Device for culturing cellular biological samples in a culture insert.

Abstract

This invention relates to an apparatus for culturing cellular biological samples in a culture insert, the apparatus comprising: a base element having a groove having a bottom formed therein, wherein the culture insert can be disposed in the groove; and a positioning element providing a first receiving position and a second receiving position for the culture insert, wherein the culture insert can be disposed on the positioning element at the first receiving position and the second receiving position such that the culture insert is disposed in the groove, and wherein the distance between the bottom of the groove and the bottom of the culture insert in the first receiving position is less than the distance in the second receiving position.

Description

Technical Field

[0001] This invention relates to an apparatus for culturing cellular biological samples. Background Technology

[0002] Today, transwell systems, or membrane insert systems, are used in cell interface experiments, such as co-culture and migration experiments. Almost all membrane inserts (also called culture inserts) are based on the same principle: a porous membrane serves as the bottom of the insert, forming a cup with a porous bottom permeable to cells and molecules. This cup can be inserted into a container holding a nutrient fluid. The porous membrane then acts as a scaffold or matrix for cell adhesion, allowing cells to form monolayers or multilayers on the membrane while allowing nutrients in the nutrient solution to diffuse through and supply nutrients to the cells. The container in which the membrane insert is placed must provide sufficient space for the nutrient solution below and around the insert, as adequate nutrient exchange is required for culturing living cells below the bottom. Therefore, during culture, the distance between the bottom of the container and the porous membrane must not fall below a certain threshold.

[0003] Meanwhile, when examining cells on the porous membrane through the bottom using an inverted microscope, this threshold of distance to the bottom of the container significantly limits the ability to examine the cells using a standard objective. One way to address this is to remove the porous membrane from the membrane insert and place it on the microscope slide. While this is possible for endpoint analysis of a single sample, it does not allow for process control and automation of the microscope.

[0004] Therefore, there is a need for a simple and user-friendly device that provides a suitable arrangement for changing the position of the membrane insert between an ideal position for culture and an ideal position for imaging. The ideal position for culture is the location where the nutrient solution is adequately supplied below the porous membrane due to the minimum distance between the bottom of the container and the porous membrane; the ideal position for imaging is the minimum possible distance between the objective lens and the cells or sample. Such a device cannot be satisfactorily achieved using currently available solutions.

[0005] In this context, one object of the present invention is to provide an apparatus for culturing cellular biological samples, the apparatus comprising a simple and user-friendly mechanism for switching between a membrane insert for culturing and a membrane insert for microscopic examination.

[0006] This objective is achieved by the subject matter described in claim 1. The dependent claims provide further embodiments. Summary of the Invention

[0007] According to the present invention, an apparatus for culturing cellular biological samples is provided, the apparatus comprising: a base element having a groove having a bottom formed therein, wherein a culture insert can be disposed in the groove; and a positioning element providing a first receiving position and a second receiving position for the culture insert, wherein the culture insert can be disposed on the positioning element in the first receiving position and in the second receiving position such that the culture insert is disposed in the groove, and wherein in the first receiving position, the distance between the bottom of the groove and the bottom of the culture insert is less than the distance in the second receiving position.

[0008] Therefore, the device provides two distinct receiving positions for the culture insert, in which it is positioned within the well slots. This means the culture insert protrudes into the well slots at both receiving positions. The first receiving position is characterized by a smaller distance between the bottom of the culture insert and the bottom of the well slot compared to the second receiving position. Therefore, the first receiving position is particularly suitable for microscopic examination, while the second receiving position is designed for culture. These two receiving positions are provided by a positioning element. Thus, switching between the two receiving positions can be achieved using the positioning element, eliminating the need to lift or remove the culture insert from the well slots. This simplifies the switching between the two receiving positions and makes it more user-friendly.

[0009] The first receiving position can be a location where the distance between the bottom of the culture insert and the bottom of the pore is zero. This means that the bottom of the culture insert rests on the bottom of the pore. Alternatively, the distance between the bottom of the culture insert and the bottom of the pore can be less than 1 mm. In the second receiving position, the distance between the bottom of the pore and the bottom of the culture insert is greater than in the first receiving position. This allows the nutrient solution to flow freely between these gaps and ensures nutrient exchange through the porous membrane. In the second receiving position, the gap can be 1 mm or greater, particularly 2 mm or greater, or even 3 mm or greater.

[0010] The culture plug can also be called a membrane plug.

[0011] The bottom of the pore can be transparent. This allows for inverted microscopy examination through the bottom. Therefore, samples located within the culture insert can be easily examined. The bottom of the pore can be transparent, particularly in the visible light wavelength range. Alternatively or additionally, transparency in the near-infrared wavelength range (e.g., between 800 nm and 1500 nm) can also be provided. Combined with a distance of less than 1 mm between the bottom of the culture insert and the bottom of the pore in the first receiving position, high resolution can be achieved in inverted microscopy examination through the bottom.

[0012] The thickness of the bottom of the pore slot can be between 100 μm and 2.0 mm, particularly between 160 μm and 190 μm, and even more particularly between 165 μm and 175 μm, for example, 170 μm. This minimizes the distance between the bottom of the culture insert and the objective lens in the first receiving position, allowing for high resolution. The bottom of the pore slot can comprise glass or plastic (such as a polymer film) or be made of either glass or plastic. The bottom of the pore slot can have optical properties such as birefringence and autofluorescence properties (e.g., Schott D 263 glass, No. 1.5H). The pore slot can have a truncated conical or cylindrical shape. The cross-section of the pore slot can be circular. The substrate element can be a porous pore plate with a plurality of pores arranged in a regular structure. An example is a plate with 24 pores arranged in six columns and four rows. A plate with six pores arranged in three columns and two rows is also possible. Compared to a single pore slot, the advantage of a porous pore plate is that multiple samples can be cultured simultaneously, thereby increasing yield. All the holes and slots in a porous grooved plate can be formed identically.

[0013] The average diameter of the slot is, for example, 16.0 mm. The depth of the slot can be approximately 18.0 mm.

[0014] The culture insert can have a basic cylindrical shape. The average outer diameter of the culture insert can be 10.0 mm. This ensures that when the culture insert is centered within the cavity, the distance between the culture insert and the inner wall of the cavity is approximately 3 mm.

[0015] In addition, the cultivation insert may include fastening devices for positioning or securing it to the positioning element.

[0016] The bottom of the culture insert may be or contains a porous membrane. This membrane may be permeable to cells or molecules and to liquids. The membrane can serve as a scaffold or matrix for cell adhesion, allowing cells to form monolayer and multilayer structures on the membrane.

[0017] The bottom of the culture insert can have a thickness between 1 μm and 500 μm. Specifically, the thickness can be between 10 μm and 100 μm. Example values ​​are 12 µm and 50 µm.

[0018] If the objective lens is placed as close as possible to the bottom of the well, the minimum distance between the objective lens and the cells on the bottom of the culture insert corresponds to the sum of the thickness of the bottom of the well and the thickness of the bottom of the culture insert.

[0019] The culture insert can be hooked into the positioning element, particularly at the first receiving position and / or the second receiving position. The culture insert may include a protrusion. The protrusion can interact with a corresponding mating part on the positioning element. In this way, the culture insert can be hooked into the positioning element.

[0020] The positioning element may have two different layers defining a first receiving position and a second receiving position, and may be configured such that the change between the first receiving position and the second receiving position is achieved by relative movement between the positioning element and the culture insert.

[0021] The change between the two receiving positions is achieved through simple relative movement. In particular, the positioning element can be fixed relative to the substrate element. The culture insert can be configured such that the change between the two receiving positions is achieved through movement of the culture insert, wherein the movement includes displacement (translation) and / or lifting or lowering and / or rotation.

[0022] For example, two layers can be formed on the upper edge of the cavity and connected by a step. Here, the change from the first receiving position to the second receiving position is achieved by rotating the culture insert and lifting it over the step between the two layers.

[0023] A ramp connecting two layers can be positioned between them. The inclination angle of the ramp can be greater than 0° and less than 90°. Specifically, the inclination angle can be between 20° and 70°, between 30° and 60°, or between 40° and 50°. Due to this ramp, the culture insert does not need to be lifted to move to the second receiving position. During rotation, the height difference between the two receiving positions is overcome by pushing upwards over the ramp.

[0024] The positioning element is movable relative to the substrate and can be switched to a first position and a second position, wherein the first position defines a first receiving position and the second position defines a second receiving position. This means that the positioning element can be switched to a first position and a second position relative to the substrate element. The first position of the positioning element defines a first receiving position of the culture insert. The second position of the positioning element defines a second receiving position of the culture insert. Therefore, the first position and the second position are different. In particular, the substrate element may have two layers that define the first position and the second position for the positioning element.

[0025] This represents an alternative way to achieve a simple change from the first receiving position to the second receiving position. In the previous example, the positioning element was fixed relative to the substrate element, and the change was achieved by moving the culture plug; in this example, the positioning element moves relative to the substrate element.

[0026] In particular, the culture insert can be fixed relative to the positioning element. This has the significant advantage that the culture insert does not need to be moved independently. This reduces the risk of accidental contamination of the sample or cells in the culture insert, which could otherwise occur due to contact between the culture insert and the user moving it.

[0027] The movement of the positioning element between the two receiving positions can be purely vertical (parallel to the longitudinal axis of the orifice). Alternatively, the movement of the positioning element can be a combination of horizontal and vertical movements. A particular advantage of purely vertical movement is that there is no strict limitation on the height difference between the first and second receiving positions. For horizontal movement, the range is instead limited by the initial distance between the sidewalls of the orifice and the culture insert.

[0028] According to the present invention, the positioning element provides two receiving positions. The positioning element itself may have devices for arranging the culture insert at two different positions on the positioning element to achieve the two receiving positions. Optionally, the positioning element may be moved relative to the substrate element to two different positions, thereby providing two receiving positions. Both configurations also achieve the added advantage of user-friendliness.

[0029] The device may also include a ramp, through which the positioning element can be transitioned from a first position to a second position. In this document, a ramp refers to a surface inclined relative to a horizontal plane at an angle greater than 0° and less than 90°. This allows the height difference to be overcome by pushing over the ramp. Here, this is the height difference between the first and second positions of the positioning element. The angle of the ramp can be between 20° and 70°, between 30° and 60°, or between 40° and 50°.

[0030] Due to the ramp, the positioning element can be moved from a first position to a second position simply by applying a lateral force. The necessary vertical displacement of the positioning element is achieved by pushing it up the ramp. This greatly simplifies changes between the two positions, thereby simplifying changes between the two receiving positions used to train the plug. Furthermore, automated changes are much easier. A robot designed for this purpose only needs to be trained to move the positioning element laterally to perform the change. This is much easier to achieve than lifting or a combination of lifting and lateral movement.

[0031] The ramp can be part of the base element. The positioning element can contact a part of the base element. The part can then be pushed over the ramp to move the positioning element from a first position to a second position. For example, it is conceivable that the positioning element includes a bracket by which the positioning element is mounted on the base element, thereby allowing the bracket to be pushed over the ramp.

[0032] A ramp can be arranged on the horizontal section of the base element. The edges of the slot can be chamfered. This beveling of the edges creates a ramp that the support can slide on.

[0033] The positioning element can also be switched from the second position to the first position via a ramp.

[0034] Alternatively, the ramp can be an additional movable element of the device that can be displaced relative to the base element, wherein the positioning element can be moved from a first position to a second position by moving the ramp. The ramp can only move horizontally.

[0035] Assuming the ramp is an additional element, it is also called a ramp element. To switch between the first and second receiving positions, only the ramp element needs to be shifted (horizontally, without vertical movement), thereby providing the switching between the first and second positions of the positioning element. In particular, there is no need to actively move the positioning element, which further simplifies the switching between the two receiving positions.

[0036] The movement of the positioning element caused by the displacement of the ramp can only be vertical, that is, along the longitudinal axis of the slot. In other words, the direction of movement of the ramp and the direction of movement of the positioning element can be perpendicular to each other.

[0037] The device may include multiple ramp elements, each of which can be displaced relative to the base element. Each ramp element can be displaced individually, i.e., independently of the other ramp elements.

[0038] In a device with a ramp, a positioning element can be arranged above a base element, with the ramp positioned between the base element and the positioning element, and the positioning element contacting the ramp. Specifically, the positioning element can be partially resting on the ramp. The ramp can also be arranged directly on the base element.

[0039] This arrangement, where the base element, ramp or ramp element, and positioning element are stacked vertically in this order, offers the advantage of allowing switching between two positions without requiring significant force. Strictly speaking, the required force is largely determined by the mass of the positioning element.

[0040] The positioning element can be disposed above the base element and can have a support having at least a partially beveled lower side, wherein when the positioning element is in a first position, the beveled portion of the lower side of the support contacts the edge of the slot and partially protrudes into the slot. Optionally, the base element may include a recess adjacent to the slot, wherein when the positioning element is in the first position, the beveled lower side of the support protrudes into the recess. The advantage of using a slot is that the positioning element can be used with conventional perforated slotted plates. This eliminates the need for additional slots.

[0041] Here, the ramp is achieved by the beveled lower side of the support. Moving the positioning element causes the beveled lower side to slide along the edge of the slot, thereby changing the distance between the base element and the positioning element located thereon. In this way, the positioning element transitions from a first position to a second position, and vice versa. In this case, neither a separate ramp element nor a ramp as part of the base element is required. Therefore, this design represents a simple and easy-to-manufacture option that allows for switching between two receiving positions for the culture insert. In this embodiment, the movement of the positioning element consists of horizontal displacement and vertical lifting or lowering.

[0042] The lower first segment may be beveled, and the second segment adjacent to the first segment may be horizontal. In the second position, the second segment may rest on the edge of the slot. In the first position, as described above, the first segment may rest on the edge of the slot.

[0043] The culture insert can be moved to a first receiving position and locked in a second receiving position by applying an increased force. The positioning element can be moved to a first position and locked in a second position by applying an increased force. For example, in the second position, a form-locking connection can exist between the positioning element and the substrate element. A form-locking connection between the culture insert and the positioning element is also possible when the culture insert is in the second receiving position.

[0044] This locking mechanism prevents the culture insert from unintentionally moving from the second receiving position to the first receiving position (e.g., through a slight impact on the device), or prevents the positioning element from unintentionally moving from the first position to the second position. For example, without the locking mechanism, the positioning element might unintentionally slide down a ramp.

[0045] The increased force required should be understood as a force that must be applied beyond the static friction between the ramp and the positioning element. For example, an additional protrusion may be provided on the ramp, which must be traversed to move the positioning element from the second position to the first position. Optionally, the ramp may include a section with higher friction (especially higher sliding friction) with the positioning element. The section of the ramp may have a surface made of rubber or another material that increases said friction.

[0046] The lower side of the bracket of the positioning element may have three sections:

[0047] The first segment is inclined at a first angle relative to the horizontal plane.

[0048] The second segment, adjacent to the first segment and inclined at a second angle relative to the horizontal plane, the second angle having the opposite sign to the first angle, and

[0049] The third segment is adjacent to the second segment and is formed parallel to the horizontal plane.

[0050] In the first receiving position, the first segment contacts the edge of the slot, and in the second receiving position, the third segment contacts the edge of the slot. Specifically, the horizontal direction is perpendicular to the vertical or longitudinal axis of the slot.

[0051] These three sections provide triangular protrusions on the underside of the bracket, which can be described as teeth. In the second position of the positioning element, the third section rests on the edge of the slot. To move the positioning element to the first position, it must pass over the teeth, which requires increased force. This locks or engages the positioning element in the second position and prevents it from accidentally falling into the first position. Once the teeth have been passed, the positioning element slides along the first section to the first position.

[0052] The third section can also be tilted at an angle of no more than 10° relative to the horizontal plane. This also ensures that, in the second position of the positioning element, the third section rests on the edge of the slot and will not slip. If the angle is small enough, the static friction between the support and the base element is sufficient to prevent slippage.

[0053] This design is particularly suitable if the base element is a porous slotted plate with adjacent slots. In the second position of the positioning element, the third section rests on a surface between the adjacent slots, which separates the two slots, and toothed protrusions extend into the adjacent slots. The surface between the adjacent slots is also called the partition wall.

[0054] The first angle can be between 50° and 60°, especially 54°. The second angle can be between 65° and 75°, especially 70°.

[0055] The specified values ​​for the first and second angles have proven to be optimal in terms of practicality. In particular, these angles provide reliable locking of the positioning element in the second position and require an appropriate amount of force to move the positioning element from the first position to the second position.

[0056] Looking at the third section, the tooth height can be between 0.7 and 0.9 mm, particularly 0.8 mm. The teeth can be rounded at their ends. This rounding ensures more uniform movement of the positioning element when displacement occurs on the teeth. Furthermore, the tooth height is chosen so that the height difference to be traversed is not too large. Looking at the second section, the third section can have a length of approximately 1 mm.

[0057] Using a ramp, the positioning element only needs to move laterally to switch between two positions. The first segment acts as a ramp, additionally converting lateral motion into vertical motion. This motion can be relatively easily automated using robots because complex motion sequences are not required.

[0058] The device may also have one or more lateral limiting elements to restrict the horizontal movement of the positioning element between a first position and a second position. At least one lateral limiting element ensures that the positioning element cannot move beyond the first and second positions. Simultaneously, the first and second positions are clearly defined by a stop at at least one lateral limiting element to allow the user to clearly identify the correct position of the positioning element. At least one lateral limiting element is also helpful in the case of robot automation, as it specifies two positions of the positioning element, and they do not need to be precisely implemented in the robot's programming.

[0059] The at least one lateral limiting element can be attached to the base element. The lateral limiting element can form a frame extending upward from the base element, thereby allowing the positioning element to move within the frame.

[0060] The positioning element may have a through-hole formed for arranging the culture insert, wherein the through-hole includes an additional protrusion. This protrusion allows the shape of the through-hole to deviate from a circle or an ellipse.

[0061] This protrusion can be used to bypass the culture insert and introduce culture medium into the well slot using a pipette, especially when the culture insert is in the second receiving position. The protrusion provides additional space for the pipette and simplifies the addition of culture medium.

[0062] The positioning element may have two supports with three segments each. The positioning element may have four supports, with at least two supports having three segments each. The remaining supports may have a flat lower side and / or a beveled lower side. The arrangement of the supports may be symmetrical with respect to the central axis of the positioning element.

[0063] The arrangement of four supports increases the stability of the device because the positioning element is stably rested on the base element. Two of the supports have three segments, ensuring that the positioning device is not obstructed on one side and does not rotate accidentally relative to the base element. Since supports with three segments are more delicate to manufacture than supports with flat or beveled lower sides, not all supports include three segments, simplifying the manufacture of the positioning element.

[0064] The base element may include or be composed of plastic. Specifically, the base element may include plastics such as COC (cyclic olefin copolymer), COP (cyclic olefin polymer), PC (polycarbonate), PS (polystyrene), PE (polyethylene), PMMA (polyethylene methacrylate), or transparent thermoplastics or elastomers. The positioning element may include or be composed of plastic materials. Specifically, the positioning element may include PS, polypropylene (PP), or polyethylene terephthalate (PET), or be composed of these. The advantage of choosing these plastics is that both elements can be sterilized and therefore can be reused.

[0065] The base element and / or positioning element can be injection-molded parts. Using the materials described above, the base element or positioning element can be efficiently and mass-produced at a consistent quality and cost. Alternatively, the positioning element and / or base element can be manufactured using 3D printing. The advantage of 3D printing is that it can reliably produce intricate and complex structures. The base element and / or positioning element can be fabricated as a single part.

[0066] Alternatively, the substrate element may include or be composed of glass.

[0067] Glass or plastic used as substrate elements can exhibit birefringence and autofluorescence properties, especially those of Schott glass slides (such as Schott D 263 glass, No. 1.5H).

[0068] The device may include a cap that can be placed on a substrate element. The cap may be shape-fitting. Specifically, when the cap is in place, a positioning element is located between the substrate element and the cap. The cap shields the culture insert and the cells contained therein from the external environment, thereby protecting them from contamination.

[0069] The present invention also provides a system comprising the aforementioned apparatus and culture plug for culturing cellular biological samples.

[0070] The culture insert may include: a bottom having a porous membrane permeable to cells or molecules; and a fastening element by means of which the culture insert is positioned on a positioning element. The culture insert may be a single-use item. Attached Figure Description

[0071] Other features and advantages of the invention are explained below with reference to the exemplary accompanying drawings.

[0072] Figure 1 A first apparatus for culturing cellular biological samples is shown;

[0073] Figure 2 A second apparatus for culturing cellular biological samples is shown;

[0074] Figure 3A , Figure 3B A third apparatus for culturing cellular biological samples is shown, along with a detailed view;

[0075] Figure 4A , Figure 4B A fourth apparatus for culturing cellular biological samples is shown, along with a detailed view;

[0076] Figure 5A , Figure 5B A fifth apparatus for culturing cellular biological samples is shown, along with a detailed view;

[0077] Figure 6 The positioning element of the sixth device for culturing cellular biological samples is shown;

[0078] Figure 7 The positioning elements of the seventh device for culturing cellular biological samples are shown;

[0079] Figure 8 A detailed view of the scaffold according to the seventh device for culturing cellular biological samples is shown; and

[0080] Figure 9A , Figure 9B A detailed view of a seventh apparatus for culturing cellular biological samples is shown, with positioning elements in a first and second position. Detailed Implementation

[0081] In the following text and the accompanying drawings, unless otherwise stated, the same reference numerals are used for the same or corresponding elements in the various embodiments.

[0082] Figure 1 A first device 10 for culturing cellular biological samples in a culture insert 100 is shown. The device includes a substrate element 11 in which wells 12 with bottoms are formed. Specifically, the substrate element may be a multi-well plate, in this embodiment having 24 wells arranged in a square array of six columns and four rows. All wells of the multi-well plate are uniformly formed. It should be understood that the substrate element 12 is not limited to the shape and number of wells shown.

[0083] In the following discussion, the perforated slot 12 and the culture insert 100 disposed therein are described, and the description given therein applies in the same manner to other perforated slots of the porous slot plate. Furthermore, other devices described according to the following figures are also based on this porous slot plate, such as the substrate element 11, and need not be mentioned further.

[0084] Furthermore, the device 10 includes a positioning element 20 that provides a first receiving position and a second receiving position for the culture insert 100. In the first receiving position, the distance between the bottom of the slot 12 and the bottom 102 of the culture insert 100 is smaller than in the second receiving position. Figure 6 A visual schematic diagram showing the distances between the culture inserts at the first and second receiving positions is provided. To achieve the aforementioned purpose, the positioning element 20 has two layers. More specifically, the positioning element 20 is formed around the aperture groove in a structure comprising two layers. The higher layer defines the second receiving position, while the lower layer defines the first receiving position. The positioning element 20 is fixed relative to the substrate element 11.

[0085] The culture insert 100 can be arranged in the slot 12 such that it protrudes into the slot 12. For this purpose, the culture insert 100 includes three fastening elements 101, which allow it to be positioned on the positioning element at three support points. This arrangement can be, in particular, a fastener or a hook. In the example shown, the fastening elements are protrusions or hooks that allow the culture insert 100 to hook onto the positioning element 20. In the second receiving position, the culture insert 100 is hooked onto the higher layer. In the first receiving position, the culture insert 100 is hooked onto the lower layer.

[0086] The first receiving position can be a position where the distance between the bottom of the culture insert and the bottom of the pore is as small as possible. Specifically, the distance is zero in the first receiving position, i.e., the bottom of the culture insert rests on the bottom of the pore. A distance of less than 1 mm is also possible. In the second receiving position, the distance between the bottom of the pore and the bottom of the culture insert is greater. This allows the nutrient solution to flow smoothly within this distance and ensures nutrient exchange through the porous membrane. This distance can be 1 mm or greater, particularly 2 mm or greater, or even 3 mm or greater. Therefore, the first receiving position can also be referred to as the microscope position. The second receiving position can be referred to as the culture position.

[0087] To move from the first receiving position to the second receiving position, the culture insert 100 must be raised vertically relative to the positioning element 20 and rotated relative to the longitudinal axis of the slot 12. The positioning element 20 is fixed relative to the base element 11. However, simply rotating the culture insert 100 to move from the second receiving position to the first receiving position is insufficient. This is because an additional protrusion is provided between the two layers on which the culture insert 100 is hooked. This obstructs the culture insert 100 at the second receiving position, and the culture insert must first be raised to allow it to be rotated. This requires a greater force than simple rotation and prevents the culture insert 100 from accidentally falling into the first receiving position.

[0088] The culture insert 100 includes a base 102 containing a porous membrane. This membrane is permeable to cells and molecules and serves as a scaffold or matrix for cell adhesion, allowing cells to form monolayer or multilayer structures on the membrane. These structures should be visible under a microscope. Simultaneously, nutrient solutions can diffuse through the porous membrane from the pores, providing nutrients to the cells. The thickness of the porous membrane is between 10 μm and 100 μm, for example, 12 μm or 50 μm.

[0089] Each aperture includes a transparent bottom in the visible and / or near-infrared range (particularly wavelengths up to 1500 nm) (see...). Figure 6 Therefore, inverted microscopy can be performed via the bottom. The bottom of the well 12 can comprise either a glass slide or plastic, such as a polymer film. The optical properties of the bottom specifically correspond to the optical properties of the glass slide (e.g., Schott D 263 glass, No. 1.5H). The thickness of the bottom is between 100 μm and 2.0 mm, particularly between 160 μm and 190 μm, and further particularly between 165 μm and 175 μm, for example, 170 μm. Therefore, the minimum distance between the cells and the microscope objective is the sum of the thickness of the bottom of the well 12 and the thickness of the porous membrane (bottom 102) of the culture insert 100. This simplifies the use of the apparatus 10 for culture and microscopy while providing higher optical resolution. Furthermore, since cells do not need to be removed from the well 12 for microscopy, the risk of potential contamination is reduced.

[0090] In the first receiving position, the smaller distance between the cells and the objective lens allows for high-resolution microscopy without having to remove the culture insert 100 from the well 12. However, in the second receiving position, the nutrient solution can flow beneath the porous membrane, effectively delivering the nutrient solution to the cells. Switching between the first and second receiving positions is particularly easy and user-friendly.

[0091] Therefore, it is not necessary to remove the culture insert 100 from the well slot to switch between the two receiving positions. This also reduces the risk of cell contamination and simplifies the user's operation.

[0092] Figure 2 A second device 10 is shown, which is largely similar to Figure 1 The first device is shown. Therefore, the same components will not be described again. The main difference lies in the design of the positioning element 20.

[0093] A positioning element 20 is formed on one edge of the slot 12 and consists of three pairs of hooks arranged regularly along the edge. In the second receiving position, the culture insert 100 is hooked onto the hooks. In the first receiving position, the culture insert 100 is positioned immediately adjacent to the hooks on the edge of the slot 12. As per [the previous sentence, the last part is incomplete and requires further context]. Figure 1 Similar to the first device 10, the change from the first receiving position to the second receiving position is achieved by lifting and rotating the culture insert 100. Likewise, the culture insert 100 is locked in the second receiving position, and hooks prevent accidental rotation of the culture insert 100, thus preventing it from falling into the first receiving position.

[0094] Typically, the positioning element 20 and the culture plug 100 work together to enable two receiving positions. The positioning element 20 is specifically designed to fit the culture plug 100.

[0095] Figure 3 illustrates a third form of the device 10, including a different design of the positioning element 21. Here, the positioning element 21 is a plate arranged above the substrate element 11. The positioning element 21 can be switched to two different positions relative to the substrate element 11, and these two positions define a first receiving position and a second receiving position for the culture plug introduced above. The first position of the positioning element 21 defines a first receiving position of the culture plug 100. This will be referred to... Figure 3B A more detailed explanation is provided. To better illustrate the various components, positioning element 21 is... Figure 3A It is shown as being far above the base element 11 (exploded view).

[0096] The positioning element 21 is also configured to allow a culture insert to be disposed on the positioning element, such that the culture insert protrudes into the slot 12 in the substrate element 11. For this purpose, the positioning element has through holes 26. When the positioning element 21 is disposed in a first position and a second position above the substrate element 11, the through holes 26 are located above the slot 12, allowing the culture insert to protrude into the slot 12 through the through holes 26. Specifically, the substrate element 11 is a porous slotted plate as described above. Therefore, the positioning element 21 has as many through holes 26 as the slots in the substrate element 11. When the positioning element 21 is correctly placed on the substrate element 11, the through holes 26 are located precisely above the slots 12.

[0097] The base element 11 has an upward-facing annular frame that serves as a lateral limiting element 14. This frame restricts the mobility of the positioning element 21 to the extent that only the necessary displacement between the first and second positions is possible. In other words, the frame prevents the positioning element 21 from shifting beyond the first and second positions.

[0098] Figure 3B A detailed view shows how the positioning element 21 is arranged above the base element 11 in a second position. The frame has a lateral groove 14a in which two planes are formed. The positioning element 21 has a lateral protrusion 21a that protrudes into the groove 14a and rests on one of the planes. In the second position, the protrusion 21a rests on the higher layer. To change to the first position (not shown), the positioning element 21 is shifted and lowered such that the protrusion rests on the lower layer. Conversely, to change from the first position to the second position, the positioning element 21 is raised and moved.

[0099] A raised section can be provided between the two layers of the groove 14a to prevent the positioning element 21 from accidentally falling into the first position. In particular, a tight fit can be established between the positioning element 21 and the base element 11 in the second position by clamping the protrusion 21a between the protrusion and the sidewall of the groove 14a.

[0100] A feature of the positioning element 21 in plate form shown is that all culture inserts can be simultaneously switched from the first receiving position to the second receiving position, and vice versa. Figure 1 and Figure 2 In the illustrated apparatus, the culture insert can be individually switched from a first receiving position to a second receiving position and vice versa. Depending on the desired application, one of the two types of positioning elements 20, 21 may be preferred.

[0101] The device 10 may also include a lid 40, which can be placed on the substrate element 11. The lid shields the orifice 12 from the environment. The lid can be placed on the substrate element 11 in a form-fitting manner. A positioning element 20 is located specifically between the substrate element 11 and the lid 40. This allows the sample in the culture insert to be effectively shielded from external environmental influences and contamination. The lid can be placed on all the devices described herein in a corresponding manner.

[0102] Figure 4A Another device 10 is shown, having an alternative embodiment for switching between a first position and a second position. The base element 11 and positioning element 21 are substantially the same as those of the third device, therefore only the differences are explained here. For better illustration of the various components, Figure 4A It is a split view.

[0103] Here, the positioning element 21 has a bracket 22 instead of a lateral protrusion (see...). Figure 3BThe support is a column that extends from the surface of the positioning element 21 and stands upright on the base element 11. The device 10 also includes a movable ramp as a ramp element 31. The ramp element 31 includes a lower layer connected to the upper layer via a ramp. The positioning element 21 is erected on the ramp element 31 via the support 22. Figure 3A As shown, the device 10 includes a lateral limiting element 14 in the form of an upward-facing annular frame arranged on the base element 11. Since the change between the two positions of the positioning element 21 is entirely vertical here, the frame can be fitted tightly against the positioning element 21. This prevents undesirable lateral displacement and possible tilting of the positioning element 21.

[0104] Figure 4B yes Figure 4A A detailed view is provided, showing the positioning element 21 in a first position. The support 22 is positioned on the lower level of the ramp element 31. As the ramp element 31 moves, the support 22 slides upwards along the ramp to a higher level, after which the positioning element 21 is in a second position. The movement performed by the positioning element 21 is entirely vertical, i.e., without any lateral displacement relative to the base element 11.

[0105] To prevent the positioning element 21 from tilting, at least in the second position, two identical ramp elements 31 are provided in the device. These ramp elements interact with two supports 22 of the positioning element 21, which are arranged on different sides of the positioning element. Therefore, the two ramp elements 31 are also arranged on different sides of the base element 11. For optimal switching between the two positions, the two ramp elements 31 must move synchronously; otherwise, the positioning element 21 may temporarily tilt. The latter situation can be largely avoided by a tightly fitted frame.

[0106] The improvement of the fourth device is Figure 5A and Figure 5B The fifth device 10 is shown. This is particularly different from the design of the ramp element 31, which will be explained in more detail below. To better illustrate the various components, Figure 5A An exploded view is shown.

[0107] The ramp element 31 has the shape of a rectangular frame and is referred to hereinafter as the positioning frame. The positioning frame is arranged on the base element 11. Similar to... Figure 4A The base element 11 has a frame-shaped lateral limiting element 14, which has a... Figure 4A The same characteristics and functions are described herein. Furthermore, the frame includes four interruptions 14b through which the positioning frame passes. This is done to ensure that the positioning element can only move along one axis, otherwise it will be blocked by the frame. The range of this movement is also limited by the frame. The plate-shaped positioning element 21 is positioned on the ramp element 31.

[0108] The corner of the ramp element 31 is characterized as a ramp, enabling the positioning element 21 to switch between a first position and a second position (see...). Figure 4B For this purpose, one surface of the positioning frame is beveled relative to the horizontal plane in the beveled area. A corresponding portion of the positioning element rests on this surface. This portion can also be beveled at the same angle to achieve a form-fit connection between the positioning element 21 and the ramp element. Alternatively, this portion can also be horizontal.

[0109] If the ramp element 31 now shifts along the aforementioned axis, the positioning element 21 slides on the ramp and switches between a first position and a second position. The movement performed by the positioning element 21 is only vertical. The movement performed by the ramp element 31 is only horizontal.

[0110] Compared to the fourth device, the fifth device 10 has the advantage that the two ramp elements do not need to move simultaneously in order to switch between the two positions. However, in this case, the ramp element 31 may be more difficult to manufacture. The ramp element 31 may be very precise, making injection molding infeasible. 3D printing offers better results.

[0111] Figure 6 It shows Figure 4A and Figure 4B Another alternative to the design shown is referred to herein as the sixth device 10. Here, the ramp (not shown) is the fixing part of the base element 11 and is arranged on its surface. Similar to Figure 4B In the scenario described, positioning element 21 includes a bracket (not shown) that slides on a ramp when positioning element 21 is switched from a first position to a second position. The bracket can also be designed as follows: Figure 4B As shown. However, since the ramp is fixed, the change between the two positions is achieved by moving the positioning element 21. The figure now shows the positioning element in a first position (left half of the figure) and a second position (right half of the figure), and the culture insert 100 in a first receiving position (left half of the figure) and a second receiving position (right half of the figure) associated with it. As shown, the culture insert 100 is arranged on the positioning element 21 such that the culture insert protrudes into the slot of the substrate element 11. The culture insert 100 is fixed relative to the positioning element 21.

[0112] Matching the base element 11 with a porous grooved plate having 24 slots, the positioning element 21 includes 24 through holes 26. When the positioning element 21 is placed on the base element, the through holes are positioned directly above the slots. Fastening elements 26a are formed at each through hole 26, designed to secure the culture insert with their fastening elements 101. An example of a through hole 26 is shown. Here, the culture insert 100 is hooked into the positioning element 21 by engaging the fastening element 101 of the culture insert 100 in the fastening element 26a at the through hole. The culture insert 100 then protrudes through the through hole 26.

[0113] A partition wall 15 is formed between two adjacent slots 12, separating the two slots 12.

[0114] Each through-hole 26 has a protrusion 27, meaning that the shape of the through-hole 26 deviates from a circular shape. The protrusion 27 is used to bypass the culture insert and fill the well with nutrient solution. This can be done, for example, with a pipette. The protrusion 27 provides extra space for the pipette, making it easier and more convenient to inject the nutrient solution. This is also shown in the figure.

[0115] In addition, the device 10 includes a lateral limiting element 14, such as Figure 3A As shown, for example, the lateral limiting element can be designed to surround the frame. The positioning element 21 can only move laterally, and its lateral movement range is limited by the limiting element 14. When the positioning element 21 touches the right side of the frame, i.e., the right side of the limiting element 14, the positioning element 21 reaches a first position. The first position defines a first receiving position of the culture insert 100, also as shown. In the first receiving position, the bottom 102 of the culture insert 100 rests on the bottom 13 of the aperture 12. As described above, this represents the microscope position.

[0116] When the positioning element 21 abuts against the left side of the frame, i.e., the left side of the limiting element 14, the positioning element 21 reaches a second position. This second position defines a second receiving position for the culture insert 100, as also shown. In this second receiving position, the bottom 102 of the culture insert 100 is spaced apart from the bottom 13 of the slot 12 by a distance, which can be, for example, 1 mm. As described above, this represents the culture position.

[0117] In order to switch between the first and second positions, the positioning element 21 must move laterally, as can be seen from a comparison of the two relevant partial views. During this movement, the support slides over the ramp, thereby achieving a vertical offset between the first and second positions.

[0118] When the positioning element 21 abuts against the left side of the frame, i.e., the left side of the limiting element 14, the positioning element 21 reaches a second position. This second position defines a second receiving position for the culture insert 100, as also shown. In this second receiving position, the bottom 102 of the culture insert 100 is spaced apart from the bottom 13 of the slot 12 by a distance, which can be, for example, 1 mm. As described above, this represents the culture position.

[0119] In order to switch between the first and second positions, the positioning element 21 must move laterally, as can be seen from a comparison of the two relevant partial views. During this movement, the support slides over the ramp, thereby achieving a vertical offset between the first and second positions.

[0120] This embodiment has several advantages over the device described above. Firstly, the change between the two positions 21 can only be achieved by laterally moving the positioning element 21, and the vertical offset between the two positions is automatically achieved by moving it up or down along the ramp. Furthermore, for each direction of change (from the first position to the second position, and vice versa), there is only one contact point on the positioning element 21. This makes the change automated for the robot, as the robot only needs to be configured to move the positioning element 21 in two opposite directions. It is not necessary to raise or lower the positioning element. Moreover, as a fixing element of the base element 11, the ramp is easy to manufacture and does not need to be provided as a separate component of the device 10. Figure 5A This is especially true compared to the fifth device shown, in which the ramp element has an ingenious design and is difficult to manufacture by injection molding.

[0121] The base element 11 may be a perforated slotted plate. To meet the requirements of the ANSI SLAS standard for perforated slotted plates (e.g., regarding dimensions and distances between adjacent slots), it may be necessary for each slot to have an elliptical cross-section.

[0122] Figure 7 Positioning element 21 is shown, which can be coupled with according to Figure 6 The base element 11 (but without a ramp) is combined to form the seventh device. The device thus obtained is combined with... Figure 6 The sixth device differs in the design of the positioning element 21 and does not include the ramp that is part of the base element 11. As in the third to sixth devices described above, the positioning element 21 is a plate that can be arranged on the base element. The through hole 26 and its protrusion 27 have also been described. However, the shape of the bracket 22 is significantly different, and will now be referred to Figure 8 This will be described in more detail. The positioning element comprises a total of four supports 22. Two are shown directly in the figure, and the other two are located on opposite sides of the positioning element 21 with the same construction.

[0123] Figure 8 A detailed view of the aforementioned bracket 22, arranged below the positioning element 21, is shown. The lower side of bracket 22 has three segments: a first segment 23, a second segment 24, and a third segment 25. The first segment 23 is obliquely cut or inclined at an angle α1 of 54° to the horizontal plane. The second segment 24 follows or is adjacent to the first segment 23, and is obliquely cut or inclined at a second angle α2 of 70° in the opposite direction to the first segment. Because these angles are arranged in opposite directions, the two segments 23 and 24 form a peak of 56°. The third segment 25 follows or is adjacent to the second segment 24, and is formed substantially horizontally. Viewed from the third segment 25, this arrangement creates a protrusion formed by the second segment 24 and the first segment 23. This protrusion is also called a tooth. The height of the tooth is 0.8 mm, opposite to the third segment 25. The ends of the tooth may be rounded.

[0124] Figure 9A and Figure 9B A cross-sectional view of the seventh device 10 is shown. As described above, the substrate element 11 is a porous grooved plate with a total of 24 slots 12. The cross-sectional view shows a row of six slots 12. Culture inserts 100 are arranged in each slot and hooked onto positioning elements 21 in sequence.

[0125] Figure 9A Positioning element 21 is now shown in a first position, with culture insert 100 correspondingly in a first receiving position. In this receiving position, the culture insert stands upright on the bottom 13 of the slot 12. In other words, the bottom 102 of each culture insert 100 contacts the bottom 13 of the corresponding slot 12. Positioning element 21 abuts against lateral limiting element 14 on the right-hand side, preventing further rightward movement. In this first position, the first segment 23 of the support 22 contacts the edge of the slot 12. Because positioning element 21 abuts against lateral limiting element 14, it cannot slide further, even though the beveled first segment 23 rests on the edge.

[0126] The positioning element 21 can be in a first position lower than the position defined by the first receiving position. The difference between the corresponding first position and the actual first position is called travel excess. This travel excess ensures that all culture inserts 100 in the porous tray are in contact with the bottom 13 of the slot 12. Therefore, any deviation of the culture inserts 100 or the positioning element 21 can be compensated for. The travel excess can be between 0.3 mm and 0.5 mm, for example, 0.4 mm.

[0127] Figure 9BPositioning element 21 in its second position is now shown, with culture insert 100 correspondingly in its second receiving position. Here, a distance is maintained between the bottom 13 of the slot 12 and the bottom 102 of the culture insert 100. This distance may be, for example, 1 mm or greater. Positioning element 21 abuts against lateral limiting element 14 on its left side, preventing further leftward movement of positioning element 21. In the second position of positioning element 21, third segment 25 rests on the edge of slot 12. More precisely, a partition wall 15 separates the two slots 12, 12', and third segment 25 rests on this partition wall 15. The tooth protrudes into the adjacent slot 12'.

[0128] In order to move the positioning element to the first position, the positioning element must move to the right to achieve this. Figure 9A The configuration is shown. For this purpose, the second segment 24 must slide past the edge of the adjacent slot 12' by moving the positioning element 21, thereby pushing the teeth out to move them from the adjacent slot 12' into the slot 12, after which the first segment 23 contacts the edge of the slot 12. Therefore, the teeth and the second segment 24 are used to ensure that the positioning element 21 does not simply move from the second position to the first position, but requires additional force.

[0129] Figure 8 The angles of the cross-section shown have been proven to be optimal. The first angle α1 is small enough to allow the positioning element 21 to move with a reasonable force, and large enough to push the teeth out within the available space for lateral movement, thereby achieving lateral offset between the first and second positions. The second angle α2 is small enough to allow the positioning element 21 to return to the first position via robot-driven movement, and large enough to prevent the positioning element 21 from accidentally moving to the first position.

[0130] The aforementioned ramp concept for easy and automatic switching between two positions of the positioning element is also included in this embodiment. The first section 23 serves as a ramp over which the positioning element 21 must be pushed upwards to switch between the first and second positions. The primary purpose of the second section 24 is to lock the positioning element 21 in the second position, preventing it from accidentally sliding into the first position. However, the change between the two positions can be achieved simply by lateral displacement of the positioning element 21. This has the advantage of being relatively easy to automate. In particular, a correspondingly configured robot can perform this change, thus eliminating the need for the robot to perform any complex movements. This also allows for the automated cultivation and observation of long-term cultures. In such long-term cultures, microscopic observation must be performed simultaneously, requiring a change from the second position to the first. After microscopic examination, the positioning element must return to the second position for further microscopic examination. This can now be fully automated, facilitating the feasibility of corresponding experimental and test series.

[0131] In the cross-sectional view shown, one of the brackets 22 has a special structure with three segments, which will be referenced Figure 8 A more detailed explanation follows. The second bracket 22' comprises only two segments, which are equivalent to the first and third segments. In general, the positioning element 21 comprises four brackets, two of which have a structure with three segments 23, 24, and 25. This is sufficient to achieve the aforementioned advantages. Since these three segments are delicate and difficult to manufacture, it is advantageous to design only two brackets 22 accordingly.

Claims

1. An apparatus (10) for culturing cellular biological samples in a culture insert (100), the apparatus (10) comprising: A substrate element (11) having a groove (12) having a bottom (13) formed therein, wherein the culture insert (100) can be disposed in the groove (12), and Positioning elements (20; 21) provide a first receiving position and a second receiving position for the culture insert (100). The culture insert (100) is positioned on the positioning elements (20; 21) at the first receiving position and the second receiving position, such that the culture insert (100) is arranged in the slot (12), and In the first receiving position, the distance between the bottom (13) of the slot (12) and the bottom (102) of the culture plug (100) is less than the distance in the second receiving position.

2. The apparatus according to claim 1, wherein, The positioning element (20) has two different layers, which define the first receiving position and the second receiving position, and The positioning element is configured such that a change between the first receiving position and the second receiving position is achieved by relative movement between the positioning element (20) and the culture plug (100).

3. The apparatus according to claim 1, wherein, The positioning element (21) is movable relative to the substrate (11) and can be switched to a first position and a second position, wherein the first position defines the first receiving position and wherein the second position defines a second receiving position for the culture plug.

4. The device (10) according to claim 3, further comprising a ramp (31). in, The positioning element (21) can be moved from the first position to the second position via the ramp (31).

5. The device (10) according to claim 4, wherein the ramp (31) is an additional movable element of the device (10) capable of displacement relative to the base element (11), and The positioning element (21) is capable of being moved from the first position to the second position by shifting the ramp (31).

6. The apparatus according to claim 4 or 5, wherein, The positioning element (21) is arranged above the base element (11). The ramp (31) is arranged between the base element (11) and the positioning element (21), and The positioning element (21) is in contact with the ramp (31).

7. The apparatus (10) according to claim 3 or 4, wherein, The positioning element (21) is arranged above the base element and has a bracket (22) having at least a partially beveled lower side. When the positioning element (21) is in the first position, the oblique portion on the lower side of the bracket (22) contacts the edge of the slot (12) and partially protrudes into the slot (12).

8. The apparatus (10) according to any one of the preceding claims, wherein, The culture insert (100) is moved to the first receiving position by requiring increased force, and the culture insert (100) can be locked in the second receiving position, and / or The positioning element (20; 21) is moved to the first position by requiring increased force, and the positioning element (20; 21) can be locked in the second position.

9. The apparatus (10) according to claim 7, wherein, The lower side of the bracket includes three sections: The first segment (23) is inclined at a first angle (α1) relative to the horizontal plane. The second segment (24), which is adjacent to the first segment (23) and inclined at a second angle (α2) relative to the horizontal plane, the second angle having the opposite sign to the first angle (α1), and The third segment (25) is adjacent to the second segment (24) and is formed parallel to the horizontal plane. At the first receiving position, the first segment (23) contacts the edge of the slot (12), and In the second receiving position, the third segment (25) contacts the edge of the slot (12).

10. The apparatus (10) according to claim 9, wherein the first angle (α1) is between 50° and 60°, particularly 54°, and / or The second angle (α2) is between 65° and 75°, especially 70°.

11. The apparatus according to any one of claims 3 to 10, further comprising one or more lateral limiting elements (14) for limiting the horizontal movement of the positioning element (21) between the first position and the second position.

12. The apparatus (10) according to any one of claims 3 to 11, wherein the positioning element (21) has a through hole (26) formed for the arrangement of the culture insert (100), and The through hole (26) includes an additional protrusion (27). In particular, the shape of the through hole (26) deviates from a circle or an ellipse due to the protrusion (27).

13. The apparatus (10) according to claim 9 or 10, wherein, The positioning element (21) has two brackets (22) with the three sections.

14. A system for culturing biological samples, the system comprising: The device (10) according to any one of the preceding claims, and Cultivation Plug-in (100).

15. The system of claim 14, wherein the culture plug (100) comprises: The substrate (102) has a porous membrane that is permeable to cells or molecules; as well as Fastening element (101), by means of which the culture insert (100) is arranged on the positioning element (20).

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