Three-dimensional cell culture device

Abstract

The invention discloses a three-dimensional cell culture device which comprises a fixing frame, a glass slide, a loading film, a nanofiber layer, a frame body and a cover, the loading film is attached to the surface of the glass slide, the nanofiber layer is coated on the side, away from the glass slide, of the loading film, and one side of the frame body abuts against the nanofiber layer; the glass slide is arranged on one side of the frame body, the cover is buckled on the other side of the frame body, a chamber for culturing cells is jointly formed among the glass slide, the frame body and the cover, and the fixing frame is used for locking the frame body and the glass slide. The three-dimensional cell culture device provided by the invention integrates culture, observation and analysis, so that errors among experimental groups are reduced, experimental operation is simplified, and time and manpower are saved.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture equipment, and in particular to a three-dimensional cell culture device. Background Art

[0002] Three-dimensional cell culture technology refers to the co-culturing of materials with three-dimensional structures and cells in vitro, allowing cells to migrate and grow in a three-dimensional spatial structure, forming a three-dimensional cell-carrier complex, making the cells' behavior closer to the body's actual physiological environment, thereby having better biological relevance and clinical predictability.

[0003] The three-dimensional cell culture device in the prior art is not convenient for observing and analyzing cells through a microscope. If one wants to observe and analyze cells through a microscope, the cells in the three-dimensional cell culture device need to be transferred, such as by cell climbing. This transfer process causes large errors between experimental groups, and the operation is cumbersome, wasting a lot of time and manpower. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides a three-dimensional cell culture device, which integrates culture and observation and analysis, reduces the error between experimental groups, simplifies experimental operations, and saves time and manpower.

[0005] The present invention is achieved through the following technical solutions:

[0006] A three-dimensional cell culture device comprises a fixing frame, a glass slide, a load film, a nanofiber layer, a frame and a cover, wherein the load film is attached to the surface of the glass slide, the nanofiber layer is coated on the side of the load film facing away from the glass slide, and one side of the frame abuts against the nanofiber layer; the cover is buckled on the other side of the frame, and the glass slide, the frame and the cover together form a chamber for culturing cells, and the fixing frame is used to lock the frame and the glass slide.

[0007] Furthermore, the fixing frame is integrally formed and includes a base plate and a pair of side plates symmetrically arranged on both sides of the base plate, the side plates extend toward the direction of the chamber to form a card block, and one end of the frame body close to the nanofiber layer is radially protruded outward to form a convex edge, the convex edge and at least part of the glass slide are located between the base plate and the card block, and the convex edge abuts the card block, and the glass slide abuts the base plate.

[0008] Furthermore, the convex edge is provided with a fixing groove, and the clamping block is convexly provided with a fixing block matched with the fixing groove.

[0009] Furthermore, a fracture is formed between the pair of side plates, and the slide includes a gripping portion that at least partially passes through the fracture, and the gripping portion is located outside the chamber.

[0010] Furthermore, a seal is provided between the frame and the nanofiber layer.

[0011] Furthermore, the nanofiber layer structure includes but is not limited to random arrangement, parallel orientation arrangement, grid arrangement, and macroporous structure arrangement.

[0012] Furthermore, the thickness of the nanofiber layer is 0.5-10 μm.

[0013] Furthermore, the diameter of the nanofibers in the nanofiber layer is 200-900 nm.

[0014] Furthermore, the raw materials for preparing the nanofiber layer can be one or more of polycaprolactone, polylactic acid, collagen, gelatin, polylactic acid-glycolic acid, polyglycolic acid, polyhydroxybutyrate, polyamide, polyacrylonitrile, polyurethane, polystyrene, polyvinyl butyral, polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene oxide, chitosan, silk protein, cellulose acetate, etc.

[0015] Furthermore, the support film is transparent, and the support film is any one of a polyethylene terephthalate film, a cycloolefin copolymer film, a polymethyl methacrylate film, a polycarbonate film, and a polystyrene film.

[0016] Compared with the prior art, the advantages of the present invention are:

[0017] 1. Compared with existing products, the three-dimensional cell culture device is designed as a detachable structure, which improves the convenience of operation. It provides greater flexibility and operating space during cell culture, fixation, staining and imaging. In addition, by setting a glass slide, and a load membrane and a nanofiber layer are sequentially attached to one side of the glass slide, when it is necessary to observe and analyze the cell condition through a microscope, the fixing frame and the frame are directly separated by disassembly, and then the frame and the cover are removed, and finally the glass slide is clamped to the placement platform of the microscope for observation and analysis using tools, which integrates culture and observation and analysis, reduces the error between experimental groups, simplifies experimental operations, improves experimental efficiency, and saves time and manpower.

[0018] 2. By setting up a nanofiber layer in the chamber, the nanofiber layer can mimic the structure and biological function of the natural extracellular matrix (ECM), providing a more physiologically relevant environment for cell growth, which helps to improve the accuracy and reliability of experimental results. In addition, by regulating the material and surface hydrophilicity and cell compatibility of the nanofiber layer, the adhesion, proliferation and functional expression of cells can be better promoted. These characteristics make their application in the fields of tissue engineering, regenerative medicine, disease modeling and drug discovery superior to traditional two-dimensional cell culture.

[0019] 3. By setting a load membrane, the load membrane has the following advantages: First, as a key support for the nanofiber layer: the load membrane has the necessary strength to ensure that it can maintain a stable shape and size during processing. This feature enables it to effectively receive nanofibers and can be cut as needed in the later stage, providing flexibility in use. Second: The load membrane has excellent optical properties: The load membrane is very thin, and its optical quality is comparable to that of glass. It has superior light transmittance and refractive index, and can meet the requirements of high-resolution microscopic imaging technology, such as phase contrast microscopy, wide-field fluorescence microscopy, confocal microscopy, and two-photon microscopy. Third: Compared with the existing patent CN202123141766.6, our design adds a layer of load membrane, which not only serves as a macroscopic framework structure, but also has effective support and fixation at the microscopic level, thereby improving the overall performance and application potential of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a schematic diagram of the structure of a three-dimensional cell culture device;

[0021] Figure 2 An exploded view of a three-dimensional cell culture device;

[0022] Figure 3 is a cross-sectional view of a three-dimensional cell culture device;

[0023] Figure 4 It is a schematic diagram of a part of the structure of a three-dimensional cell culture device;

[0024] Figure 5 is a schematic diagram of the structure of the fixing frame;

[0025] Figure 6 The structure of the frame Figure 1 ;

[0026] Figure 7 The structure of the frame Figure 2 ;

[0027] Figure 8 This is a schematic diagram of the structure of the cover.

[0028] 1. Fixing frame; 10. Bottom plate; 11. Side plate; 12. Block; 120. Fixing block; 13. Fracture; 2. Glass slide; 20. Grabbing part; 3. Loading membrane; 4. Nanofiber layer; 5. Seal; 6. Frame; 60. Convex edge; 600. Fixing groove; 61. Concave groove; 62. Positioning groove; 7. Cover; 70. Positioning block; 8. Chamber. DETAILED DESCRIPTION

[0029] The following is a further non-restrictive detailed description of the technical solution of the invention in conjunction with the preferred embodiments and the accompanying drawings. In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like indicate the orientation or position relationship based on the orientation or position relationship shown in the accompanying drawings. In addition, the terms "first" and "second" are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "multiple" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and cannot be understood as limitations on the present invention.

[0030] like Figure 1-Figure 8As shown, a three-dimensional cell culture device according to an embodiment of the present invention comprises a fixing frame 1, a glass slide 2, a load film 3, a nanofiber layer 4, a frame 6 and a cover 7, wherein the load film 3 is attached to the surface of the glass slide 2, the nanofiber layer 4 is coated on the side of the load film 3 away from the glass slide 2, and one side of the frame 6 is in contact with the nanofiber layer 4; the cover 7 is buckled on the other side of the frame 6, and the glass slide 2, the frame 6 and the cover 7 together form a chamber 8 for culturing cells, and the fixing frame 1 is used to lock the frame 6 and the glass slide 2. Compared with existing products, the three-dimensional cell culture device is designed as a detachable structure, which improves the convenience of operation. In the process of cell culture, fixation, staining and imaging, greater flexibility and operating space are provided. In addition, by setting a glass slide 2, and on one side of the glass slide 2, a load film 3 and a nanofiber layer 4 are sequentially attached. When it is necessary to observe and analyze the cell situation through a microscope, the fixed frame 1 and the frame 6 are directly separated by disassembly, and then the frame 6 and the cover 7 are removed. Finally, the glass slide 2 is clamped by a tool to the placement platform of the microscope for observation and analysis, which integrates cultivation and observation and analysis, reduces the error between experimental groups, simplifies experimental operation, improves experimental efficiency, and saves time and manpower. The nanofiber layer can imitate the structure and biological function of the natural extracellular matrix (ECM), provides a more physiologically relevant environment for cell growth, and helps to improve the accuracy and reliability of the experimental results. In addition, by regulating the material and surface hydrophilicity and cell compatibility of the nanofiber layer, the adhesion, proliferation and functional expression of cells can be better promoted. These characteristics make them better than traditional two-dimensional cell culture in the fields of tissue engineering, regenerative medicine, disease modeling and drug discovery. The nanofiber layer 4 material can be used as a scaffold material to mediate tissue formation and support cell adhesion, proliferation and differentiation.

[0031] like Figure 3 As shown, the fixing frame 1 is integrally formed and includes a base plate 10 and a pair of side plates 11 symmetrically arranged on both sides of the base plate 10, the side plates 11 extend toward the direction of the chamber 8 to form a card block 12, and the end of the frame 6 close to the nanofiber layer 4 is radially protruded outward to form a convex edge 60, the convex edge 60 and at least a part of the glass slide 2 are located between the base plate 10 and the card block 12, and the convex edge 60 is in contact with the card block 12, the glass slide 2 is in contact with the base plate 10, and the convex edge 60 is provided with a fixing groove 600, and the card block 12 is protruded with a fixing block 120 matched with the fixing groove 600.

[0032] like Figure 4 and Figure 5 As shown, a fracture 13 is formed between a pair of side plates 11, and the glass slide 2 includes a grabbing portion 20 that at least partially passes through the fracture 13. The grabbing portion 20 is located outside the chamber 8. The grabbing portion 20 can be clamped by a tool, and the glass slide 2 can be placed on the placement platform of the microscope for observation and analysis.

[0033] In this embodiment, the size of the glass slide 2 is 25mm×75mm, and the thickness is 1-1.2mm. The left end of the upper surface of the glass slide 2 is designed with a cavity code, and the right end, i.e., the upper surface of the gripping portion 20, is a single-sided frosted surface for data recording and experimental operation.

[0034] like Figure 3 As shown, a seal 5 is provided between the frame 6 and the nanofiber layer 4 to ensure that the frame 6 and the nanofiber layer 4 are sealed and abutted to maintain the airtightness of the chamber 8. Specifically, a groove 61 is provided on the frame 6, and the seal 5 is accommodated in the groove 61. The seal 5 maintains the sealing of the chamber 8 to prevent cross contamination.

[0035] In this embodiment, the nanofiber layer 4 is directly coated on the support membrane 3 through the electrospinning process, so that the nanofiber layer 4 and the support membrane 3 are integrated. The support membrane 3 is the key support of the nanofiber layer 4. Since the support membrane 3 has the necessary strength to ensure that it can maintain a stable shape and size during the processing, this feature enables it to effectively receive the nanofibers and can be cut as needed in the later stage, providing flexibility in use.

[0036] The nanofiber layer 4 combines a variety of different spinning raw materials through a layer-by-layer alternating spinning technology to obtain a layer-by-layer nanofiber film material, which not only maintains some of the advantages of each raw material component, but also produces better properties that the original components do not have. Among them, the structure of the nanofiber layer 4 includes but is not limited to random arrangement, parallel orientation arrangement, grid arrangement, and macroporous structure arrangement.

[0037] The diameter of the nanofibers in the nanofiber layer 4 is 200-900 nm. The raw materials for preparing the nanofiber layer 4 can be one or more of polycaprolactone, polylactic acid, collagen, gelatin, polylactic-glycolic acid, polyglycolic acid, polyhydroxybutyrate, polyamide, polyacrylonitrile, polyurethane, polystyrene, polyvinyl butyral, polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene oxide, chitosan, silk protein, cellulose acetate, etc. Natural raw materials and synthetic biodegradable polymer raw materials are preferred.

[0038] The thickness of the nanofiber layer 4 is 0.5-10 μm. It is worth noting that the nanofiber layer 4 is a three-dimensional structure and has a variety of nanofiber structures, which can maximally mimic the extracellular matrix (ECM) structure in the human body, play a role in guiding stem cell differentiation, guiding the axon growth of nerve cells, etc., and provide a large surface area for cell attachment.

[0039] In this embodiment, the size of the nanofiber layer 4 is 25*53 mm, and the nanofiber layer 4 can be surface-modified by low-temperature plasma, chemical, physical and other methods, for example, to change its hydrophilicity and cell compatibility.

[0040] In this embodiment, the thickness of the load film 3 is 100-200μm. The load film 3 in this thickness range provides sufficient strength to ensure that it can maintain a stable shape and size during the processing. This feature enables it to effectively receive nanofibers and can be cut as needed in the later stage, providing flexibility in use. In addition, compared with the prior art, by adding a layer of load film, it not only serves as a macroscopic framework structure, but also has an effective supporting and fixing function at the microscopic level, thereby improving the overall performance and application potential of the product. At the same time, the optical quality of the load film 3 in this thickness range is comparable to that of glass, with excellent light transmittance and refractive index, and can meet the requirements of high-resolution microscopic imaging techniques, such as phase contrast microscopy, wide-field fluorescence microscopy, confocal microscopy and two-photon microscopy.

[0041] The supporting membrane 3 is any one of a polyethylene terephthalate membrane, a cycloolefin copolymer membrane, a polymethyl methacrylate membrane, a polycarbonate membrane, and a polystyrene membrane.

[0042] The frame 6 can be divided into 9.4c㎡*single hole, 4.55c㎡*2 holes, 2.13c㎡*4 holes, and 0.98c㎡*8 holes according to the bottom surface area and number of the cell culture holes, which is convenient for multi-chamber cell culture. The frame 6 can be easily disassembled and replaced, which is convenient for cleaning and experimental operation.

[0043] The frame 6 is made of one of polyurethane, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, polyethylene terephthalate, epoxy resin, and silicone resin, preferably a medical grade material, or one that is easily sterilized by conventional processes.

[0044] like Figure 7 and Figure 8 As shown, a positioning block 70 is convexly provided on the inner wall of the cover 7, and a positioning groove 62 is concavely formed on the outer peripheral surface of the frame body 6. The positioning block 70 is matched with the positioning groove 62 to facilitate the correct closing of the cover 7.

[0045] The preparation process of the three-dimensional cell culture device of the present invention is as follows:

[0046] Medical grade polylactic acid (PLA) was dissolved in hexafluoroisopropanol to prepare a 5% PLA solution as spinning solution A; medical grade polycaprolactone (PCL) was dissolved in hexafluoroisopropanol to prepare an 8% PCL solution as spinning solution B.

[0047] Prepare an electrospinning device, which has two sets of electrospinning devices, which are respectively arranged on both sides of the roller receiver. Use a syringe to extract spinning solution A and spinning solution B respectively, and place them at the liquid supply pump position of the two sets of electrospinning devices and fix them. Connect a metal spinning nozzle to the front end of the syringe and connect it to the electrostatic positive high voltage.

[0048] A layer of cyclic olefin copolymer (COC) film is laid flat on the surface of the rotating roller receiver to receive the nanofiber layer 4 .

[0049] The spinning distance from the spinning nozzle to the roller receiver is set to 18 cm, the liquid supply rate of spinning liquid A is 0.08 mm / min, the positive high voltage is 15 kV, the liquid supply rate of spinning liquid B is 0.12 mm / min, the positive high voltage is 12.5 kV, the receiver speed is set to 40 rpm, and the electrostatic negative high voltage is connected, and the negative high voltage is set to -3.0 kV.

[0050] Under the above conditions, electrospinning is carried out, and the jets of spinning solution A and spinning solution B do not affect each other, and are respectively deposited on one side of the receiver. As the receiver rotates, alternating spinning is achieved layer by layer to obtain a layered nanofiber structure, i.e., nanofiber layer 4.

[0051] The obtained nanofiber layer 4 together with the load membrane 3 are cut into rectangular pieces with a diameter of 25*53mm, which are placed on the glass slide 2, and then the glass slide 2 is placed in the fixing frame 1 in turn, and the frame 6 containing the seal 5 is placed on the surface of the nanofiber layer 4 and locked with the fixing frame 1, and finally covered with a lid 7 to form a three-dimensional cell culture device with a nanofiber structure.

[0052] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A three-dimensional cell culture device, characterized in that: The invention comprises a fixing frame (1), a glass slide (2), a load film (3), a nanofiber layer (4), a frame (6) and a cover (7); the load film (3) is attached to the surface of the glass slide (2); the nanofiber layer (4) is coated on the side of the load film (3) facing away from the glass slide (2); one side of the frame (6) abuts against the nanofiber layer (4); the cover (7) is buckled on the other side of the frame (6); and the glass slide (2), the frame (6) and the cover (7) together form a chamber (8) for culturing cells; the fixing frame (1) is used to lock the frame (6) and the glass slide (2).

2. The three-dimensional cell culture device according to claim 1, characterized in that: The fixing frame (1) is integrally formed and comprises a bottom plate (10) and a pair of side plates (11) symmetrically arranged on both sides of the bottom plate (10); the side plates (11) extend in the direction of the chamber (8) to form a block (12); one end of the frame (6) close to the nanofiber layer (4) is radially outwardly protruding to form a convex edge (60); the convex edge (60) and at least a portion of the glass slide (2) are located between the bottom plate (10) and the block (12); the convex edge (60) abuts against the block (12), and the glass slide (2) abuts against the bottom plate (10).

3. The three-dimensional cell culture device according to claim 2, characterized in that: The convex edge (60) is provided with a fixing groove (600), and the clamping block (12) is provided with a fixing block (120) matched with the fixing groove (600).

4. The three-dimensional cell culture device according to claim 2, characterized in that: A fracture (13) is formed between the pair of side plates (11), and the glass slide (2) comprises a gripping portion (20) that at least partially passes through the fracture (13), and the gripping portion (20) is located outside the chamber (8).

5. The three-dimensional cell culture device according to claim 1, characterized in that: A sealing member (5) is provided between the frame (6) and the nanofiber layer (4).

6. The three-dimensional cell culture device according to claim 1, characterized in that: The structure of the nanofiber layer (4) includes, but is not limited to, random arrangement, parallel orientation arrangement, grid arrangement, and macroporous structure arrangement.

7. The three-dimensional cell culture device according to claim 1, characterized in that: The thickness of the nanofiber layer (4) is 0.5-10 μm.

8. The three-dimensional cell culture device according to claim 1, characterized in that: The diameter of the nanofibers in the nanofiber layer (4) is 200-900 nm.

9. The three-dimensional cell culture device according to claim 1, characterized in that: The raw materials for preparing the nanofiber layer (4) can be one or more of polycaprolactone, polylactic acid, collagen, gelatin, polylactic acid-glycolic acid, polyglycolic acid, polyhydroxybutyrate, polyamide, polyacrylonitrile, polyurethane, polystyrene, polyvinyl butyral, polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene oxide, chitosan, silk protein, cellulose acetate, etc.

10. The three-dimensional cell culture device according to claim 1, characterized in that: The supporting film (3) is transparent, and the supporting film (3) is any one of a polyethylene terephthalate film, a cycloolefin copolymer film, a polymethyl methacrylate film, a polycarbonate film, and a polystyrene film.

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