Three-dimensional cell culture device

Abstract

The utility model 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 utility model 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 utility model relates to cell culture apparatus technical field, especially in three -dimensional cell culture device. BACKGROUND

[0002] Three -dimensional cell culture technology refers to the material with three -dimensional structure and cell in vitro together with the cultivation, make cell can migrate, grow in three -dimensional space structure, constitute three -dimensional cell-carrier complex, let cell be closer to the actual physiological environment in behavior, thereby have better biological correlation and clinical predictability.

[0003] The three -dimensional cell culture device in prior art is inconvenient to observe and analyze cell through microscope, if wanting to observe and analyze cell through microscope, need to carry out transfer operation to the cell in three -dimensional cell culture device, for example, carry out cell transfer through cell climbing piece method, the error between experimental groups is big in this transfer process, and the operation is tedious, waste a lot of time and manpower. UTILITY MODEL CONTENT

[0004] In view of the deficiency of prior art, the utility model provides a three -dimensional cell culture device, the three -dimensional cell culture device integrates cultivation and observation analysis, reduces the error between experimental groups, and simplifies experimental operation, saves time and manpower.

[0005] The utility model realizes the following technical scheme:

[0006] A three -dimensional cell culture device, including fixed frame, glass slide, load film, nanometer fiber layer, frame and lid, the load film is attached to the surface of glass slide, the nanometer fiber layer is applied to one side of load film away from glass slide, and the frame is abutted on nanometer fiber layer on one side;The lid is buckled on the other side of the frame, and the glass slide, frame and lid form a cavity for culturing cells together, and the fixed frame is used to lock the frame and the glass slide.

[0007] Further, the fixed frame is integrally formed, and the fixed frame includes a bottom plate and a pair of side plates symmetrically arranged on both sides of the bottom plate, the side plates extend towards the cavity and form clamping blocks, one end of the frame close to the nanometer fiber layer is radially outwardly convex to form a convex edge, the convex edge and at least part of the glass slide are located between the bottom plate and the clamping block, the convex edge abuts against the clamping block, and the glass slide abuts against the bottom plate.

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

[0009] Further, a break is formed between a pair of side plates, the slide includes a gripping portion at least partially passing through the break, and the gripping portion is located outside the chamber.

[0010] Further, a seal is arranged between the frame and the nanofiber layer.

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

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

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

[0014] Further, the raw material for preparing the nanofiber layer can be one or more of polycaprolactone, polylactic acid, collagen, gelatin, poly-lactic-glycolic acid, polyglycolic acid, polyhydroxybutyrate, polyamide, polyacrylonitrile, polyurethane, polystyrene, polyvinyl butyral, polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene oxide, chitosan, silk fibroin, cellulose acetate, and the like.

[0015] Further, the support film is transparent, and the support film is any one of polyethylene terephthalate film, cyclic olefin copolymer film, polymethyl methacrylate film, polycarbonate film, and polystyrene film.

[0016] Compared with the prior art, the utility model has the advantages that:

[0017] 1、Compared with the prior art, the three-dimensional cell culture device is designed as a detachable structure, which improves the convenience of operation. In the cell culture, fixation, staining and imaging process, more flexibility and operation space are provided. In addition, by arranging the slide and sequentially attaching the support film and the nanofiber layer on one side of the slide, when it is necessary to observe and analyze the cell condition through the microscope, the fixing frame and the frame are directly detached and separated, then the frame and the cover are removed, and finally the slide is clamped to the placement platform of the microscope for observation and analysis by using the tool, the culture and observation and analysis are integrated, the error between experimental groups is reduced, the experimental operation is simplified, the experimental efficiency is improved, and the time and manpower are saved.

[0018] 2. By incorporating nanofiber layers within the chamber, these layers can mimic the structure and biological functions of the natural extracellular matrix (ECM), providing a more physiologically relevant environment for cell growth and contributing to improved accuracy and reliability of experimental results. Furthermore, by modulating the material and surface hydrophilicity / hydrophobicity, as well as cell compatibility, of the nanofiber layers, cell adhesion, proliferation, and functional expression can be better promoted. These properties make them superior to traditional two-dimensional cell culture in applications such as tissue engineering, regenerative medicine, disease modeling, and drug discovery.

[0019] 3. By incorporating a load film, the load film offers the following advantages: First, it serves as a crucial support for the nanofiber layer: the load film possesses the necessary strength to ensure stable shape and dimensions during processing. This characteristic allows it to effectively support nanofibers and can be cut as needed later, providing flexibility in use. Second, the load film exhibits excellent optical performance: being very thin, its optical quality rivals that of glass, possessing superior light transmittance and refractive index, meeting the requirements of high-resolution microscopic imaging technologies such as phase-contrast microscopy, wide-field fluorescence microscopy, confocal microscopy, and two-photon microscopy. Third, compared to the existing patent CN202123141766.6, our design adds a load film layer, making it not only a macroscopic framework structure but also providing effective support and fixation at the microscopic level, thus enhancing the overall performance and application potential of the product. Attached Figure Description

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

[0021] Figure 2 This is an exploded view of a three-dimensional cell culture apparatus;

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

[0023] Figure 4 This is a partial structural diagram of a three-dimensional cell culture device.

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

[0025] Figure 6 Schematic diagram of the frame structure Figure 1 ;

[0026] Figure 7 Schematic diagram of the frame structure Figure 2 ;

[0027] Figure 8 This is a schematic diagram of the lid's structure.

[0028] 1. Fixing frame; 10. Base plate; 11. Side plate; 12. Clamping block; 120. Fixing block; 13. Break; 2. Glass slide; 20. Gripping part; 3. Loading film; 4. Nanofiber layer; 5. Sealing element; 6. Frame; 60. Raised edge; 600. Fixing groove; 61. Groove; 62. Positioning groove; 7. Cover; 70. Positioning block; 8. Chamber. Detailed Implementation

[0029] The following detailed, non-limiting description of the utility model's technical solution, in conjunction with preferred embodiments and accompanying drawings, is provided. In the description of this utility model, it should be understood that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," and "circumferential," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this utility model, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this utility model, and should not be construed as limiting this utility model.

[0030] like Figures 1-8As shown, a three-dimensional cell culture device according to an embodiment of this utility model includes a fixture 1, a glass slide 2, a loading membrane 3, a nanofiber layer 4, a frame 6, and a lid 7. The loading membrane 3 is attached to the surface of the glass slide 2, the nanofiber layer 4 is coated on the side of the loading membrane 3 facing away from the glass slide 2, and one side of the frame 6 abuts against the nanofiber layer 4. The lid 7 is fastened to the other side of the frame 6, and the glass slide 2, the frame 6, and the lid 7 together form a cell culture chamber 8. The fixture 1 is used to lock the frame 6 to the glass slide 2. Compared with existing products, by designing the three-dimensional cell culture device as a detachable structure, the ease of operation is improved. Greater flexibility and operating space are provided during cell culture, fixation, staining, and imaging processes. Furthermore, by setting up a glass slide 2, with a loading membrane 3 and a nanofiber layer 4 sequentially attached to one side of the slide 2, when it is necessary to observe and analyze the cells under a microscope, the fixture 1 and frame 6 can be directly disassembled and separated, then the frame 6 and cover 7 can be removed, and finally the glass slide 2 can be used to hold it on the microscope platform for observation and analysis. This integrates culture and observation analysis, reduces errors between experimental groups, simplifies experimental operations, improves experimental efficiency, and saves time and manpower. 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 and helping to improve the accuracy and reliability of experimental results. In addition, by regulating the material and surface hydrophilicity / hydrophobicity and cell compatibility of the nanofiber layer, cell adhesion, proliferation, and functional expression can be better promoted. These properties make them superior to traditional two-dimensional cell culture in applications such as tissue engineering, regenerative medicine, disease modeling, and drug discovery. The nanofiber layer 4 can serve as a scaffold material, mediating tissue formation and supporting 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 cavity 8 to form a locking block 12. The frame 6 near the nanofiber layer 4 has a protruding edge 60 that protrudes radially outward. The protruding edge 60 and at least part of the glass slide 2 are located between the base plate 10 and the locking block 12. The protruding edge 60 abuts against the locking block 12, and the glass slide 2 abuts against the base plate 10. The protruding edge 60 has a fixing groove 600. The locking block 12 has a fixing block 120 that mates with the fixing groove 600.

[0032] like Figure 4 and Figure 5 As shown, a break 13 is formed between a pair of side plates 11. The slide 2 includes a gripping part 20 that passes at least partially through the break 13. The gripping part 20 is located outside the chamber 8. The gripping part 20 can be grasped with a tool and the slide 2 can be placed on the microscope's placement platform for observation and analysis.

[0033] In this embodiment, the slide 2 has a size of 25mm × 75mm and a thickness of 1-1.2mm. The upper surface of the slide 2 has a chamber code on the left end and the upper surface of the gripping part 20 on the right end has a single-sided frosted surface for data recording and experimental operations.

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

[0035] In this embodiment, the nanofiber layer 4 is directly coated onto the support membrane 3 via electrospinning, making the nanofiber layer 4 and the support membrane 3 an integral unit. The support membrane 3 serves as a crucial support for the nanofiber layer 4. Due to its necessary strength, the support membrane 3 ensures stable shape and size during processing. This characteristic allows it to effectively support the nanofibers and provides flexibility for subsequent trimming as needed.

[0036] The nanofiber layer 4 combines various different spinning raw materials through a layer-by-layer alternating spinning technique to obtain a layered nanofiber membrane material. This not only retains some of the advantages of each raw material component but also produces superior properties not possessed by the original components. The structure of the nanofiber layer 4 includes, but is not limited to, random arrangement, parallel orientation, mesh arrangement, and macroporous structure arrangement.

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

[0038] The thickness of nanofiber layer 4 is 0.5-10 μm. Notably, nanofiber layer 4 has a three-dimensional structure and contains various nanofiber structures, which can maximally mimic the structure of the extracellular matrix (ECM) in the human body, playing a role in guiding stem cell differentiation and guiding axonal growth of nerve cells, and providing a large surface area for cell attachment.

[0039] In this embodiment, the nanofiber layer 4 has a size of 25*53mm, and the nanofiber layer 4 can be surface modified by low-temperature plasma, chemical, physical and other methods, such as changing its hydrophilicity, hydrophobicity and cell compatibility.

[0040] In this embodiment, the thickness of the load film 3 is 100-200 μm. This thickness range provides sufficient strength to ensure stable shape and size during processing. This characteristic allows it to effectively support nanofibers and can be cut as needed later, providing flexibility in use. Furthermore, compared to existing technologies, adding a load film not only serves as a macroscopic framework structure but also provides effective support and fixation at the microscopic level, improving the overall performance and application potential of the product. Simultaneously, the optical quality of the load film 3 within this thickness range is comparable to glass, exhibiting superior transmittance and refractive index, meeting the requirements of high-resolution microscopic imaging techniques such as phase contrast microscopy, wide-field fluorescence microscopy, confocal microscopy, and two-photon microscopy.

[0041] Among them, the supported membrane 3 is any one of polyethylene terephthalate membrane, cyclic olefin copolymer membrane, polymethyl methacrylate membrane, polycarbonate membrane, and polystyrene membrane.

[0042] The frame 6 can be divided into 9.4 cm²*single well, 4.55 cm²*2 wells, 2.13 cm²*4 wells, and 0.98 cm²*8 wells according to the bottom area and number of cells culture wells, which is convenient for multi-chamber cell culture. The frame 6 can be easily disassembled and replaced, making it convenient for cleaning and experimental operations.

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

[0044] like Figure 7 and Figure 8 As shown, a positioning block 70 is protruding on the inner wall of the cover 7, and a positioning groove 62 is recessed on the outer circumferential surface of the frame 6. The positioning block 70 and the positioning groove 62 are engaged to facilitate the correct closing of the cover 7.

[0045] The preparation process of the three-dimensional cell culture device in this 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 the electrospinning equipment, which has two sets of electrospinning devices, respectively located on both sides of the rotating roller receiver. Use syringes to draw spinning solution A and spinning solution B, respectively, and place them in the supply pump positions of the two sets of electrospinning devices and secure them. Attach a metal spinning nozzle to the front end of the syringe and connect it to a positive electrostatic voltage.

[0048] A layer of cyclic olefin copolymer (COC) film is laid flat on the surface of the 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 supply speed of spinning solution A is 0.08 mm / min, the positive voltage is 15 kV, the supply speed of spinning solution B is 0.12 mm / min, the positive voltage is 12.5 kV, the receiver rotation speed is set to 40 rpm, and an electrostatic negative voltage is connected with a negative voltage of -3.0 kV.

[0050] Electrospinning is performed under the above conditions. The jets of spinning solution A and spinning solution B do not affect each other and are deposited on one side of the receiver. As the receiver rotates, the spinning is carried out layer by layer to achieve alternating spinning, resulting in a layered nanofiber structure, namely nanofiber layer 4.

[0051] The obtained nanofiber layer 4, together with the load membrane 3, is cut into a rectangular piece with a diameter of 25*53 mm. It is placed on a glass slide 2, and then the glass slide 2 is placed in the holder 1. The frame 6 containing the sealing element 5 is placed on the surface of the nanofiber layer 4 and locked with the holder 1. Finally, the cover 7 is put on to form a three-dimensional cell culture device with a nanofiber structure.

[0052] The embodiments described above are merely illustrative of several implementations of this utility model, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of this utility model patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this utility model, and these all fall within the protection scope of this utility model. Therefore, the protection scope of this utility model patent should be determined by the appended claims.

Claims

1. A three-dimensional cell culture device, characterized by, The application relates to a cell culture device, which comprises a fixing frame (1), a glass slide (2), a loading film (3), a nanofiber layer (4), a frame body (6) and a cover (7), the loading film (3) is attached to the surface of the glass slide (2), the nanofiber layer (4) is coated on the side of the loading film (3) away from the glass slide (2), one side of the frame body (6) abuts against the nanofiber layer (4), the cover (7) is buckled on the other side of the frame body (6), and the glass slide (2), the frame body (6) and the cover (7) jointly form a cell culture cavity (8), and the fixing frame (1) is used for locking the frame body (6) and the glass slide (2).

2. The three-dimensional cell culture device of claim 1, wherein, The fixing frame (1) is integrally formed, and the fixing frame (1) 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 towards the cavity (8) and are formed with clamping blocks (12), one end of the frame body (6) close to the nanofiber layer (4) is formed with a convex edge (60) outwardly protruding in the radial direction, the convex edge (60) and at least part of the glass slide (2) are located between the bottom plate (10) and the clamping blocks (12), the convex edge (60) abuts against the clamping blocks (12), and the glass slide (2) abuts against the bottom plate (10).

3. The three-dimensional cell culture device of claim 2, wherein, The convex edge (60) is provided with a fixing groove (600), and the clamping blocks (12) are provided with fixing blocks (120) matched with the fixing groove (600).

4. The three-dimensional cell culture device of claim 2, wherein, A pair of side plates (11) are formed with a break (13), the glass slide (2) comprises a grabbing part (20) penetrating through the break (13), and the grabbing part (20) is located outside the cavity (8).

5. The three-dimensional cell culture device of claim 1, wherein, A sealing element (5) is arranged between the frame body (6) and the nanofiber layer (4).

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

7. The three-dimensional cell culture device of claim 1, wherein, The thickness of the nanofiber layer (4) is 0.5-10 mu m.

8. The three-dimensional cell culture device of claim 1, wherein, The diameter of the nanofiber in the nanofiber layer (4) is 200-900 nm.

9. The three-dimensional cell culture device of claim 1, wherein, The raw material for preparing the nanofiber layer (4) can be one 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 fibroin and cellulose acetate.

10. The three-dimensional cell culture device of claim 1, wherein, The loading film (3) is transparent, and the loading film (3) is any one of a polyethylene terephthalate film, a cyclic olefin copolymer film, a polymethyl methacrylate film, a polycarbonate film and a polystyrene film.

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