DE19801763A1 - New culture apparatus useful for submerged or basal cell or tissue cultivation - Google Patents

Abstract

A culture apparatus comprises a level sensor for controlling a supply system for use as a culture medium supply, and discharge to and from a culture vessel. An Independent claim is also included for a cell or tissue cultivation method in which the liquid level of a nutrient solution lies alternately a certain distance above and below the culture surface.

Description

The invention relates to a culture device according to the Preamble of claim 1 and a method for Cultivation of cells or tissue components.

Such known culture devices are used for cultivation of isolated cells or tissue components. These well-known Culture fixtures are on use at Limited submerged culture systems, i. H. on the membrane of a Cultured cells are inserted through cell culture inserts Overlay with a culture solution with the appropriate Nutrients.

For some experimental studies, for example studies with differentiated ciliated or pulmonary epithelial cells, it would be desirable when solving specific issues the tissue growth conditions could vary. With conventional culture devices, for example Cultivation of cells of the respiratory tract on one Do not carry out air / liquid boundary layer (air liquid), even if you use porous membranes made by offered to various companies as so-called Transwell systems become.

A culture device is intended by the present invention further developed according to the preamble of claim 1 be that they have a variety of different cultural and Exposure conditions allowed.

This object is achieved by a Culture device with the features specified in claim 1.

In the culture device according to the invention, you can adjust the height the level of the culture medium inside the culture vessel adjust, especially so that they are above the top of the  Cell culture insert membrane (submersion culture) or just contacted the membrane (or something inside the same lies), creating a basal, from below nutritional supply.

The switch from submerged culture to basal culture can be done with the culture device according to the invention simply by The supply device is activated, which supplies predetermined amounts of culture medium to the culture vessel or dissipates from this until the output signal of Level sensor each set level setpoint corresponds.

This adjustability of the level of the culture medium in the Culture containers can be very precise and easy without Read off marks or the like easily. The creation of different cultural conditions necessary equipment parts are simple and not expensive.

Advantageous developments of the invention are in the Subclaims specified.

With the development of the invention according to claim 2 achieved the difference between submerged and basal Diet is very sharply adjustable. The different Areas of a cell culture carried by the cell culture insert grow under exactly the same conditions.

In a culture device according to claim 3 you can in culture medium located in the individual culture containers analyze during the growth phase and from the Composition of the culture medium Growth of the cell cultures via their metabolic products conclude.

In a culture device according to claim 4, one can larger number of cell cultures under exactly the same  Breeding conditions. The apparatus structure of a such culture device for a large number of Cell culture is easy. The culture device also needs little space.

With the development of the invention according to claim 5 achieved that only one for the different culture containers only level sensor is required for level control.

The development of the invention according to claim 6 enables it, the level inside the culture container in a simple way determined from the outside. The container can thus be a have a smooth surface and are free of internals, which would interfere with cleaning. They can also Culture containers so easily sterilized at high temperatures become.

The development of the invention according to claim 7 enables it, also manually set the target level for the culture medium adjust.

According to claim 8, it is possible to adjust the Make the desired level electrically in a simple manner. Of the Level sensor itself measures over a larger height range of the Culture container, and the level is set by Setting the level setpoint signal.

Is used for level detection according to claim 9 Photoelectric sensors, especially a forked photoelectric sensor, can to use components that are considered cheap at prices Standard components are available.

The development of the invention according to claim 10 is in With a view to increasing the number again Cell cultures that can be carried out together are an advantage. Of the apparatus expenditure for such a multi-culture device is small. This consists of standard components (modules) that  can be mass-produced.

The development of the invention according to claim 11 enables if desired, residue-free removal of the Culture medium equally from all modules of the Culture device.

With a culture device according to claim 12, one can in the grow different cell cultures in different modules, trotidem has the opportunity to monitor the progress of the Growth over the metabolic products for the different To examine cultures (the different modules) separately.

The development of the invention according to claim 13 is in With regard to equal tempering of the different cultures advantageous.

With the development of the invention according to claim 14 without the use of stirrers ensures that within of the individual temperature containers no temperature gradients form.

In a culture device according to claim 15, the Connection between the individual modules of the Culture device particularly simple. Such Module arrangement can also be easily expanded.

In a culture device according to claim 16, the Interior of the culture containers from the ambient atmosphere Cut.

With such a culture device, one can then according to Claim 17 the interior of the outer housing with one fill other gaseous medium and its influence on the Examine growth of cultures.

The development of the invention according to claim 18 is in  In terms of creating impeccable germ-free Starting conditions for the cell cultures are an advantage. If glass is used as the sterilizable material, so you also have good eye contact with the cell cultures. Also with regard to unwanted redemption of the Material of the culture container is glass as a material advantageous.

In a culture device according to claim 19 both the supply as well as the removal of culture medium forced by a bidirectional one Pump arrangement. This is quick in terms of Reaching the target level values, i.e. avoiding Control vibrations, an advantage.

In a culture device according to claim 20, the Targeted exposure of cultures that make up one Change in growth conditions results. You can also so the cultures specifically expose substances that are not in the liquid phase of the nutrient media, such as gaseous Substances or not dispersible in the liquid phase solid materials.

In a culture device according to claim 21 alternate submerged nutrition and basal nutrition periodically.

With a development of the present invention Culture device controlled by a program. This Program is especially under operating systems MS-DOS executable.

The program is suitable for all functions of the To control pump systems. The pump system is typically to the computer via a serial interface connected. It also detects and processes this Program the output signal of the photocell. In a The test sequence is a particularly preferred embodiment  logged.

The invention based on Embodiments with reference to the drawing explained in more detail. In this show:

Fig. 1 is a schematic view of a simple, only one culture container encircling culture device according to the invention with controllable level height of the culture medium;

FIG. 2 is a view similar to FIG. 1, in which a modified culture device is shown;

Fig. 3, which in turn each comprise a plan view of a further modified culture apparatus having a plurality of parallel juxtaposed culture modules, a plurality of culture containers;

FIG. 4 shows a longitudinal section through one of the culture modules of the culture device according to FIG. 3;

Figure 5 is an angled transverse section through the culture module of Figure 4 taken along the section line VV..;

FIG. 6 shows a longitudinal section through the culture device according to FIG. 3;

Fig. 7 is a transverse section through the culture apparatus of Figure 3 taken along the section line IV-IV. and

Fig. 8 is a block diagram of a level height control, which takes place in conjunction with the culture devices of Figs. 1 to 6 using.

In Fig. 1, 10 overall culture unit, respectively. The culture unit 10 comprises a culture container 12 which, roughly speaking, has the shape of an inverted bottle, a circular opening 14 being provided in the bottom of the bottle.

A cell culture insert 15 , which is made of a porous plastic material, for example of polyethylene terephthalate, is attached inside the culture container 12 . The cell culture insert 15 represents a liquid-permeable support structure for a membrane 16 which, depending on the requirements of the cells to be cultivated, can be made of different plastic materials, e.g. B. also polyethylene terephthalate. As shown in the enlarged detail in FIG. 1, the membrane 16 carries a cell culture 18 .

The culture container 12 is carried by a holding device 20 , which is only indicated schematically in FIG. 1.

The bottom connection 22 of the culture container 12 is connected via a hose 23 to the one working connection of a bidirectional pump 24 , which in practice can be a peristaltic pump. The other connection of the pump 24 is connected via a hose 26 to the interior of a storage container 28 which contains a culture liquid 30 . This contains the nutrients that are to be made available to the cell culture 18 for growth.

The two control terminals of the pump 24 are connected to the outputs of an operating circuit 36 via power amplifiers 32 , 34 . The operating circuit 36 generates depending on the output signal of a continuously operating level sensor 38 , which is shown as a sensor attached to the outer surface of the culture container 12 (but may also be attached to the inner surface of the culture container 12 ) and in practice can be an optical sensor, and depending on a setpoint generator 40 , which is shown as an adjustable resistance, a signal at one, at the other or none of its outputs in order to cause the pump 24 to supply additional culture fluid or culture fluid from the interior to the interior of the culture vessel 12 from the storage vessel 28 to return the culture container 12 into the storage container 28 .

The setpoint generator 40 can be set by a program control 42 , as indicated in FIG. 1 by a dashed line.

In the exemplary embodiment considered here, the program controller 42 switches the setpoint generator 40 between two positions. The one position of the setpoint generator 40 corresponds to a level of the culture fluid 30 inside the culture container 12 , as indicated in the drawing: the culture fluid 30 just touches the underside of the cell cut insert 15 . Under these conditions, the cell culture 18 is supplied with culture fluid only from below (basal nutrition).

The second level setpoint set by the program controller 42 corresponds to a level position as indicated by the dashed line 44 in FIG. 1. In this position the liquid level lies above the tips of the cell culture 18 ; this is thus nourished submerged.

The exemplary embodiment shown in FIG. 2 largely corresponds to that according to FIG. 1, corresponding components are again provided with the same reference numbers and are not explained in detail again below.

Instead of the bidirectional pump 24 , a unidirectional feed pump 24 'is provided, which is connected to the connection 22 of the culture container 12 via a check valve 46 . The latter is also connected to an outlet line 50 via a 2/2 solenoid valve 48 . The solenoid valve 48 is resiliently biased into the closed position and can be brought into the open position by activating its magnet. The magnet of the solenoid valve 48 and the pump 24 'are again connected to the operating circuit 36 via the power amplifiers 32 , 34 , as described above, which forms a control loop.

FIGS. 3 to 7 show a culture apparatus with which a greater number can be performed by cell cultures simultaneously.

The culture device shown in FIGS . 3 to 7 has a tightly closable outer housing 52 , which consists of a lower box-shaped housing part 54 composed of plates and a cover 56 which can be placed tightly thereon , from which a vertical down along the lines 55-1 , 55-2 and 55-3 extend three glass webs 57 to the upper edge of the culture container 12 in order to fix cell culture inserts 122 attached therein.

Four culture modules 58-1 , 58-2 , 58-3 and 58-4 are arranged in the interior of the outer housing 52 . Insofar as no differentiation of the culture modules is necessary below, reference is made to these with reference number 58 .

Each of the culture modules 58 comprises a temperature control housing, designated as a whole by 60 , which is sealed and is composed of different plates. Three culture units 10 are arranged in the interior of a culture housing 60 , as can be seen from the drawing. The connections 22 of the different culture containers 12 of these culture units are connected to a common supply line 60 .

The supply lines 62 of the various culture modules 58 are each connected to an associated outlet connection 66 of a culture medium distributor 68 via a hose 64 made of silicone, which is only shown schematically. The culture medium distributor 68 is connected to a supply connector 70 for culture medium which, similarly to the exemplary embodiment according to FIG. 1 or 2, is connected to the outlet of a culture medium pump 24 or 24 'via a silicone hose (not shown).

The supply lines 62 each have a branch 72 at their connection end lying outside of the associated temperature control housing 60, which branch is connected via a silicone hose 74 to an assigned removal nozzle 76 of the outer housing 52 . In this way, culture fluid 30 can be drawn off separately from the various modules 58 for test purposes.

The culture containers 12 of a culture module 58 are likewise flowed around by the temperature control medium (eg temperature-controlled water) supplied via a temperature control medium connecting piece 78 . The temperature control medium supplied is discharged through an overflow 80 which is provided in the vicinity of the corner of the temperature control housing 60 diametrically opposite the connecting piece 78 .

The overflow 80 is connected via an angular pipe section 82 to an outlet connection 84 of the temperature control housing 60 . The outlet port 84 of a culture module 58 is each connected to the connection port 78 of the next culture module 58 which is located next to it, e.g. B. through a hose 86 . In this way, the temperature control housing 60 is successively flowed through by the same temperature control medium.

The first connection piece 72 is connected to a bath fluid supply pipe 88 , which is carried by the outer housing 52 . The outlet port 84-1 of a module is connected via a hose 86 to the connection port of the following module. The last outlet connection 84-2 of the module arrangement communicates with a temperature control medium outlet connection 94 via a hose 90 and a pipe section 92 .

As can be seen from the drawing, a riser pipe 96 is in flow connection with the end of the supply line 62 . This is perpendicular to the drawing plane of FIG. 3, as can be seen particularly well from FIG. 7.

From Fig. 7 is further well understood that the culture medium distributor in front view has the shape of a wide-open "V" 68, wherein said supply pipe 70, which at the same time excess for withdrawing the culture fluid is used, is provided at the lowest point of the culture medium distributor 68.

The two arms of a fork light barrier, designated overall by 98 , overlap the outer wall of the riser pipe 96 . The fork light barrier 98 is adjustably supported in the vertical direction by a support 100 which extends parallel to one of the outer plates of the outer housing 52 in the vertical direction.

In the arms of the fork light barrier 98 , an IR light source 102 (cf. FIG. 8) in the form of an IR diode or an IR detector 104 in the form of an IR-sensitive phototransistor are used in the usual way. If the liquid level in the riser pipe 96 , which corresponds to the liquid level of the various culture containers 12 that are exactly horizontally aligned, lies below the IR light barrier described above, the phototransistor 104 receives current and controls. If the liquid column in the riser pipe 96 has risen to the level of the axis of the light barrier formed by the IR light source 102 and the IR detector 104 , the output signal of the phototransistor disappears. This is recognized by a differential amplifier 106 , which receives at its side input the output signal of a setpoint generator 108 , again shown as an adjustable resistor.

The output signal of the differential amplifier activates the relay coil 112 of a reed relay 114 , generally designated 114 , via a switching transistor 110 . This closes the reed contact 116 , which is a potential-free contact and controls, for example, the operation of the pump 24 in the direction of supplying further culture fluid 30 .

In a simple version of the culture device, the changeover from submerged nutrition to basal nutrition takes place, for example, by moving the fork light barrier 98 correspondingly downwards, then lowering the excess culture fluid by manual emptying to a level well below the level desired for basal nutrition and then the liquid level, under the control of the output signal of the fork light barrier 98 , which is now adjusted, can rise to the level for basal nutrition.

For an automatic switchover between these two nutritional variants, however, a second forked light barrier is preferably provided, which is fixed at the level for basal nutrition. A second controlled system is then provided for this second fork light barrier, which corresponds to that according to FIG. 8, with the difference that the reed contact 116 then controls the removal of culture fluid, that is to say the operation of a bidirectional pump in the sense of a removal of culture fluid.

The various mechanical components of the above described culture device are insofar as they are rigid Components are, preferably made of glass. This Components can therefore be used at temperatures of 120 ° C can be easily sterilized and can be carried out before of attempts into a perfect aseptic Initial state are shifted. The different above described hose connections preferably consist of  Silicone tubing, which is also at 120 ° C can be easily sterilized.

As shown above, only a single level sensor is used to regulate the level of the culture liquid in the different culture containers 12 . For this reason, it is necessary to exactly align the various culture containers 12 in such a way that the areas on which the cell cultures grow lie in a common horizontal plane. For forced positioning of the different culture modules and for locking the culture modules in the outer housing, two positioning strips 118 and 120 are provided, which cooperate with the front edges or rear edges of the lower plates of the temperature control housing 60 .

In the schematic diagrams in Figs. 1 and 2, the cell culture inserts were 15 shown schematically as a built-in substrates. In practice, the cell culture inserts 15 are not components permanently installed in the culture containers 12 , but rather parts that can be removed from them. As indicated in FIG. 4 at 122 for one of the culture containers 12 , commercial cell culture inserts have the shape of a cylindrical beaker with a flat bottom wall 124 and a cylindrical peripheral wall 126 . By inserting the cell culture inserts 122 into the culture containers 12 and fixing them by means of the glass webs 57, it is ensured that the different bottom walls 124 lie in a common horizontal plane. The cell culture inserts 122 are made entirely of a porous plastic material, as are the cell culture inserts 15 of FIGS. 1 and 2.

As shown above, in the culture device described, it is very easy to switch from submerged to basal cell culture nutrition. A further variation of the growth conditions can be carried out by changing the temperature of the bath liquid supplied. Finally, by supplying gaseous substances to the inside of the outer housing 52 via a line 128 , the cell cultures can be exposed to predetermined damaging or therapeutic conditions. Exposure to aerosols can also take place in the same way.

Analogous to direct treatment with gases and / or aerosols can particulate active ingredients directly with the Bring cell cultures into contact. For this, the particles, the impact of which is to be investigated on the cell cultures dusted. If you match the densities of the particles and the Culture liquid so that the particles are lighter than the culture fluid, so the Particles with raising the level of the culture fluid from of the cell culture. Is the culture fluid against it lowered (basal nutrition), the particles are in contact with the cell culture. Such suitable particles are very fine dusts, e.g. B. wood dust or hydrophobic particles, which are inadequate or not at all in suspension drop.

These different possibilities of cell culture are discussed in get simple apparatus structure of the culture device.

The culture devices described above work well for the universal use of commercial cell culture inserts. The modular design of the device also allows one easy adaptation of the culture containers ("wells") specific insert sizes.

The execution of the tests is very flexible, three parallel test approaches can be carried out per module. Using all the modules of the culture device according to FIGS. 3 to 7, four times three parallel test runs can be carried out. This allows, for example, cellular reactions to be determined as a function of concentration and / or time.

Various special cell biological questions that can be solved with a culture device according to the invention are, for example:

• 1. Treatment of cells with e.g. B. gases, aerosols or Dust directly at the air / liquid limit layer.
• 2. Cellular interactions through cocultivation different cell types, e.g. B. of epithelial cells and Fibroblasts.
• 3. Cell attachment behavior in a system that pulsating with culture fluid.
• 4. Differentiation of cells from cultures in basal Culture fluid supply (polarization).
• 5. Examination of cilia-bearing cells in basal and / or intermittent supply of culture fluid.
• 6. Continuous analysis of cell excretion products during the trials.
• 7. Intermittent addition of growth stimulating and growth-inhibiting agents without interruption the culture tour.
• 8. Intermittent addition of those mentioned under 6 Substances during the exposure of cells with active or Pollutants.

In FIGS. 3 to 7, a culture apparatus has been shown which has its own temperature control. However, just like culture devices without a temperature control device, as shown in FIGS. 1 and 2, it can simply be placed in a temperature control cabinet for temperature control.

The preparation of the culture device for an experiment proceeds as follows:
The culture device is autoclaved in the assembled state, but without cell culture inserts, supply device and level sensor, at 120 ° C. in an autoclave for 30 minutes, as well as other required materials, such as pump tubes, tweezers for inserting the cell culture inserts, bottles for holding the culture medium, etc. after autoclaving the level sensor is installed in a clean room workbench. Then the medium hose is connected to the pump and the pump speed is set for the intended test conditions. Before inserting the cell culture inserts, which are individually packaged and supplied sterile by the manufacturer, the culture device is filled once with the cell type-specific culture medium and emptied again (rinsed), after which the medium used is discarded. The entire culture device is then filled with the cell-type-specific medium to below the culture vessels, and the prepared cell culture inserts are inserted into the clean room workbench using tweezers. The selected pump control program is then started and the culture device is transferred to the temperature control cabinet, if necessary.

The program controls a cycle that consists of the steps (1) back pumping and culture time and (2) back pumping and waiting consists.

After initialization, the pump performs its priming function independently by. Depending on the cell type and Cultivation type selected the adequate pumping speed. The program monitors the output signal of the photocell and regulates the termination of pumping activity as soon as the desired level is reached. Typically done  the end of the pumping activity only after two or repeated signal detection monitored by the program. This allows the detection of false signals to be avoided For example, can be caused by bubbles and at one-time signal detection to a premature and would lead to undesired termination of the pumping activity. The subsequent cultivation time, is preferably selectable and adjustable.

After the culture period has ended, the medium is pumped out initialized, again selecting the pump speed and is adjustable. From the selected pumping speed and the volume of the medium results in that for the Drainage required waiting time.

The aforementioned cycle controlled by the program can be done once, be run twice or more.

The cell culture inserts can at any time in the Clean room workbench can be removed. This will be Computer program stopped. If only a few inserts the program can be started again.

The entire culture device is used several times for cleaning Aqua dest. rinsed. The glass parts can also be easily clean with alcohol or other cleaning agents.

The invention has been described above with the aid of special level sensors. It goes without saying that other level sensors are also suitable which allow the position of the current liquid level in the culture container 12 to be recognized and provide a corresponding output signal. These include shimmer-controlled sensors, sensors working according to the dielectric principle, a plurality of sensors having electrodes which are spaced apart in the vertical direction, and infrared and ultrasonic sensors.

Claims (25)

1. Culture device, with at least one culture container ( 12 ) which has at least one connection ( 22 ) for supplying and removing culture medium ( 30 ), each with a cell culture insert ( 15 ; 122 ) for each of the culture containers ( 12 ), which is inside the same is held, and with a supply device ( 24 to 28 ; 24 ', 48 , 50 ) for supplying culture medium ( 30 ) to the culture containers ( 12 ) or for removing culture medium therefrom, characterized in that the culture containers ( 12 ) a level sensor ( 38 ; 98 ) responsive to the level of the culture medium ( 30 ) is assigned and that the supply device ( 24 to 28 ; 24 ', 28 , 48 , 50 ) is controlled as a function of the output signal of the level sensor ( 38 ; 98 ) . 2. Culture device according to claim 1, characterized in that the cell culture inserts ( 15 ; 122 ) each specify a horizontal culture area. 3. Culture device according to claim 1 or 2, characterized by the individual culture containers ( 12 ) or groups of culture containers ( 12 ) associated with extraction lines ( 74 , 76 ). 4. Culture device according to one of claims 1 to 3, characterized in that a plurality of culture containers ( 12 ) are connected to a common supply line ( 62 ) such that the culture surfaces of the cell culture inserts ( 15 ; 122 ) lie in a common horizontal plane. 5. Culture device according to claim 4, characterized in that the supply lines ( 62 ) communicate with a riser pipe ( 96 ), in which the level sensor ( 38 ; 98 ) is provided. 6. Culture device according to one of claims 1 to 5, characterized in that the level sensor ( 98 ) comprises a fork light barrier. 7. Culture device according to claim 5 or 6, characterized in that the level sensor ( 98 ) on the riser ( 96 ) is adjustable. 8. Culture device according to one of claims 1 to 7, characterized in that the level sensor ( 38 ; 98 ) is a continuously measuring the level sensor. 9. Culture device according to one of claims 1 to 7, characterized in that the level sensor ( 38 ; 98 ) is a responsive level switch at a predetermined level. 10. Culture device according to one of claims 5 to 9, characterized in that it has a plurality of modules ( 58 ), which in turn each comprise a plurality of culture containers ( 12 ) which are arranged in parallel in an outer housing ( 52 ), a supply distributor ( 68 ) is provided, which is connected to supply lines ( 62 ) of the individual modules ( 58 ). 11. Culture device according to claim 10, characterized in that one with the supply device ( 24 to 28 ; 24 ', 28 , 48 , 50 ) to be connected is the only connection ( 70 ) of the supply distributor ( 68 ) at the lowest point thereof. 12. Culture device according to claim 10, characterized in that the outer housing ( 52 ) has separate connections ( 76 ) for sampling lines ( 74 ), each leading to one of the culture modules ( 58 ). 13. Culture device according to claim 12, characterized in that the culture containers ( 12 ) in at least one culture container ( 12 ) comprising modules ( 58 ) are arranged, each having a culture container arrangement surrounding the temperature housing ( 60 ) which has a temperature medium inlet ( 78 ) and a tempering medium outlet ( 84 ). 14. Culture device according to claim 13, characterized in that the temperature control medium outlet ( 84 ) is connected to an overflow ( 80 ) lying in the upper region of the temperature control housing ( 60 ), which is preferably diametrically opposite the temperature control medium inlet ( 78 ). 15. Culture device according to claim 14, characterized in that the temperature control housing ( 60 ) of the various modules ( 58 ) are connected in flow with respect to the temperature control medium in series. 16. Culture device according to one of claims 1 to 15, characterized in that the culture containers ( 12 ) are surrounded by a tightly closable outer housing ( 52 ). 17. Culture device according to claim 16, characterized in that the outer housing ( 52 ) is provided with a connection ( 128 ) for a gaseous medium. 18. Culture device according to one of claims 1 to 17, characterized in that apart from the Level sensor and parts of the supply facility in the essentially made of a sterilizable material, in particular glass and silicone material. 19. Culture device according to one of claims 1 to 18, characterized in that the supply device ( 24 to 28 ) has a bidirectional pump arrangement ( 24 ), in particular a peristaltic pump. 20. Culture device according to one of claims 1 to 19, characterized by a program control ( 42 ) which controls a set level transmitter ( 40 ) as a function of time. 21. Culture device according to claim 20, characterized in that the program control ( 42 ) periodically switches the target level between two values, one of which corresponds to a predetermined distance above the culture surface liquid level and the other a predetermined distance below the top of the cultivated area. 22. Method of culturing cells or Fabric components, characterized in that the Liquid level of a nutrient solution for the cells or tissue parts alternating a predetermined distance over the top of a culture of cells or Tissue parts (submerged cultivation) and one predetermined distance under the top of the culture (basal cultivation). 23. The method according to claim 22, characterized in that in a period when the liquid level is below the top of the culture, the culture is firm, exposed to gaseous or aerosol-like substances becomes. 24. The method according to claim 22 or 23, characterized characterized in that the location of the liquid level with With the help of a level sensor and one controlled by it Pump device is made.   25. The method according to any one of claims 22 to 24, characterized characterized that a timing of the situation the liquid level with the help of a program control is made.