A method for promoting efficient transfer of mitochondria to cells
Patent Information
- Application Number
- CN202610981420.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2026-07-02
- Publication Date
- 2026-11-03
- Estimated Expiration
- 2046-07-02
Smart Images
Figure CN122484206B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of cell engineering and regenerative medicine technology, specifically relating to a method for promoting efficient mitochondrial transfer in cells. Background Technology
[0002] Mitochondria, as the cell's "energy factories," directly determine the cell's fate through their functional state. When cells encounter adverse factors such as oxidative stress, inflammation, and aging, mitochondrial function is impaired, energy metabolism is exhausted, leading to cellular dysfunction and hindering tissue regeneration. Therefore, targeted restoration of mitochondrial function has been proposed as a cell therapy strategy for repairing various forms of tissue damage. Since the first discovery of intercellular mitochondrial transfer in 2006, numerous studies have found that mitochondrial transfer can regulate energy metabolism levels, improve cellular function, and promote tissue repair and regeneration by inhibiting apoptosis and regulating inflammatory responses. Therefore, restoring mitochondrial function in target cells through mitochondrial transfer is highly valued in the field of regeneration and shows great potential for application in novel cell therapy strategies.
[0003] Current mitochondrial transfer technologies can be broadly categorized into three types: transfer based on physiological processes, transfer assisted by physical / mechanical forces, and mitochondrial transfer mediated by biological and synthetic vectors. While all of these technologies have demonstrated therapeutic potential in experimental studies, existing techniques struggle to simultaneously achieve safe and efficient delivery of mitochondria to target cells. Low transfer efficiency directly limits therapeutic efficacy, and potential delivery-related side effects may further damage cells. Therefore, developing safe, controllable, and efficient mitochondrial transfer technologies has become a key bottleneck for their clinical translation.
[0004] Existing technologies primarily utilize methods such as co-culture, exosomes, microinjection, and microfluidics to deliver healthy, functional mitochondria into target cells to replace or supplement defective mitochondria and restore normal cellular physiological function. While these methods are effective, they suffer from limitations including low efficiency or high cost of in vitro mitochondrial transfer, complex procedures, and the potential for cell-damaging side effects.
[0005] Therefore, providing a novel method for mitochondrial transfer that is simple to operate, efficient, biosafety-free, causes no significant cell damage, and can be precisely controlled to overcome existing technological bottlenecks has become an urgent problem to be solved. Summary of the Invention
[0006] Based on the above technical background, the main objective of this invention is to provide a method for promoting efficient mitochondrial transfer in cells, which can solve the shortcomings of existing technologies such as complex mitochondrial transfer operations, low safety, easy cell damage during the transfer process, and inability to be precisely controlled.
[0007] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:
[0008] This invention provides a method for promoting efficient mitochondrial transfer in cells, the method comprising the following steps: Recipient cells were seeded on micron-sized culture media to allow them to grow under constrained conditions. Then, exogenous mitochondria were added to the constrained recipient cells for co-culture. The morphological regulation of the recipient cells promoted the transfer of mitochondria into the recipient cells.
[0009] The steps described above are described in detail below.
[0010] The surface area of the micron-sized culture medium is 350–2000 μm. 2 .
[0011] Preferably, the surface area of the micron-sized culture medium is 750 μm. 2 .
[0012] Experiments have shown that the surface area of each micrometer-sized culture medium affects the mitochondrial transfer efficiency, with each micrometer-sized culture medium having an surface area ranging from 350 to 2000 μm. 2 It can significantly promote mitochondrial transfer within the entire range. Specifically, when the area of each of the micron-sized culture media is 750 μm... 2 At this time, the corresponding mitochondrial transfer efficiency is the highest, which is much higher than that of traditional co-culture transfer efficiency. This is beneficial for improving cell function or promoting the repair of damaged cell mitochondria, thereby restoring normal cell function and achieving tissue regeneration.
[0013] Preferably, the micron-sized culture medium is circular in shape.
[0014] The micron-scale culture medium is used to constrain the morphology of recipient cells, geometrically constraining them to precisely control cell morphology and spreading area. Mild physical and mechanical stimulation can significantly promote the internalization process of free mitochondria into the cell, thereby achieving efficient and safe mitochondrial transfer and improving mitochondrial transfer efficiency.
[0015] Multiple micron-sized culture media are uniformly arranged in an array on the substrate.
[0016] The specific method for uniformly distributing multiple micron-sized culture media in an array on a substrate is as follows: The material for preparing the micro-patterned stamp is mixed with a curing agent. The resulting premix is then applied to a mold for curing. The mold is a silicon wafer with multiple grooves on its surface. These grooves are evenly distributed in an array on the surface of the silicon wafer. After curing, the mold is removed to obtain the micro-patterned stamp. The micro-patterned stamp includes multiple protrusions evenly arranged in an array. The raw materials for preparing micron-sized culture media are covered on the raised surface, and then transferred from the raised surface of the micro-patterned stamp to cover the substrate surface, thereby uniformly arranging multiple micron-sized culture media in an array on the substrate surface. The substrate is flat, such as... Figure 1 As shown.
[0017] Preferably, the substrate is made of one or more of PDMS (polydimethylsiloxane), glass (cover glass), and ultra-low adsorption water adhesive (alginate hydrogel).
[0018] The glass is preferably a cover glass.
[0019] More preferably, the substrate is made of PDMS and a coverslip.
[0020] According to a preferred embodiment of the present invention, the method for preparing the substrate includes the following steps: The coverslip was ultrasonically cleaned, and then the PDMS mixture was coated onto the cleaned coverslip surface. It was then cured at 40–60°C for 20–30 hours to obtain the substrate.
[0021] According to a further preferred embodiment of the present invention, the method for preparing the substrate includes the following steps: The coverslip was ultrasonically cleaned, and then the PDMS mixture was coated onto the cleaned coverslip surface. It was then cured at 50°C for 24 hours to obtain the substrate.
[0022] The PDMS mixture is obtained by mixing PDMS and curing agent at a mass ratio of (8-12):1.
[0023] Preferably, the PDMS mixture is obtained by mixing PDMS and curing agent at a mass ratio of 10:1.
[0024] The curing agent is selected from silicone elastomer curing agents.
[0025] Preferably, the curing agent is hydrogen-containing polydimethylsiloxane.
[0026] By limiting the depth and area of the holes in the silicon wafer as described above, this invention enables the micro-patterned stamp after demolding to have columnar protrusions of corresponding height and area, such as... Figure 1As shown, even though the surface of the micropatterned stamp has a regular array of columnar protrusions, and the height of each columnar protrusion is the same as the depth of each hole on the silicon wafer surface, and the area of each columnar protrusion is the same as the area of each hole on the silicon wafer surface, after covering the columnar protrusions on the surface of the micropatterned stamp with the preparation material of the culture medium, and then transferring the preparation material of the culture medium to the substrate through the micropatterned stamp, it is possible to distribute micron-sized culture medium with uniform shape, equal area, and regular arrangement on the substrate surface, and the area of each micron-sized culture medium is the same as the area of each hole on the silicon wafer surface. This invention adjusts the area of each micron-sized culture medium on the substrate surface by adjusting the area of the holes on the silicon wafer surface.
[0027] The silicon wafer surface has multiple regularly arranged pore structures in a micron-scale array, with a depth of 10 μm. These pore structures serve as a mold for multiple uniformly arranged protrusions on a micro-pattern stamp. The micro-pattern stamp includes multiple columnar protrusions arranged in an array. These columnar protrusions are used to adhere materials for micron-scale culture substrates, which are then transferred to the substrate, resulting in the substrate surface having multiple micron-scale culture substrates arranged in an array with specific shapes.
[0028] The holes are preferably circular, and the area of each hole is 350–2000 μm. 2 .
[0029] Preferably, the area of each of the holes is 750 μm. 2 .
[0030] The micro-patterned stamp is made of the same material as the substrate, and the preferred material for the micro-patterned stamp is PDMS.
[0031] The curing agent is selected from silicone elastomer curing agents.
[0032] Preferably, the curing agent is hydrogen-containing polydimethylsiloxane.
[0033] The mass ratio of the material used to prepare the micro-patterned stamp to the curing agent is (8-12):1.
[0034] Preferably, the mass ratio of the material used to prepare the micro-patterned stamp to the curing agent is 10:1.
[0035] The curing conditions are as follows: the curing temperature is 70-80℃, and the curing time is 10-15h.
[0036] Preferably, the curing conditions are: a curing temperature of 75°C and a curing time of 12 hours.
[0037] The micron-scale culture medium is prepared from one or more of fibronectin, collagen, and laminin.
[0038] Preferably, the micron-sized culture medium is prepared from fibronectin.
[0039] This invention cultured recipient cells in a micron-scale culture medium region on the substrate surface. By culturing individual recipient cells in a micron-scale culture medium of a specific shape and area, individual recipient cells can be constrained to a specific size and shape under culture conditions.
[0040] The recipient cells include one of mesenchymal stem cells and dental pulp stem cells.
[0041] Preferably, the recipient cell is a mesenchymal stem cell.
[0042] Recipient cells were seeded onto micron-sized culture medium and cultured under the following conditions: the culture medium was Gibco α-MEM medium supplemented with 10% fetal bovine serum, the culture temperature was 35–37°C, the culture humidity was 93–97%, and the carbon dioxide concentration was 4–6%.
[0043] Preferably, the culture conditions are as follows: the culture medium is α-MEM medium supplemented with 10% fetal bovine serum, the culture temperature is 37°C, the culture humidity is 95%, and the culture carbon dioxide concentration is 5%.
[0044] The method for isolating exogenous mitochondria involves using a glass homogenizer combined with differential centrifugation to separate them from mesenchymal stem cells.
[0045] Exogenous mitochondria were seeded onto recipient cells under restricted growth conditions and co-cultured. The co-culture conditions were as follows: the culture medium was α-MEM medium supplemented with 10% fetal bovine serum, the co-culture temperature was 35-37°C, the co-culture humidity was 93-97%, and the co-culture carbon dioxide concentration was 4-6%.
[0046] Preferably, the co-culture conditions are as follows: the culture medium is α-MEM medium supplemented with 10% fetal bovine serum, the co-culture temperature is 37°C, the co-culture humidity is 95%, and the co-culture carbon dioxide concentration is 5%.
[0047] This invention utilizes micron-sized culture media of fibronectin of varying sizes and shapes adhered to and covered a substrate surface. When single mesenchymal stem cells (MSCs) are seeded onto these micron-sized culture media, the specific shapes of the culture media formed by fibronectin constrain the individual MSCs, altering their morphology and promoting mitochondrial internalization into the recipient cells (MSCs). Existing research indicates that freely growing cells can transfer mitochondria via endocytosis. This invention, by constraining cell morphology, enhances endocytosis by regulating cell membrane tension, thereby achieving highly efficient mitochondrial transfer.
[0048] The beneficial effects of this invention are as follows: (1) The method described in this invention can significantly improve the transfer efficiency of mitochondria. This invention sets up a micron-level culture medium on the substrate. The morphology of a single recipient cell can be precisely controlled by the micron-level culture medium, thereby effectively promoting the internalization process of free mitochondria into the recipient cell. Under suitable culture medium area, the mitochondrial transfer efficiency is much higher than that of the traditional co-culture method, which greatly improves the mitochondrial delivery effect. The operation is simple, the transfer process is gentle, and it will not cause cell damage. It is conducive to the rapid recovery of mitochondrial function of damaged cells and enhances the efficacy of cell therapy and tissue repair.
[0049] (2) The mitochondrial transfer conditions of the method described in this invention are mild and have high biosafety. This invention achieves efficient mitochondrial transfer by setting up a micron-sized culture substrate with a geometric structure on the substrate and applying mild mechanical stimulation to the cells through geometric constraints. It does not require violent physical means such as electroporation or microinjection, nor does it rely on carrier materials with potential toxicity. This transfer method effectively avoids mechanical damage to the cell membrane, cytoskeleton and mitochondrial structure, significantly improves cell survival rate and operational safety, and is more suitable for clinical translation scenarios with strict safety requirements.
[0050] (3) By standardizing and limiting the area of micron-sized culture substrate, this invention can achieve precise control over the morphology and spreading area of individual cells, ensure stable and consistent experimental conditions, improve the reproducibility and reliability of mitochondrial transfer efficiency, and lay a stable technical foundation for subsequent mechanism research and clinical application.
[0051] (4) The transfer method described in this invention relies on mature micro-nano fabrication and PDMS molding technology. The preparation process is simple, the equipment dependence is low, and the cost is low. It can realize the batch preparation of micro-patterned substrates without the need for complex and expensive instruments, which greatly reduces the operational difficulty and experimental cost of mitochondrial transfer and has good industrialization and promotion potential.
[0052] (5) The technical principle of the mitochondrial transfer method does not depend on a specific cell type. It can adapt to the mitochondrial transfer needs of various cells through customized micron-level culture medium. It has important application value in the fields of stem cell function enhancement, damaged cell repair, regenerative medicine and organelle therapy. It provides a new, efficient and safe solution for the clinical application of mitochondrial transfer technology. It has a wide range of applications and broad application prospects. Attached Figure Description
[0053] Figure 1 A schematic diagram of the process for setting up micron-sized culture media in a uniform array on a substrate is shown. Figure 2 In the image, Figure A shows a patterned fluorescence image of a single mesenchymal stem cell, demonstrating that the cell morphology is highly consistent with the shape of the micron-scale culture medium; Figure B shows the experimental results of mitochondrial internalization under different patterning conditions using fluorescence microscopy. Figure 3 A schematic diagram illustrating cell proliferation following patterned mitochondrial translocation. Figure 4 This diagram illustrates the production of adenosine triphosphate (ATP) in cells after mitochondrial translocation is promoted under patterned conditions. Detailed Implementation
[0054] The present invention will now be described in detail, and its features and advantages will become clearer and more apparent from these descriptions.
[0055] This invention provides a method for promoting efficient mitochondrial transfer in cells, the method comprising the following steps: Step 1: Mix PDMS, the material for preparing the micro-patterned stamp, with hydrogen-containing polydimethylsiloxane at a mass ratio of (8-12):1. Cover the resulting premix onto a mold and cure it. The curing conditions are: curing temperature of 70-80℃ and curing time of 10-15 h. The mold is a silicon wafer with multiple grooves on its surface. The grooves are evenly distributed in an array on the surface of the silicon wafer. After curing, demold to obtain the micro-patterned stamp. The micro-patterned stamp includes multiple protrusions evenly arranged in an array. The raw materials for preparing micron-sized culture media are covered on the raised surface, and then transferred from the raised surface of the micro-patterned stamp to the substrate surface, thereby uniformly setting multiple micron-sized culture media in an array on the substrate surface, such as... Figure 1 As shown.
[0056] Each of the micron-sized culture media is spherical in shape, and the area of each micron-sized culture media is 350–2000 μm.2 .
[0057] Step 2: Seed each of the mesenchymal stem cells onto each of the micron-sized culture media, and culture the mesenchymal stem cells on the micron-sized culture media. This allows each recipient cell mesenchymal stem cell to grow under constrained conditions within each micron-sized culture media region, resulting in recipient cells (mesenchymal stem cells) with a specific morphology. The culture conditions are: Gibco α-MEM medium supplemented with 10% fetal bovine serum; culture temperature of 35–37°C; humidity of 93–97%; and carbon dioxide concentration of 4–6%.
[0058] Step 3: Obtain mitochondria from mesenchymal stem cells using differential centrifugation. Co-culture the isolated exogenous mesenchymal stem cell mitochondria with recipient cells exhibiting a specific morphology. The co-culture conditions are: Gibco α-MEM medium supplemented with 10% fetal bovine serum; co-culture temperature: 35–37°C; co-culture humidity: 93–97%; co-culture carbon dioxide concentration: 4–6%. Specific morphological regulation of the recipient cells promotes the transfer of mitochondria into the recipient cells.
[0059] Example The present invention is further illustrated below with specific examples. These embodiments are merely illustrative and not intended to limit the scope of the invention. All raw materials used in the embodiments of the present invention were commercially available.
[0060] Example 1 A method for promoting efficient mitochondrial transfer in cells, the method comprising the following steps: Step 1: Mix PDMS, the material for preparing the micro-patterned stamp, with hydrogen-containing polydimethylsiloxane at a mass ratio of 10:1. Cover the resulting premix onto a mold and cure it. The curing conditions are: curing temperature of 75°C and curing time of 12 hours. The mold is a silicon wafer with multiple grooves on its surface. The grooves are evenly distributed in an array on the surface of the silicon wafer. After curing, demold to obtain the micro-patterned stamp. The micro-patterned stamp includes multiple protrusions evenly arranged in an array. The raw materials for preparing micron-sized culture media are covered on the raised surface, and then transferred from the raised surface of the micro-patterned stamp to the substrate surface, thereby uniformly setting multiple micron-sized culture media in an array on the substrate surface, such as... Figure 1 As shown.
[0061] Each of the micron-sized culture media is spherical in shape, and each of the micron-sized culture media has an area of 750 μm. 2 ( Figure 2 Group B).
[0062] Step 2: Each of the mesenchymal stem cells is seeded onto each of the micron-sized culture media, and the mesenchymal stem cells are cultured on the micron-sized culture media. This allows each recipient cell mesenchymal stem cell to grow under constrained conditions within each micron-sized culture media region, resulting in recipient cell mesenchymal stem cells with a specific morphology. The culture medium is Gibco α-MEM medium supplemented with 10% fetal bovine serum, at a culture temperature of 37°C, a humidity of 95%, and a carbon dioxide concentration of 5%.
[0063] Step 3: Obtain mitochondria from mesenchymal stem cells using differential centrifugation. Co-culture the isolated exogenous mesenchymal stem cell mitochondria with mesenchymal stem cells of a specific morphology in Gibcoα-MEM medium supplemented with 10% fetal bovine serum. The co-culture temperature is 37℃, the humidity is 95%, and the carbon dioxide concentration is 5%. The transfer of mitochondria into the recipient cells is promoted by the specific morphological regulation of the recipient cells.
[0064] Example 2 The efficient transfer of mitochondria was performed in a manner similar to that in Example 1, the only difference being that the area of each of the micron-sized culture media was 350 μm. 2 ( Figure 2 Group A).
[0065] Example 3 The efficient transfer of mitochondria was performed in a manner similar to that in Example 1, the only difference being that the area of each of the micron-sized culture media was 1000 μm. 2 ( Figure 2 Group C).
[0066] Example 4 The efficient transfer of mitochondria was performed in a manner similar to that in Example 1, the only difference being that the area of each of the micron-sized culture media was 2000 μm. 2 ( Figure 2 Group D).
[0067] Comparative Example Comparative Example 1 The efficient transfer of cell mitochondria was carried out in a manner similar to that in Example 1, except that: instead of setting up a micron-sized culture medium on the PDMS substrate, fibronectin was uniformly coated on the surface of the PDMS substrate, and both mesenchymal stem cells and mesenchymal stem cell mitochondria were cultured on the fibronectin-coated PDMS substrate.
[0068] Experimental Example Experiment Example 1: Fluorescence Intensity Test Immunofluorescence staining with Myosin II was used to observe the transferred mitochondria in Examples 1-4 and Comparative Example 1. Differences in fluorescence intensity were observed in culture media with micropatterns of varying circular areas, suggesting that this invention promotes mitochondrial transfer by regulating cell membrane tension. The test results are as follows: Figure 2 As shown ( Figure 2 The control group in this study is the same as Comparative Example 1.
[0069] from Figure 2 As shown in Figures A and B, the area of each micrometer-sized culture substrate ranges from 350 to 2000 μm. 2 Both methods can achieve efficient mitochondrial transfer, even when the surface area of the micron-sized culture substrate is 750 μm. 2 At this time, the transfer effect on mitochondria was best and the transfer efficiency was highest. These results indicate that the surface area of the micron-sized culture substrate has a significant impact on mitochondrial transfer.
[0070] Experimental Example 2 EdU Staining Test EdU staining was used to observe the cell proliferation in Example 1 and Comparative Example 1. The test results are as follows: Figure 3 As shown ( Figure 3 The control group in this study is the same as Comparative Example 1.
[0071] from Figure 3 As can be seen from the results, there is a difference in the proportion of 5-ethynyl-2'-deoxyuridine (EdU) positive cells between Example 1 and Comparative Example 1. The proportion of EdU positive cells in Example 1 is significantly higher than that in Comparative Example 1. These results indicate that the present invention can significantly enhance cell proliferation capacity by promoting mitochondrial translocation.
[0072] Experiment Example 3: ATP Production Test The ATP production in Example 1 and Comparative Example 1 was tested using ATP production data. The test results are as follows: Figure 4 As shown ( Figure 4 The control group in this study is the same as Comparative Example 1.
[0073] from Figure 4 As can be seen from the data, the area of Example 1 is 750 μm. 2 The ATP levels in the circular micron-sized culture medium were significantly higher than those in Comparative Example 1. These results indicate that the present invention can significantly enhance ATP function by promoting mitochondrial translocation, thus providing sufficient energy for cell proliferation.
[0074] The present invention has been described in detail above with reference to specific embodiments and exemplary examples; however, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the invention, and all such modifications and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the appended claims.
Claims
1. A method for promoting efficient mitochondrial transfer in cells, characterized in that, The method includes the following steps: Recipient cells were seeded on micron-sized culture media and allowed to grow under constraint within the micron-sized culture media. Then, exogenous mitochondria were added to the constrained recipient cells for co-culture. The morphological regulation of the recipient cells promoted the transfer of mitochondria into the recipient cells. The surface area of the micron-sized culture medium is 350–2000 μm. 2 ; Multiple micron-sized culture media are uniformly arranged in an array on the substrate; The micron-sized culture medium is circular in shape; The specific method for uniformly distributing multiple micron-sized culture media in an array on a substrate is as follows: The material for preparing the micro-patterned stamp is mixed with a curing agent. The resulting premix is then applied to a mold for curing. The mold is a silicon wafer with multiple grooves on its surface. These grooves are evenly distributed in an array on the surface of the silicon wafer. After curing, the mold is removed to obtain the micro-patterned stamp. The micro-patterned stamp includes multiple protrusions evenly arranged in an array. The raw materials for preparing micron-sized culture media are covered on the raised surface, and then the raw materials for preparing micron-sized culture media are transferred from the raised surface of the micro-pattern stamp to the substrate surface, thereby so that multiple micron-sized culture media are uniformly arranged in an array on the substrate surface.
2. The method according to claim 1, characterized in that, The substrate is made from one or more of PDMS, glass, and ultra-low adsorption water adhesive.
3. The method according to claim 1, characterized in that, The micron-scale culture medium is prepared from one or more of fibronectin, collagen, and laminin.
4. The method according to claim 1, characterized in that, The obtained premix was applied to a silicon wafer and cured at 70–80°C for 10–15 h.
5. The method according to claim 1, characterized in that, The curing agent is selected from silicone elastomer curing agents; The mass ratio of the material used to prepare the micro-patterned stamp to the curing agent is (8-12):
1.
6. The method according to claim 1, characterized in that, The culture conditions for constraining the growth of recipient cells in micron-sized culture medium are: a culture temperature of 35–37°C, a culture humidity of 93–97%, and a culture carbon dioxide concentration of 4–6%.
7. The method according to claim 1, characterized in that, The recipient cells include one of mesenchymal stem cells and dental pulp stem cells.
8. The method according to claim 1, characterized in that, The co-culture conditions are as follows: co-culture temperature is 35-37℃, co-culture humidity is 93-97%, and co-culture carbon dioxide concentration is 4-6%.