Culture device for biological sample containing laminations

Abstract

The utility model relates to the technical field of bioreactors, in particular to a biological sample culture device comprising laminations. The culture device comprises a frame, and different numbers of laminations can be installed in the frame so as to adapt to different culture scales. Moreover, the laminations and the frame are designed in an inserting manner, and each lamination can be independently assembled and disassembled, so that the biological sample on each lamination can be observed in real time and in situ.

Description

Technical Field

[0001] The utility model relates to the technical field of bioreactors, in particular to a biological sample culture device comprising laminated sheets. Background Art

[0002] With the development of biomedicine, the demand for expanded production of biological samples is increasing.

[0003] Existing technologies for producing biological samples (such as cells, microorganisms, migratory bodies, exosomes, etc.) mostly use 2D culture dishes or cell factories, microcarrier systems or roller bottle-type equipment / instruments, but each has some problems. For example, 2D culture dishes are difficult to scale up, and other equipment / instruments are very expensive or require expensive reagents. Moreover, since these devices / instruments are often integrally formed or difficult to disassemble, it is impossible to observe the state of cells or biological products in situ through a microscope. In particular, some special biological samples cannot even be produced, such as migratory bodies (including engineered migratory bodies (eMig) or self-releasing migratory bodies (cMig)).

[0004] Based on this, there is an urgent need for a convenient, low-cost, and efficient culture device for expanding the production of biological samples. Utility Model Content

[0005] The utility model provides a culture device for biological samples including stacks. The culture device includes a frame, and different numbers of stacks can be installed in the frame to adapt to different culture scales. In addition, the stacks and the frame adopt a plug-in design, and each stack can be loaded and unloaded separately, so as to realize real-time and in-situ observation of biological samples on each layer of stacks. The plug-in design also allows the stack to be taken out and flipped over before being inserted into the groove of the stack holder. Of course, the rotating body can also be directly flipped over for culture, so as to complete double-sided culture on the front and back sides of the stack. And the stack spacing can be adjusted to control the size of the volume of culture fluid contacted per unit area of ​​each layer of stacks. It is possible to produce biological samples conveniently, at low cost and efficiently. The specific scheme is as follows:

[0006] The utility model provides a biological sample culture device containing laminates, the culture device comprising a tank body and a tank cover; a rotating body is arranged in the tank body, the rotating body is connected to the tank cover through a connecting rod, the rotating body comprises a frame, and at least one layer of laminates is arranged in the frame; the laminates are sheet-like structures, the laminates are divided into an A region, a B region and a C region, the A region and the C region are located on both sides of the B region, and the A region and the C region are asymmetric relative to the B region; wherein the A region and the C region are arc-shaped, and the B region is rectangular; the distance between the two end points of the A region is different from the distance between the two end points of the C region; wherein the distance between the two end points of one arc is greater than the side length of the B region and the distance between the two end points of the other arc, and is greater than the length of the frame; the laminates can be detachably mounted on the frame; spacer grilles are symmetrically arranged on both sides of the frame, and the laminates are placed in the spacer grilles.

[0007] Preferably, the laminations are placed horizontally in the spacer grid.

[0008] Preferably, the laminations are placed horizontally on the protrusions of the spacer grid.

[0009] It should be understood that the distance between the two end points of an arc is greater than the length of the frame to ensure that when the rotating body rotates, the stack can rotate together with the frame and the stack will not be thrown out.

[0010] Preferably, the distance between two adjacent protrusions of the spacer grid is 1-20 mm (preferably 2-10 mm, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mm), which can control the spacing between the laminates, and for different purposes, the distance between two adjacent protrusions of the spacer grid arranged in the frame can be the same or different to adjust the spacing between the laminates. Of course, the laminates can be placed with one or more protrusions spaced apart to further adjust the spacing.

[0011] Preferably, the distance between the two end points of the other arc is less than (slightly less than) or equal to the length of the frame.

[0012] Preferably, the B region forms a limiting surface, which can provide torque obtained by rotation.

[0013] In a specific embodiment of the present invention, the side length of the side where the B region is connected to the A region is less than the distance between the two endpoints of the A region, and the side length of the side where the B region is connected to the C region is greater than or equal to the distance between the two endpoints of the C region.

[0014] In a specific embodiment of the present invention, the side length of the side where the B region is connected to the C region is less than the distance between the two endpoints of the C region, and the side length of the side where the B region is connected to the A region is greater than or equal to the distance between the two endpoints of the A region.

[0015] Preferably, the can cover can be made of any material, preferably the can cover can be obtained by 3D printing or injection molding, such as ABS (acrylonitrile butadiene styrene terpolymer, acrylonitrile butadiene styrene, plastic material), PETG (polyethylene terephthalate copolymer).

[0016] Preferably, the tank body can be made of any transparent material, such as plastic or glass. In a specific embodiment of the present invention, the tank body is made of glass.

[0017] Preferably, the frame can be made of any material, preferably the frame is obtained by 3D printing, such as any one of ABS, polylactic acid (PLA), stainless steel or resin (VeroClearTM).

[0018] Preferably, the culture device further comprises a stack fixture (eg, at least two stack fixtures). The stack fixture is provided with stack spacers, and grooves are formed between adjacent stack spacers.

[0019] Preferably, the stack holder may be made of any material, for example the same material as the frame.

[0020] Preferably, the thickness of the stack spacer plate provided on the stack fixer is the same as the thickness of the protrusion at the corresponding position of the spacer grid provided on the frame.

[0021] Preferably, the height of the grooves formed between the stack spacers provided on the stack fixer is the same as the distance between two adjacent protrusions at corresponding positions of the spacer grid provided on the frame.

[0022] Preferably, the frame is provided with 1-100 layers of laminates, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 layers. It should be understood that, according to the needs of the specific implementation, the laminate may also include more layers.

[0023] Preferably, the material of the stack includes but is not limited to one or more of silicon material, fluorine material, glass quartz material, metal material, ceramic material or organic high molecular polymer material.

[0024] Further preferably, the silicon material includes but is not limited to polydimethylsiloxane (PDMS) or glass.

[0025] More preferably, the fluorine material includes but is not limited to polytetrafluoroethylene (PTFE) and the like.

[0026] Further preferably, the glass quartz material includes but is not limited to quartz and / or glass.

[0027] Further preferably, the metal material includes but is not limited to stainless steel and / or titanium alloy.

[0028] Further preferably, the ceramic material includes but is not limited to alumina and / or hydroxyapatite and the like.

[0029] Further preferably, the organic high molecular polymer material includes but is not limited to polymethyl methacrylate (PMMA), polystyrene (PS), plastic and / or polycarbonate (PC) and the like.

[0030] Preferably, the laminate is made of a transparent material.

[0031] In a specific embodiment of the present invention, the laminate is made of plastic, polystyrene (sheet material) or polycarbonate. Preferably, the laminate can be obtained by laser cutting.

[0032] Preferably, the laminate has a thickness of 0.1-5 mm, preferably 1-3 mm, more preferably 1-2 mm, for example 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 or 5 mm.

[0033] In a specific embodiment of the present invention, more than two laminates are included, and the sizes, shapes and materials of the two or more laminates may be the same or different.

[0034] Preferably, the stack is provided with a limiting hole, and the limiting hole is used to fix the position of the stack, for example, to fix the position of the stack relative to the rotating body or the frame.

[0035] Preferably, in order to increase the stability of the culture device, the limiting hole can be provided at any position of the stack.

[0036] In a specific embodiment of the utility model, a limiting hole is provided at the center of the stack, and the stack is connected to the hole at the center of the frame through the limiting hole at the center, and the connection includes a positioning pin.

[0037] It should be understood that the limiting hole provided at the center position of the stack means that the distance from the limiting hole to the vertex of the A region is equal to the distance to the vertex of the C region.

[0038] In a specific implementation of the present invention, the limiting holes are arranged at the edge of the A region and / or the edge of the C region.

[0039] Preferably, the stack fixer is connected to the stack through limiting holes provided on the edge position of the A region and / or the edge position of the C region, and the connection includes through positioning pins.

[0040] Preferably, the positioning pin can be made of any material, such as the same material as the frame, and can also be made of stainless steel, such as 304 stainless steel.

[0041] In a specific embodiment of the present invention, the spacer grids provided in the frame and the stacking plate holder are used in combination, which not only ensures that there is a certain spacing between the stacking plates, but also ensures that the spacing is accurate and fixed.

[0042] Preferably, the spacing between the stacked sheets may vary according to the type of biological sample to be cultured.

[0043] Preferably, the culture device is provided with a magnetic stirring bar.

[0044] Preferably, a fixing device is provided below the frame, and the magnetic stirring bar is provided below the frame through the fixing device. Preferably, there are at least two fixing devices.

[0045] Preferably, a limiting device is provided below the frame.

[0046] Preferably, the magnetic stirring bar is arranged at a central position below the frame.

[0047] Preferably, the fixing device is a hollow structure (such as a hole or a channel), and the magnetic stirring bar can pass through the hollow structure and be fixed below the frame. It should be understood that the distance between the fixing devices is less than the length of the magnetic stirring bar.

[0048] Preferably, the limiting device is arranged on one side of the direction in which the magnetic stirring bar passes through the fixing device, and preferably the limiting device is a baffle, which can limit the magnetic stirring bar to the central position below the frame. It should be understood that the thickness of the limiting device (preferably the baffle) is sufficient to limit the further movement of the magnetic stirring bar.

[0049] In a specific implementation of the present invention, the magnetic stirring bar is fixed at a central position below the frame by the fixing device and the limiting device.

[0050] Preferably, the connecting rod may be made of stainless steel, such as 304 stainless steel.

[0051] Preferably, the connecting rod comprises two parts, an upper end and a lower end.

[0052] Preferably, at least a portion of the upper end of the connecting rod is provided with a thread, the tank cover is provided with a hole, the upper end of the connecting rod passes through the hole provided on the tank cover, and the upper end of the connecting rod is fixed to the tank cover by a nut.

[0053] Preferably, there are at least two nuts, and in a specific embodiment of the present invention, there are two nuts, one of which is arranged above the tank cover, and the other is arranged below the tank cover.

[0054] Preferably, the lower end of the connecting rod is connected to the center position of the upper part of the frame in a hanging manner.

[0055] Preferably, the lower end of the connecting rod includes a boss, a straight rod and a chassis.

[0056] Preferably, the diameter of the straight rod is smaller than the diameter of the chassis.

[0057] Preferably, a slide groove is provided at the center position of the upper part of the frame, and the slide groove includes an upper and lower part, wherein the opening distance of the lower part is greater than the diameter of the chassis, ensuring that the lower end of the connecting rod can slide into the slide groove, and the opening distance of the upper part is less than the diameter of the chassis and the opening distance of the lower part, and is greater than or equal to the diameter of the straight rod, ensuring that the connecting rod will not slide out when lifting; a rounded corner is provided at the connection between the upper and lower parts, and the chassis rotates within the rounded corner provided at the connection between the upper and lower parts.

[0058] Preferably, the radius of the fillet is the same as the radius of the chassis, and the angle of the fillet is the same as the angle of the chassis.

[0059] Preferably, in order to reduce friction and wear during assembly, during installation, the connecting rod is easier to align with the frame and the structure is more stable. The bottom of the slide groove is chamfered, and the chamfer angle can be optionally in the range of 10°-80°, preferably 20°-60°, for example 10, 20, 30, 40, 45, 50, 60, 70 or 80°.

[0060] It should be understood that in specific use, the lower end of the connecting rod can slide into the sliding groove, and after the connecting rod is lifted, the chassis can be stuck in the rounded corner, which can ensure that the connecting rod will not slide out during use (rotation) and can also ensure that the frame can rotate.

[0061] Preferably, a liquid adding bottle mouth and a liquid discharging bottle mouth are obliquely arranged on the edge of the tank cover, the openings of the liquid adding bottle mouth and the liquid discharging bottle mouth are oriented in the same direction, and the liquid adding bottle mouth and the liquid discharging bottle mouth are provided with ventilation covers, and the ventilation covers are provided with ventilation holes.

[0062] Preferably, the liquid adding bottle mouth and / or the liquid discharging bottle mouth is a cylinder, and further preferably, the liquid adding bottle mouth and / or the liquid discharging bottle mouth is a thin-walled cylinder.

[0063] Preferably, the angle between the opening direction of the liquid adding bottle mouth and the plane where the tank cover is located and the center direction of the tank cover is an acute angle, and the angle between the opening direction of the liquid discharging bottle mouth and the plane where the tank cover is located and the center direction of the tank cover is an obtuse angle.

[0064] Preferably, the acute angle is 30-75°, for example 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75°.

[0065] Preferably, the obtuse angle is 120-165°, for example 120, 125, 130, 135, 140, 145, 150, 155, 160 or 165°.

[0066] Preferably, the liquid adding bottle opening and the liquid discharging bottle opening are asymmetric relative to the axis of the tank body, wherein the opening protruding out of the side wall of the tank body is the liquid discharging bottle opening.

[0067] Preferably, the distance between the center position of the liquid discharge bottle opening and the center position of the tank cover is greater than the distance between the center position of the liquid filling bottle opening and the center position of the tank cover.

[0068] Preferably, when adding liquid, the liquid flows into the tank body along the inner wall of the tank body to avoid directly flushing the stacking sheets.

[0069] Preferably, the distance between the frame and the inner wall of the tank body is 1-10 mm, more preferably 1.5-5 mm, for example 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mm.

[0070] It should be understood that setting a suitable distance between the frame and the inner wall of the tank body can maximize the use efficiency of the biological sample when planting the biological sample and the material use efficiency when carrying out the production process, while not causing excessive liquid flow shear force (avoiding damage to the attachment of the biological sample). For example, when the biological sample is a cell, the greater the distance, the more culture medium is wasted when planting the cell, and the smaller the distance, the greater the shear force generated by the rotation, which will have a negative effect on the cell (such as cell shedding).

[0071] Preferably, a sealing device is provided between the tank cover and the tank body, between the nut and the tank cover, between the vent cover and the liquid adding bottle opening, and / or between the vent cover and the liquid discharging bottle opening.

[0072] It should be understood that the sealing device can prevent liquid leakage during drainage and avoid various contaminations.

[0073] Preferably, the sealing device includes one or more of a sealing sheet, a sealing strip, a sealing ring, a gasket, a filter screen or a filter paper.

[0074] Preferably, the sealing device can be made of any material as long as it can perform a sealing function, such as silicone.

[0075] In a specific embodiment of the present invention, a sealing ring, such as an O-ring, is provided between the tank cover and the tank body.

[0076] In a specific embodiment of the present invention, a gasket is provided between the nut and the tank cover.

[0077] In a specific embodiment of the present invention, a filter screen or filter paper is provided between the vent cover and the liquid adding bottle opening, or between the vent cover and the liquid discharging bottle opening.

[0078] Beneficial effects of the utility model:

[0079] 1) The asymmetric shape of the stack and the limiting holes ensures that the stack and the frame are installed stably and can rotate together. The parallel limiting surfaces in the stack cooperate with the frame to provide torque obtained by rotation. In addition, the stack and the frame adopt a plug-in design, and each stack can be loaded and unloaded separately, so as to realize real-time and in-situ observation of biological samples on each layer of the stack. In addition, the plug-in design can also take out the stack, flip it over, and then insert it into the spacer to complete double-sided culture on the front and back sides of the stack.

[0080] 2) A connecting rod passing through the tank cover, wherein the upper end of the connecting rod has a thread, and the connecting rod and the tank cover are fixed by a nut, and can be adapted to rotating bodies with different numbers of laminations.

[0081] 3) In the preferred embodiment, the edge of the tank cover is provided with an opening facing the same liquid adding bottle opening and liquid discharging bottle opening, so as to facilitate liquid adding and liquid discharging. Meanwhile, a certain distance is set between the frame and the inner wall of the tank body. Such a design ensures that when liquid is added, the liquid will flow into the tank body through the inner wall of the tank body, and will not directly wash the stack, thereby avoiding washing off the biological sample attached to the stack. When discharging, the liquid can be discharged at a small tilt angle.

[0082] 4) In the preferred solution, the rotating body and the connecting rod are detachable, and the rotating body and the connecting rod can be assembled in a hanging manner: replacing the assembly method of connecting rods passing through holes commonly used in existing commercial products, making it convenient to assemble and unload the rotating body structure, so as to achieve inversion of the rotating body, plant cells on both sides of the stack, and increase the culture density. By designing chamfers, the connecting rod and the frame are matched with each other.

[0083] 5) In the preferred embodiment, the spacing between the multiple stacks can be adjusted according to the spacing grids and stack holders set in the frame to adjust the culture medium coefficient, thereby saving culture medium and other production process materials; in addition, this adjustable design makes the spacing between each layer of stacks precise and fixed. The precise and fixed spacing can ensure the uniform planting density of biological samples, including (1) the single layer of stacks is uniform in the XY direction; (2) the planting density of each layer of stack is similar (of course, the planting density of a certain layer of stack can also be increased or decreased by setting different spacing); (3) the planting density on the front and back of the single layer of stack is uniform.

[0084] 6) The spacing between the rotating body (frame) and the inner wall of the tank is optimized, which maximizes the efficiency of biological sample utilization and material utilization during the production process without causing excessive liquid flow shear force (to avoid damaging the attachment of biological samples).

[0085] 7) The rotating body speed is precisely controlled by a magnetic table or a motor-driven magnetic stirring bar to provide sufficient nutrient convection; and the liquid flow shear force to which the cells are subjected is controllable, thus avoiding the uncontrolled turbulence problem of the culture method in the existing technology (such as microcarrier culture system). Furthermore, the controllable liquid flow shear force can more effectively wash the biological samples and their products (such as migratory bodies) from the surface of the laminate, and can also provide additional physical factors to stimulate the cells.

[0086] 8) The culture device of the present application can achieve one-time complete drainage / replacement of liquid, thereby realizing a complex process, thereby avoiding the problem of the prior art (such as microcarrier culture system) requiring several complete liquid changes when producing engineered migratory bodies.

[0087] 9) In the preferred embodiment, an O-ring is provided at the connection position between the tank cover and the tank body to fix and seal the tank cover and the tank body to prevent liquid leakage during discharge. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0089] Figure 1 : Schematic diagram of the structure of the culture device;

[0090] Figure 2 : Schematic diagram of lamination structure;

[0091] Figure 3 : Schematic diagram of the lamination and frame installation structure;

[0092] Figure 4 : Schematic diagram of the installation of the magnetic stirring bar and the frame;

[0093] Figure 5 : Schematic diagram of connecting rod structure;

[0094] Figure 6 : Schematic diagram of the installation of connecting rod and frame;

[0095] Figure 7 : Schematic diagram of the connecting rod and rotating body structure;

[0096] Figure 8 : Schematic diagram of partial enlargement of the framework;

[0097] Fig. 9 : Schematic diagram of the distance between the frame and the inner wall of the tank;

[0098] Fig.10 : Schematic diagram of adding and draining liquid in the culture device;

[0099] Fig.11 : Schematic diagram of the culture device with added laminated structure;

[0100] Fig.12 : Schematic diagram of the method of using the culture device;

[0101] Fig.13 : Cell mapping diagrams on the front and back sides of the laminate;

[0102] Fig.14 : Comparison chart of the effects of the culture dish and the culture device of the present application.

[0103] In each figure, the numbers respectively represent: 1-tank body, 2-tank cover, 201-liquid filling bottle mouth, 202-liquid discharge bottle mouth, 203-vent cover, 204-gasket, 205-nut, 3-frame, 301-spacer, 4-connecting rod, 401-boss, 402-straight rod, 403-chassis, 5-laminate, 501-A area, 502-B area, 503-C area, 504-limiting hole, 6-laminate fixer, 601-laminate spacer, 7-magnetic stirring rod, 8-locating pin, D-spacer distance, L-spacer distance between frame and inner wall of tank body. DETAILED DESCRIPTION

[0104] The following will be combined with the drawings in the embodiments of the utility model to clearly and completely describe the technical solutions in the embodiments of the utility model. Obviously, the described embodiments are only some embodiments of the utility model, not all. Based on the embodiments in the utility model, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the utility model.

[0105] Example 1 Structural design and assembly of a culture device

[0106] 1. The culture device described in this application includes the following components (see Figure 1-3 ): tank cover 2 (3D printed acrylonitrile butadiene styrene, acrylonitrile butadiene styrene, ABS, plastic material), connecting rod 4 (304 stainless steel), tank body 1 (glass material), frame 3 (3D printed ABS plastic), stack 5 (divided into A area 501, B area 502 and C area 503, the structural schematic diagram of stack 5 can be seen in Figure 2 , the stack 5 is laser cut, 1 mm thick, polystyrene, plate), magnetic stirring bar 7, vent cover 203, nut 205 (304 stainless steel), gasket 204 (silicone material), stack fixture 6 (the stack fixture 6 is provided with stack spacers 601, and grooves are formed between adjacent stack spacers 601. The stack fixture 6 is made of 3D printed resin (VeroClearTM) material), positioning pin 8 (304 stainless steel), O-ring (silicone material), liquid filling bottle mouth 201 and liquid discharge bottle mouth 202.

[0107] 2. Assembly of each structure:

[0108] (1) Assembling the "rotating body": The "rotating body" of the bioreactor is assembled by assembling the frame 3, the stack 5 (different numbers of stacks 5 can be assembled according to the height of the frame 3, for example, 10 to 60 layers, and this embodiment has 10 layers of stacks 5), the magnetic stirring bar 7, the stack fixture 6 (two), and the positioning pins 8 (three). Figure 3-4 ,in, Figure 3 (1)-(2) insert the stack 5 into the groove between the two protrusions of the spacer 301 set in the frame 3, (3) insert a positioning pin 8 into the center limit hole 504, (4) insert two stack fixers 6 into the stack 5, (5) insert the two positioning pins 8 into the limit hole 504 to fix the stack fixers 6.

[0109] (2) Assemble the tank cover 2 and the tank body 1 together to form the "tank body" of the bioreactor; assemble the connecting rod 4 together with the nuts 205 (two), the gaskets 204 (two) and the tank cover 2 to form the connecting part between the tank cover 2 and the rotating body.

[0110] Among them, the structure of the connecting rod 4 is as follows Figure 5 As shown, the lower end of the connecting rod 4 includes a boss 401, a straight rod 402 and a chassis 403. The connection method of the connecting rod 4 and the rotating body is shown in FIG. Figure 6 The internal structure of the tank 1 (connecting rod 4 and rotating body) is as follows: Figure 7 shown.

[0111] 3. Design details (see Figure 2-7 ):

[0112] 1) Asymmetrically shaped laminations 5 (see Figure 2 ,in, Figure 2 (A) is a schematic diagram of the structure of the laminate 5, Figure 2 (B) is a schematic diagram of dividing the A area 501, the B area 502 and the C area 503) and the limiting holes 504 make the installation of the stack 5 and the frame 3 stable, and enable the stack 5 and the frame 3 to rotate together. The parallel limiting surfaces in the stack 5 cooperate with the frame 3 to provide the torque obtained by the rotation. In addition, the stack 5 and the frame 3 adopt a plug-in design (see Figure 3 ), each stack 5 can be loaded and unloaded separately, so as to realize real-time and in-situ observation of biological samples on each layer of stack 5; in addition, the plug-in design can also take out the stack 5, turn it over and then insert it into the spacer 301, so as to complete double-sided culture on the front and back sides of the stack 5.

[0113] 2) A connecting rod 4 passing through the tank cover 2, wherein the upper end of the connecting rod 4 has a thread, and the connecting rod 4 and the tank cover 2 are fixed by a nut 205, which can be adapted to a rotating body with different numbers of laminations 5 (see Fig.11 ).

[0114] 3) The edge of the tank cover 2 is provided with a liquid filling bottle opening 201 and a liquid discharge bottle opening 202 with the same opening direction, so as to facilitate liquid filling and liquid discharge. At the same time, there is a certain distance between the frame 3 and the inner wall of the tank body 1. Such a design ensures that when liquid is added, the liquid will flow into the tank body 1 through the inner wall of the tank body 1, and will not directly wash the stack 5, so as to avoid washing off the biological sample attached to the stack 5. When discharging liquid, it can also be completed at a small tilt angle (see Fig.10 ).

[0115] 4) The rotator and the connecting rod 4 are detachable, and the rotator and the connecting rod 4 can be assembled in a hanging manner: replacing the assembly method of the connecting rod 4 penetrating the hole commonly used in existing commercial products, making it convenient to assemble and unload the rotator structure, so as to achieve the inversion of the rotator, and plant cells on both sides of the stack 5 to increase the culture density (see Fig.12 ). By designing the chamfer, the connecting rod 4 and the frame 3 are matched with each other. The chamfer angle can be selected in the range of 10°-80°, preferably in the range of 20°-60°, and in this embodiment 45°, Figure 6 In the embodiment, (1) the connecting rod 4 is slid into the slide groove of the frame 3, and (2) the connecting rod 4 is lifted to fix the positions of the frame 3 and the connecting rod 4, and the gravity of the rotating body is used to ensure the concentric position relationship between the rotating body and the connecting rod 4. The rounded contact between the connecting rod 4 and the slide groove of the frame 3 limits the relative displacement between the two parts, so that the frame 3 can only rotate on the connecting rod 4 around the axial direction of the connecting rod 4.

[0116] 5) The spacing distance D between the multiple stacking sheets 5 can be adjusted according to the spacing grid 301 and the stacking sheet holder 6 provided in the frame 3 (see Figure 8 ), to adjust the culture fluid coefficient (the culture fluid coefficient represents the efficiency of the culture fluid, the unit is mL / cm 2 ). Taking cells as an example, because cells are planted on both sides, the culture medium coefficient = stacking distance (cm) / 2. The recommended coefficient for general 2D culture dishes is 0.2 mL / cm 2 , that is, the thickness of the culture medium is 0.2 cm. The spacing between the laminates 5 of the utility model can be as low as 1 mm, that is, the culture medium coefficient can be as low as 0.05 mL / cm 2 , this culture medium coefficient is equivalent to that of the microcarrier system, so culture medium and other production process materials can be saved; in addition, this adjustable design makes the spacing between each layer of stacked sheets 5 precise and fixed. The precise and fixed spacing can ensure the uniform planting density of biological samples, including (1) the single layer of stacked sheets 5 is uniform in the XY direction; (2) the planting density of each layer of stacked sheets 5 is similar (of course, the planting density of a certain layer of stacked sheets 5 can also be increased or decreased by setting different spacings); (3) the planting density on the front and back sides of the single layer of stacked sheets 5 is uniform.

[0117] 6) The spacing distance L between the rotating body (frame 3) and the inner wall of the tank 1 is optimized (see Fig. 9 ), which can maximize the efficiency of biological sample use and material use during the production process without causing excessive liquid flow shear force (avoiding damage to biological sample attachment). Taking cells as an example, the greater the distance, the more culture medium and seed cells are wasted when seeding cells, and the smaller the distance, the greater the shear force generated by the rotation, which will have a negative effect on the cells (such as cell shedding). The recommended spacing range is 1-10 mm, preferably 1.5-5 mm. In this embodiment, 3 mm.

[0118] 7) The magnetic stirring bar 7 is driven by a magnetic table or a motor to precisely control the rotation speed of the rotating body to provide sufficient nutrient convection; and the liquid flow shear force to which the cells are subjected is controllable, thereby avoiding the uncontrolled turbulence problem of the culture method in the prior art (such as the microcarrier culture system). Furthermore, the controllable liquid flow shear force can more effectively wash the biological sample and its products (such as the migratory body) from the surface of the laminate 5, and can also provide additional physical factors to stimulate the cells. This avoids the problem that the prior art (such as cell factories and similar products) cannot provide controllable liquid flow shear force.

[0119] 8) The culture device of the present application can achieve one-time complete drainage / replacement of liquid, thereby realizing a complex process, thereby avoiding the problem of the prior art (such as microcarrier culture system) requiring several complete liquid changes when producing engineered migratory bodies.

[0120] 9) An O-ring is provided at the connection position between the tank cover 2 and the tank body 1 to fix and seal the tank cover 2 and the tank body 1 to prevent liquid leakage during discharge.

[0121] 4. Working principle:

[0122] In the culture device described in the present application, the rotating body rotates with the magnetic table through the magnetic stirring rod 7, and the adjustable speed completes the uniform culture of the biological sample at a low speed, and forms a liquid flow shear force at a high speed, thereby washing down the biological sample attached to the stack 5 and completing the collection. The spacing between the stacks 5 is controllable, and the culture fluid supply coefficient can be increased to a level close to that of the microcarrier system. The stack 5 can be surface treated (such as protein coating, plasma treatment, etc.) to meet the requirements of different cells and different production processes.

[0123] 5. Usage:

[0124] The utility model can be used for culturing various biological samples, such as cells, migratory bodies (such as eMig or cMig).

[0125] This example takes cell culture as an example (see the operation flow chart for details) Figure 10-12 ).

[0126] 1. Double-sided cell planting: Place the sterilized assembled rotating body at the bottom of the sterilized tank body 1 (with the magnetic stirring bar 7 on top); prepare a cell suspension according to the required cell planting density, pour the cell suspension into the culture device along the inner wall of the tank body 1, and the cell suspension needs to be immersed in the top layer of the laminated sheet 5, and the liquid level is about 1 mm higher than the top layer of the laminated sheet 5, and cover the tank cover 2; place it in the incubator and wait for the cells to attach; pour out the cell suspension from the drain bottle 202, open the tank cover 2, and add the required number of cells; take out the rotating body (sterile state) and invert it at the bottom of the tank body 1 (with the magnetic stirring bar 7 at the bottom); add the cell suspension again (the same as the liquid addition step in the previous step), and place it in the incubator and wait for the cells to attach.

[0127] 2. Assemble the tank cover 2, tank body 1, rotating body, and connecting rod 4 together, add cell culture solution through the liquid adding bottle opening 201, control the speed (RPM, turns / minute) of the magnetic stirring bar 7 through the magnetic platform, and culture the cells at a low speed (e.g., less than 20 RPM) (culture according to the culture time and temperature of different cells disclosed in the prior art). After the culture is completed, rotate at a high speed (e.g., 50-150 RPM) to throw the cells attached to the stack 5 into the liquid environment, pour them out through the liquid discharge bottle opening 202, and collect them.

[0128] Example 2: Cells can be uniformly grown on the culture device described in this application

[0129] In order to prove the distribution of cells on the culture device described in the present application, an experimental microscope is used to observe and evaluate the cells cultured on each stack 5 and the front and back sides and / or different positions of the single-layer stack 5 of the culture device.

[0130] The results are as follows Fig.13 As shown, the cells are cultured uniformly on each stacked sheet 5 and on the front and back sides and / or different positions of the single-layer stacked sheet 5 of the culture device described in the present application.

[0131] Example 3: Comparison of production of engineered migratory bodies (eMig) using the culture device described in this application and using 2D culture dishes

[0132] To prove that the culture device described in this application can replace 2D culture dishes to efficiently produce biological samples, especially biological samples with high culture difficulty, this example takes the production of engineered migratory bodies (eMig) as an example, and refers to the methods disclosed in Examples 1, 2 and 7 in the patent document (CN116964192A), and uses the culture device described in this application to produce eMig of HEK293T cells (front and back double-sided seeding cells) as the experimental group. Take a T175 culture dish (area 175cm 2) was used as a control group to produce eMig using the same method as the traditional 2D culture dish. Finally, the cell planting density of both groups was ensured to be 4.57E4 cells / cm 2 The eMig produced by the experimental group and the control group were normalized to the area of ​​a T175 culture dish in the control group (175 cm 2 ), thus obtaining comparable mass per unit area (dimension: ug / 175 cm 2 ).

[0133] The results are as follows Fig.14 As shown, Fig.14 (A) in the middle indicates that the eMig produced using the culture device described in the present application and the eMig produced using the culture dish are very similar in morphology. Fig.14 (B) indicates that the production efficiency of eMig produced using the culture device described in the present application is higher than the production efficiency using a culture dish. Fig.14 (C) indicates that the eMig produced by the culture device described in the present application and the eMig produced by the culture dish have similar markers (e.g., T4-GFP (i.e., Tspan4-GFP), Na + K + ATP (i.e., cell membrane Na + -K + -ATPase)).

[0134] The above conclusions prove that the culture device of the present invention can be used to successfully and efficiently produce biological samples (such as eMig).

[0135] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, and these simple modifications all belong to the protection scope of the present invention.

[0136] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present utility model will not further describe various possible combinations.

Claims

1. A biological sample culture device comprising a laminate, characterized in that: The culture device comprises a tank body and a tank cover; A rotating body is arranged in the tank body, and the rotating body is connected to the tank cover through a connecting rod. The rotating body includes a frame, and at least one layer of laminate is arranged in the frame; the laminate is a sheet structure, and the laminate is divided into an A region, a B region and a C region, and the A region and the C region are located on both sides of the B region, and the A region and the C region are asymmetric with respect to the B region; wherein, The A area and the C area are arc-shaped, and the B area is rectangular; The distance between the two end points of the A region is different from the distance between the two end points of the C region; Wherein, the distance between two end points of one arc is greater than the side length of the B region and the distance between two end points of another arc, and is greater than the length of the frame; The stack is detachably mounted on the frame; Spacer grilles are symmetrically arranged on both sides of the frame, and the laminated sheets are placed in the spacer grilles.

2. The culture device according to claim 1, characterized in that: The culture device also includes a stack fixer, on which stack spacers are arranged, and grooves are formed between adjacent stack spacers.

3. The culture device according to claim 1, characterized in that: The stack is provided with a limiting hole, and the limiting hole is used to fix the position of the stack.

4. The culture device according to claim 1, characterized in that: The culture device is provided with a magnetic stirring bar; A fixing device and a limiting device are provided below the frame, and the magnetic stirring bar is fixed at a central position below the frame by the fixing device and the limiting device.

5. The culture device according to claim 1, characterized in that: At least a portion of the upper end of the connecting rod is provided with a thread, the tank cover is provided with a hole, the upper end of the connecting rod passes through the hole provided on the tank cover, and the upper end of the connecting rod is fixed to the tank cover by a nut.

6. The culture device according to claim 1, characterized in that: The lower end of the connecting rod is connected to the center position of the upper part of the frame in a hanging manner.

7. The culture device according to claim 6, characterized in that: The lower end of the connecting rod includes a boss, a straight rod and a chassis; A slide groove is provided at the center position of the upper part of the frame, and the slide groove includes an upper and a lower part, wherein the opening distance of the lower part is greater than the diameter of the chassis, and the opening distance of the upper part is less than the diameter of the chassis and the opening distance of the lower part, and is greater than or equal to the diameter of the straight rod; a rounded corner is provided at the connection between the upper and lower parts, and the chassis rotates within the rounded corner provided at the connection between the upper and lower parts.

8. The culture device according to claim 1, characterized in that: A liquid adding bottle opening and a liquid discharging bottle opening are obliquely arranged on the edge of the tank cover, the openings of the liquid adding bottle opening and the liquid discharging bottle opening are oriented in the same direction, the liquid adding bottle opening and the liquid discharging bottle opening are provided with ventilation covers, and the ventilation covers are provided with ventilation holes.

9. The culture device according to claim 8, characterized in that: The angle between the opening direction of the liquid adding bottle mouth and the plane where the tank cover is located and the center direction of the tank cover is an acute angle, and the angle between the opening direction of the liquid discharging bottle mouth and the plane where the tank cover is located and the center direction of the tank cover is an obtuse angle.

10. The culture device according to claim 1, characterized in that: The distance between the frame and the inner wall of the tank body is 1-10 mm.

Images