Cell stretching device and method for promoting cells to secrete exosomes
By designing a device for cell stretching and applying mechanical force to stimulate cells, the problem of insufficient secretion of exosomes under conventional culture conditions was solved, and the effect of efficient secretion and simplification of the separation process was achieved.
Patent Information
- Application Number
- CN202411948996.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-27
- Publication Date
- 2025-02-07
AI Technical Summary
The ability of cells to secrete exosomes under conventional culture conditions is limited, and the yield is insufficient, making it difficult to meet the needs of high quality and quantity.
A cell stretching device is designed to stimulate cells by applying mechanical force to promote the formation and release of exosomes through the combination of flexible polyester membrane and adjustment rod.
Through mechanical force stimulation, the efficiency and yield of cells secreted exosomes are improved, the isolation and purification process of exosomes is simplified, and the application prospects are broad in the field of biomedical science.
Smart Images
Figure CN119391542A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of exosomes, and specifically relates to a cell stretching device and a method for promoting cell secretion of exosomes. Background Art
[0002] Exosomes are tiny membrane vesicles secreted by cells and widely present in various biological fluids. Usually, after exosomes are formed inside cells, they are released outside the cells through the lipid bilayer structure of the cell membrane. This process is regulated by cellular mechanical forces and intracellular signaling pathways.
[0003] The cytoskeleton is composed of microfilaments, intermediate filaments and microtubules, which form a support network through cross-linking and dynamic regulation to maintain the stability and morphology of the cell. The extracellular matrix is composed of complex molecules such as collagen and elastin, which bind to receptors on the cell surface and regulate physiological processes such as cell growth, differentiation, and migration through signal pathways.
[0004] During the cell culture process, cells secrete exosomes into the cell supernatant under normal growth conditions. In order to obtain high-quality and quantitative exosomes, it is usually necessary to ensure healthy and normal cell growth. However, the ability of cells to secrete exosomes under conventional culture conditions is limited and the output is insufficient. Therefore, it is necessary to develop a new cell stretching device and a method for promoting cell secretion of exosomes to solve the existing problems. Summary of the invention
[0005] The object of the present invention is to provide a cell stretching device and a method for promoting cell secretion of exosomes to solve the problem of low efficiency of cell secretion of exosomes.
[0006] To achieve the above object, the present invention provides the following technical solution: a cell stretching device, comprising: main body; a plurality of cell stretching components disposed in the main body; The cell stretching component comprises: a culture chamber fixed in the main body; A flexible polyester film disposed in the culture chamber for stretching cells; An adjusting rod connected to the flexible polyester film and capable of deforming the flexible polyester film; Wherein, sterile compartments corresponding to the plurality of cell stretching components are arranged in the main body.
[0007] Preferably, the adjusting rod is provided with a fine-tuning button for rotating the adjusting rod.
[0008] Preferably, rotating the fine-tuning button by 0.5 degrees increases or decreases the length of the flexible polyester film by 0.5 cm.
[0009] Preferably, scale marks are provided on the periphery of the fine-tuning button.
[0010] Preferably, one end of the flexible polyester film is fixed on the inner wall of the culture chamber; the other end of the flexible polyester film is fixed to the adjusting rod and deforms as the adjusting rod rotates.
[0011] Preferably, the main body is provided with a rotation hole for connecting the adjusting rod.
[0012] Preferably, the culture chamber is provided with an opening for feeding and discharging materials; the opening is arranged above the flexible polyester film.
[0013] Preferably, the inner wall of the sterile compartment maintains a steady-state space for the existence of liquid, and the sterile compartment is a rectangular parallelepiped or cube structure; The length of the sterile compartment is 1 / 4 of the length of the culture chamber, and the distance between two adjacent cell stretching components is 1 / 5 of the width of the culture chamber.
[0014] The present invention further provides a method for promoting cell secretion of exosomes using a cell stretching device, comprising: The cells are introduced into the flexible polyester film from the opening and cultured on the flexible polyester film; Attach and grow, observe cell growth; If the fusion degree is 50%-60%, rotate the fine-tuning button; Until the appropriate density is reached for digestion operation; After digestion is complete, the pellets are poured out from the opening.
[0015] Preferably, the digestion operation method comprises: Use trypsin to rinse. First rinse the cells with PBS, then rinse with trypsin for 5 minutes. The operating temperature is 37 degrees.
[0016] The technical effects and advantages of the present invention are as follows: the cell stretching device and the method for promoting cell secretion of exosomes thereof stimulate cells by applying mechanical force, causing changes in the cell membrane structure, thereby promoting the formation and release of exosomes. The design is based on the mechanical properties of cells, that is, cells will produce corresponding responses to mechanical force stimulation; it has broad application prospects in the biomedical field, including exosome separation and purification, biomarker detection and drug delivery, etc.; the morphological changes of cells under external physical and chemical stimulation have an important influence on the physiological functions and pathological processes of cells, and therefore has broad application value in cell research. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 It is a structural schematic diagram of the present invention; Figure 2 It is a rear view of the present invention; Figure 3 It is a front view of the present invention; Figure 4 This is a schematic diagram of the fine-tuning buttons and scale markings of the present invention; Figure 5 The particle size distribution of exosomes obtained in Example 3 of the present invention Figure 1 ; Figure 6 The particle size distribution of exosomes obtained in Example 3 of the present invention Figure 2 ; Figure 7 This is a microscope photograph of exosomes obtained in Example 3 of the present invention.
[0018] In the figure: 100, main body; 200, cell stretching component; 300, sterile compartment; 210, flexible polyester film; 220, adjustment rod; 230, fine-tuning button; 240, scale mark; 250, opening. DETAILED DESCRIPTION
[0019] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Embodiment 1
[0020] The present invention provides Figure 1 A cell stretching device as shown in , comprising: The main body 100 is a frame structure, and its outer wall is used to isolate air and bacteria.
[0021] A plurality of cell stretching components 200 disposed in the main body 100; The cell stretching assembly 200 comprises: A culture chamber fixed in the main body 100; the culture chamber is a rectangular parallelepiped or cube structure; A flexible polyester film 210 for stretching cells disposed in the culture chamber; the flexible polyester film 210 is in a rectangular shape; The adjusting rod 220 connected to the flexible polyester film 210 is capable of deforming the flexible polyester film 210; the adjusting rod 220 is provided with a fine-tuning button 230 for rotating the adjusting rod 220. Each rotation of the fine-tuning button 230 is 0.5 degrees, and a rotation of 0.5 degrees increases or decreases the length of the flexible polyester film 210 by 0.5 cm.
[0022] The main body 100 is provided with a sterile compartment 300 corresponding to the plurality of cell stretching components 200; the sterile compartment 300 is a rectangular parallelepiped or cube structure. The length of the sterile compartment 300 is 1 / 4 of the length of the culture chamber, and the distance between two adjacent cell stretching components 200 is 1 / 5 of the width of the culture chamber.
[0023] The sterile compartment 300 includes: an inner wall of a steady-state space for maintaining the presence of liquid; One end of the flexible polyester film 210 is fixed to the inner wall of the culture chamber; the other end of the flexible polyester film 210 is fixed to the adjustment rod 220 and deforms as the adjustment rod 220 rotates.
[0024] like Figure 2 As shown, the culture chamber is provided with an opening 250, and the opening 250 is used for feeding and discharging materials and is arranged above the flexible polyester film 210; like Figure 3 As shown, the main body 100 is provided with a rotation hole for connecting the adjustment rod 220 .
[0025] like Figure 4 As shown, the periphery of the fine-tuning button 230 is provided with a scale mark 240 .
[0026] In this embodiment, six cell stretching assemblies 200 are arranged in a straight line in the main body 100, the structure of the culture chamber is a rectangular parallelepiped structure, the adjustment rod 220 is rotatably connected in the rotation groove of the culture chamber, and the adjustment rod 220 is equipped with a fine-tuning button 230 at the end after extending out of the rotation hole; Embodiment 2
[0027] The present invention provides a cell stretching device and a method for promoting cell secretion of exosomes, which specifically comprises the following steps: The classification of cells in this application includes, but is not limited to: immune cells include: dendritic cells, T cells, B cells, macrophages; Tumor cells include: breast cancer cells, lung cancer cells, and colorectal cancer cells; Stem cells include: mesenchymal stem cells, neural stem cells; In this embodiment, the cells selected are mesenchymal stem cells; Cultivate cells in appropriate culture medium until logarithmic growth phase; When the cells grow to a certain degree of confluence, continue culturing for 48 hours and collect the culture medium containing exosomes: Remove cell debris in the culture medium by low-speed centrifugation to obtain the cell supernatant; Exosomes were extracted by ultracentrifugation, kit extraction, density gradient centrifugation, size exclusion chromatography, etc.; The separated exosomes are further purified to remove impurities to obtain the final preserved exosomes. Embodiment 3
[0028] On the basis of Example 2, cells to be cultured are input from the opening 250 into the flexible polyester film 210 and cultured on the flexible polyester film 210; adhere and grow, and observe cell growth; if the degree of fusion is 50%-60%, rotate the fine-tuning button 230, and continue to observe cell growth until a suitable density is reached for digestion operation; after digestion is completed, the formed particles are poured out from the opening 250; the cell secretion of exosomes is promoted by the cell stretching device, which can simplify the separation and purification process of exosomes. Since the concentration of exosomes in the cell culture medium is low, the yield of exosomes can be increased by stretching stimulation, thereby reducing the difficulty of separation and purification; The collected exosomes were analyzed by electrophoresis and Brownian motion characteristics using a microscope. The microscope was a laser scattering microscope with a laser wavelength of 520 nanometers, a filter wavelength of scattering, a sensitivity of 80, and a shutter of 100. The results are shown in Table 2: quantity concentration volume Median diameter (X50) 106.9 106.9 131.6 StdDev 31.9 31.9 59.0 Concentration: 9.6E+7 particles / mL, dilution factor: 7000; original concentration: 6.7E+11 particles / mL; Quality: Average number of counted particles: 235, number of tracked particles: 1811; Figure 7 As shown; Peak concentration analysis: Figure 5 and Figure 6 As shown; Diameter 106.3nm, particles: 6.2E+6 / mL, full width at half maximum: 56.6nm, percentage: 100; X values Table 3: All dimensions are in nm; number concentration volume X10 79.5 79.5 95.4 X50 106.9 106.9 131.6 X90 146.6 146.6 229.8 Span 0.6 0.6 1.0 Mean 113.5 113.5 150.6 StdDev 31.9 31.9 59.0 Embodiment 4
[0029] The digestion operation method is to use trypsin to rinse, for example, first use PBS to rinse the cells, then rinse with trypsin for 5 minutes, the operating temperature is 37 degrees, and the pH value is 8 to digest the cells; Trypsin is replaced by ethylenediaminetetraacetic acid, or a trypsin / EDTA mixture is used; EDTA solution (preferably 0.02% - 0.05%) can effectively dissociate cells from the flexible polyester film 210, and the damage to the cells is relatively small; after digesting the cells with EDTA solution, the cells need to be washed with a buffer solution containing appropriate concentrations of Ca²⁺ and Mg²⁺ (PBS -Ca²⁺ / Mg²⁺); Embodiment 5
[0030] Based on Example 4, the steps of using EDTA solution to digest cells include: A 0.02% EDTA solution was used; Preheat PBS buffer and cell culture medium in a 37°C water bath to ensure that the cells are in a suitable temperature environment during the digestion process.
[0031] Culture the cells to be digested to the logarithmic growth phase to ensure that the cells are in good condition and the cell density reaches a level suitable for digestion and subculture; Use a pipette to remove the culture medium; Wash the cells 1-2 times with preheated sterile PBS, Hanks solution or serum-free culture medium, adding an appropriate amount of solution each time to cover the cell surface, and then aspirate and discard the washing solution; Add a small amount of EDTA solution or trypsin-EDTA digestion solution to just cover the cells; Incubate at room temperature for 0.5 to 5 minutes. Observe the cell status under a microscope. When the cells shrink significantly, the connections between cells become loose, and more importantly, the cell morphology changes significantly, the digestive fluid is removed; The cells are blown to make them fall off the flexible polyester film 210 and dispersed into a single cell suspension to proceed to the next step. Embodiment 6
[0032] Based on Example 4, the steps of using EGTA to digest cells include: Choose 0.1-0.5mM EGTA solution; Preheat the cell culture medium, PBS buffer, etc. in a 37°C water bath; Select cells in the logarithmic growth phase for digestion; Use a pipette to remove the culture medium to avoid residual serum affecting the digestion process; Wash the cells 1-2 times with sterile PBS, adding an appropriate amount of solution each time to cover the cell surface, and then aspirate and discard the washing solution; EGTA solution was added to cover the cells; Incubate at 37°C for 1-10 minutes, 7 minutes in this example, and observe the cell state under a microscope. When the cells begin to shrink and become round, and the connections between cells become loose, add cell culture medium or a buffer containing Ca²⁺ to terminate the digestion reaction; The cells are blown to fall off the flexible polyester film 210 and dispersed into a single cell suspension, which is then collected in the opening 250 .
[0033] The cell stretching device and the method for promoting cell secretion of exosomes can mechanically stimulate cells during the culture process through periodic mechanical stretching; can imitate the physical stimulation in the in vivo environment to promote the secretion of exosomes; cause changes in the cell membrane structure, thereby promoting the formation and release of exosomes. The design of this device is based on the mechanical properties of cells, that is, cells will respond accordingly to mechanical force stimulation; it has broad application prospects in the biomedical field, including exosome separation and purification, biomarker detection and drug delivery; at the same time, the morphological changes of cells under external physical and chemical stimulation have an important influence on the physiological functions and pathological processes of cells, so it has broad application value in cell research.
[0034] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. A cell stretching device, characterized in that: include: body(100); A plurality of cell stretching components (200) disposed in the main body (100); The cell stretching component (200) comprises: a culture chamber fixed in the main body (100); A flexible polyester film (210) disposed in the culture chamber for stretching cells; An adjustment rod (220) connected to the flexible polyester film (210) and capable of causing the flexible polyester film (210) to deform; Wherein, sterile compartments (300) corresponding to the plurality of cell stretching components (200) are arranged in the main body (100).
2. A cell stretching device according to claim 1, characterized in that: The adjusting rod (220) is provided with a fine adjustment button (230) for rotating the adjusting rod (220).
3. A cell stretching device according to claim 2, characterized in that: The fine-tuning button (230) is rotated 0.5 degrees to increase or decrease the length of the flexible polyester film (210) by 0.5 cm.
4. A cell stretching device according to claim 1, characterized in that: The outer periphery of the fine-tuning button (230) is provided with scale marks (240).
5. A cell stretching device according to claim 1 or 4, characterized in that: One end of the flexible polyester film (210) is fixed to the inner wall of the culture chamber; the other end of the flexible polyester film (210) is fixed to the adjustment rod (220) and deforms as the adjustment rod (220) rotates.
6. A cell stretching device according to claim 5, characterized in that: The main body (100) is provided with a rotation hole for connecting the adjustment rod (220).
7. A cell stretching device according to claim 1, characterized in that: The culture chamber is provided with an opening (250) for feeding and discharging materials; the opening (250) is arranged above the flexible polyester film (210).
8. The cell stretching device according to claim 1, characterized in that: The inner wall of the sterile compartment (300) maintains a steady-state space in which liquid exists, and the sterile compartment (300) is a rectangular parallelepiped or cube structure; The length of the sterile compartment (300) is 1 / 4 of the length of the culture chamber, and the distance between two adjacent cell stretching components (200) is 1 / 5 of the width of the culture chamber.
9. A method for promoting cell secretion of exosomes using the cell stretching device according to any one of claims 1 to 8, characterized in that: include: The cells are introduced into the flexible polyester film (210) from the opening (250) and cultured on the flexible polyester film (210); Attach and grow, observe cell growth; If the fusion degree is 50%-60%, rotate the fine-tuning button (230); Until the appropriate density is reached for digestion operation; After digestion is complete, the formed particles are poured out from the opening (250).
10. The method for promoting cell secretion of exosomes using a cell stretching device according to claim 9, characterized in that: The digestion operation method comprises: Use trypsin to rinse. First rinse the cells with PBS, then rinse with trypsin for 5 minutes. The operating temperature is 37 degrees.