DEVICE AND METHOD FOR CELL CULTURE

Description

[0001] The present invention relates to an apparatus, the use of the apparatus for cell culture, and a method for cell culture using the apparatus. The apparatus is characterized in that it has separate wells for receiving cells, the wells being in contact with a larger volume, thus allowing fluid exchange. The wells have a large aspect ratio (depth to diameter), which retains cells within the wells and / or suppresses the release of cells from the wells into the larger volume in contact with them. The method has the advantage of retaining cells, which can be single or multiple cells, within their wells, with several wells being in contact with a common volume of medium.By keeping cells in wells with a high elongation ratio, the method has the advantage that cells can be identified based on the wells they contain and observed over a longer cultivation period, e.g., 1 to 10 days. Due to the high elongation ratio of the wells, in conjunction with a variation in the diffusion rate, e.g., by varying the inflow and outflow of medium, either metabolic products can accumulate separately in each well and be assigned to the well and the cells contained therein, or particularly constant conditions can be created through rapid exchange of medium and removal of metabolic products.

[0002] Conventionally, microtiter plates are used for cell culture and biological experiments in such procedures. Microtiter plates have wells with a volume of, for example, approximately 300 µl. One biological experiment can be performed per well, therefore a large quantity of reagents is required per well. Furthermore, microtiter plates are relatively large and typically only offer space for 96 and up to 1536 parallel experiments. Locating individual cells in these wells, for example using a light microscope, is time-consuming.

[0003] This poses major challenges for high-throughput experiments in which specific cell types are to be cultured and studied, e.g. cell line development, development of monoclonal antibodies, synthetic biology, cell therapy, immunotherapy, stem cell research.

[0004] The use of highly miniaturized wells, e.g., volumes reduced by a factor of 100,000, is technically feasible, but presents new challenges, particularly regarding evaporation, nutrient supply, concentration of metabolic products, and cell migration. The latter arises from the movement of cells by their own force or from turbulence during refilling of reagents or media, movement of the supports, heating, and convection.

[0005] For most biomedical developments, consistent conditions must be ensured in each individual experiment. Monoclonality must be demonstrated for the approval of medical products. Therefore, the purpose of this invention is to solve these challenges. State of the art

[0006] DE 10 2020 209 825 A1, which was published after the priority date of the present application and can therefore only be considered with regard to novelty, describes a method for producing a plastic part having a glass insert, from a prefabricated plastic part with a circumferential welded rib and a glass insert which has spaced-apart through-holes in a circumferentially closed contact area, and heating the welded rib and pressing the glass insert with its contact area against the welded rib of the plastic part to produce a connection area in which plastic rests in the contact area and has passed through the through-holes of the glass insert.

[0007] DE 10 2019 217 466 A1, which according to Article 54(3) EPC can only be considered with regard to novelty, describes glass reaction vessels formed as recesses of at least 30 µm depth in a single glass plate or in two interconnected glass plates. The recesses may have a section extending only to a first thickness section in the glass plate, from which further smaller recesses may extend to a deeper second thickness section.

[0008] WO 2016 / 041544 A1 generally describes a preferred method for producing recesses in glass in the present invention.

[0009] WO 2016 / 076795 A1 describes a microfluidic plate made of plastic with fully enclosed channels for cell cultivation, with a connection at each end for an inlet or outlet.

[0010] CN 107338183 A shows a carrier with recesses suitable for cells, each having a bore in the base for connection to an outlet pipe opposite the recess, the recesses being covered by a spaced cover enclosing a common supply line.

[0011] DE 20 2014 105 173 U1 shows a frame open on both sides for covering the wells of a microtiter plate, which is covered and encompassed by the protruding edge of a cover plate in order to cover one side of the frame when a microtiter plate is clamped into the frame.

[0012] EP 2 011 857 A1 describes the manufacture of a container for cell culture with recesses, the bottoms of which have even smaller depressions. Object of the invention

[0013] The invention aims to provide an alternative method for cultivating cells and a carrier suitable for use in the method, with recesses for cells, in order to hold cells in their separate wells and yet allow several wells to be connected to a common volume of medium, forming a common reservoir of medium. The carrier is designed to allow liquid, in particular cell culture medium, to drain or be extracted from the recesses in a controlled manner. Description of the invention

[0014] Cell cultivation, as described here, is an in vitro procedure, specifically the cultivation of cells in a medium that keeps them alive and / or is suitable for cell proliferation. The cells can be, for example, animal cells, especially human cells, plant cells, fungal or yeast cells, or bacteria. Cultivation can be performed, for example, for the analysis and / or isolation of cells, optionally with the addition of an active substance to the medium.

[0015] The invention solves the problem with the features of the claims, and in particular by a support suitable for cell cultivation, preferably a plate-shaped support, e.g., made of glass, silicon, or plastic, or a combination thereof, in which at least one first recess is formed extending over a first thickness section of the support, wherein an arrangement of second recesses extends from the first recess into a second thickness section of the support adjacent to the first thickness section. The first thickness section can have a thickness of a few micrometers to several centimeters. The second recesses, extending from a first recess into the second thickness section and each forming a second arrangement, constitute wells suitable for receiving cells and / or synthetic particles, e.g., made of plastic or glass.The second set of recesses, also called cups, have a high aspect ratio of depth to diameter, which is determined in the plane of the first thick section adjacent to the second thick section or at half the height of the second thick section. The cups have, for example,a depth-to-diameter ratio 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 cups 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 a maximum of 20 µm, preferably a maximum of 10 µm, preferably a maximum of 5 µm.

[0016] The diameter of the wells can be adapted to the size of the cells, for example, to arrange cells in a stack or to bring them into 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 also have additional microstructures.

[0017] The first thickness section is bounded in a plane by the first surface of the support, in which the cross-sectional openings of the first recesses lie, and in a plane spaced apart from this by the cross-sectional openings of the second recesses. The cross-sectional openings of the second recesses are spaced apart by surface areas of the support, which is in particular a second partial support. More preferably, these surface areas and the cross-sectional openings of the second recesses lie in a common plane. According to the invention, the cross-sectional openings of the second recesses border directly on the cross-section of a first recess.

[0018] The arrangement of the second recesses can be arbitrary, but is preferably arranged regularly, e.g. in parallel rows, at nodes of a grid and / or at equal intervals, which is e.g. a square or hexagonal grid. An arrangement of the second recesses, extending adjacent to a first arrangement, can be, for example, 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 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 10,000, preferably at least 50,000, preferably at least 100,000, preferably at least 500,000, preferably at least 1,000.000, preferably at least 10,000,000, preferably at least 50,000,000, preferably at least 100,000.000 second recesses, spaced at a maximum distance of 10 mm, preferably a maximum of 9 mm, preferably a maximum of 8 mm, preferably a maximum of 7 mm, preferably a maximum of 6 mm, preferably a maximum of 5 mm, preferably a maximum of 4 mm, preferably a maximum of 3 mm, preferably a maximum of 2 mm, preferably a maximum of 1 mm, preferably a maximum of 900 µm, preferably a maximum of 800 µm, preferably a maximum of 700 µm, preferably a maximum of 600 µm, preferably a maximum of 500 µm, preferably a maximum of 400 µm, preferably a maximum of 300 µm, preferably a maximum of 200 µm, preferably a maximum of 100 µm, preferably a maximum of 90 µm, preferably a maximum of 80 µm, preferably a maximum of 70 µm, preferably a maximum of 60 µm, preferably a maximum of 50 µm, preferably a maximum of 40 µm, preferably a maximum of 30 µm, preferably a maximum of 20 µm, preferably a maximum of 10 µm, preferably a maximum of 9 µm, preferably a maximum of 8 µm, preferably a maximum of 7 µm, preferably a maximum of 6 µm, preferably a maximum of 5 µm, preferably a maximum of 4 µm, preferably a maximum of 3 µm, preferably a maximum of 2 µm, preferably a maximum of 1 µm apart.

[0019] The cross-sectional opening of the first recess lies in the plane of a first surface of the beam, so that the first recess opens into the plane of the first surface of the beam. The beam is characterized by an outlet whose cross-section opens into or adjacent to the first thickness section, wherein the cross-section of the outlet preferably adjoins the plane of the first surface of the beam, or is open to the plane of the first surface of the beam, and the outlet is connected to the first recess. The outlet is formed in the beam, e.g., in the form of a groove that is open to the plane of the first surface of the beam. Alternatively, the outlet can be a conduit that extends into the first recess. Generally, the outlet is designed to discharge liquid, in particular a medium, from the first recess.The outlet can be configured to discharge liquid solely by the action of gravity and / or by means of a pump and / or by pressurizing the first recess. The outlet can have a cross-section extending into the support to a depth that is, for example, 1 to 95% of the first thickness section. Such an outlet serves to drain or extract liquid that extends in the first recess up to the cross-section of the outlet, particularly when the liquid level in the first recess reaches the plane of the first surface of the support. The outlet can extend circumferentially around at least one, preferably exactly one, first recess, so that the outlet forms a circumferential recess in the support around a first recess, the circumferential recess being bounded by the support, which extends at a distance from this outlet up to the plane of its first surface.In the embodiment of the outlet as a circumferential recess or hydrophilic surface formed around a first recess, exactly one or at least two connecting lines can be connected to this outlet. Optionally, the outlet can additionally form an inlet connected to the first recess, particularly for liquid, e.g., medium, or gas.

[0020] The rate at which fluid exits or is drawn off at the outlet can be controlled by the thickness of the first thickness section, the rate of fluid supply, or the inclination of the support. The outlet can be formed by multiple openings, for example, holes of different diameters. The outflowing fluid can be collected and analyzed or reused.

[0021] In a further embodiment, the carrier can have a hydrophilic surface as an outlet, comprising exactly one or at least two first recesses, wherein this outlet is surrounded by a hydrophobic surface or coating of the first surface of the carrier. In this embodiment, the hydrophilic surface comprising a first recess as an outlet can lie in the same plane as the first surface of the carrier, which is preferably adjacent to it and hydrophobic. In this embodiment as well, a connecting line can be arranged in the outlet, which consists of a hydrophilic surface section on the carrier, optionally without a recess in the carrier.In this process, the hydrophilic surface of this outlet, even if it lies in the plane of the first surface of the support, allows liquid to pass from the first recess into the connecting line, whereby the spread of the liquid onto the first surface is limited by the hydrophobicity of the first surface adjacent to this outlet.

[0022] The support structure is equipped with an outlet and preferably a connecting line for a cell cultivation process in which liquid, in particular cell culture medium, can be selectively drained or extracted from recesses. A connecting line links the outlet to a remote extraction point, which may be, for example, a container for liquid or an analytical device. Generally, a connecting line can be designed as a recess in the support structure, having a cross-section open to the surface of the support structure, such as a groove, or a fully enclosed cross-section, such as a bore through the support structure.

[0023] In one embodiment, a connecting line is formed within the support and / or on an end face of the support, extending from the first surface of the support across its entire cross-section to its second surface. The connecting line is connected to the outlet opposite the second surface of the support, allowing fluid from the outlet to flow through the connecting line into the plane of the second surface of the support, particularly when the support is arranged horizontally with its first surface above the second surface.

[0024] In certain embodiments, the connecting line directs liquid, whose level in the first recess reaches the cross-section of the outlet and flows through it, to the opposite second surface of the support. Therefore, when liquid is introduced into the first recess, liquid whose level reaches the cross-section of the outlet is discharged in a controlled manner, and the level will not exceed the cross-section of the outlet and, in particular, the first surface of the support. The opening of the connecting line in the second surface of the support allows the liquid emerging there to be drawn off, e.g., for subsequent analysis, without an additional sampling device, e.g., a pipette, coming into contact with the liquid in the first recess.

[0025] The surfaces of the connecting pipe and the outlet are preferably hydrophilic; more preferably, the first surface of the support and / or the opposing second surface of the support is hydrophobic, e.g., provided with a hydrophobic coating. Optionally, a groove is formed in the second surface of the support at a distance around the opening of the connecting pipe, framing the opening. A groove in the second surface of the support framing the opening of the connecting pipe reduces the spread of liquid exiting the opening across the second surface of the support.

[0026] Preferably, a carrier has at least two first recesses, each with an arrangement of second recesses adjoining its first thick section, each first arrangement being connected to at least one or exactly one connecting line by means of at least one outlet. In this embodiment, at least two first recesses are independently connected to a connecting line, so that liquid exiting from the first recess exits separately for each first recess on the second surface from a connecting line and can be separately sampled and / or analyzed.

[0027] Optionally, a carrier has at least two first recesses, at the first thickness section of each of which an arrangement of second recesses is attached, wherein the outlet connected to one first recess opens into another first recess, so that the outlet is configured to direct liquid exiting from one first recess into the other first recess.

[0028] In general, at least one supply line can optionally be formed adjacent to the first surface of the support, opening into at least one first recess. A supply line can, for example, be designed as a trench in the support, with the cross-section of the trench being open in the plane of the first surface of the support. At a distance from a first recess, a reservoir recess extending into the support can be connected to the supply line. A reservoir recess, for example with a depth equal to or less than the depth of the supply line, can serve to hold liquid, such as cell culture medium, which is metered into the reservoir recess.

[0029] The first and second recesses can be formed in a single-piece carrier, preferably a glass carrier. Recesses in a single-piece carrier can be produced by removing material from the original single-piece carrier, e.g., by laser irradiation followed by etching. Alternatively, the first recesses can be through holes in a first sub-carrier to which a second sub-carrier is attached, in which the second recesses are formed. If the carrier is formed from at least two sub-carriers, the carrier is two-piece, and the sub-carriers can be directly connected to each other, e.g., by bonding, or connected to each other by a bonding agent, e.g., molten glass frit or adhesive.The second recesses can be through holes in the second sub-support, the cross-sectional opening of which is covered by a third sub-support relative to the first sub-support, or blind holes in the second sub-support. Optionally, the second sub-support is made of colored glass, the optional third sub-support of transparent glass, and the first sub-support is made of uncolored or transparent glass. Transparent glass is transparent to light that is shone onto the support for optical detection and to light that can emanate from the second recesses for optical detection. Colored glass is preferably opaque to light that is shone onto the support for optical detection and to light that can emanate from the second recesses for optical detection. Preferably, the support or all sub-supports are made of glass.

[0030] The large elongation ratio of the second cavities ensures that cells introduced into them remain within the second cavity, enabling a cell culture method in which fluid is introduced into or removed from the first cavity while the cells remain in the second cavities. The internal volumes of the second cavities are in contact with the internal volume of the first cavity, allowing for an exchange between them, for example, through diffusion of metabolic products from the second cavity into the first cavity, and of nutrients, dissolved oxygen, and any added active substances from the first cavity into the second cavity.

[0031] Optionally, a sensor is arranged in a first recess, in one of the second recesses, in the outlet and / or inlet, e.g., in a connecting line. A sensor can be a gas sensor, e.g., for oxygen or carbon dioxide, a pH sensor, a temperature sensor, or a binding molecule specific to an analyte, preferably immobilized, e.g., an antibody specific to an analyte in the medium. Optionally, an identical sensor can be arranged in both the inlet and the outlet, connected to an evaluation unit configured to detect changes in the sensor signals.

[0032] The introduction of liquid into the first recesses can be achieved by dropping it or by pouring it through a line that is in contact with the liquid in the first recess, in each case through the cross-sectional opening of the first recess in the plane of the first surface of the support. Alternatively, the support can have a supply line that extends directly from its second surface into the first recess, such that the supply line is configured to convey liquid and / or gas from the second surface into the first recess. Generally, if the support is arranged horizontally, its second surface is arranged horizontally and below its first surface, with the first and second surfaces preferably being parallel to each other. Alternatively or additionally, a supply line can extend through a wall section within the first thickness section in which a first recess is formed, so that liquid, e.g., gas, can be supplied through the supply line.B. Medium for cell culture, can be introduced into the first recess in the first thick section.

[0033] In the first thickness section of the support, an outlet can be designed as a conduit, e.g. in a first recess in a wall that is opposite a supply line.

[0034] In one embodiment, the wall extending over the first thickness section and comprising or including the first recesses can be formed by a first partial beam adjoining a second partial beam in which second recesses are formed. The first partial beam can be placed onto the second partial beam, for example, by the first and second partial beams overlapping in a slidable and liquid-tight manner at their edges. Optionally, the first partial beam can be slidably perpendicular to the second partial beam in a liquid-tight manner, so that the first thickness section can be adjusted by the slidability of the first partial beam relative to the second partial beam. If first recesses are formed in a first partial beam that is liquid-tight and can be placed on top of the second partial beam, the first partial beam can be slidably and liquid-tightly overlapped.The carrier is arranged to be slidable relative to the second sub-carrier, which has second recesses in it. The carrier is designed to adjust the liquid level in the first recess depending on the position of the first sub-carrier relative to the second. This is because the outlet, especially if it lacks a valve, drains liquid from the first recess when its level reaches its cross-section. The first sub-carrier can be made of plastic and the second of glass, or both the first and second sub-carriers can be made of glass, optionally with a sliding seal in the overlap area of ​​the sub-carriers. Alternatively, a valve can be arranged in the inlet and / or outlet to adjust the liquid level in the first recess, allowing, for example, the liquid level in the first recess to be adjusted using a specific method.

[0035] In one embodiment, inlets and / or outlets can have one or more valves that regulate one or more connecting lines. With multiple valves and multiple connecting lines, it is possible to meter different liquids into the first recess in a controlled manner.

[0036] In one embodiment, the support can consist of a second sub-support with second recesses arranged therein and a first sub-support attached to it, the first sub-support having exactly one first recess that extends over all second recesses. Generally, and particularly in this embodiment, the first sub-support can be made of plastic or glass, and the second sub-support can be made of glass with second recesses therein. In embodiments where the first sub-support is made of plastic and the second sub-support is made of glass, it is preferred that the second sub-support has through-holes in the area of ​​its connection with the first sub-support, and that plastic from the first sub-support extends integrally through these through-holes, for example, by pressing the plastic from the first sub-support into and, if necessary, through these through-holes after heating the first and / or second sub-support in the connection area.Preferably, the first partial support has an outlet at a distance from the area covered by the second partial support, optionally with a connecting line attached to it.

[0037] Cutouts in glass carriers are preferably produced by irradiating the glass with laser radiation and subsequent etching.

[0038] In general, in any embodiment, a first recess can be subdivided by a membrane, which is preferably arranged perpendicular to the first surface of the support, so that, for example, it is arranged vertically when the support or its first surface is arranged horizontally. A membrane that subdivides a first recess can, for example, be stretched by a frame that, for example, abuts surfaces of the support located between the cross-sectional openings of the second recesses.

[0039] In this process, liquid is generally introduced into the first recess, while the second recesses contain liquid with at least one cell. This is achieved, for example, by selectively introducing liquid containing cells into the second recesses, e.g., by means of a droplet generator or a dosing device, which is in particular a nozzle. Alternatively, a cell-containing liquid can be introduced into the first recess so that it spreads into the second recesses. In this way, for example, cells can disperse from the liquid into the second recesses. Optionally, the cell-containing liquid remaining in the first recess can then be removed. The liquid, which is preferably a cell culture medium, can be in mixture with suspended cells and be used as a mixture or...Suspension can be introduced into the recesses, or the liquid containing the cells can be introduced into recesses in a separate step using a separate droplet generator or a separate dosing device, and a cell culture medium that does not contain cells can be introduced into recesses in a separate step, preferably subsequently, preferably using a separate dosing device.

[0040] In the present case, a cell culture medium comprises a medium that keeps cells, in particular plant cells, yeast, bacteria or animal cells, preferably human cells, viable, e.g. for at least 1h, preferably for at least 12h or at least 24h or at least two days or at least three days or at least four days or at least five days or at least six days or at least seven days.

[0041] In an embodiment of the method, in which the carrier optionally has only recesses in the form of the second recesses and further optionally at least one outlet, optionally at least one connecting line connected thereto, or consists thereof, liquid can be applied directly to the cross-sectional openings of the second recesses, which lie in the plane of the first surface of the carrier. In an embodiment in which the carrier has only second recesses and medium projects beyond the plane of the cross-sectional openings of the second recesses, this projecting layer of medium forms the volume of the first recess and extends over the first thickness section, so that this medium covers the second recesses. The medium can extend over a first thickness section of, for example, 1 µm to 3 mm, or, for example, 10 µm to 200 µm above the second recesses.The liquid extending beyond the plane of the first surface of the support can flow over the edge of the support, preferably along the at least one outlet and optionally along the connecting line. This liquid can be set in motion by tilting the support, by applying a gas stream, and / or by vibration, e.g., ultrasound. Particularly in this embodiment, the first surface can be poorly wettable for aqueous liquids, e.g., hydrophobic, e.g., made of plastic or a structured, functionalized, or coated glass surface. A liquid droplet applied to the first surface or to the plane in which the cross-sectional openings of the second recesses are located can undergo mass exchange with any liquid present there as it moves across this plane. For example, a droplet of medium applied to this plane can, as a rolling droplet, interact with any liquid already present, e.g.,The carrier absorbs and / or partially replaces the consumed medium. Optionally, the surface of the carrier, which lies in the plane where the cross-sectional openings of the second recesses are located, can have channels incorporated into the carrier, the cross-section of which is open to this plane, in order to direct the fluid extending across this plane as it moves. The fluid can be applied to this plane of the carrier in pulses or in batches as at least two drops. When the volume of fluid above the plane in which the carrier extends exceeds the limit, fluid will drain off, in particular through an outlet.

[0042] In general, fluid, preferably a medium, extending across the first thickened section prevents the evaporation of medium from the second cavities. The first thickened section can range in size from a few micrometers to several centimeters. Its large volume provides sufficient space for fluid to mitigate the effects of nutrient consumption and metabolic product accumulation in the second cavities through diffusion. The large elongation ratio of the second cavities ensures that fluid movement within the adjacent first thickened section or the first cavity has only a minimal impact on the cells in the second cavities, preventing them from being flushed out.

[0043] For example, a first cavity can be connected to 100,000 second cavities, where each second cavity has a diameter of 54 µm, a depth of 436 µm, a depth-to-diameter ratio of 8:1, and a volume of 1 nl. The cavities are spaced, for example, 10 µm apart. To encompass all second cavities, the first cavity must have an area of ​​at least 410 mm². Conventionally, cups have significantly smaller aspect ratios, for example, 0.16:1. With the same volume of 1 nl per cup and a diameter of 200 µm, the depth is approximately 32 µm. In this case, 100,000 cavities occupy an area of ​​4410 mm², ten times larger than the area of ​​the cups with the large aspect ratio.

[0044] Due to the smaller surface area for the same volume per bowl, it is possible to monitor the contents of several bowls in parallel using an optical microscope.

[0045] The large elongation ratio of the second cavities offers several advantages. For example, cell products, such as proteins or antibodies, can accumulate in these cavities with little or no removal of cell products. This can be used to analyze these cell products and their production rate, for example, by attaching fluorescent markers and evaluating the fluorescence intensity. If cell products, such as metabolic products, are removed faster than they are produced by the cells, ideal cultivation conditions can be created. Switching between slow and fast removal of cell products can be achieved, for example, by varying the inflow and outflow rates of the culture medium or other fluids.

[0046] The first recess can be formed in a plastic, metal, silicon, or glass component, for example, in the form of a microtiter plate, a Petri dish, a sealing ring, or a frame, even by combining different materials. The carrier can be connected to existing inlets and outlets in a cultivation station, particularly for long-term cultivation. Furthermore, the temperature, the gas atmosphere (e.g., CO₂ content), the composition of the liquids, and the concentration of biomolecules or other substances can be adjusted at the inlets or measured at the outlets. Sensors can be attached to the outlets to measure pH, fluorescence, concentrations, temperature, metabolic products, cell-produced biomolecules, and so on.

[0047] Various liquids, such as culture medium, buffer solutions, reagents, or dyes, can be introduced into the first well via one or more inlets, for example, in the form of capillaries. These liquids may contain cells. The liquids can be withdrawn through one or more outlets to ensure continuous, intermittent, or demand-driven exchange. Inlets can be positioned so that the cells are not affected during liquid introduction. When using a capillary as an inlet, liquid can be introduced, in particular, via a second well that does not contain cells.

[0048] Samples can be taken from the liquid in the first recess, for example to detect cell products (proteins, antibodies, metabolites) at intervals or continuously, to control the concentration of added reagents, or to analyze exosomes or extracellular vesicles (EVs) produced by cells, in particular the loading with the type and quantity of proteins and nucleic acids, in order to determine parameters such as heterogeneity of the EVs in their biological and physical properties (size, composition in type and quantity, density, refractive index).

[0049] If multiple inlets are used, one part can be used to add non-cell-containing liquids such as medium, water, etc., and another part can be used to add cell-containing liquids.

[0050] When liquid is introduced into the first or second recesses, a liquid with a lower density than the cell-containing liquid, preferably immiscible with the cell-containing liquid, can serve as a temporary evaporation barrier. If the first thickened section is sufficiently thin, cells can be introduced through the liquid in this first thickened section into the second recesses. A second supply line can be used in parallel with the introduction of the cell-containing liquid to compensate for evaporation. Optionally, the composition of the dispensed liquid can be continuously varied to replace the medium surrounding the cells over a period of time.

[0051] The carriers in various embodiments are designed so that cells can be cultured in the secondary recesses. Additionally, procedures for cell analysis can be performed. In particular, cells can be stained with a dye. Because an exchange of fluids in the cell environment is ensured, the dye can subsequently be washed out without removing or otherwise affecting the cells.

[0052] Various cell culture methods can be used. In particular, cells can be printed into the second wells with a very small amount of liquid, followed by the addition of medium. Alternatively, medium can be added first, followed by the cell-containing liquid. Another option is to add the cell-containing medium in a single step. To ensure that the cells settle in the second wells, a waiting period can be observed between the process steps. Additionally, cell settling can be aided by centrifugation or agitation, especially shaking or vibrating the substrate. This also helps to remove gas bubbles from the second wells. Subsequently, cell-containing medium can be withdrawn until cells are present almost exclusively within the second wells.

[0053] The invention will now be described in more detail with reference to examples and the figures shown schematically in Fig. 1 one embodiment of the invention, Fig. 2 an embodiment of the invention with a supply line from the second surface of the carrier, Fig. 3 an embodiment of the invention with inlet and outlet from the second surface of the carrier, Fig. 4 Embodiments of the invention in top view, Fig. 5 an embodiment of the invention with inlet and outlet, Fig. 6 another embodiment of the invention with inlet and outlet, Fig. 7 another embodiment of the invention, Fig. 8 another embodiment of the invention, Fig. 9 another embodiment of the invention, Fig. 10 another embodiment show.

[0054] The Figure 1Figure 1 shows a cross-section of a beam 1 consisting of a first sub-beam 2 and an adjacent second sub-beam 3. A first recess 4 is formed in the first sub-beam 2, covering the cross-sectional openings 5 ​​of the adjacent second recesses 6. Therefore, the internal volumes of the second recesses 6 are in contact with the internal volume of the first recess 4 along their cross-sectional openings 5, allowing for mass exchange between them, particularly by diffusion. The second recesses 6 extend over a second thickness section 8 of the beam 1, specifically a second thickness section 8 in the second sub-beam 3. In the first sub-beam 2, the first recess 4 extends over the first thickness section 7, which is equal to the thickness of the first sub-beam 2.The support 1 has an outlet 9 in its first thickness section, here corresponding to the first sub-support 2, through which fluid F, whose level reaches the cross-section of the outlet 9, can exit. A supply line 10 opens at a distance above the plane over which the outlet 9 extends. The supply line 10 is not necessarily connected to the support, but can be routed independently of the support, e.g. by means of a traversing device (not shown).

[0055] The large elongation ratio of the second recesses 6 allows the fluid to move in the first recess 4 without flushing cells Z, which are located at the bottom 14 or in the volume of the second recess 6, out of the second recesses, or minimizing the influence of turbulence on the cells.

[0056] The Figure 2 shows a beam 1 in cross-section, which is like the one in Figure 1The beam 1 shown consists of a first sub-beam 2 with a first recess 4 and a second sub-beam 3 attached to it, in which second recesses 6 extend adjacent to the first recess 4 over the second thickness section 8. An outlet 9 is formed in the first sub-beam 2. The one in the Figures 1 and 2 The outlet 9 shown extends along an outer end face 11 of the support 1. A supply line 10 extends from the second surface 12 of the support 1 or its second sub-support 3 into the first recess 4. Preferably, the supply line 10 has a controlled valve 13 to control the flow of liquid F into the first recess 4.

[0057] The Figure 3Figure 1 shows an embodiment of the carrier 1 in which a connecting line 15, forming an outlet 9 and, in the opposite direction of flow, an inlet 10, extends through the carrier 1 from the first recess 4 to its second surface 12. Such a connecting line 15, forming both an inlet 10 and an outlet 9, preferably has a controlled valve 13, which further preferably has two or more connected lines, one of which, for example, is connected to a reservoir for the medium and another to a collection container for liquid F discharged from the first recess 4. This embodiment can also be provided with a cover that covers the first recess. Preferably, the cover then has a valve with which the composition of the gas atmosphere can be controlled.

[0058] The Figure 4Figure 1 shows embodiments of the invention in a one-piece carrier 1 in a top view of the first surface 20 of the carrier 1 and of the cross-sectional opening of each of a first recess 4. The first recess 4, here rectangular, extends over the cross-sectional openings of the adjacent second recesses 6, which are designed as an arrangement of 3 x 3 second recesses 6 for each first recess 4.

[0059] In one embodiment A of Figure 4The first recess 4 is surrounded by connecting lines 15 passing through the support 1, which are connected to or form an outlet 9. The connecting lines 15 extend from the first recess through the support 1 to its second surface 12, which is opposite the cross-sectional opening of the first recess 4. The connecting lines 15 passing through the support 1 direct the liquid exiting the first recess precisely onto the opposite second surface 12 of the support 1, so that the liquid can be selectively drawn off there and does not contaminate a further first recess 4 formed in the same support 1.

[0060] In one embodiment B of Figure 4A first recess 4 is connected to an outlet 9, which is formed in the support 1 in the form of a trench whose cross-section extends into the support 1. The outlet 9 is connected at the circumference of the support 1 to a connecting line 15, which extends along the circumferential end wall of the support 1 onto its second surface 12.

[0061] In one embodiment C of Figure 4A first recess 4a is enclosed by a circumferential outlet 9, which, via a conduit section 9a, forms an inlet to a further first recess 4b, so that liquid F can flow through the outlet 9 and its conduit section 9a from a first recess 4a into a further first recess 4b. The first recess 4b is enclosed by connecting lines 15, through which liquid F, exiting this first recess 4b, is directed to the opposite second surface 12 of the support 1. This embodiment also shows, as an example of a storage recess 21, a first recess 4a that is connected to a further first recess 4b by a conduit 10.

[0062] In one embodiment C of Figure 4Cells that produce specific biomolecules, e.g., cytokines, antibodies, proteins, can be cultured in the second recesses 6, which are associated with a first recess 4. These molecules can then be transported via the conduit section 9a to another first recess 4, where they can be detected or act upon other cells.

[0063] In one embodiment D of Figure 4 As also described with reference to embodiment B, a first recess 4 is connected to an outlet 9, which is formed in the support 1 in the form of a groove whose cross-section extends into the support 1. The outlet 9 is connected at the circumference of the support 1 to a connecting line 15, which extends along the circumferential end wall 11 of the support 1 onto its second surface 12. On the wall of the first recess 4 opposite the outlet 9, a supply line 10 is formed in the support 1, also in the form of a groove.

[0064] The carrier 1 can have one or more first recesses 4 of the same or different shape, e.g. Figs. 4A to 4D exhibiting multiple first recesses 4. Different first recesses 4 may contain different liquids, or the same liquids in different concentrations, or combinations thereof. The second recesses 6 belonging to different first recesses 4 may contain different cell types.

[0065] The Figure 5Figure 1 shows an embodiment in which the wall extending over the first thickness section 7 and comprising or including a first recess 4 is formed by a first partial support 2, which adjoins a second partial support 3 in which an arrangement of second recesses 4 is formed. A supply line 10, preferably connected to a connecting line 15, can open into the first partial support 2, and, preferably opposite the supply line 10, an outlet 9, preferably also connected to a connecting line 15, can be formed. The supply line 10 and the connecting line 15 shown here are each provided with a controlled valve 13, which allows control of the supply and discharge of liquid F into and out of the first recess 4.

[0066] As in Figure 6As shown, the first sub-support 2 can be displaceable relative to the second sub-support 3, for example, by the first sub-support 2 and the second sub-support 3 overlapping in a displaceable and liquid-tight manner at their edge regions. A seal 16 can be arranged between the first sub-support 2 and the second sub-support 3, which is, for example, a burr on the first sub-support 2 or the second sub-support 3, which is made of plastic, or a rubber ring. Further shown Figure 6 that the first partial beam 2 can cover the first recess 4. The covering part of the first partial beam 2 can be designed as a cover that is reusable.

[0067] The Figure 7 shows a method for separately introducing cell-containing liquid by means of a first metering device 17 and, before, after or simultaneously, introducing cell-free medium by means of a second metering device 18, into recesses 4, 6 of the carrier 1.

[0068] The Figure 7 Figure 1 shows a carrier 1 whose recesses consist of an arrangement of secondary recesses 6, and a layer of liquid covering these recesses, forming the first thickness section 7, is held in place by surface tension. Preferably, the enclosure of the arrangement of secondary recesses 6 is hydrophobic, e.g., has a hydrophobic coating. The carrier 1 has, for example, a hydrophilic region as an outlet 9, which adjoins a connecting line 15 extending along the end face 11 of the carrier 1 to its second surface 12, which is opposite the cross-sectional openings of the recesses 6. The hydrophobic coating encompassing the secondary recesses 6 is preferably designed such that drops of liquid applied to the carrier coalesce into a continuous layer and only then run off at the outlet 9. Alternatively, the outlet 9 can be formed by microstructures in the surface of the carrier 1.

[0069] In this process, the flow of liquid through outlet 9 can be controlled by tilting the carrier 1 or by bringing the liquid into contact with a device with capillary action.

[0070] The Figure 8 Figure 1 shows a carrier 1 in whose first sub-carrier 2 optionally no outlet 9 is provided, but rather the inlet 10 serves simultaneously as a metering device 17 and as an outlet 9. Alternatively, the inlet and outlet can be implemented as two metering devices 17, 18. The first recess is subdivided perpendicular to the cross-sectional opening of the first recess by a membrane 19, which is preferably semipermeable, thereby dividing the common liquid volume of the first recess 4 above the second recesses 6. The membrane 19 can also be combined analogously with other embodiments and there subdivide the first recess 4 or the first thickness section 7.

[0071] The Figure 9 Figure 1 shows an embodiment in which the carrier 1 consists of a first sub-carrier 2, preferably made of plastic, and a second sub-carrier 3, preferably made of glass, wherein the first sub-carrier 2 is connected to the second sub-carrier 3 along its circumference. A first recess 4 extends over second recesses 6 formed in the second sub-carrier 3. The first sub-carrier 2 has an outlet 9 at a distance from the region of its first recess 4, preferably at a distance from the region covered by the second sub-carrier 3. A supply line 10 opens at a distance above the first recess 4 to meter drops of liquid F into the first recess 4. The first sub-carrier 2 preferably has a cross-sectional opening opposite the second sub-carrier 3, into or against which the supply line 10 is directed. Preferably, a connecting line 15 is connected to the outlet 9.

[0072] In this method, the second recesses 6 and the first recess 4 are filled with liquid F, which is preferably a medium, and are in contact with each other via the through-holes 6c. Particularly in an embodiment in which a supply line 10 opens below the second recesses 6, or below the second sub-support 3 in the first sub-support 2, a gas, e.g., an inert gas or air, e.g., air or oxygen for cell cultivation, optionally air or O₂ with 5% CO₂, can be introduced through the supply line 10 in addition to or as an alternative to liquid F. The gas can be supplied without bubbles by ensuring a sufficiently low gas flow rate. Alternatively, the gas can be supplied with a flow rate sufficient to form bubbles, whereby liquid F is moved in the second recesses and, optionally, cells Z are floated by means of the gas bubbles G.Gas bubbles can alternatively be generated by cavitation triggered with laser radiation.

[0073] Pressure changes can move cells Z away from the through-holes 6c to aid fluid exchange.

[0074] The Fig. 10Figure 1 schematically shows an embodiment in which the support 1 consists of a second sub-support 3 with second recesses 6, the cross-sectional openings 5 ​​of which and areas of the surface of the support 1, 3 between these cross-sectional openings 5 ​​lie in a common plane E. In particular, if these surface areas between the cross-sectional openings 5 ​​have a surface energy that does not allow wetting by the liquid F or allows only minimal wetting, e.g., surface areas made of plastic or functionalized, coated, or hydrophobically structured glass, a liquid F moving over the plane E, e.g., in the form of a rolling droplet Fr, can absorb used medium from the second recesses 6 and at least partially replace it. A rolling droplet Fr can be created, e.g., by applying liquid F in the form of several drops to the plane E or into second recesses 6, e.g., by a supply line 10 opening into a second recess 6, as e.g., in [reference to figure]. Fig. 2 The fluid F projecting above the plane E, particularly in the form of a rolling droplet Fr, can be driven by tilting the support 1 towards the horizontal, by accelerating and / or vibrating the support, or by applying a gas stream.

[0075] In general, in embodiments in which the support consists of a second sub-support 3 with second recesses 6 therein, the second sub-support can be referred to as support 3 with recesses 6 therein. Reference symbol:

[0076] 1 carrier 16 seal 2 first partial carrier 17 first dosing device 3 second sub-carrier 18 second dosing device 4, 4a, 4b first extraction 19 membrane 5 Cross-sectional opening of a second 20 first surface of the carrier Exclusion 21 Storage removal 6 second recess 22 material 6b bottom of a second recess 23 Transitional management 6c through hole 24 Floor recess in second partial beam 7 first thickness section 8 second thickness section 25 spacers 9 Outlet E level 9a Pipe section of the outlet F liquid 10 supply line Fr rolling drop 11 Front wall, front surface Z cell 12 second surface of the carrier G Gas bubble 13 controlled valve 14 bottom of the second recess 15 Connection line

Claims

1. Carrier (1) for cultivating cells (Z), the carrier having at least one first recess (4, 4a, 4b) extending over a first thickness section (7) from a first surface (20) of the carrier (1) up to a second thickness section (8) which is adjacent to the first thickness section (7) and in which an arrangement of at least 90 second recesses (6) extends from the first recess (4, 4a, 4b), wherein the cross-sectional opening of the first recess (4, 4a, 4b) extends in the plane of the first surface (20) of the carrier (1), characterized in that the second recesses (6) form wells suitable for receiving cells, which wells have an aspect ratio of depth to diameter of at least 1:1 and a diameter of at most 1 mm, and in that the carrier (1) has an outlet (9) whose cross-section opens in or adjacent to the first thickness section (7), and in that that a supply conduit (10) is formed in the first surface (20) or in the first thickness section (7) of the carrier (1), which supply conduit (10) opens into the first recess (4, 4a, 4b).

2. Carrier (1) according to claim 1, characterized in that the outlet (9) has a cross-section projecting into the carrier (1), which cross-section is open to the plane of the first surface (20) of the carrier (1).

3. Carrier (1) according to one of the preceding claims, characterized by a connection conduit (15) which is connected to the outlet (9) and which extends to the second surface (12) that lies opposite to the first surface (20) of the carrier (1).

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

5. Carrier (1) according to one of claims 3 to 4, characterized in that the outlet (9) and the connection conduit (15) adjacent thereto have a hydrophilic surface, and in that regions of the first surface (20) and / or regions of the second surface (12) of the carrier (1) are hydrophobic.

6. Carrier (1) according to one of the preceding claims, characterized in that it is made single-pieced of glass.

7. Carrier (1) according to one of the preceding claims, characterized in that it consists of a second partial carrier (3) extending over the second thickness section (8) and of a first partial carrier (2) extending over the first thickness section (7), the first (2) and second (3) partial carriers being arranged in a liquid-tight manner relative to one another.

8. Carrier (1) according to one of the preceding claims, characterized in that it consists of a second partial carrier (3) extending over the second thickness section (8) and of a first partial carrier (2) extending over the first thickness section (7), the first (2) and the second (3) partial carriers being arranged in a liquid-tight manner to one another and displaceably against one another.

9. Carrier (1) according to one of the preceding claims, characterized in that the first partial carrier (2) consists of glass, plastic and / or silicon.

10. Carrier (1) according to one of claims 7 to 9, characterized in that the second partial carrier (3) consists of glass and / or silicon.

11. Carrier (1) according to one of the preceding claims, characterized in that in the carrier (1) at least two first recesses (4, 4a, 4b) are formed, each of which is enclosed by circumferentially arranged connection conduits (15) extending through the carrier (1).

12. Carrier (1) according to one of the preceding claims, characterized in that in the carrier (1) at least two first recesses (4, 4a, 4b) are formed, at least one of which is enclosed by an outlet (9) in the form of a trench that opens into another one of said first recesses (4, 4a, 4b), which is enclosed by circumferentially arranged connection conduits (15) extending through said carrier (1).

13. Carrier (1) according to one of the preceding claims, characterized in that a controlled valve (13) is arranged each in an outlet (9), in a connection conduit (15) and / or in a supply conduit (10).

14. Method for cultivating cells, characterized by introducing liquid (F) in which cells (Z) are suspended into second recesses (6) of a carrier (1) according to one of the preceding claims, with subsequent removal of liquid from the first thickness section (4, 4a, 4b) along the outlet (9) and the connection conduit (15).

15. Method according to claim 18, characterized in that the first partial carrier (2) is formed only by the surface of the second partial carrier (3) by which the second recesses (6) are spaced apart, and the first recess (4, 4a, 4b) adjacent to the arrangement of second recesses (6) is formed solely by a liquid layer generated by surface tension.

16. Method according to one of claims 14 to 15, characterized in that the surface of the second partial carrier (3) by which the second recesses (6) are spaced apart, is a hydrophobic surface.

17. Method according to one of claims 14 to 16, characterized in that the carrier (1) has only second recesses (6) and medium forms a layer projecting above the plane of the cross-sectional openings of the second recesses (6), which layer forms the volume of the first recess.

18. Method according to one of claims 14 to 17, characterized in that the carrier (1, 3) has recesses (6) whose cross-sectional openings (5) are spaced apart by surface regions of the carrier (1, 3), these surface regions and the cross-sectional openings (5) of the recesses (6) lying in a common plane (E), wherein liquid (F) is applied onto this plane (E) and the liquid (F) is moved along the carrier (1, 3) by tilting the carrier (1, 3) against the horizontal, by vibrating the carrier (1, 3), and / or by applying pressurized gas to the plane (E).

19. Method according to claim 18, characterized in that the liquid (F) is applied to this plane (E) by means of a supply conduit (10) opening in the bottom (6c) of a recess (6).