the enriched culture from the cone remains in the tank, which is then used again to inoculate the subsequent batch.

The (target) cell culture obtained in this way can be used in a further process, for example fermentation. For example, a (target) yeast cell culture obtained in this way can be used to ferment a beer wort in the context of the production of a type of beer for which a high proportion of yeast cells with a strong tendency to float is desired.

For a process following the propagation, in which it is desirable to use cells with a strong tendency to sedimentation, the propagated cell culture will be withdrawn from below via the lower inlet / outlet line 21, as shown in FIG. 3G. A comparatively larger residual volume of around 10 to 20% should be retained for the subsequent batch. The (target) cell culture obtained by pulling off from below not only has an increased tendency to sedimentation, but also a higher concentration than that obtained by pulling off via the upper inlet / outlet line 23 and / or the middle inlet / outlet Exit 23 is obtained. In this way, (target) cell cultures with different concentrations can optionally be provided for further processes.

When transferring the (target) cell culture obtained to a further process, for example for making wort with yeast cells, the real amount of yeast added can be determined over the transfer time by also taking into account fluctuations in the number of cells (measured for example via the turbidity) in relation to the mass transfer will. If the mixture is more turbid than usual, less yeast is used for fermentation, as the turbidity suggests higher cell numbers and fermentation should always take place with a similar number of cells.

For cleaning, the propagator is completely emptied, and a conventional CIP process takes place, in which, however, a certain part of the media can always linger in the lower tank area (sump), so that all lines (lower, middle and upper inlet / outlet 21 , 22, 23, the distribution station 24 and the circulation line 25) flows through at a high flow rate and can thus be cleaned.

As described above, the method according to the invention makes it possible in particular to carry out the process from inoculation to removal of the finished (target) cell culture to be used in one and the same propagator, for example in the propagator 10 shown in FIG FIGS. 3A to 3G propagator 20 shown. The propagation speed can for example be suitably adjusted by a suitable control / regulation of the temperature, circulation speed, selection of the circulation zones in the propagator 20, ventilation rates, etc. Before or during the propagation, nutrients and / or growth-promoting substances can also be added to the medium in the propagator via at least one of the three inlet / outlet lines 21, 22, 23.

An application of voltage to a zinc and / or manganese electrode, for example located in the circulation line 13, causes its accelerated dissolution and ionization, so that the content of free zinc / manganese in the liquid required for the cells increases, thus increasing the vitality of the culture . In the case of a copper electrode, on the other hand, copper ions are released, which means that they can react with sulphurous compounds, resulting in a reduced sulfur content in the product.

Claims

1. Method for providing a cell culture with the steps:

Filling a substrate into a propagator (10, 20);

Inoculating the substrate in the propagator (10, 20) with a first cell culture;

Propagating the first cell culture in the propagator (10, 20) in order to obtain a desired amount of a second cell culture with a desired concentration in the propagator (10, 20), the second cell culture having a sedimented portion and a floating portion; and optionally withdrawing at least a portion of the sedimented portion of the desired amount of the second cell culture from a lower area of the propagator (10, 20) and / or at least a portion of the floating portion of the desired amount of the second cell culture from an upper area of the propagator (10 , 20).

2. The method according to claim 1, in which the propagation comprises the following steps carried out one after the other in the specified order:

1) a first propagation of the first cell culture in the propagator (10, 20) until a first concentration of cells to substrate is reached;

2) adding further substrate to the propagator (10, 20);

3) a second propagation in the propagator (10, 20) until a second concentration of cells to substrate is reached, which in particular can be the same as the first concentration; and

4) Repeat steps 2) and 3) until the desired amount of the second cell culture with the desired concentration of cells to substrate is obtained.

3. The method according to any one of the preceding claims, in which the propagator (10, 20) has a first inlet / outlet line (21) with a first opening in one, in particular the lower, end region of the propagator (10, 20) and a second inlet / outlet line (22) with a second opening in one Area of the propagator (10, 20), which is arranged further than the first opening from the, in particular lower, end area of the propagator (10, 20), and wherein the optional removal of the part of the sedimented portion of the desired amount of second cell culture through the first opening of the first inlet / outlet line (21) and the optional removal of the part of the floating portion of the desired amount of the second cell culture through the second opening of the second inlet / outlet line (22).

4. The method according to claim 3, in which the second inlet / outlet line (22) is guided within the first inlet / outlet line (21).

5. The method according to claim 4, wherein the propagator (20) has a third inlet / outlet line (23) with a third opening which is guided within the first inlet / outlet line (21) and extends further into the propagator (10, 20) extends as the second inlet / outlet line (22), and the optional withdrawal of the part of the floating portion of the desired amount of the second cell culture optionally also through the third opening of the third inlet / outlet line (23) or instead of through the second opening the second inlet / outlet line (22) takes place through the third opening of the third inlet / outlet line (23).

6. The method according to any one of the preceding claims, further with at least temporary circulation of the inoculated substrate and / or the propagating first cell culture and / or the second cell culture in at least a portion of the propagator (10, 20).

7. The method according to claim 6, in which the at least temporary circulation takes place via a circulation line (13, 25) connected to the propagator (10, 20) and with the aid of a circulation pump (14, 26) connected in the circulation line.

8. The method according to claim 7, wherein the at least temporary circulation takes place via at least one of the first inlet / outlet line (21), second inlet / outlet line (22) and third inlet / outlet line (23).

9. The method according to claim 8, in which the at least temporary circulation at least temporarily, in particular after sedimentation or for sedimentation of the per paging cell culture in a lower area of the propagator (10, 20), only in an area between the second opening of the second Inlet / outlet line (22) and the third opening of the third inlet / outlet line (23) takes place.

10. The method according to any one of the preceding claims, further comprising supplying zinc, manganese and / or copper to the substrate during and / or after the propagation, in particular via a zinc / manganese electrode or electrode and / or copper anode or electrode, which is arranged in particular in the circulation line (13, 25).

11. The method according to any one of the preceding claims, further comprising feeding nutrient and / or growth-promoting substances into the propagator (10, 20) before and / or during the propagation.

12. The method according to any one of the preceding claims, further comprising feeding air, oxygen and / or another gas / gas mixture into the propagator (10, 20) before and / or during the propagation with the aid of a circulation line connected to the propagator ( 10, 20), connected gas / air supply device (31).

13. A method for fermenting a beer wort, with filling a fermentation tank with the beer wort and delivering the cell culture provided according to one of the methods according to one of the preceding claims into the fermentation tank.

14. A method for retrofitting a propagator and / or previously otherwise used tank, which has an inlet / outlet line at one end area, with routing a first additional and in particular additionally a second additional inlet / outlet line through the inlet / outlet line into the propagator and / or previously otherwise used tank and to the supply / discharge line, the first further supply / discharge line and in particular the second further supply / discharge line via a distribution station to a circulation line into which a circulation pump is connected, with the help of which a content of the propagator and / or tanks previously used otherwise via the inlet / outlet line, and / or the first further Feed / discharge and / or in particular the second further feed / discharge can be circulated.

15. A device for providing a cell culture, comprising a propagator (10, 20) and a control / regulating unit, the control / regulating unit being designed to carry out the method according to one of claims 1 to 13.