Specimen container for culturing biological specimens, device for its operation and microscope

Abstract

A sample container for culturing a biological sample, an apparatus for its operation, and a microscope are provided. [Solution] The present invention relates to a sample container (1) for culturing a biological sample (9), comprising: a cavity (2) for accommodating a medium (8); at least one access opening (10) for delivering the medium (8) into the cavity (2); and a sample space (3) arranged within the cavity (2) for accommodating the sample (9), the sample space (3) being separated from the rest of the cavity (2) by at least one side wall (4), the at least one side wall (4) having a hole (6) by which the medium (8) present in the cavity (2) can communicate with the sample space (3). The sample container (1) is characterized in that at least one side wall (4) stands on a bottom (5) of the sample space (3), and that the bottom (5) is transparent to wavelengths in at least one wavelength range of visible light, thereby allowing illumination of the sample space (3) and / or detection of detection radiation leaving the sample space (3) through the bottom (5) of the cavity (2). The invention further relates to an apparatus (12) for operating the sample container (1) and to a microscope (17).

Description

[Technical Field]

[0001] The invention relates to a sample container for culturing biological samples according to the preamble of the main claim, and to a device for operating the sample container and a microscope comprising such a device according to the additional independent claims. [Background technology]

[0002] In addition to culturing substantially planar cell cultures (2D cell cultures), the culture of three-dimensional organisms (3D cell cultures, 3D cultures), such as organoids and spheroids, is becoming increasingly important. In environments where 3D culture is possible, the spatial extent of biological tissues can usually be considered. The organisms (hereinafter also referred to as "biological samples" or simply "samples") generated using in vitro 3D cultures can be attached to a surface, placed on such a surface, embedded in a gel matrix (such as Matrigel and related products), or freely floating in the surrounding medium ("free-floating").

[0003] The spatial extent of an organism and its presence in a surrounding medium poses considerable technical challenges when the medium is exchanged in whole or in part. The purpose of such an exchange is to deliver nutrients, basic building blocks for (protein) biosynthesis and signaling substances, to supply oxygen, and to remove metabolites and depleted medium. When the medium is exchanged, for example, by using a pipette tip, the pipette tip may inadvertently touch or damage the sample. Furthermore, a sample floating freely in the medium may inadvertently swirl or even become co-absorbed.

[0004] In contrast to 2D cell cultures, medium changes are therefore usually performed manually in 3D cultures, which is very labor-intensive. Sample-friendly medium changes are particularly important for in vitro 3D cultures, as they need to be cultivated for long periods of time. Any interference can negatively impact sample development and quality.

[0005] For 2D cell cultures, methods for (semi-)automatically changing the medium used are known from the prior art. However, these methods are specifically designed for adherent 2D cell cultures and cannot be directly applied to free-floating samples in 3D cultures. On the other hand, when samples of 3D cultures are cultured in Matrigel droplets, the samples are localized, but the Matrigel droplets can vary in height and occupy most of the well of a sample container, such as a (micro)titer plate. This poses a risk of the sample being damaged by the pipette tip.

[0006] To reduce the aforementioned risks to the sample, medium changes can be performed by carefully tilting the sample vessel or by pipetting a large excess of medium, in either case leaving a significant proportion of the medium in the sample vessel.

[0007] Optimized culture plates that minimize sample volume and thus facilitate medium exchange are also known. An example is the multiwell plate (Akura™ 96 Spheroid Microplate) manufactured by Insphero AG, Schlieren, Switzerland. Within each well serving as a sample container, a channel is formed that narrows toward the bottom of the container. As a result of this narrowing, the area where the sample resides is defined, and the pipette can be moved to a defined position within the well without touching the sample. However, this plate is limited to being suitable only for cultures in Matrigel. For cultures that require a larger surface area for growth, the plate is not compatible.

[0008] To perform automated medium changes, slides such as the Fluidic 480 and Fluidic 983 chips manufactured by Chipshop, Jena, Germany, can be connected to pumps. However, such designs are not compatible with Matrigel and are only suitable for relatively small 3D cell cultures. Furthermore, each slide must have its own pump, making it difficult to scale up. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention is based on the object of proposing a method for cultivating and optically detecting 3D cell cultures that is improved with respect to the prior art. [Means for solving the problem]

[0010] The object is achieved by the subject matter of the independent and additional independent claims. Advantageous developments are the subject matter of the dependent claims.

[0011] The object is achieved by a sample container for culturing a biological sample, the sample container comprising a cavity for accommodating a medium, at least one access opening for delivering the medium into the cavity, and at least one sample space arranged in the cavity for accommodating the sample, the at least one sample space being separated from the rest of the cavity by at least one side wall. The at least one side wall has holes, by which a medium present in the cavity can communicate with the sample space. The medium can either come into contact with another medium present in the sample space or enter the sample space, e.g., flow through the sample space.

[0012] The sample container according to the invention is characterized in that at least one side wall stands on a bottom of the sample space, which is additionally transparent to wavelengths in at least one wavelength range of visible and / or infrared light, thereby allowing illumination of the sample space and / or detection of detection radiation leaving the sample space through the bottom of the cavity.

[0013] In a further embodiment of the sample container according to the invention, in addition to at least one side wall, the bottom of the sample space may be provided with holes, by means of which the medium present in the cavity can communicate with the sample space.

[0014] The basic idea behind the present invention is to physically and functionally divide the cavity in such a way that the sample is localized in one area while, for example, a pipette tip can be used elsewhere in the cavity without endangering the sample. In contrast to the prior art, the sample is positioned in the sample space, and by appropriately selecting the number, size, and arrangement of holes, high flow rates or high transmission capacities can be advantageously avoided, further preventing the sample from being inadvertently expelled. Furthermore, the sample container according to the present invention allows optical detection, visual monitoring, and / or visual display of processes within the sample container.

[0015] To achieve the advantages outlined above, the holes have a maximum internal width of at most 1000 μm, advantageously at most 500 μm, preferably at most 200 μm. In further embodiments of the sample container, the holes may have an internal width of less than 200 μm, for example between 100 μm and 50 μm. To retain individual cells within the sample space, the holes may have an internal width of at most 10 μm.

[0016] The side walls of the sample space may have holes of different internal widths. For example, in one embodiment of a sample container according to the invention, the internal width of the holes may become larger with increasing distance from the bottom of the sample space. This makes it possible to minimize fluid stress on the sample in the region where it is located, without compromising the supply of, for example, oxygen and / or nutrients to the sample.

[0017] The side walls used may be flat structures with holes, closely spaced bars and / or grids. The cross section of the sample space in plan view may be circular, elliptical, polygonal or semicircular.

[0018] In one embodiment of the invention, the sample container is open-topped when oriented in use. Optionally, the sample container may be fully or partially equipped with a lid to reduce the risk of contamination and unintentional evaporation of the contents of the cavity. The lid may be removable or may have an opening that is optionally reclosable. The access opening used may be open-topped or a cross-section of an open-topped sample container. In a further embodiment, the access opening may be formed by a separate channel that terminates in the cavity. The same applies to any exit openings present.

[0019] In a further possible embodiment of the invention, only the sample space is partially or completely covered by the lid, whereas at least part of the cross section (plan view) of the cavity serving as an access opening remains free.

[0020] For example, by guiding a pipette tip into the cavity, a gap space into which the medium can be delivered advantageously remains between the side wall of the sample space and the wall of the cavity, in order to create an area in the cavity for the delivery of the medium. This gap space advantageously serves as an access opening in at least the upper part of its extent. In the context of this description, the delivery or withdrawal of the medium is usually carried out by using a pipette tip as an example. Corresponding conduits, tubing and / or channels have the same meaning.

[0021] The interstitial space may extend laterally around the periphery of the sample space, in which case the sample space is completely surrounded by side walls and the interstitial space. The sample space may be formed in the middle of the cavity such that the interstitial space between the sample space and the walls extending around the cavity is substantially constant. Such an embodiment supports full exchange or full contact of the medium within the cavity and, optionally, uniform flow through the cavity and / or sample space.

[0022] In other embodiments of the sample container according to the invention, the interstitial space may be present over at least a lateral angular range or sector of the sample space, where the walls of the sample space are formed by the walls of the cavity on one side, while the side walls with holes define the remaining sector of the cavity. This makes it possible to create large interstitial spaces.

[0023] In addition to the access opening, there may be an outlet opening through which the medium can be discharged from the cavity in order to exchange the medium present in the cavity. The medium can be removed at the open-top side, for example, by bringing a (further) pipette tip into contact with the medium there and removing part of the medium using an aspirator (pipette, pump) connected to the pipette tip.

[0024] To achieve uniform flow above and / or through the sample space, the outlet opening can be formed at or near the bottom of the cavity, where flow through the cavity and sample space occurs as medium is delivered to the upper region of the cavity and medium is discharged near the bottom, advantageously reducing remaining dead space where little or no medium exchange occurs.

[0025] The outlet opening may further comprise a closure that is operated in a controlled manner to affect the rate of flow of the medium through the cavity as well as the amount of medium delivered. The access opening may comprise a closure that is similarly operated in a controlled manner.

[0026] The closure may be implemented, for example, in the form of a valve, a gate valve or a bladed shutter. When using pipette tips or the like, a controlled closure or a drive for operating said closure may also mean a corresponding technical element, for example a pipette head, a multipipette or the like, with which, for example, the contents of the pipette tip can be expelled or drawn into said pipette tip.

[0027] The outlet opening may be formed in the bottom of the cavity, in particular together with the outlet nozzle. The conduit may, for example, be associated with or connected to the outlet nozzle.

[0028] Furthermore, the outlet opening can be closed by a porous matrix, the flow resistance of which can prevent the medium from escaping the cavity. To expel the medium, negative pressure can be applied to the outlet opening, and the medium is expelled through the porous matrix due to the effect of said negative pressure. Such an embodiment allows the sample container to be used without the need to establish an interlocking connection with a channel, piping, or the like. One of the ends of the channel can press against the bottom of the sample container, surrounding the outlet opening. Advantageously, at the end face of the channel that presses against the bottom (channel end), there is a seal to support the generation of negative pressure and prevent any leakage of the medium beyond the channel. Instead of a porous matrix, in a further embodiment of the sample container according to the invention, the closure can be made of a flexible material, for example a flap or star-shaped closure made of a rubber compound.

[0029] In a simple embodiment of the sample container according to the invention, at least one side wall stands on the bottom of the sample container. In one advantageous development, the sample support can be formed, for example, on the bottom of the sample space. The sample to be cultured can be placed thereon. The sample support can have, for example, a surface structure that serves for the adhesion of the sample or of components from which the sample develops or which will further develop over a certain period of time. The surface structure can be the physical configuration of the surface, for example, by having a particular roughness value or a regular or irregular texture. The sample support can alternatively or additionally comprise molecules (linkers) that can specifically bind molecules of the sample or molecules present in the medium to the sample support. In a further embodiment of the invention, the sample space, particularly in at least one region of its bottom and / or side wall, can have such a surface structure.

[0030] In addition to or instead of the above possible embodiments, the shape of the sample support may influence the sample. In one advantageous embodiment, the sample support is concavely curved in the direction of the sample space, thus forming a hollow. Such a shape has been found to support the formation of spheroids, i.e., aggregates of individual cells and / or cell clusters.

[0031] To enable better optical detection, visual monitoring and / or visual indication of processes in the sample vessel, in particular in the sample space, the bottom of the sample vessel and / or the sample space may be formed by at least two flat or curved side walls that form an angle of less than 180°. Such an embodiment is advantageous for optical detection, visual monitoring and / or visual indication through the bottom of the sample vessel, i.e., due to the opposite arrangement of the illumination beam path and the detection beam path. This advantageously reduces imaging errors that occur when the illumination radiation and / or the detection radiation pass obliquely through the bottom of the sample vessel.

[0032] For the operation of the sample vessel of the invention according to one of the aforementioned embodiments, an apparatus for operating the sample vessel is advantageous, comprising a first pump for delivering a medium into the cavity or into the interstitial space of the cavity. The first pump is connected to a control device in such a way that it is able to exchange data and transmit control commands. The control device can be, for example, a computer, a microcontroller or an FPGA (Field Programmable Gate Array).

[0033] Thus, in a further embodiment, there may be a second pump which serves to expel the medium through the outlet opening, the second pump being advantageously likewise controlled by the control device.

[0034] The device for operating the sample container may be part of a microscope. The microscope includes a light source for providing illumination radiation, which is guided along an illumination beam path and may optionally be shaped by optical elements, such as optical lenses, arranged therein. Furthermore, the microscope includes a detection objective for detecting detection radiation leaving the sample space and a detector for converting the detected detection radiation into an electronic signal (image data).

[0035] The light source and the detection objective may be configured for transmitted light illumination. The sample space and the sample present therein are illuminated by the illumination radiation. The detection radiation used may be reflected and / or attenuated illumination radiation. The action of the illumination radiation may also trigger the emission of detection radiation within the sample, for example through labeling of components of the sample with fluorophores (markers) that can be excited by the illumination radiation to emit fluorescence.

[0036] Transmitted light illumination can be used especially for quantitative evaluation of current properties of a process or sample in the sample space.

[0037] In a further embodiment of the microscope according to the invention, the light source and the detection objective are configured for inverted illumination and detection through the bottom of the sample container.

[0038] In a further possible use, the device for operating the sample container can be part of an arrangement for an imaging method that does not directly illuminate the sample. For example, such an arrangement can be designed for optical coherence tomography. The sample container according to the invention can therefore be used not only in optical microscopy, but also in other imaging methods, such as scanning methods and / or illumination of the sample with invisible light.

[0039] The above-mentioned possible embodiments of the sample container can be realized, mutatis mutandis, when two or more sample spaces are formed in the cavity. In such cases, multiple samples can, for example, interact via the medium surrounding the samples and occupying the cavity, exchanging messengers and growth factors, for example, without the samples directly touching each other. Such a sample container can, for example, have a base and a standardized support, for example, the dimensions of a standard plate in laboratory operation, for example, an SBS plate.

[0040] Such embodiments of the present invention allow for the cultivation of multiple, even different samples, such as organoids, in a sample container, allowing chemical communication between said samples.

[0041] Alternatively, multiple sample vessels can be advantageously arranged on a common support, which may advantageously have the dimensions of a standardized plate, such as an SBS plate or the like, and may have, for example, 6, 12, 24, 48, 96 or 384 sample vessels, allowing the invention to be used with existing laboratory equipment and, if necessary, easily automated.

[0042] When there are multiple sample vessels per support, the sample vessels may rest on a common base plate that forms the bottom of each of the sample vessels.

[0043] The sample support according to the invention may of course advantageously be provided in sterile form.

[0044] The invention is explained in more detail below on the basis of exemplary embodiments and with reference to the drawings. [Brief explanation of the drawings]

[0045] [Figure 1] 1 shows a schematic diagram of a first exemplary embodiment of a sample vessel according to the invention in a perspective view in the form of a wire model; FIG. [Figure 2]1 shows a schematic diagram of a second exemplary embodiment of a sample vessel according to the invention in a perspective view in the form of a wire model; [Figure 3] 1 shows a schematic diagram of a third exemplary embodiment of a sample vessel according to the invention in a perspective view in the form of a wire model; [Figure 4] 1 shows a schematic illustration of a first exemplary embodiment of a device according to the invention and a fourth exemplary embodiment of a sample container according to the invention in a side cross-sectional view. [Figure 5] 1 shows a schematic illustration of a first exemplary embodiment of a device according to the invention and a fifth exemplary embodiment of a sample container according to the invention in a side cross-sectional view. [Figure 6] 1 shows a schematic diagram of a second exemplary embodiment of a device according to the invention and a sixth exemplary embodiment of a sample container according to the invention in a side cross-sectional view. [Figure 7] 1 shows a schematic diagram of a third exemplary embodiment of a device according to the invention in a side cross-sectional view. [Figure 8] 1 shows a schematic illustration of a first exemplary embodiment of a microscope according to the invention as a transmitted light microscope in a side cross-sectional view; [Figure 9] 1 shows a schematic diagram of a first exemplary embodiment of a microscope according to the invention as a transmitted light microscope and a seventh exemplary embodiment of a sample container according to the invention with a concave sample support in a side cross-sectional view. [Figure 10] 1 shows a schematic diagram of a second exemplary embodiment of a microscope according to the invention as an inverted microscope in a side cross-sectional view. [Figure 11] 1 shows a schematic diagram of a second exemplary embodiment of a microscope according to the invention as an inverted microscope and an eighth exemplary embodiment of a sample container according to the invention in a side cross-sectional view. [Figure 12] 1 shows a schematic diagram of a second exemplary embodiment of a microscope according to the invention as an inverted microscope and a ninth exemplary embodiment of a sample container according to the invention in a side cross-sectional view. [Figure 13] 1 shows a schematic representation of a support in the form of a plate with a plurality of sample vessels according to the invention in a perspective view; FIG. [Figure 14]1 shows a schematic view of a sample vessel according to the invention in the form of a support having a plurality of sample spaces in fluid communication with one another via a medium; DETAILED DESCRIPTION OF THE INVENTION

[0046] The drawings of the exemplary embodiments are schematic and not to scale. For better visualization, Figures 1 to 3 are shown as so-called wire models, which show only the outer contours of the respective structures and omit showing the closure surfaces, e.g., the walls of the sample container 1. In Figures 7 to 12, the technical elements used for delivering and removing the medium 8 have been omitted for clarity.

[0047] In the exemplary embodiments shown, the sample vessel 1 according to the invention is in the form of a hollow cylinder that is open at the top and has a bottom 5 (FIG. 1). The internal volume of the sample vessel 1, called cavity 2, contains a sample space 3, at least one side wall 4 of which has a plurality of holes 6 and stands on the bottom 5. In all exemplary embodiments, the bottom 5 is transparent at least over the extent of the sample space 3, and in particular for at least certain wavelength ranges of visible and / or infrared light.

[0048] The sample space 3 has a diameter smaller than that of the cavity 2, such that a clearance space 7 remains between the side wall 4 and the wall of the sample container 1. In a first exemplary embodiment, the sample space 3 is centrally arranged within the cavity 2, such that the clearance space 7 extends with a constant extent around the sample space 3. The clearance space 7 serves as an access opening 10 and / or an exit opening 11.

[0049] Optionally, there is a lid 27 with which the sample vessel 1 can be closed, but which covers it at least as needed. In a further embodiment, the lid 27 may also cover only a part of the cavity 2 (see Figure 3). Furthermore, the lid 27 may have an opening for delivering and / or removing the medium 8 present therein (see also Figure 2). The opening may be able to be opened or closed again actively or passively.

[0050] In the second exemplary embodiment, the sample space 3 is positioned off-center within the cavity 2, thus creating a larger interstitial space 7 in one direction compared to the first exemplary embodiment, despite the sample space 3 being the same size (FIG. 2). In the variant embodiment shown, there is optionally a lid 27 having an access opening 10 through which the medium 8 can be introduced into the interstitial space 7. The opening 10 can of course also serve as an exit opening 11 through which all or part of the medium 8 can be removed, if desired.

[0051] In a third exemplary embodiment, the side wall 4 may extend from one, in particular vertically extending, contact line on the wall of the sample container 1 to another vertical contact line on said wall and may be joined to said wall at both contact lines (FIG. 3). Here, the optionally used lid 27, for example, closes only the top of the sample space 3, whereas the interstitial space 7 remains substantially freely accessible from above and serves as an access opening 10 and / or an outlet opening 11.

[0052] Given the technical requirements, the exemplary embodiments of the lid 27 can be freely combined with the embodiments of the sample container 1 described above or below.

[0053] The sample container 1 can be used in an apparatus 12 for operating the sample container 1. Figure 4 shows a vertical section of the sample container 1, which is partially filled with a medium 8 by way of example. Suspended in the medium 8 within the sample space 3 are individual cells, cell clusters and / or aggregates which serve as the sample 9. The dimensions of the holes 6 (see above) in the side wall 4 are such that the sample 9 does not itself leave the sample space 3 when there is a flow in the medium 8.

[0054] The medium 8 is delivered to the cavity 2 by a conduit 13, for example in the form of a pipette tip, directed towards the interstitial space 7 at the open side of the sample container 1, which acts as an access opening 10. Starting from the conduit 13, the medium 8 flows along the interstitial space 7 and can enter or leave the sample space 3 through the holes 6. The generation of a directed flow (shown by the arrows) is made possible by an outlet opening 11 present in the region of the interstitial space 7 and an optional closure 14 used to close the outlet opening 11, which opens as needed. The outflowing medium 8 leaves the sample container 1 again through the conduit 13, for example in the form of a connecting nozzle for connecting tubing or pipes. The medium 8 is delivered at the access opening 10 by means of a first pump 25 of the conduit 13 and is pumped out via the outlet opening 11 by means of a second pump 26.

[0055] Operating the device 12, and in particular controlling the closure 14 and the pumps 25, 26, is a control device 15, which is in each case connected to a drive device 16. As already discussed above, it should also be understood that a technical unit for dispensing an amount of medium 8 is encompassed by the term "controllable closure". The associated drive device 16 receives control commands from the control device 15 and, when executing said control commands, adjusts the degree of opening of the respective closure 14. The delivery rate of medium 8 can be adjusted and controlled by controlling the pumps 25, 26.

[0056] Based on the first exemplary embodiment of the device 12 according to the invention, a fifth exemplary embodiment of the sample container 1 according to the invention is shown, in which several sample spaces 3, in this case two, are present in the cavity 2 (FIG. 5), which are in fluid communication with each other. Such an embodiment of the sample container 1 can be used, for example, when a reaction of a sample 9 in a fluid-downstream sample container 1 is to be tested, for example, for a physiological process in a fluid-upstream sample container 1. For example, hormones, growth factors and / or metabolites can be carried with the flowing medium 8 from the upstream sample space 3 to the downstream sample space 3, which can potentially trigger a reaction.

[0057] The device 12 according to the invention can be used with a further exemplary embodiment of a sample container 1 according to the invention (FIG. 6). A plurality of sample spaces 3 are formed on the support 28. Each sample space 3 is laterally delimited with respect to its neighboring sample spaces 3 by a partition 29, which prevents the exchange of liquid or gaseous media. Each sample space 3 can be individually filled via a conduit 13 and emptied via an outlet opening 11. In a further embodiment, a common conduit 13 can be used. For this purpose, the support 28 and the conduit 13 are subjected to controlled movement relative to each other, for example, through the presence and use of a pipette head and / or a movable sample table. The relative movement is likewise controlled using the control device 15 and can be closed-loop or open-loop controlled. A further alternative has a common outlet channel 30 (see FIG. 13) through which media can be removed from some or all of the sample containers 1.

[0058] In a second exemplary embodiment of the device 12 according to the invention (FIG. 7), the side of the sample container 1, which is open at the top, is used both as an access opening 10 for the conduit 13 and as an outlet opening 11. The sample 9 is, by way of example, an organoid or spheroid having a three-dimensional extent.

[0059] The apparatus 12 may be part of a microscope 17 configured for transmitted light illumination (FIG. 8). The microscope 17 comprises a light source 18 providing illumination radiation that is directed towards the sample space 3 along an illumination beam path 19. Optical elements may be present in the illumination beam path 19 for guiding and / or shaping the illumination radiation, such as an illumination objective 24 (see, for example, FIG. 10), which is not shown here for clarity. Components of the illumination radiation passing through the sample 9 and / or fluorescence radiation emitted as a result of the action of the illumination radiation are guided along a detection beam path 20, collected by means of a detection objective 21, and forwarded to a detector 22, where they are detected as measured values ​​(image signals, image data).

[0060] In an exemplary embodiment, the sample space 3 and the sample 9 present therein are illuminated from above the sample container 1. In a further embodiment not shown, transmitted light illumination can be achieved by arranging a light source 18 below the sample container 1 and illuminating through the transparent bottom 5. In such an embodiment, the detection objective 21 and the detector 22 can be above the sample space 3. In addition to the functions of the control device 15 already described above, the control device 15 can also control the light source 18 and / or the detector 22.

[0061] A fifth exemplary embodiment of the sample container 1 according to the invention has a concave sample support 23 on its bottom 5 (FIG. 9). The shape and dimensions of the sample support 23 advantageously support, for example, cell aggregates and relatively small cell clusters to give spheroids forming the sample 9. The sample support 23 is transparent for the detection radiation and / or illumination radiation to be detected. If, for example, only the presence of fluorescence radiation of a specific wavelength is detected as detection radiation, without aiming to image the sample 9 and / or identify the origin of the detection radiation, any light scattering effect of the sample support 23 is harmless. The materials of the bottom 5 and of the sample support 23 advantageously have a high degree of transparency (transmittance) for the wavelength of the detection radiation used. The working distance between the detection objective 21 and the sample 9 to be detected is advantageously kept as short as possible.

[0062] A microscope 17 with an inverted illumination and detection arrangement is shown in Figure 10. Illumination is achieved using an illumination objective 24 that is directed towards the bottom 5 from the outside of the sample container 1 at an angle other than 90°, preferably in the range of 30° to 60°. Detection radiation is detected using a detection objective 21 that is also directed towards the bottom 5 from the outside of the sample container 1 at an angle in the range of 30° to 60°, the optical axes of the illumination objective 24 and the detection objective 21 preferably forming an angle of 90° with respect to each other.

[0063] In a further embodiment of the sample container 1 according to the invention in combination with an inverted microscope 17, the bottom 5 can advantageously be formed by two or more walls that form an angle other than 180° with respect to one another in order to reduce aberrations that occur when illumination and detection radiation pass obliquely through the bottom 5 (FIG. 11). The illumination objective 24 and the detection objective 21 are arranged in relation to the bottom 5 such that their respective optical axes are directed perpendicular to the associated wall of the bottom 5. This makes it possible to avoid to a large extent aberrations that occur at the interface of the bottom 5. At the same time, the shape of the wall of the bottom 5 can advantageously support the formation of spheroids.

[0064] The modification of the sample container 1 shown with reference to Figure 11 has a sample support 23 above the lowest point of the sample space 3 (Figure 12). The sample support 23 is used to position the sample 9 with respect to the beam paths of the objectives 21, 24. Just as in the example above, the sample support 23 may be provided with a surface structure that is beneficial for adhesion and / or aggregation. The inverted embodiment of the microscope 17 may of course also be used to examine a free-floating sample 9.

[0065] 13 provides a simplified view of a support 28 in the form of a plate containing a plurality of sample vessels 1 (shown in simplified form) arranged in rows and columns. The sample vessels 1 are open on the face of the support 28 facing upwards. A common base plate 5.1 of the support 28 closes the underside of the sample vessels 1 and forms the bottom 5 of each sample vessel 1 (see above). The base plate 5.1 consists of a material that is transparent for the detection radiation and / or the illumination radiation. The support 28 can consist of a single material. In a further embodiment, the base plate 5.1 is made of a different material than the rest of the support 28.

[0066] The sample containers 1 may optionally be provided with individual lids 27. In a further possible embodiment, there may be a common lid 27 (not shown) with or without access openings 10 and covers for some or all of the sample containers 1 present as needed.

[0067] The individual sample vessels 1 each have an outlet opening 11 in the base plate 5.1 or in the face of the support 28 shown facing upwards. The outlet openings 11 of all or some of the sample vessels 1 can be connected to outlet channels 30 through which the medium 8 can be removed from the associated sample vessels 1 (shown as an option by broken solid lines). Closures 14 of the sample vessels 1 may optionally be present. Thus, there may be a common closure 14 of the outlet channels 30 (not shown).

[0068] The outlet channels 30 may, for example, connect the sample vessels 1 arranged on the support 28 in rows and / or columns. In further embodiments, selected sample vessels 1 may be connected to each other in different arrangements.

[0069] In a further embodiment of the support 28, it is in the form of a sample container 1 by having a base plate 5.1 with a peripheral wall 31 standing on the base plate 5.1 (FIG. 14). In a cavity 2 surrounded by the wall 31, a plurality of sample spaces 3 standing on the base plate 5.1 are arranged. When the cavities 2 are filled with a medium 8, the sample spaces 3 are thereby fluidly connected to one another. Via an outlet opening 11, the medium 8 can be introduced into the cavity 2 and removed therefrom. Here again, a lid 27 may optionally be present and optionally further has an access opening 10. This embodiment advantageously allows different cell types or organoid types to be cultured in the different sample spaces 3, which can exchange substances via a common medium 8 without themselves coming into direct contact. [Explanation of symbols]

[0070] 1. Sample container 2 cavities 3. Sample space 4 side wall 5 Bottom 5.1 Base Plate 6 holes 7. Interstitial Space 8 Medium 9 Samples 10 Access opening 11 Exit opening 12 Equipment 13 Pipette tips, conduits, and tubing connectors 14 Closing part 15 Control device 16 Drive unit 17 Microscope 18 light source 19 Illumination beam path 20 Detection beam path 21 Detection objective lens 22 Detector 23 Sample support 24 illumination objective lens 25 First pump unit 26 Second pump unit 27 Lid 28 Support 29 Bulkhead 30 Exit Channel 31 Wall

Claims

1. A sample container (1) for culturing a biological sample (9), comprising: a cavity (2) for containing a medium (8), at least one access opening (10) for delivering said medium (8) into said cavity (2); at least one sample space (3) arranged in said cavity (2) for containing a sample (9), said sample space (3) being separated from the remaining space of said cavity (2) by at least one side wall (4) and said at least one side wall (4) having holes (6) by means of which said medium (8) present in said cavity (2) can communicate with said sample space (3); In a sample container (1) comprising - said at least one side wall (4) stands on the bottom (5) of said sample space (3), and - said base (5) is transparent to wavelengths in at least one wavelength range of visible light; - a sample vessel (1), characterized in that it allows illumination of said sample space (3) and / or detection of detection radiation emerging from said sample space (3) through said bottom (5) of said cavity (2).

2. 2. Sample vessel (1) according to claim 1, characterized in that the holes (6) have a maximum internal width of at most 1000 μm, advantageously at most 500 μm, preferably at most 200 μm.

3. 3. A sample container (1) according to claim 1 or 2, characterized in that a gap space (7) through which the medium (8) can be delivered remains between the side wall (4) of the sample space (3) and the wall of the cavity (2).

4. 4. A sample vessel (1) according to claim 3, characterized in that the interstitial space (7) extends around the periphery of the sample space (3).

5. 4. The sample vessel (1) according to claim 3, characterized in that the interstitial space (7) is present at least over the lateral angle range of the sample space (3).

6. 6. A sample container (1) according to any one of claims 1 to 5, characterized in that the cavity (2) has an outlet opening (11) through which the medium (8) can be discharged from the cavity (2).

7. 7. Sample vessel (1) according to claim 6, characterized in that the outlet opening (11) has a closure (14) which is operated in a controlled manner.

8. 7. The sample container (1) according to claim 6, characterized in that the outlet opening (11) comprises a porous matrix through which the medium (8) can be drawn out of the cavity (2) by negative pressure generated on the side facing away from the sample space (3).

9. 9. The sample container (1) according to claim 1, characterized by a sample support (23) present at the bottom (5) of the sample space (3) and concavely curved towards the sample space (3).

10. A sample vessel (1) according to any one of claims 1 to 9, characterized in that the sample vessel (1) and / or the bottom (5) of the sample space (3) are formed by at least two flat walls enclosing an angle of less than 180°.

11. A device (12) for operating a sample vessel (1) according to any one of claims 1 to 10, comprising: a first pump unit (25) for delivering said medium (8) into said cavity (2); a control device (15) for controlling said first pump unit (25); An apparatus (12) comprising:

12. 12. The device (12) according to claim 11, characterized by a second pump unit (26) for discharging the medium (8) through the outlet opening (11), the second pump unit (26) being controlled by the control device (15).

13. A microscope (17) comprising a device (12) according to claim 11 or 12, a light source (18) for providing illumination radiation along an illumination beam path; a detection objective (21) for detecting the detection radiation leaving said sample space (3); a detector (22) for converting the detected radiation into an electronic signal; A microscope (17) including:

14. 14. The microscope (17) according to claim 13, characterized in that the light source (18) and the detection objective (21) are configured for transmitted light illumination.

15. 14. The microscope (17) according to claim 13, characterized in that the light source (18) and the detection objective (21) are configured for inverted illumination and detection through the bottom (5) of the sample container (1).

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