Apparatus and method for culturing cells

Abstract

The present invention relates to a carrier for culturing cells, the carrier being, for example, a plate-shaped carrier made of glass, silicon, plastic, or a combination thereof, having at least one first recess formed therein, the first recess extending across a first thickness of the carrier, and an array of second recesses extending from the first recess to a second thickness of the carrier adjacent to the first thickness. The first thickness may have a thickness of several micrometers to several centimeters. The second recesses extend from the first recess to the second thickness, forming a second arrangement, respectively. The second recesses form wells, which are suitable for containing cells and / or synthetic particles made of plastic or glass.

Description

[Technical field]

[0001] The present invention relates to a device for culturing cells, to the use of the device for culturing cells and to a method for culturing cells using the device. According to a feature of the device, it has mutually separated wells for containing cells, which are in contact with a larger volume, so that liquid exchange can take place. The wells have a high aspect ratio of depth to diameter, which holds the cells in the wells or prevents them from flowing out of the wells into the larger volume in contact with the wells. The method has the advantage of holding cells, which may be individualized cells or multiple cells, in the wells, where multiple wells are in contact with a common volume of medium. By holding the cells in wells with a high aspect ratio, the method has the advantage that the cells can be distinguished based on the well that contains them and that the cells can be observed over a longer culture period, for example from 1 to 10 days. The high aspect ratio of the wells, together with the change in diffusion rate, for example due to the change in inflow and outflow of the medium, allows metabolites to be collected therein separately in each well and assigned to the well and the cells contained therein, or allows the creation of particularly constant conditions by a fast exchange of medium and the extraction of metabolites.

[0002] For such methods, so-called microtiter plates are customarily used as devices for culturing cells and for carrying out biological experiments. Microtiter plates comprise wells with a volume of, for example, about 300 μl. A biological experiment can be carried out per well, and therefore a large amount of reagents is required per well. Moreover, although relatively large, microtiter plates typically only provide space for 96 to 1536 parallel experiments. Finding individual cells in these wells, for example with an optical microscope, is tedious. This poses a great challenge for high-throughput experiments in which certain types of cells are to be cultured and investigated, for example cell line development, monoclonal antibody development, synthetic biology, cell therapy, immunotherapy, stem cell research. The use of significantly miniaturized wells, for example with volumes reduced by a factor of 100,000, is technically possible, but entails new challenges, in particular evaporation and supply of nutrients, concentration of metabolites and cell migration. The latter occurs due to the movement of the cells under their own forces or due to turbulence when refilling reagents or medium, or when the carriers are moved by heating and convection. For many biomedical developments, certain conditions must be ensured for each individual experiment. For drug approval, monoclonality must be proven. The object of the present invention is therefore to solve the abovementioned challenging problems. [Background technology]

[0003] DE 10 2020209 825 A1 was published after the priority date of the present application and therefore only has to be considered as to novelty, and describes a method for producing a plastic part with a glass insert from a prefabricated plastic part with a circumferentially extending welding web and a glass insert with a plurality of through holes arranged at a distance from one another in a circumferentially closed abutment area, in which the welding web is heated and the glass insert is pressed with its abutment area against the welding web of the plastic part to form a bonding area, in which the plastic is placed in the abutment area and passes through the through holes of the glass insert.

[0004] DE 10 2019 217 466 A1, which according to § 3 para. 2 of the Patent Act has only to be considered as novel, describes a reaction vessel made of glass, which is formed as recesses of at least 30 μm deep in one glass sheet or in two glass sheets joined together, The recesses may have a portion which extends into the glass sheet exclusively up to a first thickness, from which another smaller recess may extend up to a deeper second thickness.

[0005] WO 2016 / 041544 generally describes a method suitable for the present invention for forming recesses in glass. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] DE 102020209825 A1 [Patent Document 2] DE 102019217466 [Patent Document 3] International Publication No. 2016 / 041544 Summary of the Invention [Problem to be solved by the invention]

[0007] The object of the present invention is to provide a carrier with recesses for cells suitable for use in alternative cell culturing methods and methods, in which cells are held in separate wells, but which are nevertheless in contact with a common volume of medium forming a common medium stock. Preferably, the carrier should be constructed so that liquid, in particular cell culture medium, can flow out or be removed from the recesses in an appropriate manner.

[0008] Description of the invention Here, the culturing of cells is an in vitro method, in particular the culturing of cells in a medium containing the cells in a living state and / or suitable for cell growth. The cells are for example animal, in particular human, plant, fungal or yeast cells, or bacteria. The culturing can be carried out, optionally with the addition of agents to the medium, for example for the analysis and / or isolation of the cells. [Means for solving the problem]

[0009] The invention solves the problem by the features of the claims, in particular by a carrier, which is suitable for culturing cells and is preferably a plate-shaped carrier, for example made of glass, silicon, plastic or a combination thereof, in which at least one first recess is formed, which extends over a first thickness of the carrier, from which an array of second recesses extends into a second thickness of the carrier adjacent to the first thickness. The first thickness may have a thickness of a few micrometers to a few centimeters. The second recesses extend from the first recess into the second thickness, respectively forming a second array, the second recesses forming wells suitable for receiving cells and / or synthetic particles, for example made of plastic or glass. The second recesses, also called wells, have a high aspect ratio of depth to diameter. The aspect ratio is determined in the plane of the first thickness adjacent to the second thickness or at half height of the second thickness. The wells may for example have an aspect ratio of depth to diameter of at least 1:1, preferably at least 2:1, preferably at least 5:1, preferably at least 10:1, preferably at least 20:1, preferably at least 40:1, preferably at least 50:1, wherein the wells have a diameter of at most 1000 μm, preferably at most 900 μm, preferably at most 800 μm, preferably at most 700 μm, preferably at most 600 μm, preferably at most 500 μm, preferably at most 400 μm, preferably at most 300 μm, preferably at most 200 μm, preferably at most 100 μm, preferably at most 90 μm, preferably at most 80 μm, preferably at most 70 μm, preferably at most 60 μm, preferably at most 50 μm, preferably at most 40 μm, preferably at most 30 μm, preferably at most 20 μm, preferably at most 10 μm, preferably at most 5 μm.

[0010] The diameter of the wells can be adapted to the size of the cells, so that, for example, the cells can be stacked or in contact with each other. The cross section of the individual wells can be rectangular, A-shaped, V-shaped or tapered. The bottom of the wells can be flat, tapered (conical) or rounded. The bottom can have other microstructures.

[0011] The first thickness portion is defined in one plane by a first surface of the carrier in which the cross-sectional opening of the first recess is located, and in one plane spaced from the first plane by the cross-sectional opening of the second recess. The cross-sectional opening of the second recess is spaced from one another by a surface area of ​​the carrier, in particular the second part carrier. More preferably, this surface area and the cross-sectional opening of the second recess are located in a common plane. Preferably, the cross-sectional opening of the second recess is directly adjacent to the cross-section of the first recess. Alternatively, the second recess may be spaced from the first recess and connected to the first recess via a transition path. Such a transition path is formed in a preferred embodiment as a partial space between the first part carrier and the second part carrier, in which case the first recess and the second recess are formed in the first part carrier and their bottoms are respectively formed by the second part carrier. The second part carrier has a recess, which forms a partial spacing between the first part carrier and the second part carrier over the cross section of the transition path.

[0012] The arrangement of the second recesses may be arbitrary, but is preferably regular, for example in parallel rows, in the form of lattice nodes and / or equally spaced apart from one another, the lattice being, for example, a square or hexagonal lattice.A second array of recesses extending adjacent to the first array may for example have at least 1, preferably at least 10, preferably at least 20, preferably at least 30, preferably at least 40, preferably at least 50, preferably at least 60, preferably at least 70, preferably at least 80, preferably at least 90, preferably at least 100, preferably at least 200, preferably at least 300, preferably at least 400, preferably at least 500, preferably at least 600, preferably at least 700, preferably at least is at least 800, preferably at least 900, preferably at least 1000, preferably at least 2000, preferably at least 3000, preferably at least 4000, preferably at least 5000, preferably at least 6000, preferably at least 7000, preferably at least 8000, preferably at least 9000, preferably at least 10000, preferably at least 50000, preferably at least 100000, preferably at least 500000, preferably at least 1000000, 0000000, preferably at least 50000000, preferably at least 100000000 second recesses, the second recesses being at most 10mm, preferably at most 9mm, preferably at most 8mm, preferably at most 7mm, preferably at most 6mm, preferably at most 5mm, preferably at most 4mm, preferably at most 3mm, preferably at most 2mm, preferably at most 1mm, preferably at most 900μm, preferably at most 800μm, preferably at most 700μm, preferably at most 600μm, preferably at most 500μm, preferably at most 400μm, preferably at most are spaced apart from each other by at most 300 μm, preferably at most 200 μm, preferably at most 100 μm, preferably at most 90 μm, preferably at most 80 μm, preferably at most 70 μm, preferably at most 60 μm, preferably at most 50 μm, preferably at most 40 μm, preferably at most 30 μm, preferably at most 20 μm, preferably at most 10 μm, preferably at most 9 μm, preferably at most 8 μm, preferably at most 7 μm, preferably at most 6 μm, preferably at most 5 μm, preferably at most 4 μm, preferably at most 3 μm, preferably at most 2 μm, preferably at most 1 μm.

[0013] The cross-sectional opening of the first recess is located in the plane of the first surface of the carrier, so that the first recess opens in the plane of the first surface of the carrier. The carrier features an outlet, the cross-section of which opens in the first thickness or adjacent to the first thickness, in which case the cross-section of the outlet is preferably adjacent to or opens towards the plane of the first surface of the carrier, in which case the outlet is connected to the first recess. The outlet is formed in the carrier, for example in the form of a groove that opens towards the plane of the first surface of the carrier. Alternatively, the outlet may be a channel that extends into the first recess. In general, the outlet is configured to drain the liquid, in particular the medium, from the first recess. In this case, the outlet may be configured to drain the liquid exclusively by the action of gravity and / or by using a pump and / or by applying a positive pressure to the first recess. The outlet may have a cross-section that extends into the carrier at a depth that is, for example, 1 to 95% of the first thickness. Such an outlet is used for the outflow or removal of liquid in the first recess, which is present up to the cross section of the outlet and extends, in particular at the level of the liquid in the first recess, up to the plane of the first surface of the carrier. The outlet can extend around at least one, preferably exactly one, first recess, so that the outlet forms a circumferential recess in the carrier around the first recess, in which case the circumferential recess is defined by the carrier, which extends at the interval of this outlet up to the plane of its first surface. In the embodiment of the outlet as a recess or hydrophilic surface formed around the first recess, exactly one or at least two connecting channels may be connected to this outlet. Optionally, the outlet may additionally form an inlet, in particular for a liquid, for example a medium or a gas, connected to the first recess.

[0014] The rate at which the liquid exits or is removed at the outlet can be controlled by the thickness of the first thickness, by the liquid feed rate or by the inclination of the carrier. The outlet can be implemented by multiple openings, for example holes with different diameters. The exiting liquid can be collected, analyzed or reused.

[0015] In another embodiment, the carrier may have a hydrophilic surface as an outlet, the hydrophilic surface having exactly one or at least two first recesses, respectively, in which case the outlet is surrounded by a hydrophobic surface or coating of the first surface of the carrier. In this embodiment, the hydrophilic surface having the first recess as an outlet may be located in the same plane as the first surface of the carrier, which is preferably adjacently hydrophobic. In this embodiment, a connecting channel may also be arranged at the outlet, the outlet being formed from a hydrophilic surface portion on the carrier, optionally configured without a recess in the carrier. In the method, the hydrophilic surface of this outlet allows the overflow of liquid from the first recess to the connecting channel, even if the outlet is located in the plane of the first surface of the carrier, in which case the spreading of the liquid on the first surface is limited by the hydrophobicity of the first surface adjacent to this outlet.

[0016] The carrier is configured for use in a method for culturing cells by means of an outlet and preferably by means of a connecting channel connected to the outlet, in which case liquid, in particular cell culture medium, can flow out or be removed precisely from the recess. The connecting channel connects the outlet to a removal point remote from the outlet. The removal point can be, for example, a container for the liquid or an analytical device. Typically, the connecting channel can be configured as a recess in the carrier, which has a cross section that is open towards the surface of the carrier, for example a groove, or which has a cross section that is closed around the periphery, for example a hole passing through the carrier.

[0017] In one embodiment, a connecting passage is formed in and / or on an end face of the carrier, the connecting passage extending from a first surface of the carrier over its entire cross section to its second surface, the connecting passage being connected to an outlet on the side of the carrier opposite the second surface, so that liquid can flow from the outlet through the connecting passage into the plane of the second surface of the carrier, particularly in a horizontal position of the carrier with its first surface above the second surface.

[0018] In an embodiment, the connecting channel directs the liquid flowing through the outlet when the liquid level in the first recess reaches the cross section of the outlet to the opposite second surface of the carrier. Thus, when liquid is supplied to the first recess, the liquid whose level reaches the cross section of the outlet is led off in a controlled manner, such that the liquid level does not exceed the cross section of the outlet and in particular the first surface of the carrier. The opening of the connecting channel on the second surface of the carrier allows the liquid flowing out there to be removed, for example for subsequent analysis, without an additional removal device, for example a pipette, coming into contact with the liquid in the first recess.

[0019] The surfaces of the connecting passage and the outlet are preferably hydrophilic, more preferably the first surface of the carrier and / or the opposite second surface of the carrier are hydrophobic, e.g. coated with a hydrophobic coating. Optionally, the second surface of the carrier is provided with grooves surrounding the openings of the connecting passage at predetermined intervals. The grooves surrounding the openings of the connecting passage in the second surface of the carrier reduce the spread of liquid flowing out of the openings over the second surface of the carrier.

[0020] Preferably, the carrier has at least two first recesses, each connected to a first thickness with an array of second recesses, each first array being connected to at least one or exactly one connecting channel by at least one outlet. In this embodiment, the at least two first recesses are each connected to a connecting channel independently of one another, so that the liquid flowing out of the first recesses flows out of the connecting channel at the second surface separately for each first recess and can be removed and / or analysed separately.

[0021] Optionally, the carrier has at least two first recesses, each having an array of second recesses connected to its first thickness portion, in which case an outlet connected to a first recess opens into another first recess, such that the outlet is configured to direct liquid flowing out of a first recess to the other first recess.

[0022] Typically, optionally adjacent to the first surface of the carrier, at least one supply channel may be formed in the carrier, the supply channel opening into at least one first recess. The supply channel may be formed in the carrier, for example, as a groove, in which case the cross section of the groove is open in the plane of the first surface of the carrier. In this case, a reservoir recess extending into the carrier at a distance from the first recess may be connected to the supply channel. For example, the reservoir recess having a depth equal to or smaller than the depth of the supply channel may be used to contain a liquid, for example a cell culture medium, which is metered into the reservoir recess as a stock.

[0023] The first and second recesses may be formed in a one-piece carrier, preferably a glass carrier. The recess in the one-piece carrier can be produced by removing material from the original one-piece carrier, for example by laser irradiation and subsequent etching. Alternatively, the first recess may be a through hole in the first part carrier, in which the second part carrier is arranged and in which the second recess is formed. When the carrier is formed from at least two part carriers, the carrier is two-piece, in which case the part carriers may be directly connected to each other, for example by bonding, or by a bonding material, for example by using molten glass frit or adhesive. The second recess may be a through hole in the second part carrier, the cross-sectional opening of which is covered by a third part carrier on the side opposite the first part carrier, or may be a blind hole in the second part carrier. Optionally, the second part carrier is made of colored glass, the optional third part carrier is made of transparent glass, and the first part carrier is made of non-colored or transparent glass. In this case, the transparent glass is transparent to the light that is incident on the carrier for optical detection and is transparent to the light that may originate from the second recess for optical detection. The colored glass is preferably opaque to the light that is incident on the carrier for optical detection or is opaque to the light that may originate from the second recess for optical detection. Preferably, the carrier or all the partial carriers consist of glass.

[0024] The high aspect ratio of the second recess allows for a method of culturing the culture in which the cells introduced therein remain in the second recess while liquid is introduced into or discharged from the first recess, the internal volume of the second recess being in contact with the internal volume of the first recess, and exchanges can occur between them, for example by diffusion of metabolic products from the second recess to the first recess, and of nutrients, dissolved oxygen and possibly additional agents from the first recess to the second recess.

[0025] Optionally, a sensor is arranged in the first recess, in one of the second recesses, for example at the outlet and / or at the inlet in the connecting channel. The sensor may be, for example, a gas sensor for oxygen or carbon dioxide, a pH sensor, a temperature sensor, or a sensor for a binding molecule specific for an analyte, which may be suitably immobilized, for example an antibody specific for an analyte in the medium. Further optionally, identical sensors may be arranged in the inlet and at the outlet, respectively, which are connected to an evaluation unit configured to determine a change in the sensor signal.

[0026] The introduction of liquid into the first recess can be done by dripping or by introduction through a channel in contact with the liquid present in the first recess through a cross-sectional opening of the first recess, which is respectively provided in the plane of the first surface of the carrier. Alternatively, the carrier can have a supply channel extending from its second surface directly into the first recess, so that the supply channel is configured to guide liquid and / or gas from the second surface into the first recess. Typically, in a horizontal arrangement of the carrier, the second surface of the carrier is horizontal and arranged below the first surface of the carrier, in which case the first and second surfaces are preferably located parallel to each other. Alternatively or additionally, the supply channel can extend through a wall section within the first thickness, in which the first recess is formed, so that liquid, for example a medium for cell culture, can be introduced into the first recess in the first thickness through the supply channel.

[0027] In the carrier, in the first thickness, the outlet may be formed as a channel, for example in the first recess, in a wall located opposite the supply channel.

[0028] In one embodiment, the wall extending over the first thickness and having or surrounding the first recess may be formed by a first part carrier, which is adjacent to a second part carrier, in which the second recess is formed. In this case, the first part carrier may be mounted on the second part carrier, for example by a slidable and liquid-tight overlap of the first and second part carriers in an edge region. Optionally, the first part carrier may be liquid-tight and vertically slidable relative to the second part carrier, so that the first thickness is adjustable by the slidability of the first part carrier relative to the second part carrier. When the first recess is formed in the first part carrier, which is arranged liquid-tight and mountable or slidably relative to the second part carrier in which the second recess is formed, the carrier is configured to adjust the level of the liquid in the first recess depending on the position of the first part carrier relative to the second part carrier. Thus, the outlet, in particular when it does not have a valve, leads the liquid out of the first recess, the liquid level reaching the cross section of the first recess. In this case, the first part carrier can be made of plastic and the second part carrier can be made of glass, or the first and second part carriers can be made of glass, optionally with a slidable seal in the overlapping area of ​​the part carriers. Alternatively, in order to adjust the liquid level in the first recess, a valve can be arranged at the inlet and / or at the outlet, by means of which the liquid level in the first recess can be adjusted, for example in the method.

[0029] In one embodiment, the inlet and / or outlet may have one or more valves regulating one or more connections. Multiple valves and multiple connections allow different liquids to be metered in a controlled manner into the first recess.

[0030] In one embodiment, the carrier may consist of a second part carrier in which the second recess is arranged and a first part carrier adjacent to the second part carrier, the first part carrier having exactly one first recess and the first recess extending over all the second recesses. In general, and in this embodiment in particular, the first part carrier may consist of plastic or glass, and the second part carrier may consist of glass and have the second recess therein. In an embodiment in which the first part carrier consists of plastic and the second part carrier consists of glass, the second part carrier preferably has a number of through holes in the bonding area with the first part carrier, for example by the plastic of the first part carrier being forced into and possibly through these through holes after heating of the first and / or second part carrier in the bonding area, so that the plastic of the first part carrier extends through these through holes in one piece. Preferably, the first part carrier has an outlet at a distance from the area covered by the second part carrier and optionally has a connecting line connected to the outlet.

[0031] The recesses in the glass carrier are preferably produced by irradiating the glass with laser radiation and subsequent etching.

[0032] Generally, in each embodiment, the first recess may be divided by a diaphragm that is preferably arranged perpendicular to the first surface of the carrier, so that the diaphragm is arranged vertically, for example in a horizontal arrangement of the carrier or the first surface of the carrier. The diaphragm dividing the first recess may be tensioned, for example, by a frame that abuts against a surface of the carrier that is located between the cross-sectional openings of the second recess.

[0033] In the method, typically, a liquid is introduced into a first well, while a second well contains a liquid containing at least one cell. This is achieved by precisely introducing the liquid containing the cells into the second well, for example by using a drop generator or a metering device, in particular a nozzle. Alternatively, the liquid containing the cells can be introduced into the first well, so that the liquid containing the cells is distributed into the second well. Thus, for example, cells can be distributed from the liquid into the second well. Optionally, the liquid containing the cells remaining in the first well may be subsequently removed. In this case, the liquid, which is preferably a cell culture medium, is provided as a mixture with cells dispersed therein and can be introduced into the well as a mixture or suspension, or the liquid containing the cells can be introduced into the well in a separate step using a separate drop generator or a separate metering device, and the cell culture medium without cells can be introduced into the well in a separate step, preferably subsequently, preferably using a separate metering device.

[0034] Here, a cell culture medium comprises a medium that keeps cells, in particular plant cells, yeast, bacterial or animal cells, preferably human cells, viable for e.g. at least 1 hour, preferably at least 12 hours, or at least 24 hours, or at least 2 days, or at least 3 days, or at least 4 days, or at least 5 days, or at least 6 days, or at least 7 days.

[0035] In an embodiment of the method, in which the carrier only has a recess, optionally in the form of a second recess, and further optionally has or consists of at least one outlet, optionally connected thereto, the liquid can be supplied directly to the cross-sectional opening of the second recess, which is located in the plane of the first surface of the carrier. In an embodiment in which the carrier only has a second recess and the culture medium protrudes beyond the plane of the cross-sectional opening of the second recess, this protruding layer of culture medium forms the volume of the first recess and extends over a first thickness, so that this culture medium covers the second recess. In this case, the culture medium may extend over a first thickness, for example of 1 μm to 3 mm, for example of 10 μm to 200 μm, above the second recess. The liquid protruding beyond the plane of the first surface of the carrier can flow out over the edge of the carrier, preferably along at least one outlet and optionally along a connecting path. The liquid can then be excited and moved by tilting the carrier, by supplying a gas flow and / or by vibration, for example ultrasonic waves. In particular, in this embodiment, the first surface may have poor wettability, for example with respect to aqueous liquids, may be hydrophobic, for example, and may consist of a plastic or a structured, functionalized or coated glass surface. A drop of liquid applied to the first surface or to the plane in which the cross-sectional opening of the second recess is located can exchange material with the liquid present there when moving beyond this plane. For example, a drop of medium applied to this plane can absorb and / or partially replace the liquid already present as a rolling drop, for example the used medium. Optionally, the surface of the carrier lying in the plane in which the cross-sectional opening of the second recess is located can have a channel worked into the carrier, the cross-section of the channel being open towards the plane in order to guide the liquid extending across the plane during the movement. The liquid can then be applied to this plane of the carrier intermittently or in predetermined amounts as at least two drops. If the volume of the liquid exceeds the volume of the liquid on the plane in which the carrier extends, the liquid flows out through the outlet in a particularly precise manner.

[0036] Typically, the liquid, preferably the medium, spreads over the first thickness, preventing evaporation of the medium from the second recess. The first thickness may then be several micrometers to several centimeters in size. In this case, the large volume of the first thickness provides enough space for the liquid to reduce the effects of nutrient consumption and the concentration of metabolites in the second recess by diffusion. The high aspect ratio of the second recess has the effect that the movement of the liquid in the adjacent first thickness or first recess only slightly moves the cells in the second recess, and the cells are not washed out of the second recess.

[0037] For example, a first recess may be connected to 100,000 second recesses, where each second recess has a diameter of 54 μm, a depth of 436 μm, an aspect ratio of depth to diameter of 8:1, and a volume of 1 nl. The recesses are spaced from each other by, for example, 10 μm. To enclose all the second recesses, the first recesses must be at least 410 mm 2 Conventionally, the wells have a significantly lower aspect ratio, e.g., 0.16:1. For the same volume of 1 nl per well and a diameter of 200 μm, the depth is about 32 μm. In this case, 100,000 recesses would fit over an area of ​​4410 mm 2 and has an area ten times that of a high aspect ratio well.

[0038] The smaller area for the same volume per well allows viewing in more wells in parallel using an optical microscope.

[0039] The high aspect ratio of the second wells provides many advantages. For example, cell products, such as proteins or antibodies, can be collected in the second wells without or with slow export of the cell products. This can be used to analyze these cell products and their production rates, for example by attaching a fluorescent marker and evaluating the intensity of the fluorescence. Ideal culture conditions can be formed when cell products, such as metabolic products, are exported faster than they can be produced by the cells. The change between slow and fast export of cell products can be achieved, for example, by changing the inflow and outflow rates of the nutrient medium or other liquids.

[0040] The first recess can be formed in the plastic, metal, silicon or glass part, for example in the form of a microtiter plate, in the form of a petri dish, as a sealing ring or as a frame, and also by bonding of different materials. The carrier can be connected to inlets and outlets present in the culture station, especially for long-term culture. Furthermore, the temperature, the gas atmosphere, e.g. the CO2 content, the composition of the liquid, the content of biomolecules or other substances can be adjusted in the inlets or measured at the outlets. At the outlets, sensors can be attached for measuring the pH value, the fluorescence, the concentration, the temperature, the metabolic products, the biomolecules produced by the cells, etc.

[0041] Various liquids, for example nutrient media, buffer solutions, reagents, stains, can be provided to the first well through one or more supply channels, for example in the form of capillaries. These liquids may contain cells. To ensure a continuous, intermittent or on-demand controlled exchange, the liquids can be removed again through one or more outlets. The supply channels can be arranged such that the cells are not affected when the liquid is provided. When capillaries are used as supply channels, the liquid may be provided through a second well, which in particular does not contain cells.

[0042] Samples can be taken from the liquid in the first well for example to detect cellular products (proteins, antibodies, metabolites) at time intervals or continuously, to control the concentration of added reagents, or for the analysis of exosomes or extracellular vesicles (EVs) produced from the cells, including in particular the type and amount of proteins and nucleic acids, thereby identifying characteristic values ​​such as the heterogeneity of EVs in biological and physical properties (size, composition as type and amount, density, refractive index).

[0043] When multiple inlets are used, some of them can be used for adding cell-free liquids such as medium, water, etc., and some can be used for adding cell-containing liquids.

[0044] When a liquid is supplied to the first or second recess, for example a liquid having a lower density than the liquid containing the cells (preferably immiscible with the liquid containing the cells) may be used as a temporary evaporation prevention means. When the first thickness is small enough, the cells can be introduced into the second recess through the liquid in the first thickness. In parallel with the introduction of the liquid containing the cells, a second supply line can be used to compensate for evaporation. Optionally, the dispensed liquid may continuously change its composition, thereby allowing the medium surrounding the cells to be replaced over a period of time.

[0045] The carrier is in various embodiments configured to allow cells to be cultured in the second wells. Additionally, a method for analyzing the cells can be performed. In particular, the cells can be stained with a staining agent. As the exchange of liquid is ensured around the cells, the staining agent can subsequently be washed away without removing or otherwise affecting the cells.

[0046] Various methods of culturing the cells can be used. In particular, the cells in the second wells can be provided with a very small amount of liquid followed by the supply of medium. Alternatively, the medium can be supplied first, followed by the supply of the liquid containing the cells. Alternatively, the medium containing the cells can be supplied in one step. A wait can be made between method steps to ensure that the cells have settled in the second wells. Additionally, the settlement of the cells can be assisted by centrifugation or agitation, in particular by shaking or rocking the carrier. This allows additionally to remove air bubbles in the second wells. Following this, the medium containing the cells can be removed until the cells are essentially only present in the second wells.

[0047] The invention will now be explained in more detail by way of example and with reference to the drawings, in which: [Brief description of the drawings]

[0048] [Figure 1] 1 illustrates one embodiment of the present invention. [Diagram 2] 13 shows an embodiment of the present invention with feed channels coming from a second surface of the carrier. [Diagram 3] 13 illustrates one embodiment of the present invention with feed channels and outlets coming from a second surface of the carrier. [Figure 4] 1 shows an embodiment of the present invention in plan view. [Diagram 5] 1 shows an embodiment of the present invention having a supply and an outlet. [Figure 6] 13 shows another embodiment of the present invention having a supply channel and an outlet. [Figure 7] 2 illustrates another embodiment of the present invention. [Figure 8] 2 illustrates another embodiment of the present invention. [Figure 9] A shows an embodiment in a cross-sectional view of the carrier, B shows an embodiment in a plan view of the carrier, and C shows a first partial carrier in a plan view. [Figure 10] 2 illustrates another embodiment of the present invention. [Figure 11] A and B show another embodiment of the present invention. [Figure 12] Another embodiment is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] FIG. 1 shows a carrier 1 in a cross-sectional view. The carrier 1 consists of a first part carrier 2 and an adjacent second part carrier 3, in which a first recess 4 is formed, which covers the cross-sectional opening 5 of a number of adjacent second recesses 6. The internal volume of the second recess 6 is thus in contact with the internal volume of the first recess 4 along its cross-sectional opening 5, and by contact, in particular by diffusion, a material exchange between these recesses is possible. The second recess 6 extends over a second thickness 8 of the carrier 1, which here extends over the second thickness 8 in the second part carrier 3. In the first part carrier 2, the first recess 4 extends over a first thickness 7, which here is equal to the thickness of the first part carrier 2. In its first thickness 7, which here is identical to the first part carrier 2, the carrier 1 has an outlet 9, through which a liquid F can flow out, whose liquid level reaches the cross-section of the outlet 7. The feed channels 10 open at a given distance above the horizontal plane in which the outlets 9 extend. The feed channels 10 are not necessarily connected to the carrier but may be guided independently of the carrier, for example by means of a moving device (not shown).

[0050] The high aspect ratio of the second recess 6 allows liquid movement within the first recess 4 without cells Z within the bottom 14 or the volume of the second recess 6 flowing out of the second recess, or in that case, minimizing the effects of turbulence on the cells.

[0051] FIG. 2 shows a carrier 1 in a cross-section, which, like the carrier 1 shown in FIG. 1, consists of a first part carrier 2 with a first recess 4 and a second part carrier 3 attached to the first part carrier 2, in which a second recess 6 extends over a second thickness portion 8 adjacent to the first recess 4. An outlet 9 is formed in the first part carrier 2. The outlet 9 shown in FIGS. 1 and 2 extends along the outer end face 11 of the carrier 1. A supply channel 10 comes from a second surface 12 of the carrier 1 or of the second part carrier 3 of the carrier 1 and extends to the first recess 4. Preferably, the supply channel 10 has a control valve 13, by which the supply of the liquid F to the first recess 4 is controlled.

[0052] 3 shows an embodiment of the carrier 1, in which a connection 15, forming the outlet 9 and, when the flow is reversed, the supply 10, extends through the carrier 1 from the first recess 4 to the second surface 12 of the carrier 1. Such a connection 15, forming both the supply 10 and the outlet 9, preferably comprises a control valve 13, which more preferably comprises two or more connected passages, for example one of which is connected to a storage vessel for the culture medium and another of which is connected to a collection vessel for the liquid F led out of the first recess 4. In such an embodiment, a cover may be provided covering the first recess. In this case, the cover preferably comprises a valve, by means of which the composition of the gas atmosphere can be controlled.

[0053] 4 shows an embodiment of the invention in a one-piece carrier 1 in plan view of the first surface 20 of the carrier 1 and the cross-sectional opening of each one of the first recesses 4. Here, a rectangular first recess 4 extends across the cross-sectional opening of an adjacent second recess 6. The second recesses 6 are arranged in a 3×3 array of second recesses 6 per first recess 4.

[0054] In embodiment A of Fig. 4, the first recess 4 is surrounded by a number of connecting passages 15 extending through the carrier 1. The connecting passages 15 are connected to or form the outlet 9. The connecting passages 15 extend from the first recess through the carrier 1 to a second surface 12 of the carrier 1 opposite the cross-sectional opening of the first recess 4. The connecting passages 15 extending through the carrier 1 guide the liquid flowing out of the first recess accurately to the opposite second surface 12 of the carrier 1, so that the liquid can be accurately removed there and without contamination of other first recesses 4 formed in the same carrier 1.

[0055] In embodiment B of figure 4, the first recess 4 is connected to an outlet 9 formed in the carrier 1 in the form of a groove, the cross section of which extends into the carrier 1. The outlet 9 is connected to a connecting passage 15 around the periphery of the carrier 1. The connecting passage 15 extends along the peripheral end wall of the carrier 1 to a second surface of the carrier 1.

[0056] In embodiment C of Fig. 4, the first recess 4a is surrounded by a surrounding outlet 9 forming a supply channel through a channel portion 9a to another first recess 4b, so that the liquid F can flow from the first recess 4a to the other first recess 4b through the outlet 9 and the channel portion 9a of the outlet 9. This first recess 4b is surrounded by a connecting channel 15 through which the liquid F exiting this first recess 4b is led to the opposite second surface 12 of the carrier 1. This embodiment also shows a first recess 4a connected to another first recess 4b through a supply channel 10 as an example of a storage recess 21.

[0057] In embodiment C of figure 4, cells producing specific biomolecules, e.g. cytokines, antibodies, proteins, can be cultured in second recesses 6 assigned to first recesses 4. Through tract section 9a, these molecules are transported to another first recess 4 where they can be detected or act on other cells.

[0058] In embodiment D of FIG. 4, as also explained with reference to embodiment B, the first recess 4 is connected to an outlet 9 formed in the carrier 1 in the form of a groove, the cross section of which extends into the carrier 1. The outlet 9 is connected to a connecting passage 15 at the periphery of the carrier 1. The connecting passage 15 extends along a peripheral end wall 11 of the carrier 1 to a second surface 12 of the carrier 1. In the wall of the first recess 4 facing away from the outlet 9, a supply passage 10 is formed in the carrier 1, also in the form of a groove.

[0059] The carrier 1 may have one or more first wells 4 of the same or different configurations, e.g., A to D in Fig. 4. In the multiple first wells 4, different first wells 4 may contain different liquids or different concentrations of the same liquid or a combination thereof. Second wells 6 belonging to different first wells 4 may contain different types of cells.

[0060] 5 shows an embodiment, in which a wall extending over the first thickness 7 and having or including first recesses 4 may be formed by a first part carrier 2, which adjoins a second part carrier 3, in which an array of second recesses 6 is formed. A supply channel 10 opens into the first part carrier 2, which is preferably connected to a connecting channel 15, and an outlet 9 may preferably be formed opposite the supply channel 10, which outlet 9 is preferably connected to the connecting channel 15. The supply channel 10 and the connecting channel 15 shown here are each provided with a control valve 13, which allows the supply of liquid F into the first recess 4 and the withdrawal of liquid F from the first recess 4 to be controlled.

[0061] As shown in Fig. 6, the first part carrier 2 can be slidable relative to the second part carrier 3, for example by a slidable and liquid-tight overlap of the first and second part carriers 2, 3 in their edge regions. A seal 16 can be arranged between the first part carrier 2 and the second part carrier 3. The seal 16 is, for example, a protrusion or a rubber ring on the first part carrier 2 or on the second part carrier 3 made of plastic. Furthermore, Fig. 6 shows that the first part carrier 2 can cover the first recess 4. The covering of the first part carrier 2 can be configured as a reusable cover.

[0062] FIG. 7 shows a method in which a cell-containing liquid is introduced separately into recesses 4, 6 of a carrier 1 using a first metering device 17, and before, subsequently or simultaneously a cell-free medium is introduced using a second metering device 18.

[0063] FIG. 7 shows a carrier 1, the recesses of which consist of an array of second recesses 6, a layer of liquid forming a first thickness 7 covering the second recesses 6 is held by surface tension. For this purpose, the border of the array of second recesses 6 is preferably hydrophobic, e.g. has a hydrophobic coating. The carrier 1 has, as outlet 9, e.g. a hydrophilic area adjacent to a connecting passage 15 which extends along an end face 11 of the carrier 1 to a second surface 12 of the carrier 1 located opposite the cross-sectional opening of the recesses 6. The hydrophobic coating surrounding the second recesses 6 is preferably configured such that a drop of liquid applied on the carrier collects in a continuous layer and then flows out at the outlet 9. Alternatively, the outlet 9 may be formed by a microstructure of the surface of the carrier 1.

[0064] In the method, the outflow of liquid through outlet 9 can be controlled by tilting the carrier 1 or by contacting the liquid with a device that has capillary action.

[0065] 8 shows a carrier 1, in which the first part 2 of the carrier 1 is optionally fitted with no outlet 9, instead the inlet 10 is used simultaneously as metering device 17 and outlet 9. Alternatively, the inlet and outlet may be realised as two metering devices 17, 18. The first recess is divided perpendicularly to the cross-sectional opening of the first recess by a preferably semi-permeable diaphragm 19, which divides the common liquid volume of the first recess 4 above the second recess 6. The diaphragm 19 may likewise be combined with other embodiments, in which it divides the first recess 4 or the first thickness portion 7.

[0066] FIG. 9A shows a two-piece embodiment of the carrier 1 consisting of a first part carrier 2 and a second carrier part 3 attached liquid-tight to the first part carrier 2. With the first part carrier 2 and the second part carrier 3 made of glass or silicon, these part carriers can be joined to each other, for example by bonding. In this embodiment, the second recess 6 is spaced apart from the first recess 4 and is connected to the first recess 4 by a transition path 23. The first recess 4 may have any diameter, regardless of the second recess 6. In this case, the transition path 23 is formed by a bottom recess 24, which partially forms a spacing between the first part carrier 2 and the second part carrier 3. The first recess 4 and the second recess 6 are formed as through holes in the first part carrier 2, the bottom of the first recess 4 and the second recess 6, respectively, being formed by the second part carrier 3. In this case, the first thickness 7 is preferably identical to the second thickness 8. The second part carrier 3 has a bottom recess 24, which extends into the second part carrier by the height of the cross section of the transfer channel 23. The first part carrier 2 is connected to the second part carrier 3 along the area of ​​the second part carrier 3 that protrudes from the bottom recess 24. The bottom recess 24 forms a connecting channel with a diameter smaller than that of the cells in order to hold the cells in the second recess 6. Via the bottom recess 24, liquid exchange takes place, which minimizes the impact on the cells.

[0067] This embodiment has the advantage that the cross-sectional opening 5 of the second recess 6 is directly open and accessible, and the second recess 6 is also fluidly connected to the first recess 4 via the transition path 23.

[0068] Figure 9B shows the carrier 1 in a plan view of the cross-sectional opening 5 of the second recess 6 and the cross-sectional opening of the first recess 4. The transition path 23, shown here hatched, is covered by the first part carrier 2. A cross section along the line AA through the first part carrier 2 is shown at the first part carrier 2 in Figure 9A.

[0069] 9C shows a plan view of the first part carrier 2, in which both the first recess 4 and the second recess 6 are configured as spaced apart through holes. The second part carrier 3 has one or more bottom recesses 24, which extend to a certain depth and are covered by a cross section of at least one first recess 4 and at least two second recesses 6, forming a transition path 23 connecting the first recess 4 and the second recess 6.

[0070] FIG. 10 shows an embodiment, in which the carrier 1 consists of a first part carrier 2, preferably made of plastic, and a second part carrier 3, preferably made of glass, in which the first part carrier 2 is connected to the second part carrier 3 along the circumference of the second part carrier 3. The first recess 4 extends over a number of second recesses 6 formed in the second part carrier 3. The first part carrier 2 has an outlet 9 at a distance from the area of ​​the first recess 4, preferably at a distance from the area covered by the second part carrier 3. A supply channel 10 opens above the first recess 4 at a defined distance, by means of which the droplets F are metered into the first recess 4. The first part carrier 2 preferably has a cross-sectional opening, towards which the supply channel 10 is directed or which the supply channel 10 faces, on the side opposite the second part carrier 3. A connecting channel 15 is preferably connected to the outlet 9.

[0071] 11A and 11B show several embodiments in which the second part carrier 3 is arranged at least partially or completely in the first recess 4 of the first part carrier 2, with the cross-sectional openings of the first recess 4 and the cross-sectional openings of the second recess 6 being arranged parallel to one another and preferably lying in a common plane. In a horizontal arrangement of the first part carrier 2 and the second part carrier 3, the cross-sectional openings of the first recess 4 and the cross-sectional openings of the second recess 6 are horizontal and open upwards. The second recesses 6 have at least one through-hole 6c in each of their bottoms 6b, so that even if the second part carrier 3 protrudes above the cross-sectional openings of the first recess 4 and / or above the level of the liquid F in the first recess 4, each second recess 6 is still connected to the first recess 4 via at least one through-hole 6c. The bottom 6b of the second recess 6 formed in the second part carrier 3 is remote from the first part carrier 2, and preferably a spacer 25 is arranged between the bottom 6b of the second part carrier 6 and the first part carrier 2. The through-holes 6c preferably have a cross-section smaller than the respective cell Z. The through-holes 6c may for example be coated hydrophobically, which prevents or supports the exchange of certain liquids.

[0072] As shown in Fig. 11B, the first part carrier 2 may have an outlet 9, which is arranged, for example, below the second part carrier 3. A connecting line 15 may be connected to the outlet 9. Optionally, the connecting line 15 may form a supply line 10. Alternatively or additionally, the supply line 10 may open above the first part carrier 2, by means of which the liquid F is metered into the first recess 4.

[0073] The embodiment in Figures 11A and 11B is particularly suitable for separating cells and cell products from each other.

[0074] In the method, the second recess 6 and the first recess 4 are filled with a liquid F, in particular a culture medium, and are in contact with each other via the through holes 6c. In particular in the embodiment in which the supply channel 10 opens in the first part carrier 2 below the second recess 6 or below the second part carrier 3, a gas can be introduced through the supply channel 10 additionally or alternatively to the liquid F, for example an inert gas or air, for example air or oxygen for cell culture, optionally air or O2 with 5% CO2. By a sufficiently low volumetric flow rate of the gas, the gas can be supplied without bubbles. Alternatively, the gas can be supplied with a volumetric flow rate sufficient for the formation of bubbles, whereby the liquid F moves in the second recess and optionally the cells Z are suspended by the bubbles G. The bubbles can alternatively be generated by cavitation caused by laser radiation.

[0075] The pressure change can cause the cells Z to move away from the through-holes 6c, thereby promoting the exchange of liquid.

[0076] 12 shows an embodiment in which the carrier 1 consists of a second part carrier 3 with a second recess 6, the cross-sectional openings 5 ​​of the second recess 6 and the area of ​​the surface of the carrier 1, 3 between the cross-sectional openings 5 ​​lie in a common plane. If these surface areas between the cross-sectional openings 5 ​​in particular have a surface energy that does not or hardly allows wetting by the liquid F, for example surface areas made of plastic or made of functionalized, coated or hydrophobically structured glass, then the liquid F moving on the plane E, for example in the form of a rolling drop Fr, can absorb and at least partially replace the used medium from the second recess 6. The rolling drop Fr can be introduced by providing the liquid F in the form of a number of drops on the plane E or in the second recess 6, for example via a supply channel 10 opening into the second recess 6, as shown in FIG. 2. The liquid F, particularly in the form of a rolling drop Fr, protruding beyond the plane E, can be set in motion by tilting the carrier 1 relative to the horizontal, by accelerating and / or vibrating the carrier, by applying a gas flow.

[0077] Generally, in the embodiment where the carrier consists of a second part carrier 3 having a second recess 6 therein, the second part carrier may be referred to as a carrier 3 having a recess 6 therein. This application relates to the invention described in the claims, but also includes the following as other aspects. 1. A carrier (1) for culturing cells (Z), comprising: A carrier (1) comprising at least one first recess (4, 4a, 4b), the first recess (4, 4a, 4b) extending across a first thickness (7) from a first surface (20) of the carrier (1) to a second thickness (8) adjacent the first thickness (7), and an array of a plurality of second recesses (6) extending from the first recess (4, 4a, 4b) in the second thickness (8); The second recess (6) has a depth to diameter aspect ratio of at least 1:1 and a maximum diameter of 1 mm, and the carrier (1) has an outlet (9) whose cross-section opens into or adjacent to the first thickness portion (7). 2. The carrier (1) as described above, wherein the outlet (9) has a cross section protruding into the carrier (1) and opening toward the plane of the first surface (20) of the carrier (1). 3. The carrier (1) according to claim 1 or 2, characterized in that a connecting passage (15) is provided, the connecting passage (15) being connected to the outlet (9) and extending to a second surface (12) opposite the first surface (20) of the carrier (1). 4. 4. The carrier (1) according to any one of claims 1 to 3, characterized in that the connecting passage (15) extends along the end wall (11) of the carrier (1) or through the carrier (1). 5. 5. Any one of the carriers (1) as defined in claims 1 to 4, characterized in that the outlet (9) and the connecting channel (15) connecting to the outlet (9) have hydrophilic surfaces, and a region of the first surface (20) and / or a region of the second surface (12) of the carrier (1) are hydrophobic. 6. 6. Any one of the carriers (1) above, 1 to 5, characterized in that the carrier (1) is one piece of glass. 7. Any one of the carriers (1) described above in 1 to 6, characterized in that the carrier (1) comprises a second partial carrier (3) extending across the second thickness portion (8) and a first partial carrier (2) extending across the first thickness portion (7), and the first partial carrier (2) and the second partial carrier (3) are arranged liquid-tight relative to each other. 8. Any one of the carriers (1) as set forth above in 1 to 7, characterized in that the carrier (1) comprises a second partial carrier (3) extending across the second thickness portion (8) and a first partial carrier (2) extending across the first thickness portion (7), and the first partial carrier (2) and the second partial carrier (3) are arranged liquid-tightly and slidably relative to each other. 9. 9. The carrier (1) according to any one of claims 1 to 8, characterized in that the first part carrier (2) is made of glass, plastic and / or silicon. 10. 10. The carrier (1) according to any one of claims 1 to 9, characterized in that the second part carrier (3) consists of glass and / or silicon. 11. Any one of the carriers (1) as set forth in 1 to 10 above, characterized in that a supply channel (10) is formed in the first surface (20) or the first thickness portion (7) of the carrier (1), and the supply channel (10) opens into the first recess (4, 4a, 4b). 12. 12. Any one of the carriers (1) as defined in claims 1 to 11, characterized in that at least two first recesses (4, 4a, 4b) are formed in the carrier (1), each of which is surrounded by a connecting path (15) arranged around the periphery and extending through the carrier (1). 13. 13. The carrier (1) according to any one of claims 1 to 12, characterized in that the carrier (1) is formed with at least two first recesses (4, 4a, 4b), at least one of these first recesses (4, 4a, 4b) being surrounded by an outlet (9) in the form of a groove opening into another one of the first recesses (4, 4a, 4b), the other one of the first recesses (4, 4a, 4b) being surrounded by a connecting path (15) arranged around the periphery and extending through the carrier (1). 14. 14. The carrier (1) according to any one of claims 1 to 13, characterized in that a control valve (13) is arranged in the outlet (9), in the connecting line (15) and / or in the supply line (10), respectively. 15. 15. Any one of carriers (1) as defined in claim 1 to 14, wherein the second partial carrier (3) is at least partially arranged in the first recess (4) of the first partial carrier (2), and the second recesses (6) each have at least one through hole (6c) in their bottom (6b), such that each second recess (6) is connected to the first recess (4) via at least one through hole (6c). 16. Any one of the carriers (1) above from 1 to 15, characterized in that the first recess (4) and the second recess (6) are formed in the first partial carrier (2) as through holes located apart from each other and are connected to each other via a transition path (23) formed by the bottom recess (24), the bottom recess (24) being formed as a partial space between the first partial carrier (2) and the second partial carrier (3). 17. The carrier (1) of claim 16, characterized in that the first thickness portion (7) is preferably identical to the second thickness portion (8), the second part carrier (3) has a bottom recess (24) which extends into the second part carrier (3) by the height of the cross section of the transition path (23), and the first part carrier (2) is connected to the second part carrier (3) along the area of ​​the second part carrier (3) protruding from the bottom recess (24). 18. 1. A method for culturing cells, comprising: A method for culturing cells, comprising introducing a liquid (F) having cells (Z) dispersed therein into a second recess (6) of the carrier (1) of any one of 1 to 17 above, followed by discharging the liquid from the first thickness portion (4, 4a, 4b) along the outlet (9) and the connecting path (15). 19. 19. The method according to claim 18, characterized in that the second recesses (6) are spaced apart from one another via the surface of the second partial carrier (3), the first carrier (2) is formed exclusively by the surface of the second partial carrier (3), and the first recesses (4, 4a, 4b) adjacent to the array of second recesses (6) are formed exclusively by a liquid layer generated by surface tension. 20. 20. The method according to claim 18 or 19, characterized in that the second recesses (6) are spaced apart via a surface of the second part carrier (3), and the surface of the second part carrier (3) is a hydrophobic surface. 21. 21. The method according to any one of claims 18 to 20, characterized in that the carrier (1) has only the second recess (6) and the culture medium forms a layer that protrudes beyond the plane of the cross-sectional opening of the second recess (6), the layer forming the volume of the first recess. 22. 22. The method according to any one of claims 18 to 21, characterized in that the carrier (1, 3) has recesses (6) whose cross-sectional openings (5) are mutually spaced apart by a surface area of ​​the carrier (1, 3), said surface area and the cross-sectional openings (5) of the recesses (6) being located in a common plane (E), and a liquid (F) is supplied onto said plane (E) and the liquid (F) is moved along the carrier (1, 3) by tilting the carrier (1, 3) relative to the horizontal, vibrating the carrier (1, 3) and / or applying compressed gas to said plane (E). 23. 23. Any one of the methods described above in 18 to 22, characterized in that the liquid (F) is supplied onto the plane (E) through a supply channel (10) opening into the bottom (6c) of the recess (6). [Explanation of symbols]

[0078] 1. Career 2 First Part Carrier 3 Second Part Carrier 4, 4a, 4b First recess 5 Cross-sectional opening of second recess 6 Second recess 6b Bottom of second recess 6c through hole 7 First thickness part 8 Second thickness section 9 exit 9a Exit road section 10 Supply route 11 End wall, end face 12 Second Surface of the Carrier 13 Control valve 14 Bottom of second recess 15 Connecting Road 16 Seal 17 First metering device 18 Second metering device 19 Diaphragm 20 First surface of carrier 21 Reservoir recess 22 Culture medium 23 Transitional Path 24 Second section carrier floor recess 25 Spacer E plane F liquid Fr Rotating Drops Z cell G. Air bubbles

Claims

1. A carrier (1) for culturing cells (Z), comprising: The carrier (1) comprises at least one first recess (4, 4a, 4b), the first recess (4, 4a, 4b) extending across a first thickness (7) from a first surface (20) of the carrier (1) to a second thickness (8) adjacent the first thickness (7), and in the second thickness (8) extending from the first recess (4, 4a, 4b) an array of a plurality of second recesses (6) forming wells having bottoms; A carrier (1), wherein the second recess (6) has a depth to diameter aspect ratio of at least 1:1 and a maximum diameter of 1 mm, the carrier (1) having an outlet (9) whose cross section opens into or adjacent to the first thickness portion (7), The carrier (1) is one piece and made of glass, or the carrier (1) comprises a second part carrier (3) made of glass extending over a second thickness (8) and a first part carrier (2) made of glass extending over a first thickness (7), the first part carrier (2) and the second part carrier (3) being arranged liquid-tight with respect to one another and being directly connected to one another by bonding or by a bonding material, A carrier (1), characterized in that a supply channel (10) is formed in a first surface (20) or a first thickness portion (7) of the carrier (1), the supply channel (10) opening into a first recess (4, 4a, 4b).

2. 2. The carrier (1) according to claim 1, characterized in that the outlet (9) has a cross section projecting into the carrier (1) opening towards the plane of the first surface (20) of the carrier (1).

3. 2. The carrier (1) according to claim 1, characterized in that a connecting passage (15) is provided, which is connected to the outlet (9) and extends to a second surface (12) of the carrier (1) opposite the first surface (20).

4. 4. The carrier (1) according to claim 3, characterized in that the connecting passage (15) extends along an end wall (11) of the carrier (1) or through the carrier (1).

5. 5. The carrier (1) according to claim 3 or 4, characterized in that the outlet (9) and the connecting channel (15) connecting to the outlet (9) have a hydrophilic surface, and an area of ​​the first surface (20) and / or an area of ​​the second surface (12) of the carrier (1) are hydrophobic.

6. 2. The carrier (1) according to claim 1, characterized in that at least two first recesses (4, 4a, 4b) are formed in the carrier (1), each of which is surrounded by a connecting path (15) arranged around its periphery and extending through the carrier (1).

7. 2. The carrier (1) according to claim 1, characterized in that in the carrier (1) at least two first recesses (4, 4a, 4b) are formed, at least one of these first recesses (4, 4a, 4b) being surrounded by an outlet (9) in the form of a groove opening into another one of the first recesses (4, 4a, 4b), the other one of the first recesses (4, 4a, 4b) being surrounded by a circumferentially arranged connecting path (15) extending through the carrier (1).

8. 2. The carrier (1) according to claim 1, characterized in that a control valve (13) is arranged at the outlet (9), in the connection (15) and / or in the supply line (10), respectively.

9. 2. The carrier (1) according to claim 1, wherein the second part carrier (3) is at least partially arranged in a first recess (4) of the first part carrier (2), and the second recesses (6) each have at least one through hole (6c) in their bottom (6b), so that each second recess (6) is connected to the first recess (4) via at least one through hole (6c).

10. 2. The carrier (1) according to claim 1, characterized in that the first recess (4) and the second recess (6) are formed in the first partial carrier (2) as through holes located at a distance from each other and are connected to each other via a transition path (23) formed by a bottom recess (24), the bottom recess (24) being formed as a partial gap between the first partial carrier (2) and the second partial carrier (3).

11. 2. The carrier (1) according to claim 1, characterized in that the first thickness portion (7) is identical to the second thickness portion (8), the second part carrier (3) has a bottom recess (24) which extends into the second part carrier (3) by the cross-sectional height of the transition path (23), and the first part carrier (2) is connected to the second part carrier (3) along the area of ​​the second part carrier (3) which protrudes from the bottom recess (24).

12. 1. A method for culturing cells, comprising: A method for culturing cells, characterized in that a liquid (F) in which cells (Z) are dispersed is introduced into the second recess (6) of the carrier (1) according to claim 1, 2, 3, 6, 7, 8, 9, 10 or 11, followed by discharging the liquid from the first thickness portion (4, 4a, 4b) along the outlet (9) and the connecting passage (15).

13. 13. The method according to claim 12, characterized in that the second recesses (6) are spaced apart via a surface of the second part carrier (3), the first carrier (2) is formed exclusively by the surface of the second part carrier (3), and the first recesses (4, 4a, 4b) adjacent to the array of second recesses (6) are formed exclusively by a liquid layer caused by surface tension.

14. 13. The method according to claim 12, characterized in that the second recesses (6) are spaced apart via a surface of the second part carrier (3), the surface of the second part carrier (3) being a hydrophobic surface.

15. 13. The method according to claim 12, characterized in that the carrier (1) only has a second recess (6) and the culture medium forms a layer which projects beyond the plane of the cross-sectional opening of the second recess (6), the layer forming the volume of the first recess.

16. 13. The method according to claim 12, characterized in that the carrier (1, 3) has recesses (6) whose cross-sectional openings (5) are mutually spaced apart by a surface area of ​​the carrier (1, 3), said surface area and the cross-sectional openings (5) of the recesses (6) being located in a common plane (E), that a liquid (F) is supplied onto said plane (E) and that the liquid (F) is moved along the carrier (1, 3) by tilting the carrier (1, 3) relative to the horizontal, by vibrating the carrier (1, 3) and / or by applying compressed gas to said plane (E).

17. 13. A method according to claim 12, characterized in that the liquid (F) is supplied onto said plane (E) through a supply channel (10) which opens into the bottom (6c) of the recess (6).

Images