Systems and methods for high efficiency quantitative mitochondrial transfer based on droplet microfluidics

Abstract

A system for quantitatively controlling mitochondrial transfer based on droplet microfluidics, comprising a generation module for generating droplets containing free mitochondria and single cells; an observation module for observing the generated droplets under a microscope; and a collection module for collecting the generated droplets. The required number of mitochondria transferred into recipient cells is an important issue in precision medicine, and the present application can accurately control the number of transferred mitochondria at the single cell level, helping to determine the required number of exogenous mitochondria to significantly improve the function of recipient cells before cell therapy for mitochondrial gene-related diseases.

Description

Technical Field

[0001] This invention relates to mitochondrial transfer based on a droplet microfluidic system, and more specifically, to highly efficient quantitative mitochondrial transfer based on a droplet microfluidic system. Background Technology

[0002] The ratio of mutant to wild-type mitochondrial DNA (mtDNA) is called the heterogeneity coefficient, which determines the severity of mitochondrial-related diseases. In muscle tissue, when the heterogeneity coefficient exceeds a certain level or mitochondrial dysfunction occurs, less ATP and excessive reactive oxygen species (ROS) are produced, leading to muscle atrophy, weakness, and loss of endurance. Previous clinical and preclinical animal studies have shown a link between increased mitochondrial damage and poor skeletal muscle health. Since the 1990s, cell therapies (especially myoblast transplantation) have been proposed to improve the regeneration of injured skeletal muscle. However, early clinical trials of myoblast transplantation have mostly failed, mainly due to the presence of host immune cells (such as CD8+ T lymphocytes), leading to massive cell death. The accumulation of immune cells not only causes sarcolemma damage and activates caspase-3 in myofibrils but also further induces myofibril apoptosis. Therefore, new methods for treating skeletal muscle diseases need to be developed, such as modulating macrophages and chemically induced stem cells, and restoring or improving mitochondrial function to promote muscle regeneration is an attractive approach.

[0003] Besides providing energy for cells, mitochondria are also involved in cell proliferation, aging, apoptosis, innate immunity, calcium homeostasis, and even stem cell differentiation potential. Mutations in mtDNA can impair cell and tissue function. In nature, spontaneous mitochondrial transfer can occur between healthy and damaged cells through various mechanisms to protect damaged cells and restore their cellular function. Mitochondrial transfer is a technique for altering mtDNA in cells, and it has attracted increasing attention since it was first disclosed by Clark and Shay. Mitochondrial transfer is currently used as a cell therapy to treat mtDNA-related diseases. Compared to other techniques for modifying the mitochondrial genome, such as mitoZFNs and mitoTALENs, mitochondrial transfer is easier to perform and is actually more effective. By transferring exogenous mitochondria into recipient cells, the ratio of mutant to wild-type mtDNA can be reduced, and cell and tissue function can be restored or improved. Previous studies have shown that exogenous free mitochondria can be delivered into cells via co-culture or microinjection. In the co-culture method with free mitochondria, recipient cells engulf the free mitochondria through endocytosis, a cellular activity in which nanometers to micrometers of matter are taken up from the surrounding environment. Free mitochondria move randomly around the recipient cells and have the opportunity to be engulfed when they come into contact with the cells. Therefore, this phenomenon is a random and accidental process. The transfer efficiency of the co-culture method is affected by the number of extracellular free mitochondria. Although it has reached up to 28% in previous studies, even with equal amounts of free mitochondria, the number of mitochondria transferred into the cells is quite uneven (some recipient cells have 1 while others have 60). Although the co-culture method is a fairly simple process, its success depends on many uncontrollable factors, which may be one of the potential reasons for the unsatisfactory cell metabolic recovery rate (about 0.2%) of recipient cells in previous experiments. To reduce the impact of uncontrollable factors, automated optical tweezers-based manipulation systems (OT-based manipulation systems) have been used for mitochondrial transfer with controlled quality and quantity. OT-based manipulation systems can precisely acquire healthy mitochondria and deliver them to target recipient cells. However, this method is limited by low throughput, making OT-based manipulation systems difficult to use in clinical applications.

[0004] Unlike co-culture methods, microinjection involves directly injecting free mitochondria pre-loaded into recipient cells using microneedles; therefore, it may damage recipient cells because the cell membrane must be opened during delivery. Furthermore, microinjection technology has very low throughput.

[0005] The methods described above provide useful technical solutions for studying the mechanisms of cell function recovery or improvement after mitochondrial transfer; however, they still cannot meet the large demand from the cell therapy industry for cells transfected into mitochondria. Co-culture technology has considerable advantages due to its harmlessness, but its low efficiency and heterogeneity remain major bottlenecks.

[0006] Droplet microfluidics is a technique that continuously disperses chemical reagents, cells, or other biological materials into discrete volumes (called droplets) at the micrometer scale. These droplets serve as the basic units for further chemical reactions, cellular activities, target detection, and material synthesis. Droplet microfluidics provides a smaller and more confined environment than large-volume analytical methods, allowing for faster reactions and detection of molecules / particles and their interactions with encapsulated cells. Previous studies have demonstrated droplet generation rates of up to thousands of droplets per second, making droplet microfluidics inherently a high-throughput technique. A key application of droplet microfluidics is single-cell analysis, where a single cell is encapsulated within a droplet for analyzing cellular activities or performing cell modifications such as antibody analysis or gene editing.

[0007] U.S. Patent Application Publication No. 2017 / 159017A1, entitled "Method for introducing exogenous mitochondria into a mammalian cell", and U.S. Patent Application Publication No. 2013 / 149778A1, entitled "Method and Applications of Peptide-Mediated Mitochondrial Delivery System", utilize the natural cell membrane phagocytosis process (also known as endocytosis) to transfer free mitochondria.

[0008] U.S. Patent Application Publication No. 2019 / 276852A1, entitled "Method for delivering exogenous mitochondria into cells," and European Patent Application Publication No. EP3169338A1, entitled "Methods for the intercellular transfer of isolated mitochondria in recipient cells," both utilize centrifugation to transfer mitochondria. In these methods, the isolated mitochondria are centrifuged together with the cells, thereby increasing the efficiency of mitochondrial transfer by forcing the mitochondria into the cells.

[0009] US Patent No. 10,760,040B1, entitled "Mechanical transfection devices and methods", describes the use of fluid-induced mechanical force to open cell membranes and deliver free mitochondria into cells.

[0010] Traditional microinjection methods can cause physical damage to recipient cells, while co-culture techniques can produce the number of cells needed for cell therapy. However, co-culture techniques cannot control the number of mitochondria transferred to each recipient cell, so cells without sufficient mitochondria may not be able to perform their full therapeutic function. Summary of the Invention

[0011] Therefore, a droplet microfluidic-based mitochondrial transfer system provides highly efficient and quantitative control. In this invention, the droplet microfluidic-based mitochondrial transfer method does not cause physical damage to the recipient cells (which is unavoidable in microinjection) and exhibits high throughput advantages. Compared with traditional co-culture methods, the method proposed in this invention can also control the number of mitochondria transferred to the recipient cells at the single-cell level, achieving high efficiency and high throughput.

[0012] This invention provides a highly efficient quantitative control system for mitochondrial transfer based on droplet microfluidics. The system includes a generation module for generating droplets containing free mitochondria and single cells; an observation module for observing the generated droplets under a microscope; and a collection module for collecting the generated droplets.

[0013] In another embodiment, the droplet generation module and the droplet observation module are connected through a conduit.

[0014] In other embodiments, the system is mounted on a chip.

[0015] In yet another embodiment, the length of the chip is less than 8 cm.

[0016] In another embodiment, the droplet generation module includes three inlets.

[0017] In another embodiment, the droplet generation module further includes a mitochondrial receptor cell suspension, a free mitochondrial suspension, and a fluorinated oil with added surfactant.

[0018] In other embodiments, the mitochondrial receptor cell is the receptor C2C12 cell.

[0019] In another embodiment, the droplet generation module includes a flow focusing structure, wherein the flow focusing structure can separate the mitochondrial receptor cell suspension and the free mitochondrial suspension into droplets.

[0020] In another embodiment, the droplet generation module includes a wavy structure, wherein the flow focusing structure can arrange randomly distributed cells into a straight line from the inlet.

[0021] In another embodiment, the wavy structure can increase the single-cell encapsulation rate to over 47%.

[0022] In another embodiment, the wavy structure can suppress the multi-cell encapsulation rate to less than 6%.

[0023] In other embodiments, the droplet comprises free mitochondria and a single cell.

[0024] In another embodiment, the mitochondrial transfer efficiency is at least 75%.

[0025] In other embodiments, the system can generate at least 2 × 10⁻⁶ units within 30 minutes. 6 A cell encased in a droplet for mitochondrial transfer.

[0026] The present invention also provides a quantitative control method for mitochondrial transfer based on droplet microfluidics, comprising the following steps: preparing a first suspension, a second suspension, and an oil; injecting the first suspension, the second suspension, and the oil into a quantitative control system for mitochondrial transfer based on droplet microfluidics; encapsulating the first and second suspensions together in a droplet; collecting the droplet; and culturing the first and second suspensions together in the droplet.

[0027] In another embodiment, the first suspension is a mitochondrial receptor cell suspension.

[0028] In yet another embodiment, the second suspension is a free mitochondrial suspension.

[0029] In other embodiments, the droplet diameter is 40 μm.

[0030] In other embodiments, the system includes a wave-like structure.

[0031] In another embodiment, the droplet contains mitochondria and mitochondrial receptor cells. Attached Figure Description

[0032] Figure 1A-1G The diagram shows the system setup, the droplet microfluidic-based mitochondrial transfer technology, and the experimental evaluation process. Figure 1A This shows the system setup for droplet microfluidic-based mitochondrial transfer technology; Figure 1B The image shows the wavy structure used for cell pairing before encapsulation and the wavy structure used for mixing mitochondria and cell suspension after encapsulation. Figure 1C The cells and mitochondria are shown enclosed together in the droplet; Figure 1D Demonstration showing how the wavy structure focuses the cells; Figure 1E Demonstration of mitochondrial transfer via cellular endocytosis within a droplet; Figure 1F Displaying the chip used for droplet generation and observation; Figure 1G This demonstrates the procedure for in vitro myogenic differentiation experiments using recipient cells that have completed mitochondrial transfer.

[0033] Figure 2A-2I This demonstrates a mitochondrial transfer system based on droplet microfluidics. Figure 2A The cell suspension and free mitochondrial suspension flow together toward the wavy structure; Figure 2B The wavy structure is shown to improve the efficiency of single-cell encapsulation; Figure 2C Showing the flow focusing structure used for droplet generation; Figure 2D This shows free mitochondria isolated from donor cells (C2C12 myoblasts) (previously isolated using MitoTracker). TM Green FM staining); Figure 2E The image shows free mitochondria absorbed by the recipient C2C12 cells, and further analysis of a portion of the droplets using confocal imaging to confirm whether the mitochondria were successfully transferred (recipient cells are in red, and transferred mitochondria are in green). Figure 2F The efficiency of cell encapsulation using a wavy structure is shown in cell suspensions of different concentrations, where 1 and 2 represent one and two or more cells encapsulated in a droplet, respectively, and L represents 0.85 × 10⁻⁶ cells / mL. 7 Cells / mL, and H represents 1.7 × 10⁻⁶ cells / mL. 7 Cells / mL; Figure 2G This demonstrates the effect of the oil / water flow rate ratio on droplet size. Figure 2H This demonstrates the effect of droplet size on mitochondrial transfer efficiency; Figure 2I This demonstrates the effect of cell suspension flow rate on cell viability. Figure 2F-2IAll data are expressed as mean ± standard deviation and were analyzed by one-way ANOVA and Dunn's multiple comparisons test. *p < 0.05 and **p < 0.01. The red arrows from A to C indicate the flow direction of fluid in the microfluidic chip channel.

[0034] Figures 3A-3B This shows the number of free mitochondria encapsulated in each droplet under different concentrations of free mitochondrial suspensions. Figure 3A This shows the number of free mitochondria in a single droplet (using MitoTracker) when using three different concentrations of free mitochondrial suspensions. TM 3D reconstructed images (marked with Green) (by turning off the red channel of the confocal microscope to avoid showing cells stained with Cell MaskDeep Red), where each box contains a droplet and the number of free mitochondria is indicated in the upper left corner; Figure 3B This shows the number of free mitochondria encapsulated in each droplet under three different concentrations of free mitochondrial suspensions. All data are expressed as mean ± standard deviation.

[0035] Figures 4A-4C This demonstrates the quantitative control of mitochondrial transfer using a droplet-based method. Figure 4A This image shows representative confocal images of recipient cell suspensions after mitochondrial transfer at concentrations of 0.25, 0.5, or 1.0 units, where one unit concentration of mitochondria represents a concentration from 1 × 10⁻⁶. 6 The concentration of mitochondria isolated from cells and suspended in 10 μL of mitochondrial storage reagent; the transferred mitochondria were analyzed using MitoTracker before isolation. TM Green FM labeling, recipient cells were labeled with Cell Mask Deep Red before being encapsulated into droplets, and three recipient cells were selected from each unit to indicate the location of the transferred mitochondria within the cell (bottom); Figure 4B The average number of translocated mitochondria per cell was shown at different mitochondrial concentrations, and the number of mitochondria was counted in the 3D reconstructed images under a confocal fluorescence microscope. Figure 4C The transfer efficiency is shown, which is defined as the ratio of the number of free mitochondria transferred to the recipient cell to the total number of free mitochondria encapsulated in the droplet. Figure 4B and Figure 4C All data are expressed as mean ± standard deviation.

[0036] Figures 5A-5D An in vitro study demonstrating the effect of mitochondrial transfer on myoblastic differentiation of C2C12 myoblasts. Figure 5AThis image shows representative images of C2C12 cells translocated via mitochondria during myoblast induction. C2C12 cells underwent mitochondrial translocation at different concentrations before myoblast induction (8, 14, and 31 exogenously isolated free mitochondria were translocated per cell, defined as low-mitochondrial, medium-mitochondrial, and high-mitochondrial translocation groups, respectively). Cell morphology and myotube formation were imaged before induction and on days 3 and 7 of the induction process. Figures 5B-5C The results show that on day 7, the area and length of the myotubes were measured using ImageJ, with three FOVs taken from each well; Figure 5D The proliferation rate of C2C12 cells was measured by MTT assay on days 1, 2, 3, and 4 after mitochondrial transfer. All values ​​were normalized relative to day 0. Data are expressed as mean ± standard deviation and analyzed by one-way ANOVA followed by Dunn's multiple comparison test, N = 3. * (or #) indicates p < 0.05, ** (or ##) indicates p < 0.01, *** (or ###) indicates p < 0.001, and **** (or ####) indicates p < 0.0001. Detailed Implementation

[0037] See attached diagram. Figure 1A A schematic depiction of a highly efficient quantitative mitochondrial transfer system based on a droplet microfluidic system is shown. A droplet generation module 10 generates droplets 40 containing free mitochondria and single cells. The droplet generation module 10 also includes a wavy structure that focuses randomly distributed cells into a row from the inlet, improving single-cell encapsulation efficiency and inhibiting multi-cell encapsulation efficiency. Element 20 is a droplet observation module, which allows observation of the generated droplets under a microscope (not shown). Element 30 is a droplet collection module, which collects the generated droplets 40. The droplet generation module and the droplet observation module can be connected via a conduit 50. Optionally, as... Figure 1F As shown, system 1 is set on chip 60.

[0038] Microfluidic chip fabrication and manipulation:

[0039] The designed chip was fabricated using soft lithography. Before the experiment, the chip channels were coated with a surface modifier to make them hydrophobic, so as to stably generate and transport water-in-oil droplets.

[0040] Cell culture:

[0041] C2C12 myoblasts were cultured at 37°C and 5% CO2 in a high-glucose (Gibco) environment. TM ,11965084) and contains 10% fetal bovine serum (Gibco) TM ,12800058) and 1% antibiotic-antimycin (Gibco) TM, in Dulbecco modified Eagle medium (DMEM) of 15240096.

[0042] Mitochondrial isolation:

[0043] The mitochondria used were freshly isolated from C2C12 myoblasts prior to each mitochondrial transfer experiment, following the protocol outlined in the mitochondrial isolation kit (Beyotime, C3601). First, they were isolated using MitoTracker... TM Green FM (Invitrogen) TM Mitochondria of donor C2C12 cells were stained with PBS (M7514), followed by washing the stained cells three times with trypsin / EDTA solution (Gibco). TM Cells stained with MIRV (R001100) were isolated from the culture flask and centrifuged at 500g for 5 minutes. After removing the supernatant, the collected cells were resuspended in 1 ml of cell lysis reagent (Beyotime, C3601-1) and placed on ice for 15 minutes. The lysed cells were then homogenized 30 times using a glass homogenizer. The homogenized cells were then centrifuged at 1,000g for 10 minutes at 4°C. The supernatant was resuspended in 1 ml of cell lysis reagent (Beyotime, C3601-1) and centrifuged again at 1,000g for 10 minutes at 4°C to obtain higher purity. Finally, the supernatant was collected and centrifuged at 3,500g for 10 minutes at 4°C. The collected centrifuged block was the isolated free mitochondria. The isolated free mitochondria were resuspended at the required concentration using mitochondrial storage reagent (Beyotime, C3601-3) for further experiments. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) 6 Mitochondria isolated from cells and suspended in 10 μL of mitochondrial storage reagent are defined as one unit of free mitochondrial suspension.

[0044] 3D reconstruction of cells and mitochondria under confocal fluorescence microscopy:

[0045] After co-culturing the recipient cells and exogenously isolated free mitochondria in a droplet for 2 hours, the droplet was loaded into a droplet observation module, such as... Figure 1A As shown; then, images containing single cells (labeled with Cell Mask Deep Red) and free mitochondria (labeled with MitoTracker) were captured using confocal fluorescence microscopy (LEICASP8LIA++TRUE confocal laser scanning microscope). TMImages of the droplets (labeled with Green FM) were captured; then, 3D reconstruction of the images was performed using confocal microscopy software; finally, free mitochondria transferred to the cells were counted as green particles within the red cell region, while green particles outside the red cell region were counted as untransferred free mitochondria. The mitochondrial transfer efficiency was the ratio of transferred mitochondria to the total mitochondria in the droplet. The same process was repeated for cells recovered from the droplets to count the number of mitochondria transferred at different concentrations of free mitochondria.

[0046] Droplet breakup and cell collection:

[0047] The collected droplets floated on top of the fluorinated oil. Before the droplets broke apart, excess oil was removed from the bottom of the test tube. Then, 1 ml of 50% 1H,1H,2H,2H-perfluorooctanol (PFO, Thermo Scientific) was added. TM Add (AAB2015609) to a test tube containing the collected droplets and gently vortex for 1 minute to combine the droplets into the bulk solution. Then, carefully transfer the upper bulk solution to a new test tube and centrifuge at 300g for 3 minutes to collect the cells.

[0048] C2C12 myoblast differentiation:

[0049] To assess C2C12 differentiation, C2C12 cells were cultured at a density of 5,000 cells / cm². 2 The concentration was inoculated into six-well plates and cultured in growth medium until 80% confluence was achieved, then seeded with 2% horse serum (Gibco). TM DMEM (Gibco) of 16050130 TM (11965084) Replace the culture medium and keep the cells in the differentiation medium until the end of the experiment, usually between day 5 and day 7. Monitor myotube formation every two days at day 0, day 3 and day 7.

[0050] MTT analysis method:

[0051] Cell proliferation rate of each C2C12 cell group was determined by MTT assay. In short, C2C12 cells were cultured at a density of 5,000 cells / cm². 2 Cells were seeded at a density of 100 μL in 96-well plates and incubated for 24 hours. Then, the cells were treated with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazole bromide (MTT, 100 μL, 0.5 mg / mL) at 37°C for 3 hours. The resulting deep blue formazan crystals were then dissolved in 100 μL LDMSO. The absorbance at 570 nm was measured using a microplate reader.

[0052] In the experiment, the following methods were used: Figure 1AA droplet-based mitochondrial transfer system was developed. Three solutions, namely (1) a mitochondrial receptor C2C12 cell suspension, (2) a free mitochondrial suspension, and (3) a fluorinated oil with added surfactant, were injected into the droplet generation module 10 through three inlets, respectively. The mitochondrial receptor C2C12 cell suspension, the isolated mitochondrial suspension, and the fluorinated oil with added surfactant (Sphere Fluidics, CO21) were then collected in the droplet collection module 30. The droplets included mitochondria and mitochondrial receptor cells.

[0053] like Figure 1B and 1C As shown, the flow focusing structure is used to separate two suspensions into droplets: a mitochondrial receptor C2C12 cell suspension and a free mitochondrial suspension. Figure 1D As shown, the wavy structure can arrange randomly distributed cells into a straight line from the entrance, thus improving the efficiency of single-cell encapsulation; for example... Figure 1E As shown, free mitochondria are absorbed by receptor C2C12 cells within the droplet via endocytosis; through PE tubes (BD Intramedic... TM The BD 427406 chip connects the droplet generation module and the droplet observation module; the entire chip is only about 8 cm long. Figure 1F As shown; Figure 1G As shown, after mitochondrial transfer is completed, recipient cells are collected from the droplets by droplet disruption and functional experiments can be performed immediately to evaluate the effect of different numbers of mitochondrial transfers on in vitro myogenic differentiation of C2C12 cells.

[0054] To enable observation and 3D reconstruction under confocal fluorescence microscopy (LEICA SP8LIA++TRUE confocal laser scanning microscope), mitochondria were isolated from donor cells using a 2 μM MitoTracker solution. TM GreenFM (Invitrogen) TM Mitochondria were stained using M7514.

[0055] like Figure 2A-2C As shown, after mitochondrial separation, mitochondrial recipient cells and freshly separated free mitochondria are immediately injected into the microfluidic chip for encapsulation and mitochondrial transfer. Figure 2D As shown, the labeled free mitochondria in the suspension are spherical structures with a diameter of approximately 1 μm. After co-culturing with the generated droplets for 2 hours, Figure 2E The results show that recipient cells absorb free mitochondria through endocytosis.

[0056] As mentioned above, by using a wavy structure, the cell encapsulation efficiency is improved beyond that of the Poisson distribution. Figure 2F The results showed that when using a cell concentration of 0.85 × 10⁻⁶, 7 With a cell density of 100 cells / mL and a cell suspension flow rate of 300 μL / 30 min (the flow rate of the free mitochondrial suspension was kept consistent), the single-cell encapsulation efficiency reached approximately 47.8%, while the multi-cell encapsulation efficiency was suppressed to approximately 5.9%. This means that the single-cell to multi-cell encapsulation ratio was increased to 8.1, or in other words, 292% of the Poisson distribution (under the condition that an average of 0.6 cells are encapsulated per droplet). This improved single-cell encapsulation efficiency helps to increase throughput. This system can produce 2 × 10⁶ cells encapsulated in droplets for mitochondrial transfer within 30 min. 6 Each receptor cell, such as Figure 2H As shown, mitochondrial transfer efficiency was defined as the ratio of free mitochondria transferred to the cell to the total number of free mitochondria encapsulated in the droplet. This ratio decreased slightly from 75% to 70% as the droplet diameter increased from 40 μm to 52 μm. Figure 2G As shown, by setting the oil / water phase flow rate ratio to 6 and the droplet diameter to 40 μm; Figure 2I As shown, after treatment with this system, the recipient cells can still maintain a relatively high survival rate, such as 95% at a flow rate of 300 μL / 30 min.

[0057] The enclosed microenvironment of the droplets restricts the movement distance of free mitochondria, increasing the probability of contact between free mitochondria and cells, thus facilitating mitochondrial uptake and improving mitochondrial transfer efficiency. Furthermore, because free mitochondria are smaller than droplets (1-40 μm in diameter), they are uniformly encapsulated within each droplet, and the number of free mitochondria encapsulated within each droplet can be controlled by adjusting the concentration of the free mitochondrial suspension. Figures 3A-3B The results showed that when using free mitochondrial suspensions at concentrations of 0.25, 0.5, and 1.0 units, each droplet contained 8, 22, and 41 free mitochondria, respectively. A concentration of 1.0 unit refers to a concentration from 1 × 10⁻⁶. 6 Mitochondria isolated from cells and suspended in 10 μL of mitochondrial storage reagent (Beyotime, C3601-3).

[0058] Example

[0059] In one embodiment, three different concentrations of free mitochondrial suspensions (0.25, 0.5, and 1.0 unit concentrations) were used to verify the transfer efficiency of the present invention. Figure 4A and 4B The results showed that, at concentrations of 0.25, 0.5, and 1.0 units, an average of 8, 14, and 31 free mitochondria were transferred to recipient cells, respectively. Figure 4CThe results showed that at the concentrations of 0.25, 0.5, and 1.0 units used, the mitochondrial transfer efficiency was approximately 75%.

[0060] The differentiation capacity of C2C12 myoblasts after mitochondrial transfer was tested by inducing myoblasts to differentiate into myotubes. Seven days after induction, Figures 5A-5C The results showed that, compared with the control group, the low mitochondrial transfer group, and the medium mitochondrial transfer group (corresponding to the transfer of 0, 8, and 14 exogenously isolated free mitochondria per cell, respectively), the high mitochondrial transfer group (transfer of 31 exogenously isolated free mitochondria per cell) had significantly increased myotube area and length (indicators of myogenesis). Furthermore, MTT assay showed that on day 4, cell proliferation increased by 2.5-fold and 1.5-fold in the high mitochondrial transfer group and the medium mitochondrial transfer group, respectively. Figure 5D As shown. Industrial applicability:

[0061] The mitochondrial transfer based on a droplet microfluidic system provided by this invention is offered as a highly efficient quantitative mitochondrial transfer system because it has the following advantages:

[0062] Determining the number of mitochondria that need to be transferred to recipient cells is a crucial issue in precision medicine. This invention enables precise control over the number of transferred mitochondria at the single-cell level, allowing for the determination of the number of foreign mitochondria required to significantly improve recipient cell function prior to cell therapy for mitochondrial gene-related diseases.

[0063] Compared to existing mitochondrial transfer methods, the method of this invention can produce a large number of cells with quantitative mitochondrial transfer for cell therapy purposes. While co-culturing with free mitochondria can also produce the number of cells required for cell therapy, it cannot control the number of mitochondria transferred to each recipient cell. Therefore, some cells without sufficient mitochondrial transfer may not fully realize their therapeutic potential. However, using the system of this invention, the transfer of mitochondria to each recipient cell is controllable; therefore, cells used for cell therapy can fully realize their therapeutic potential.

[0064] While this disclosure has been described and illustrated with reference to specific embodiments thereof, such descriptions and illustrations are not limiting. Those skilled in the art will understand that various changes can be made and equivalents can be substituted without departing from the true spirit and scope of this disclosure as defined by the appended claims. Illustrations may not be drawn to scale. Due to manufacturing processes and tolerances, there may be differences between the process reproduction in this disclosure and actual equipment. This disclosure may also include other embodiments not specifically described. This specification and drawings should be considered illustrative rather than limiting. Modifications can be made to adapt particular circumstances, materials, composition, methods, or processes to the objectives, spirit, and scope of this disclosure. All such modifications are intended to fall within the scope of the appended claims. While the methods disclosed herein are described with reference to specific operations performed in a particular order, it should be understood that these operations can be combined, subdivided, or reordered to form equivalent methods without departing from the teachings of this disclosure. Therefore, unless specifically indicated herein, the order and grouping of operations are not limiting.

Claims

1. A quantitative control system for mitochondrial transfer based on droplet microfluidics, characterized in that, The droplet microfluidic-based mitochondrial transfer quantitative control system includes: A droplet generating module for generating droplets containing free mitochondria and single cells, wherein the single cells in the droplets absorb the free mitochondria through endocytosis; The droplet generation module includes: The facility has three inlets, which are used to receive: a single-cell suspension containing the single cell, a free mitochondrial suspension, and a fluorinated oil containing a surfactant, respectively. A wavy structure is used to arrange randomly distributed single cells into a straight single-cell flow to improve single-cell encapsulation rate and inhibit multi-cell encapsulation rate. The wavy structure is connected to an inlet for receiving a single-cell suspension containing the single cells and an inlet for receiving a free mitochondrial suspension, allowing the single-cell suspension and the free mitochondrial suspension to flow together towards the wavy structure. A flow focusing structure for generating the droplets comprising the single cell and the free mitochondria, wherein the wavy structure is located upstream of the flow focusing structure, and the flow focusing structure is connected to the inlet for receiving the fluorinated oil containing a surfactant. An observation module for observing the droplets under a microscope; and A collection module for collecting the droplets; The system is located on the chip.

2. The system according to claim 1, characterized in that, The droplet generation module and the droplet observation module are connected by a conduit.

3. The system according to claim 1, characterized in that, The single cell mentioned is the receptor C2C12 cell.

4. The system according to claim 1, characterized in that, The wavy structure can increase the single-cell encapsulation rate to over 47%.

5. The system according to claim 1, characterized in that, The wavy structure can suppress the multi-cell encapsulation rate to less than 6%.

6. The system according to claim 1, characterized in that, Mitochondrial transfer efficiency is at least 75%.

7. The system according to claim 1, characterized in that, The system described above generates at least 2 × 10⁻⁶ units within 30 minutes. 6 A cell encased in a droplet for mitochondrial transfer.

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