Sample vessel for cultivating biological samples, device for operating same, and microscope

Abstract

The invention relates to a sample vessel (1) for cultivating biological samples (9), comprising a cavity (2) for receiving a medium (8); at least one inlet opening (10) for feeding the medium (8) into the cavity (2); and a sample chamber (3) arranged within the cavity (2) for receiving a sample (9), wherein the sample chamber (3) is separated by at least one lateral wall (4) from a remaining chamber of the cavity (2) and the at least one lateral wall (4) has openings (6) by means of which the medium (8) contained in the cavity (2) can become connected to the sample chamber (3). The sample vessel (1) is characterised in that the at least one lateral wall (4) stands on a floor (5) of the sample chamber (3); and the floor (5) is permeable for wavelengths of at least one wavelength range of visible light and therefore it is possible to illuminate the sample chamber (3), and / or capture a detection radiation coming out of the sample chamber (3), through the floor (5) of the cavity (2). The invention also relates to a device (12) for operating the sample vessel (1), and to a microscope (17).

Description

[0001] Sample vessel for cultivating biological samples, device for its operation and microscope

[0002] The invention relates to a sample vessel for cultivating biological samples according to the preamble of the main claim, as well as a device for operating the sample vessel and a microscope with such a device according to the independent claims.

[0003] In addition to the cultivation of essentially flat cell cultures (2D cell cultures), the importance of cultivating three-dimensional biological objects (3D cell cultures, 3D culture), such as organoids and spheroids, is increasing. In an environment that allows 3D cultivation, the usual spatial extent of biological tissues can be accommodated. The biological objects produced by 3D in vitro culture (hereinafter also referred to as biological samples, or simply as samples) can be attached to a surface, rest on such a surface, be embedded in a gel-like matrix (such as Matrigel and related materials), or float freely in a surrounding medium ("free-floating").

[0004] The spatial extent of biological objects, or rather their presence in the surrounding medium, poses considerable technical challenges when the medium is to be exchanged in whole or in part. Such an exchange serves to supply nutrients, basic building blocks for (protein) biosynthesis, signaling substances, as well as the supply of oxygen and the removal of metabolic products and used medium. If pipette tips are used to exchange the medium, for example, they can inadvertently touch or damage the sample. Samples floating freely in the medium can also be accidentally disturbed or even aspirated.

[0005] In contrast to 2D cell cultures, medium changes in 3D cultures are usually performed manually, which is quite laborious. A gentle medium change is particularly important for 3D in vitro cultures, as these must be cultivated over a long period of time. Any disruption can have negative effects on the development and quality of the sample.

[0006] State-of-the-art options for (semi-)automated medium changes are known for 2D cell cultures. However, these are designed primarily for adherent 2D cell cultures, which cannot be directly transferred to freely floating samples from a 3D culture. If, on the other hand, the sample from a 3D culture is cultivated in a Matrigel droplet, the sample is localized, but the Matrigel droplet can vary in height and fill a large portion of a sample container, for example, a well of a (micro)titer plate. This poses the risk of the sample being damaged by the pipette tip.

[0007] To reduce the above-mentioned risks to the sample, a media change can be performed by gently tilting the sample container or by pipetting with a large excess of medium. In both cases, a significant portion of the medium remains in the sample container.

[0008] Optimized culture plates are also known that minimize the sample volume and thus facilitate media exchange. One example is a multiwell plate from Insphero AG, Schlieren, Switzerland (Akura™ 96 Spheroid Microplate). Each well serving as a sample vessel has a channel that narrows toward the bottom of the vessel. This narrowing defines the sample retention area, allowing a pipette to be moved to a defined position in the well without touching the sample. However, this plate is only of limited use for cultures in Matrigel. It is not compatible with cultures that require a larger growth area.

[0009] To perform automated medium changes, slides, such as the Fluidic 480 and Fluidic 983 chips from Chipshop, Jena, Germany, can be connected to pumps. However, such a design is not compatible with Matrigel and is only suitable for relatively small 3D cell cultures. Furthermore, scaling is difficult because each individual slide must be equipped with its own pump.

[0010] The invention is based on the object of proposing a possibility for cultivating and optically detecting 3D cell cultures that is improved compared to the prior art.

[0011] The problem is solved by the subject matter of the independent claim and the subordinate claims. Advantageous further developments are the subject matter of the dependent claims.

[0012] The problem is solved with a sample vessel for cultivating biological samples, comprising a cavity for receiving a medium; at least one access opening for supplying the medium into the cavity; and at least one sample chamber arranged within the cavity for receiving a sample, wherein the sample chamber is separated from a remaining space of the cavity by at least one side wall. The at least one side wall has openings through which a medium contained in the cavity can communicate with the sample chamber. The medium can contact another medium located in the sample chamber or penetrate into the sample chamber and, for example, flow through it.

[0013] A sample vessel according to the invention is characterized in that at least one side wall rests on a floor of the sample chamber. Furthermore, the floor is permeable (transparent) to wavelengths of at least one wavelength range of visible and / or infrared light, thus enabling illumination of the sample chamber and / or detection of detection radiation coming from the sample chamber through the floor of the cavity.

[0014] In further embodiments of the sample vessel according to the invention, in addition to the at least one side wall, a bottom of the sample chamber can be provided with openings by means of which a medium contained in the cavity can communicate with the sample chamber.

[0015] The basic idea of ​​the invention is to physically and functionally subdivide the cavity so that the sample is located in one area, while, for example, a pipette tip can be used at another location in the cavity without endangering the sample. In contrast to the prior art, the sample is also protected from accidental flushing, as the sample is positioned in the sample chamber, and high flow velocities or high transport capacity can be advantageously avoided by a suitable selection of the number, size, and arrangement of the openings. Furthermore, the sample vessel according to the invention allows for optical recording, monitoring, and / or display of the processes in the sample vessel.

[0016] To achieve the advantage outlined above, the apertures have a maximum clear width of 1000 pm at most, advantageously 500 pm at most, preferably 200 pm at most. In further embodiments of the sample vessel, the apertures can have clear widths of less than 200 pm, for example, 100 pm or 50 pm. To retain individual cells in the sample space, the apertures can have clear widths of 10 pm at most.

[0017] A side wall of the sample chamber can have openings of varying clear widths. Thus, in one embodiment of the sample vessel according to the invention, the clear width of the openings can increase with increasing distance from the bottom of the sample chamber. In this way, the flow load in the area where a sample is located can be kept to a minimum without jeopardizing the supply of oxygen and / or nutrients to the sample, for example.

[0018] The side wall(s) can be flat or perforated structures, closely spaced rods, and / or grids. The cross-section of the sample chamber can be circular, oval, n-sided, or semicircular in plan view.

[0019] In one embodiment of the invention, the sample vessel is open at the top in its orientation when in use. Optionally, it can be fully or partially covered with a lid to reduce the risk of contamination and accidental evaporation of the contents of the cavity. The lid can be removable or have an opening, which can optionally be closed. The cross-section of the sample vessel that is open at the top or can be opened can serve as the access opening. In further embodiments, the access opening can be formed by a separately designed channel ending in the cavity. The same applies to an existing outlet opening.

[0020] In a further possible embodiment of the invention, only the sample space is partially or completely covered by a lid, while at least one section of the cross-section of the cavity (top view) serving as an access opening remains free.

[0021] To create an area within the cavity for the supply of the medium, for example, using a pipette tip inserted into the cavity, a free space into which the medium can be supplied is advantageously left between the side wall of the sample chamber and a wall of the cavity. This free space advantageously serves as an access opening, at least over sections of its extent. In this description, the medium is usually supplied or removed using pipette tips, for example. Corresponding lines, hoses, and / or channels are equivalent.

[0022] The free space can extend laterally around the sample chamber. The sample chamber is then enclosed by the side walls and the free space all around. The sample chamber can be located in the center of the cavity, so that the free space between the sample chamber and the cavity wall remains approximately constant all the way around. This design supports all-round exchange or contact of the media in the cavity and, optionally, a uniform flow through the cavity and / or sample chamber.

[0023] In other embodiments of the sample vessel according to the invention, the free space can be present at least around a lateral angle or sector of the sample chamber. The wall of the sample chamber is formed over a section by the wall of the cavity, while the side wall with the openings delimits the remaining sector from the cavity. In this way, a large free space can be created.

[0024] To exchange the medium present in the cavity, an outlet opening can be provided next to the access opening through which the medium can be drained from the cavity. The medium can be removed from the open-top side, for example, by bringing another pipette tip into contact with the medium and removing a portion of the medium using a suction device (pipette, pump) connected to the pipette tip.

[0025] To achieve a uniform flow into and / or through the sample chamber, the outlet opening can be located in or near the bottom of the cavity. With medium supplied in an upper area of ​​the cavity and discharged near the bottom, the cavity and sample chamber are flowed through, advantageously reducing any remaining dead spaces where little or no media exchange occurs.

[0026] The outlet opening can also have a controlled closure to influence the volume flow of the medium through the cavity in conjunction with the amount of supplied medium. The access opening can also be provided with a controlled closure.

[0027] The closure can be implemented, for example, in the form of a valve, a slide, or a lamella closure. When using pipette tips or the like, the corresponding technical elements, such as a pipette head, a multi-pette, or the like, are also considered to be controlled closures or a drive for their operation. These elements can be used to dispense the contents of a pipette tip or to draw them into it.

[0028] The outlet opening can be formed, in particular, at the bottom of the cavity together with an outlet nozzle. A line can be connected to this outlet nozzle, for example.

[0029] Furthermore, it is possible for the outlet opening to be closed with a porous matrix, the flow resistance of which prevents the medium from flowing out of the cavity. To drain the medium, a negative pressure can be applied to the outlet opening, the effect of which draws the medium through the porous matrix. Such a design allows the use of the sample vessel without the need for a positive connection to a channel, hose, or the like. One end of a channel could be pressed against the bottom of the sample vessel, with the end of the channel surrounding the outlet opening. Advantageously, a seal is provided on one end (end of the channel) of the channel, which is pressed against the bottom, to support the buildup of negative pressure and to prevent the medium from escaping outside the channel.Instead of a porous matrix, in further embodiments of the sample vessel according to the invention, there may be a closure made of a flexible material, for example a flap or a star-shaped closure made of a rubber mixture.

[0030] In a simple embodiment of the sample vessel according to the invention, at least one side wall rests on the bottom of the sample vessel. In an advantageous further development, a sample support can be formed, for example, on the bottom of the sample chamber. The sample to be cultivated can rest on this. The sample support can have a surface structure that serves, for example, to promote the adhesion of the sample or of components from which the sample develops or evolves over a period of time. The surface structure can be a physical design of the surface, for example by having certain roughnesses or regular or irregular structures. Alternatively or additionally, the sample support can be provided with molecules (linkers) that allow specific binding of molecules of the sample or of molecules contained in the medium to the sample support.In further embodiments of the invention, the sample chamber, in particular its floor and / or at least one region of the side wall, can have such surface structures.

[0031] In addition to or as an alternative to the above-mentioned design options, the sample support can exert effects on a sample through its shape. In an advantageous design, the sample support is concavely curved toward the sample chamber, thus forming a depression. It has been found that such a shape promotes the formation of spheroids, i.e., clumps of individual cells and / or cell clusters.

[0032] In order to better optically record, observe, and / or display the processes in the sample vessel, particularly in the sample chamber, the bottom of the sample vessel and / or the sample chamber can be formed by at least two flat or curved side walls enclosing an angle of less than 180°. Such a design is advantageous for optical recording, observation, and / or display through the bottom of the sample vessel, i.e., by means of an inverse arrangement of the illumination and detection beam paths. This advantageously reduces imaging errors that occur when illumination radiation or detection radiation passes obliquely through the bottom of the sample vessel.

[0033] To operate a sample vessel according to the invention according to one of the aforementioned embodiments, a device for operating the sample vessel is advantageous, which allows a first pump to supply the medium into the cavity or into the free space of the cavity. The first pump is connected to a controller in a manner suitable for exchanging data and transmitting control commands. The controller can be, for example, a computer, a microcontroller, or an FPGA (field programmable gate array).

[0034] Accordingly, in a further embodiment, a second pump can be provided, which serves to discharge the medium through the outlet opening. The second pump is also advantageously controlled by the controller.

[0035] The device for operating the sample vessel can be part of a microscope. This includes a light source for providing illumination radiation. This radiation is guided along an illumination beam path and can optionally be shaped by optical elements arranged therein, such as optical lenses. Furthermore, the microscope includes a detection objective for capturing detection radiation coming from the sample chamber and a detector for converting the detected detection radiation into electronic signals (image data).

[0036] The light source and the detection lens can be arranged in a transmitted-light configuration. The sample chamber and the sample contained therein are illuminated by the illumination radiation. Reflected and / or attenuated illumination radiation can be used as detection radiation. The effect of the illumination radiation can also trigger the emission of detection radiation in the sample, for example, by coating sample components with fluorophores (markers) that can be excited by the illumination radiation to emit fluorescent light.

[0037] Transmitted light illumination can be used in particular for a quantitative evaluation of the processes in the sample space or the current properties of the sample.

[0038] In a further embodiment of a microscope according to the invention, the light source and the detection objective are arranged in a configuration for inverse illumination and detection through a bottom of the sample vessel.

[0039] In further possible applications, the device for operating the sample vessel can be part of an arrangement for an imaging method that does not rely on direct illumination of the sample. For example, such an arrangement can be designed for optical coherence tomography. The sample vessel according to the invention can thus be used not only in light microscopy methods, but also in other imaging methods, for example, scanning methods and / or with illumination of the sample with invisible light.

[0040] The aforementioned design options for the sample vessel can also be implemented accordingly if more than one sample chamber is formed in a cavity. In such a case, for example, several samples can interact via the medium surrounding the samples and filling the cavity, exchanging messenger substances and growth factors, for example, without the samples coming into direct contact. Such a sample vessel can, for example, have the base area and dimensions of a standardized carrier, for example, a plate commonly used in laboratory operations, such as an SBS plate.

[0041] Such an embodiment of the invention allows, for example, to cultivate several, even different samples such as organoids in one sample vessel and to enable chemical communication between them.

[0042] On the other hand, it is advantageously possible to arrange a plurality of sample vessels on a common carrier. Such a carrier can advantageously have the dimensions of standardized plates, for example, SBS plates or the like, and can accommodate, for example, 6, 12, 24, 48, 96, or 384 sample vessels. In this way, the invention can be used with existing laboratory equipment and easily automated if necessary.

[0043] If there are several sample vessels per carrier, these can be placed on a common base plate, which forms the respective bottoms of the sample vessels.

[0044] The sample carriers according to the invention can of course advantageously be provided in sterilized form.

[0045] The invention is explained in more detail below using exemplary embodiments and figures. They show:

[0046] Fig. 1 is a schematic representation of a first embodiment of a sample vessel according to the invention in a perspective view as a wire model;

[0047] Fig. 2 is a schematic representation of a second embodiment of a sample vessel according to the invention in a perspective view as a wire model;

[0048] Fig. 3 is a schematic representation of a third embodiment of a sample vessel according to the invention in a perspective view as a wire model;

[0049] Fig. 4 is a schematic representation of a first embodiment of a device according to the invention and a fourth embodiment of a sample vessel according to the invention in a lateral sectional view; Fig. 5 is a schematic representation of the first embodiment of a device according to the invention and a fifth embodiment of a sample vessel according to the invention in a lateral sectional view;

[0050] Fig. 6 is a schematic representation of a second embodiment of a device according to the invention and a sixth embodiment of a sample vessel according to the invention in a lateral sectional view;

[0051] Fig. 7 is a schematic representation of a third embodiment of a device according to the invention in a lateral sectional view;

[0052] Fig. 8 is a schematic representation of a first embodiment of a microscope according to the invention as a transmitted light microscope in a lateral sectional view;

[0053] Fig. 9 is a schematic representation of the first embodiment of a microscope according to the invention as a transmitted light microscope and a seventh embodiment of a sample vessel according to the invention with a concave sample support in a lateral sectional view;

[0054] Fig. 10 is a schematic representation of a second embodiment of a microscope according to the invention as an inverted microscope in a lateral sectional view;

[0055] Fig. 11 is a schematic representation of the second embodiment of a microscope according to the invention as an inverted microscope and of an eighth embodiment of a sample vessel according to the invention in a lateral sectional view;

[0056] Fig. 12 is a schematic representation of the second embodiment of a microscope according to the invention as an inverted microscope and a ninth embodiment of a sample vessel according to the invention in a lateral sectional view; and

[0057] Fig. 13 is a schematic representation of a carrier in the form of a plate with a plurality of sample vessels according to the invention in a perspective view^ and

[0058] Fig. 14 is a schematic representation of a sample vessel according to the invention in the form of a carrier with a plurality of sample chambers fluidically connected to one another via a medium. The illustrations of the exemplary embodiments are schematic and not to scale. For clarity, Figures 1 to 3 are shown as so-called wireframe models, in which only the outer contours of the respective structures are shown and closed surfaces, such as the wall of the sample vessel 1, are omitted. For the sake of clarity, technical elements used for the supply and removal of a medium 8 have been omitted in Figures 7 to 12.

[0059] In the illustrated embodiment, a sample vessel 1 according to the invention is designed as an upwardly open hollow cylinder with a base 5 (Fig. 1). The inner volume of the sample vessel 1, referred to as the cavity 2, accommodates a sample chamber 3, at least one side wall 4 of which has a plurality of openings 6 and rests on the base 5. In all embodiments, the base 5 is transparent at least over the extent of the sample chamber 3 and at least for certain wavelength ranges, in particular visible and / or infrared light.

[0060] The sample chamber 3 has a diameter that is smaller than the diameter of the cavity 2, so that a free space 7 remains between the side wall 4 and the wall of the sample vessel 1. In the first embodiment, the sample chamber 3 is arranged centrally in the cavity 2, so that the free space 7 surrounds the sample chamber 3 with a constant extension. The free space 7 serves as an access opening 10 and / or an outlet opening 11.

[0061] Optionally, a lid 27 is provided, with which the sample vessel 1 can be closed, or at least covered if necessary. In further embodiments, the lid 27 can also cover only a portion of the cavity 2 (see Fig. 3). Furthermore, the lid 27 can have openings for the supply and / or removal of a medium 8 contained therein (see also Fig. 2). The openings can be actively or passively opened, or they can be reclosed.

[0062] In a second embodiment, the sample chamber 3 is arranged decentrally within the cavity 2, so that a larger free space 7 is created in one direction compared to the first embodiment, despite the same size of the sample chamber 3 (Fig. 2). In the embodiment shown, the optionally provided cover 27 has an access opening 10 through which medium 8 can be introduced into the free space 7. The opening 10 can, of course, also optionally serve as an outlet opening 11 through which the medium 8 can be removed in whole or in part.

[0063] In a third embodiment, the side wall 4 can extend from one, in particular vertical, contact line to another vertical contact line on the wall of the sample vessel 1 and be connected to the wall at each point (Fig. 3). The optionally used lid 27, for example, only closes the sample chamber 3 at its top, while the free space 7 remains essentially freely accessible from above and serves as an access opening 10 and / or outlet opening 11.

[0064] The exemplary embodiments of the lid 27 can be freely combined with the embodiments of the sample vessel 3 described above or below, taking into account the technical requirements.

[0065] The sample vessel 1 can be used in a device 12 for operating the sample vessel 1. Figure 4 shows, by way of example, a vertical section of a sample vessel 1 partially filled with a medium 8. Within the sample chamber 3, individual cells, cell clusters, and / or aggregations are present floating in the medium 8, serving as the sample 9. The dimensions of the openings 6 (see above) in the side wall 4 are such that the sample 9 is not flushed out of the sample chamber 3, even in the presence of a flow in the medium 8.

[0066] In order to supply the medium 8 to the cavity 2, a line 13, designed, for example, as a pipette tip, is provided, which is directed into the free space 7 at the open side of the sample vessel 1 acting as the access opening 10. The medium 8 can flow from the line 13 along the free space 7 and through the openings 6 into the sample chamber 3 and out again therefrom. A directed flow (indicated by arrows) can be generated by providing an outlet opening 11 in the region of the free space 7 and opening an optional closure 14 serving to selectively close the outlet opening 11. The outflowing medium 8 exits the sample vessel 1 again through a line 13, designed, for example, as a connecting piece for connecting a hose or a pipe.The medium 8 is fed to the line 13 at the access opening 10 by means of a first pump 25 and pumped out via the outlet opening 11 by means of a second pump 26.

[0067] To operate the device 12, in particular to control the closures 14 and the pumps 25, 26, a controller 15 is provided, each of which is connected to a drive 16. As already mentioned above, the term "controllable closure" also refers to a technical unit for dispensing a quantity of the medium 8. The respective drive 16 receives control commands from the controller 15 and, upon execution, adjusts the degree of opening of the respective closure 14. The delivery rate of the medium 8 can be adjusted and regulated by controlling the pumps 25, 26.

[0068] Based on the first embodiment of the device 12 according to the invention, a fifth embodiment of the sample vessel 1 according to the invention is shown, in which a plurality of sample chambers 3, in this case two, are present in the cavity 2 (Fig. 5). These are fluidically connected to one another. Such a design of the sample vessel 1 can be used, for example, if reactions of the sample 9 in a fluidically downstream sample vessel 1 to, for example, physiological processes in a fluidically upstream sample vessel 1 are to be investigated. For example, hormones, growth factors and / or metabolic products can be transported from the upstream sample chamber 3 to the downstream sample chamber 3 with the flowing medium 8 and, if necessary, trigger a reaction there.

[0069] The device 12 according to the invention can be used with a further embodiment of the sample vessel 1 according to the invention (Fig. 6). A plurality of sample chambers 3 are formed on a carrier 28. Each of the sample chambers 3 is laterally separated from its neighboring sample chambers 3 by a partition 29, wherein the partition 29 does not allow any exchange of liquid or gaseous media. Each of the sample chambers 3 can be individually filled via a line 13 and supplied via an outlet opening 11. In further embodiments, a common line 13 can be used. For this purpose, the carrier 28 and line 13 are moved relative to one another in a controlled manner, for example by providing and using a pipetting head and / or a movable sample table. The relative movement can also be monitored and regulated or controlled by the controller 15.Further alternatives have a common outlet channel 30 (see Figure 13), via which medium can be led away from several or all sample vessels 1.

[0070] In a second embodiment of a device 12 according to the invention (Fig. 7), the upwardly open side of the sample vessel 1 is used both as an access opening 10 for a line 13 and as an outlet opening 11. The sample 9 is exemplified by an organoid or a spheroid that has a three-dimensional extension.

[0071] The device 12 can be part of a microscope 17 configured for transmitted-light illumination (Fig. 8). The microscope 17 comprises a light source 18, by means of which illumination radiation can be provided, which is directed along an illumination beam path 19 into the sample chamber 3. Optical elements, for example an illumination objective 24 (see, for example, Fig. 10), can be present in the illumination beam path 19 for the purpose of guiding and / or shaping the illumination radiation; these elements are not shown here for reasons of clarity. The portions of the illumination radiation passing through the sample 9 and / or fluorescent radiation emitted by the action of the illumination radiation are guided along a detection beam path 20, collected by a detection objective 21, directed onto a detector 22, and recorded by the detector as measured values ​​(image signals, image data).

[0072] In this embodiment, the sample chamber 3 and a sample 9 located therein are illuminated from above the sample vessel 1. In further embodiments not shown, transmitted light illumination can be achieved by arranging the light source 18 below the sample vessel 1 and illuminating it through the transparent base 5. In such an embodiment, the detection objective 21 and the detector 22 can be located above the sample chamber 3. In addition to the functions of the controller 15 already described above, the controller can also control the light source 18 and / or the detector 22.

[0073] In a fifth embodiment of a sample vessel 1 according to the invention, the vessel has a concave sample support 23 on its bottom 5 (Fig. 9). The shape and dimensioning of the sample support 23 advantageously supports the clumping of, for example, cells and smaller cell clusters into a spheroid forming the sample 9. The sample support 23 is transparent to the detection radiation to be recorded and / or to the illumination radiation. Any light-scattering effect of the sample support 23 is harmless if, for example, the detection radiation is only intended to detect the presence of, for example, fluorescent radiation of a specific wavelength, without aiming to image the sample 9 and / or localize the source of the detection radiation.The material of the base 5 and the sample support 23 advantageously exhibits high transparency (transmissivity) for the wavelengths of the detection radiation to be used. The working distance between the detection objective 21 and a sample 9 to be detected is advantageously kept as small as possible.

[0074] A microscope 17 with an inverse illumination and detection arrangement is shown in Fig. 10. Illumination is provided by means of an illumination objective 24 directed from outside the sample vessel 1 onto the base 5 at an angle other than 90°, advantageously at an angle in a range of 30 to 60°. The detection radiation is captured by the detection objective 21, which is also directed from outside the sample vessel 1 onto the base 5 at an angle in a range of 30 to 60°, wherein the optical axes of the illumination objective 24 and the detection objective 21 advantageously enclose an angle of 90° with one another.

[0075] In order to prevent the aberrations that occur when the illumination and detection radiation passes obliquely through the base 5, the base 5 can advantageously be formed, in a further embodiment of the sample vessel 1 according to the invention in combination with the inverted microscope 17, by two or more walls that enclose an angle other than 180° with each other (Fig. 11). The illumination objective 24 and the detection objective 21 are arranged relative to the base 5 such that their respective optical axes are directed perpendicularly to the respective wall of the base 5. In this way, a significant proportion of the aberrations that occur at the interface of the base 5 can be avoided.

[0076] At the same time, the shape of the walls of the base 5 can advantageously support the formation of spheroids.

[0077] A modification of the sample vessel 1 shown in Fig. 11 has a sample support 23 above the lowest point of the sample chamber 3 (Fig. 12). This serves to position the sample 9 relative to the beam paths of the objectives 21, 24. The sample support 23, like that of the example described above, can be provided with a surface structure advantageous for adhesion and / or agglomeration. The inverted design of the microscope 17 can, of course, also be used for the examination of freely floating samples 9.

[0078] Fig. 13 shows a simplified illustration of a carrier 28 in the form of a plate containing a plurality of sample vessels 1 arranged in rows and columns (shown in a simplified manner). The sample vessels 1 are open on the upward-facing side surface of the carrier 28. A common base plate 5.1 of the carrier 28 closes off the sample vessels 1 at their underside and forms the base 5 (see above) of the respective sample vessel 1. The base plate 5.1 is made of a material that is transparent to detection radiation and / or illumination radiation. The carrier 28 can be made of a single material. In further embodiments, the base plate 5.1 is made of a different material than the rest of the carrier 28.

[0079] The sample vessels 1 can optionally be individually provided with a lid 27. In another possible embodiment, a common lid 27 (not shown) with or without an access opening 10 can be provided, which covers several or all of the sample vessels 1 present as needed.

[0080] The individual sample vessels 1 each have an outlet opening 11 in the base plate 5.1 or in the upwardly facing side surface of the carrier 28. It is possible for all or a number of the sample vessels 1 to be connected at their outlet openings 11 to an outlet channel 30, via which the medium 8 can be led away from the respective sample vessels 1 (shown as an option with a broken solid line). A closure 14 for the sample vessels 1 can optionally be present. Accordingly, a common closure 14 (not shown) for the outlet channel 30 can be present. An outlet channel 30 can, for example, connect the sample vessels 1 arranged in a row and / or in a column on the carrier 28. In further embodiments, selected sample vessels 1 can be connected to one another in a different arrangement.

[0081] In a further embodiment of a carrier 28, the latter is designed as a sample vessel 1 in that it has a base plate 5.1 with a circumferential wall 31 standing on the base plate 5.1 (Fig. 14). Within a cavity 2 enclosed by the wall 31, a plurality of sample chambers 3 are arranged standing on the base plate 5.1. If a medium 8 is filled into the cavity 2, the sample chambers 3 are fluidically connected to one another. The medium 8 can be filled into the cavity 2 and removed from it via an outlet opening 11. Here, too, a lid 27 can optionally be present, which also optionally has an access opening 10. The advantage of this embodiment is that different cell types or organoid types can be cultivated in the various sample chambers 3, which can exchange substances via the shared medium 8 without coming into direct contact themselves.

[0082] Reference symbol

[0083] 1 sample container

[0084] 2 Cavity

[0085] 3 rehearsal room

[0086] 4 side wall

[0087] 5 Floor

[0088] 5.1 Base plate

[0089] 6 Breakthrough

[0090] 7 Free space

[0091] 8 Medium

[0092] 9 Sample

[0093] 10 Access opening

[0094] 11 Outlet opening

[0095] 12 Device

[0096] 13 Pipette tip, cable, hose connection

[0097] 14 Closure

[0098] 15 Control

[0099] 16 Drive

[0100] 17 Microscope

[0101] 18 Light source

[0102] 19 Illumination beam path

[0103] 20 Detection beam path

[0104] 21 Detection lens

[0105] 22 Detector

[0106] 23 Sample support 24 Illumination lens

[0107] 25 first pump unit

[0108] 26 second pump unit

[0109] 27 Lid 28 Carrier

[0110] 29 Partition wall

[0111] 30 exhaust channel

[0112] 31 Wall

Claims

1. A sample vessel (1) for cultivating biological samples (9), comprising a cavity (2) for receiving a medium (8); at least one access opening (10) for supplying the medium (8) into the cavity (2); at least one sample chamber (3) arranged within the cavity (2) for receiving a sample (9), wherein the sample chamber (3) is separated from a remaining space of the cavity (2) by at least one side wall (4), and the at least one side wall (4) has openings (6) by means of which the medium (8) contained in the cavity (2) can communicate with the sample chamber (3); characterized in that the at least one side wall (4) stands on a floor (5) of the sample chamber (3);and the bottom (5) is transparent to wavelengths of at least one wavelength range of visible light, so that illumination of the sample space (3) and / or detection of detection radiation coming from the sample space (3) through the bottom (5) of the cavity (2) is possible.; 2. Sample vessel (1) according to claim 1, characterized in that the openings (6) have a maximum clear width of at most 1000 pm, advantageously of at most 500 pm, preferably of at most 200 pm.

3. Sample vessel (1) according to claim 1 or 2, characterized in that a free space (7) remains between the side wall (4) of the sample chamber (3) and a wall of the cavity (2), into which space the medium (8) can be fed.

4. Sample vessel (1) according to claim 3, characterized in that the free space (7) runs around the sample space (3).

5. Sample vessel (1) according to claim 3, characterized in that the free space (7) is present at least around a lateral angular range of the sample space (3).

6. Sample vessel (1) according to one of the preceding claims, characterized in that the cavity (2) has an outlet opening (11) through which the medium (8) can be drained from the cavity (2).

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

8. Sample vessel (1) according to claim 6, characterized in that the outlet opening (11) is provided with a porous matrix through which the medium (8) can be sucked out of the cavity (2) by means of a generated negative pressure on the side facing away from the sample space (3).

9. Sample vessel (1) according to one of the preceding claims, characterized by a sample support (23) provided on the bottom (5) of the sample chamber (3), which is concavely curved in the direction of the sample chamber (3).

10. Sample vessel (1) according to one of the preceding claims, characterized in that the bottom (5) of the sample vessel (1) and / or of the sample chamber (3) is 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 one of claims 1 to 10, comprising a first pump unit (25) for supplying the medium (8) into the cavity (2); and a controller (15) for controlling the first pump unit (25).

12. Device (12) according to claim 11, characterized by a second Pump unit (26) for discharging the medium (8) through the outlet opening (11), wherein the second pump unit (26) is controlled by means of the controller (15).

13. A microscope (17) with a device (12) according to one of claims 11 and 12, comprising a light source (18) for providing illumination radiation along an illumination beam path; a detection objective (21) for detecting detection radiation coming from the sample chamber (3); and a detector (22) for converting detected detection radiation into electronic signals.

14. Microscope (17) according to claim 13, characterized in that the light source (18) and the detection objective (21) are arranged in a configuration for transmitted light illumination.

15. Microscope (17) according to claim 13, characterized in that the light source (18) and the detection objective (21) are arranged in a configuration for inverse illumination and detection through the bottom (5) of the sample vessel (1).

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