A mitochondrial storage system and a preparation method and application thereof

Abstract

This invention relates to the field of mitochondrial drug preparation technology, specifically to a mitochondrial storage system, its preparation method, and its applications. The mitochondrial storage system is prepared by the following method: culturing recipient cells in a culture medium containing a mitochondrial DNA debinding agent; then culturing the recipient cells in a culture medium containing a mitophagy inducer; extracting mitochondria from cells or tissues; mixing the mitochondria with the recipient cells, centrifuging the mixture to obtain cells infused with exogenous mitochondria; and culturing these cells in a culture medium containing a mitophagy inducer and a mitochondrial antioxidant to obtain the mitochondrial storage system. This method induces mitophagy to clear mitochondrial shells and optimizes the culture conditions of hybrid cells, providing conditions for the uptake and proliferation of exogenous mitochondria. This method can solve the technical problems of unsatisfactory activity of isolated mitochondria and the difficulty in achieving effective in vitro expansion in existing technologies, and has ideal application and promotion prospects.

Description

Technical Field

[0001] This invention relates to the field of mitochondrial drug preparation technology, specifically to a mitochondrial storage system, its preparation method, and its application. Background Technology

[0002] Mitochondria participate in the occurrence and development of various pathophysiological processes, and research on mitochondria is attracting increasing attention from researchers. Maintaining the in vitro activity and function of mitochondria is a necessary condition for conducting mitochondrial research. Currently, the main methods for preserving isolated mitochondria in clinical and experimental settings are mitochondrial storage solutions or phosphate-buffered saline (PBS) buffer. These two methods primarily maintain the activity of isolated mitochondria by providing an appropriate osmotic pressure and neutral pH environment. However, because these two storage solutions are mainly composed of inorganic salts and lack antioxidants, their ability to protect the activity of important enzymes in mitochondria is limited, failing to meet the requirements for long-term in vitro storage of mitochondria and restricting mitochondrial-related research and applications. On the other hand, current technology cannot achieve in vitro amplification of mitochondria. The demand for mitochondria in scientific research and clinical practice is enormous, currently requiring the acquisition from fresh tissues or blood. If mitochondria obtained from tissues could be amplified in vitro, the cost of mitochondrial-related research and clinical applications would be greatly reduced.

[0003] To enhance the activity of isolated mitochondria and achieve their in vitro expansion, researchers have conducted numerous attempts. Existing technologies include methods for introducing mitochondria into nucleated cells, where mitochondria are introduced into cells with intact structures. The main purpose is to restore the function of recipient cells through mitochondrial transplantation. Current techniques for introducing mitochondria into cells often involve introducing mitochondria into functionally damaged cells. The primary goal is to restore the function of target cells through the transplantation of healthy mitochondria. Since the recipient cells themselves have intact mitochondrial structures, the introduced mitochondria will contain two different types of mitochondria with different DNA. In this case, the introduced mitochondria will gradually be cleared by the cell. In addition, introducing mitochondria into erythrocytes is a relatively common method for in vitro mitochondrial preservation. While erythrocytes provide a cellular environment and extend the in vitro preservation time, erythrocytes lack a nucleus, and mitochondrial proliferation requires not only an intracellular environment but, more importantly, nuclear regulation. Furthermore, erythrocytes are terminally differentiated cells and do not continue to proliferate; therefore, mitochondria cannot continuously proliferate in erythrocytes. Some have attempted to amplify the full mitochondrial DNA sequence, but this method only yields the mitochondrial genome, without the complete mitochondrial structure, and cannot directly perform mitochondrial functions.

[0004] In summary, how to effectively ensure the activity of isolated mitochondria and further achieve in vitro expansion of mitochondria to increase mitochondrial production are problems that need to be solved by existing technologies. Summary of the Invention

[0005] The present invention aims to provide a method for preparing a mitochondrial storage system to solve the technical problems of unsatisfactory activity of isolated mitochondria and difficulty in achieving effective in vitro expansion in the prior art.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing a mitochondrial storage system includes the following steps: treatment of recipient cells, acquisition of mitochondria, and mitochondrial introduction and culture. Treatment of recipient cells: Recipient cells were cultured using conditioned medium to obtain p-cells. 0 Cells; then cultured in a second conditioned medium. 0 Cells, obtaining p-type cells with damaged mitochondria removed 0 cell; The first conditioned medium contains a mitochondrial DNA decomposition agent; the second conditioned medium contains a first mitochondrial autophagy inducer or a mitochondrial depolarizer. Mitochondrial acquisition: Extracting mitochondria from cells or tissues; Mitochondrial introduction and culture: Introducing mitochondria with p-type mitochondria after removing damaged mitochondria. 0 Cells were mixed and centrifuged to obtain cells infused with exogenous mitochondria; the cells infused with exogenous mitochondria were cultured in a third-conditioning medium to obtain a mitochondrial storage system; the third-conditioning medium contained a second mitophagy inducer and / or mitochondrial antioxidant.

[0007] Furthermore, in the recipient cell treatment step, the mitochondrial DNA decomposition agent includes at least one of ethidium bromide, ammonium chloride, chloramphenicol, 4',6-diamidinyl-2-phenylindole, and FCCP; The first mitophagy inducer includes at least one of MTK458, rapamycin, niclosamide, urolithiasis A, MFI8, CCCP, ATP, MFI8, and antimycin A; The mitochondrial depolarizer includes at least one of ddC, antimycin A, FCCP, valproic acid, oligomycin, and nigrain.

[0008] Furthermore, in the recipient cell treatment step, the concentration of mitochondrial DNA decomposition agent in the first conditioned medium was 1-1 × 10⁻⁶. 5 ng / mL; the concentration of the first mitophagy inducer or mitochondrial depolarizer in the second conditioned medium is 10 ng / mL-1000 ng / mL; Preferably, the concentration of the mitochondrial DNA decomposition agent is 100 ng / mL, and the concentration of the first mitophagy inducer or mitochondrial depolarizer is 100 ng / mL. The recipient cells were cultured in the first conditioned medium for 1-2400 h; the p-cells were cultured in the second conditioned medium. 0 The cell duration is 12-96 hours; Preferably, the recipient cells are cultured in the first-conditioning medium for 7-14 days; p is cultured in the second-conditioning medium. 0 The cell duration is 48-72 h.

[0009] Furthermore, in the mitochondrial introduction and culture step, the second mitophagy inducer includes at least one of ATP, CCCP, rapamycin, AICAR, Mdivi-1, and urolithin A; The mitochondrial antioxidants include CoQ10, α-tocopherol, ascorbic acid, MitoQ, resveratrol, Mito-TEMPO, and MitoVit-E.

[0010] Furthermore, in the mitochondrial introduction and culture steps, the ratio of mitochondria to cells used is 1 × 10⁻⁶. 5 -1×10 7 1 cell; 10-500 μg of mitochondria in terms of protein content.

[0011] Furthermore, in the mitochondrial introduction and culture steps, the concentration of the second mitophagy inducer in the third conditioned medium is 10-500 ng / mL; the concentration of the mitochondrial antioxidant in the third conditioned medium is 10-1000 ng / mL; and the culture time of the cells with introduced exogenous mitochondria in the third conditioned medium is 1-240 h.

[0012] Furthermore, in the mitochondrial introduction and culture step, the mitochondria are combined with p-type mitochondria from which damaged mitochondria have been removed. 0 The cells were mixed and centrifuged at 100-5000g for 5-30 minutes to obtain cells with introduced exogenous mitochondria.

[0013] Furthermore, in the mitochondrial acquisition step, mitochondria are derived from cells or tissues, including platelets and other cells; other cells include HeLa cells, 143B cells, and A549 cells; When extracting mitochondria from tissues and other cells, first obtain a single-cell suspension of cells or tissues, then perform lysis and homogenization, and finally filter the suspension through a 40-100 μm filter sieve and a 10-40 μm filter sieve to obtain mitochondria. When extracting mitochondria from platelets, the platelets are first homogenized, and then filtered sequentially through a 40-100 μm filter sieve and a 10-40 μm filter sieve to obtain mitochondria.

[0014] This technical solution also provides a method for preparing a mitochondrial storage system and the resulting mitochondrial storage system.

[0015] This technical solution also provides an application of a mitochondrial storage system in the preparation of drugs for treating mitochondrial defect-related diseases.

[0016] The technical principle of this technical solution is as follows: This invention constructs a mitochondrial storage system based on engineered recipient cells, the principle of which involves two-step processing of recipient cells and functional maintenance culture after the introduction of exogenous mitochondria.

[0017] This invention first employs a two-step process to treat recipient cells, aiming to construct a cell factory that efficiently accepts and supports the expansion of exogenous mitochondria: The first step is mitochondrial DNA clearance: recipient cells are cultured in a first-conditioning medium containing a mitochondrial DNA decomposition agent. By continuously inhibiting mitochondrial DNA replication or inducing its degradation, ρ cells lacking mitochondrial DNA are obtained. 0 Cells. This step removes endogenous mitochondrial genetic material, eliminating competitive barriers for the colonization of exogenous mitochondrial DNA. This technical solution prepares a p 0 Cell, ρ 0 Cells are a special type of cell line characterized by the lack of mitochondrial DNA (mtDNA), that is, the lack of a mitochondrial genome. Although such cells can serve as a host for exogenous mitochondria, in practice, it has been found that the uptake and amplification of exogenous mitochondria remain quite limited when they are used as a site for the replication of exogenous mitochondria.

[0018] The second step is the removal of damaged mitochondrial structures: after obtaining p... 0 Based on the cells, further treatment was performed using a second conditioned medium containing either a first mitophagy inducer or a mitochondrial depolarizer. Through extensive experimentation, the inventors discovered that treating cells with a mitochondrial DNA decomposition agent followed by further treatment with a mitophagy inducer or a mitochondrial depolarizer significantly enhanced the recipient cells' ability to take up exogenous mitochondria. Based on these experimental results, the inventors analyzed the mechanism of action of this step as follows: Although p 0The cell lacks mtDNA, but still retains nonfunctional mitochondrial shell structures within its cell. These residual mitochondrial membrane structures may occupy intracellular space, consume energy resources, and interfere with the uptake and transport of exogenous mitochondria. By inducing mitophagy or depolarization, these nonfunctional mitochondrial remnants can be specifically removed, providing a purer cellular environment for the efficient introduction and colonization of exogenous mitochondria.

[0019] After co-incubating purified exogenous mitochondria with treated recipient cells and centrifuging them, this invention employs a third-conditioning medium containing a second mitophagy inducer and / or mitochondrial antioxidants for culture. Appropriate amounts of mitophagy inducers can promote the removal of residual damaged mitochondrial debris from recipient cells, thereby optimizing intracellular mitochondrial homeostasis and improving the survival rate and functional integration efficiency of exogenous mitochondria. Mitochondria are susceptible to oxidative stress damage during isolation and introduction. Mitochondrial antioxidants can scavenge reactive oxygen species at the mitochondrial level, protecting the integrity of exogenous mitochondrial membrane structure and functional proteins, maintaining their electron transport chain activity and membrane potential, thereby prolonging the survival time of functional mitochondria and supporting their subsequent expansion.

[0020] This technical solution can further eliminate empty mitochondrial shells lacking mitochondrial DNA by inducing mitophagy, providing a purer cell factory for preserving exogenous mitochondria. On the other hand, the growth efficiency of hybrid cells determines the amplification efficiency of exogenous mitochondria. This technical solution optimizes the culture conditions of hybrid cells, thereby improving their proliferation efficiency and achieving higher amplification efficiency. Through the above treatment (ρ... 0 (Chemicalization → Autophagy Purification → Post-Introduction Protection) The mitochondrial storage system constructed in this invention achieves efficient colonization, functional maintenance, and continuous expansion of exogenous mitochondria.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) Significantly improves the uptake efficiency of exogenous mitochondria By analyzing ρ 0 Cellular autophagy induction or depolarization treatment effectively cleared non-functional mitochondrial shell structures from the cell, eliminating physical space occupation and metabolic competition. This resulted in significantly higher uptake efficiency of exogenous mitochondria in the treated recipient cells compared to conventional p-cell treatment. 0 Cells. Experiments have shown that the uptake rate of exogenous mitochondria in recipient cells was effectively increased after two-step treatment (see Experimental Example 3).

[0022] (2) Achieving long-term in vitro storage and unlimited expansion of mitochondria The mitochondrial storage system of this invention is essentially a living cell factory. Engineered cells, after the introduction of exogenous mitochondria, can proliferate and be passaged like ordinary immortalized cell lines. With each division, the exogenous mitochondria within the cells also replicate and expand. Compared to existing storage solutions or PBS buffers that can only preserve exogenous mitochondria for short periods, this invention achieves long-term storage and unlimited expansion. Engineered cells can be cryopreserved in liquid nitrogen, and after thawing, they retain the function and expansion capacity of the exogenous mitochondria, enabling long-term use after a single isolation. With cell passage, the number of exogenous mitochondria increases exponentially, solving the problems of limited mitochondrial source and insufficient yield, and greatly reducing the cost of mitochondrial-related research and clinical applications.

[0023] (3) Maintain the high activity and functional integrity of exogenous mitochondria This invention introduces mitophagy inducers and / or antioxidants during the culture phase (see Experimental Example 4), providing synergistic protective effects of antioxidant protection and homeostasis regulation. Mitochondrial antioxidants (such as MitoQ, Mito-TEMPO, MitoVit-E, etc.) can target and scavenge reactive oxygen species at the mitochondrial level, protecting mitochondrial membrane potential and electron transport chain function, and maintaining the ATP synthesis capacity of stored mitochondria at a high level. Moderate autophagy induction helps to clear residual damaged mitochondrial debris from the cell itself, maintaining the homogeneity and high quality of the exogenous mitochondrial population.

[0024] (4) The preparation method is simple and has wide applicability. The preparation method of this invention does not rely on complex equipment and can be completed through conventional cell culture, drug treatment, and centrifugation, making it easy to standardize and scale up production. Various common cell lines (such as HeLa, 143B, A549, etc.) can be used as recipient cells, and platelets or other readily available cells can also be used as the mitochondrial source, offering high flexibility. The prepared mitochondrial storage system can be directly used in cell therapy model studies of mitochondrial defect-related diseases, or as a standardized tool for mitochondrial function research.

[0025] (5) Provides a brand-new platform for mitochondrial-related research and applications This invention is the first to combine a two-step purification of recipient cells with post-transplant autophagy / antioxidant culture, constructing a mitochondrial storage system with both storage and amplification functions. This technology platform not only solves the two major challenges of maintaining the activity and insufficient yield of ex vivo mitochondria, but also provides a novel platform for constructing cell models for mitochondrial transplantation therapy, screening and evaluating the toxicity of mitochondrial drugs, studying the mechanisms of mitochondrial-related diseases, and pre-validating personalized mitochondrial therapy regimens in vitro.

[0026] In summary, compared with existing technologies, the mitochondrial storage system constructed in this invention has significant advantages in terms of storage time, mitochondrial amplification capacity, exogenous mitochondrial uptake efficiency, mitochondrial function maintenance, and sampling convenience, providing a practical solution for the long-term preservation and large-scale application of mitochondria. More specifically, existing storage solutions or PBS buffers can only maintain mitochondrial activity for several hours to several days, and red blood cell preservation methods can extend this to several days or weeks, while this invention achieves long-term storage for months or more through liquid nitrogen cryopreservation. Existing technologies cannot achieve in vitro mitochondrial amplification, and the number of mitochondria gradually decreases with storage time; this invention introduces mitochondria into proliferating engineered cells, allowing mitochondria to proliferate synchronously with cell division, achieving sustainable mitochondrial production. Existing storage solutions lack antioxidant protection, leading to rapid decline in mitochondrial function; this invention effectively protects mitochondrial membrane potential and electron transport chain function by adding mitochondrial antioxidants and autophagy inducers. Existing technologies require repeated isolation of mitochondria from organisms; this invention allows for unlimited use after a single isolation, and cryopreserved engineered cells can be thawed and used at any time without repeated isolation operations. Attached Figure Description

[0027] Figure 1 For ρ in Experimental Example 1 0 Microscopic images comparing a cell (left) with a normal cell (right).

[0028] Figure 2 The normal HeLa cells from Experiment 1 and the HeLa-derived p-cells obtained by induction with ethionide bromide were compared. 0 Statistical plot of mtDNA copy number in cells (mean±SD, n=4).

[0029] Figure 3 For Experiment 2, different concentrations of antimycin A induced ρ 0 Statistical graph of mitochondrial numbers in cells at different induction times (mean±SD, n=4).

[0030] Figure 4 For Experiment 2, different concentrations of antimycin A induced ρ 0 Statistical graph of cell survival rate at different induction times (mean±SD, n=4).

[0031] Figure 5 For ρ in Experimental Example 2 0 Cell (A) and p after being cleared by mitochondrial autophagy 0 Contrast micrograph of cell (B).

[0032] Figure 6The graph shows the efficiency of exogenous mitochondria entering recipient cells after autophagy-induced mitochondrial clearance in Experiment 3 (mean ± SD, n = 3; A represents the efficiency of ρ cells treated with autophagy inducer). 0 The cell is the recipient cell; B is the cell using p 0 (The cell is the recipient cell).

[0033] Figure 7 The graph shows the proliferation efficiency of hybrid cells in Experiment 4 under different culture conditions (mean±SD, n=4).

[0034] Figure 8 The statistical graph shows the mitochondrial DNA levels in hybrid cells under different culture conditions in Experiment Example 4 (mean±SD, n=4). Detailed Implementation

[0035] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0036] The general process for establishing a mitochondrial storage system is as follows: (1) ρ 0 Establishment of cell lines Selected cell lines (HeLa, 4T1, HepG2, 143B, A549, etc.) were cultured in basal medium to maintain their normal growth. A final concentration of 1-1 × 10⁻⁶ cells was added to the medium. 5 ng / mL of mtDNA decombinant (mitochondrial DNA decombinant) was used to prepare the first-conditioning medium, and incubated for 1-2400 h to obtain p. 0 Cells. The first conditioned medium is formed by adding a mitochondrial DNA decombinant to the basal medium, which is the standard medium for the corresponding cell line. Mitochondrial DNA decombinants include, but are not limited to, ethidium bromide (CAS No. 1239-45-8), ammonium chloride (CAS No. 12125-02-9), chloramphenicol (CAS No. 56-75-7), 4',6-diamidinyl-2-phenylindole (CAS No. 28718-90-3), and FCCP (CAS No. 370-86-5, trifluoromethoxyphenylhydrazone carbonyl cyanide), etc.

[0037] (2) Removal of functionally impaired mitochondrial shells that do not contain mtDNA Recipient cells treated with the mtDNA removal agent still retain functionally impaired mitochondria that do not contain mtDNA. Cells are cultured and screened using a second-conditioning medium for 12-96 hours (preferably 24-72 hours, more preferably 48-72 hours). After this culture process, p-cells with removed damaged mitochondria are obtained. 0 Cells. Further removal of residual mitochondria from recipient cells. The second conditioned medium includes basal medium and supplementary components. The supplementary components are mitophagy inducers or mitochondrial depolarizers. The basal medium is the standard medium for the corresponding cell line. Mitophagy inducers (first mitophagy inducers) include MTK458 (CAS No. 2499962-58-0), rapamycin (CAS No. 53123-88-9), niclosamide (CAS No. 50-65-7), urolithiasis A (CAS No. 1143-70-0), MFI8 (CAS No. 694488-83-0), CCCP (CAS No. 555-60-2, mitochondrial oxidative phosphorylation uncoupling agent), ATP (CAS No. 9000-83-3, adenosine triphosphate), MFI8 (CAS No.: 694488-83-0), antimycin A (CAS No. 1397-94-0), etc. Mitochondrial depolarizers include ddC (CAS No. 7481-89-2, 2', 3'-dideoxycytidine), antimycin A (CAS No. 1397-94-0), FCCP (CAS No. 370-86-5, trifluoromethoxyphenylhydrazone carbonyl cyanide), valinemycin (CAS No. 2001-95-8), oligomycin (CAS No. 1404-19-9), and nigrain (CAS No. 28380-24-7), etc. The concentration of mitophagy inducers or mitochondrial depolarizers in the basal culture medium is 50-200 ng / mL.

[0038] (3) To ρ 0 Mitochondria introduced into cells Mitochondria can be derived from platelets, cells, or tissues. When using cells as the source, usable cell lines include, but are not limited to, THP-1 cells and HeLa cells.

[0039] For cell and tissue samples, the first step is to prepare a single-cell suspension. The cell or tissue single-cell suspension is centrifuged at 100-2000g for 1-20 minutes at 4℃-25℃. After centrifugation, the supernatant is removed to obtain a cell pellet. The cell pellet is resuspended in 1-100 times the volume of cell lysis buffer, and then homogenized using an electric or manual homogenizer to obtain a cell homogenization product. The homogenization is then performed by filtering through a 40-100μm filter 2-5 times and then through a 10-40μm filter 2-5 times to obtain a filtered cell homogenization product (enriched with mitochondria). The filtered cell homogenization product (1×10⁻⁶) is then added to the cells obtained in (1). 5 1×10 7 One recipient cell; 10-500 μg of cell homogenate. Centrifuge at 100-5000 g for 10-20 min, and the precipitate is the cell line infused with exogenous mitochondria.

[0040] For platelet samples, cells were homogenized directly using an electric or manual homogenizer to obtain a homogenized cell product. The homogenized cell product was then filtered through a 40-100 μm filter 2-5 times, followed by a 10-40 μm filter 2-5 times, to obtain a filtered homogenized cell product. The filtered homogenized cell product (1×10⁻⁶) was then added to the cells obtained in (1). 5 1×10 7 One recipient cell; 10-500 μg of cell homogenate), centrifuged at 100-5000g for 5-30 min, the precipitate is the cell line infused with exogenous mitochondria.

[0041] (4) Screening and culture of hybrid cell lines The p-type mitochondrial cells were introduced. 0Cells (the aforementioned cell lines infused with exogenous mitochondria) require further conditioned medium selection to obtain hybrid cell lines that can stably proliferate and be passaged. The aforementioned cell lines with exogenous mitochondria are cultured in a third conditioned medium (without uracil) for 1-240 hours to obtain hybrid cell lines that can stably proliferate and be passaged. At the end of the culture, donor mitochondria expanded 2-2000 times. The third conditioned medium includes a basal medium and supplementary components, including mitophagy inducers and mitochondrial antioxidants, or mitochondrial antioxidants alone. The basal medium is the standard medium for the corresponding cell lines without uracil. Secondary mitophagy inducers include ATP (CAS No. 9000-83-3, adenosine triphosphate), CCCP (CAS No. 555-60-2, mitochondrial oxidative phosphorylation uncoupling agent), rapamycin (CAS No. 53123-88-9), AICAR (CAS No. 2627-69-2), Mdivi-1 (CAS No. 338967-87-6), and Urolithin A (CAS No. 1143-70-0); mitochondrial antioxidants include CoQ10 (CAS No. 303-98-0), α-tocopherol (CAS No. 10191-41-0), ascorbic acid (CAS No. 50-81-7), MitoQ (CAS No. 845959-50-4), resveratrol (CAS No. 501-36-0), and Mito-TEMPO (CAS No. 10191-41-0). Mitovit-E (Mito-Vitamin E, CAS No. 439146-24-0; see reference: Anuradha Dhanasekaran; Supplementation of Endothelial Cells with Mitochondria-targeted Antioxidants Inhibit Peroxide-induced Mitochondrial Iron Uptake; Oxidative Damage, and Apoptosis, Volume 279, Issue 36, 3 September 2004, Pages 37575-37587; its structural formula is shown in fig. 1 of that reference). The concentration of the second mitophagy inducer in the third-condition medium is 10-200 ng / mL (optional range is 10-500 ng / mL); the concentration of the mitochondrial antioxidant in the third-condition medium is 10-500 nM (optional range is 10-1000 ng / mL).

[0042] Example 1 More specifically, the overall process flow will be demonstrated using the HeLa cell line as an example: HeLa cell lines (cervical cancer cells) were cultured in basal medium (HeLa / 143B basal medium was DMEM high-glucose medium containing 10% FBS; A549 cells basal medium was F-12K medium containing 10% FBS) to maintain their normal growth state; then, ethidium bromide was added to the medium to a final concentration of 100 ng / mL (to form the first conditioned medium), and the cells were incubated for 7 days to obtain HeLa-ρ 0 cell.

[0043] Subsequently, cells were cultured for 24 hours in a second-conditioning medium containing 10 ng / mL rapamycin (rapamycin was added to the basal medium to form the rapamycin), to further remove damaged mitochondria from the recipient cells. After culture, HeLa-ρ cells with damaged mitochondria removed were obtained. 0 cell.

[0044] THP-1 cells (human monocytic leukemia cells) were collected and centrifuged at 800g for 20 min at 25°C. The supernatant was discarded after centrifugation. Cells were then treated with 100 times the volume of cell pellet in cell lysis buffer (the cell lysis buffer formulation was: 210 mM mannitol, 70 mM sucrose, 5 mM HEPES, 1 mM EDTA, pH 7.4) at 4°C for 20 min, and the cells were resuspended to form a cell suspension. After treatment, the cell suspension was homogenized 30 times at 4°C using a homogenizer to obtain a homogenized cell product. This homogenized product was then filtered through a 100 μm filter 5 times and a 40 μm filter 5 times to obtain the filtered homogenized cell product. This homogenized product was then transferred to HeLa-ρ, a culture medium to remove damaged mitochondria. 0 The filtered homogenized cell product was added to the cells. The filtered homogenized cell product was diluted with basal medium to a protein concentration of 50 μg / mL, and HeLa-ρ protein with damaged mitochondria removed was added. 0 The cell concentration was adjusted to 1×10 6 Cells / mL. The homogenized product dilution was added to HeLa-ρ at a 1:1 volume ratio. 0 Mixed in cell suspension (i.e., 1×10) 6 Each cell corresponds to 50 μg of cell homogenate. Centrifuge at 2000g for 10 min, and the precipitate is the cell line infused with exogenous mitochondria.

[0045] Subsequently, the cells were cultured for 48 hours using cell culture medium supplemented with 100 ng / mL MitoQ (100 ng / mL MitoQ in DMEM high glucose medium containing 10% FBS, third condition medium). After the culture was completed, dead cells were removed to obtain hybrid cells that could be stably inherited.

[0046] Example 2 The overall process of this embodiment is basically the same as that of Embodiment 1. The differences are in the type of cell line, the selection of reagents such as mitochondrial autophagy inducers, and some specific technical parameters, as follows: The 143B cell line (human sarcoma cells) was cultured in basal medium (DMEM high-glucose medium containing 10% FBS) to maintain its normal growth state; then chloramphenicol was added to the medium to a final concentration of 100 μg / mL, and the cells were incubated for 14 days to obtain 143B-ρ. 0 cell.

[0047] Subsequently, the cells were cultured in a medium containing 100 ng / mL ATP for 24 h to further remove damaged mitochondria. After the culture was completed, 143B-ρ cells with damaged mitochondria removed were obtained. 0 cell.

[0048] HeLa cells were collected and centrifuged at 800 g for 20 min at 25°C. The supernatant was discarded after centrifugation. Cells were treated with 10 times the volume of cell pellet in cell lysis buffer at 25°C for 10 min. After treatment, the cell suspension was homogenized using an electric homogenizer at 4°C for 5 min. The homogenate was then filtered twice through a 40 μm filter and twice through a 10 μm filter. Finally, the cells were transferred to 143B-ρ... 0 Add filtered cell homogenate (to 1×10⁻⁶ cells) to the cells 6 143B-ρ 0 Add 50 μg of filtered homogenized cell product to the cells, centrifuge at 3000g for 5 min, and the precipitate is the cell line introduced with exogenous mitochondria; then use cell culture medium supplemented with 100 ng / mL Mito-TEMPO (followed by DMEM medium containing 10% FBS and supplemented with 100 ng / mL Mito-TEMPO) for 72 h for selection and culture, and remove dead cells after culture to obtain hybrid cells that can be stably inherited.

[0049] Example 3 The overall process of this embodiment is basically the same as that of Embodiment 1. The differences are in the type of cell line, the selection of reagents such as mitochondrial autophagy inducers, and some specific technical parameters, as follows: The A549 cell line (human non-small cell lung cancer cell line) was cultured in basal medium (F-12K medium containing 10% FBS) to maintain its normal growth status; then ammonium chloride was added to the medium to a final concentration of 100 ng / mL, and the cells were incubated for 48 h to obtain A549-ρ. 0 Cells were then cultured for 24 hours in a medium containing 100 ng / mL ATP to further remove damaged mitochondria from the recipient cells.

[0050] HeLa cells were collected and centrifuged at 800 g for 20 min at 20 °C. The supernatant was discarded after centrifugation. Cells were treated with 20 times the volume of cell pellet in cell lysis buffer at 20 °C for 15 min. After treatment, the cell suspension was homogenized using an electric homogenizer at 4 °C for 10 min. The homogenate was then filtered through a 70 μm sieve five times and through a 20 μm sieve twice. Finally, the cells were transferred to A549-ρ... 0 Add filtered cell homogenate (to 1×10⁻⁶ cells) to the cells 6 A549-ρ 0 Add 50 μg of filtered homogenized cell product to the cells, centrifuge at 2500g for 10 min, and the precipitate is the cell line introduced with exogenous mitochondria; then use cell culture medium (F-12K medium containing 10% FBS) supplemented with 100 ng / mL Urolithin A and 50 ng / mL Mito-Vitamin E (Mito-Vitamin E is a derivative of ordinary vitamin E coupled with triphenylphosphine cation, which can specifically target into the mitochondria along the potential gradient to exert antioxidant effects compared to ordinary vitamin E) for 72 h for selection and culture. After the culture is completed, remove dead cells to obtain hybrid cells that can be stably inherited.

[0051] Example 4 The overall process of this embodiment is basically the same as that of Embodiment 1. The differences are in the type of cell line, the selection of reagents such as mitochondrial autophagy inducers, and some specific technical parameters, as follows: The 143B cell line was cultured in basal medium (MEM medium containing 10% FBS) to maintain its normal growth state; then, FCCP was added to the medium to a final concentration of 100 ng / mL, and the cells were incubated for 24 h to obtain 143B-ρ. 0 Cells were then cultured for 48 hours in medium containing 100 ng / mL MFI8 to further remove damaged mitochondria from the recipient cells.

[0052] HeLa cells were collected and centrifuged at 800 g for 20 min at 25°C. The supernatant was discarded after centrifugation. Cells were treated with 10 times the volume of cell pellet in cell lysis buffer at 25°C for 10 min. After treatment, the cell suspension was homogenized using an electric homogenizer at 4°C for 5 min. The cells were then homogenized twice through a 40 μm filter and twice through a 10 μm filter. Finally, the cells were transferred to 143B-ρ... 0 Add filtered cell homogenate (to 1×10⁻⁶ cells) to the cells 6 143B-ρ 0 Add 50 μg to the cells, centrifuge at 3000g for 5 min, and the precipitate is the cell line introduced with exogenous mitochondria; then use cell culture medium (DMEM medium containing 10% FBS) with 100 ng / mL α-tocopherol and 100 ng / mL ascorbic acid to select and culture for 72 h, and remove dead cells after culture to obtain hybrid cells that can be stably inherited.

[0053] Experimental Example 1: ρ 0 Construction and validation of cell lines HeLa cells were cultured in basal medium to maintain normal growth. Ethylene bromide, an mtDNA decomposition agent, was added to the medium to a final concentration of 100 ng / mL (forming the first conditioned medium), and the cells were incubated for 14 days. After incubation, cells were collected, fixed with glutaraldehyde, and observed under a transmission electron microscope. Figure 1 As can be seen, compared with normal cells, ρ 0 The morphology of mitochondria in the cells underwent significant changes, with the inner crest disappearing and the cells swelling and rupturing. Cells were collected, lysed, and mitochondria were collected. Mitochondrial DNA was then collected using a kit, and the intracellular mitochondrial DNA copy number was detected. ρ... 0 The number of mitochondrial DNA copies in cells decreased significantly. Figure 2 This indicates that this method can obtain cell lines without mitochondria.

[0054] Experimental Example 2: Study on Deep Mitochondrial Clearance Using Mitochondrial Autophagy Inducers ρ was prepared according to the method in Experimental Example 1. 0 Cells, and then using these p 0 Cells were used in this experimental study. Medium supplemented with different concentrations of antimycin A (second-conditioning medium) was used to study ρ... 0 Cells were cultured for a period of time, and p-values ​​were statistically analyzed. 0 The number of mitochondria in cells at different induction times, and p 0 Cell survival rate at different induction times.

[0055] To obtain recipient cells with cleared autologous mitochondria, cells were continuously cultured in a medium containing a mitophagy inducer. Careful selection of the inducer concentration and induction time was necessary. Too low a concentration would not achieve deep mitochondrial clearance, while too high a concentration would lead to cell death. Through selection of culture time and concentration, it was found that induction concentrations of 100 ng / ml, 200 ng / ml, and 500 ng / ml all achieved mitochondrial clearance. Figure 3 However, using concentrations of 200 ng / ml and 500 ng / ml resulted in cell death. Figure 4 Therefore, the concentration of the inducer needs to be carefully adjusted to achieve the ideal mitochondrial clearance effect, with 100 ng / ml being optimal. This effectively clears mitochondria (achieving almost 100% mitochondrial removal rate after more than 40 hours of culture) while having almost no impact on cell viability.

[0056] After screening the induction conditions, the rhodopsin was continuously cultured in a medium containing 100 ng / mL antimycin A. 0 After 48 hours of incubation, cells were observed to be different from ordinary ρ cells. 0 Compared to the target cells, the mitochondrial structure in the recipient cells was largely eliminated. This indicates that this method can successfully disrupt the mitochondrial double membrane structure, thereby obtaining recipient cells that contain neither mitochondrial DNA nor their own mitochondrial double membrane structure. Figure 5 ).

[0057] Experimental Example 3: Study on the efficiency of exogenous mitochondria entering recipient cells Mitochondria were extracted from the HL-60 cell line (human promyelocytic leukemia cells), and the filtered cell homogenate was enriched with HL-60 cell line mitochondria. The filtered cell homogenate was labeled using Mito Tracker Red CMX Ros before being mixed with recipient cells.

[0058] HL-60 cell line (human promyelocytic leukemia cells) was collected and centrifuged at 800g for 20 min at 25°C. The supernatant was discarded after centrifugation. Cells were then treated with 100 times the volume of cell pellet in cell lysis buffer (210 mM mannitol, 70 mM sucrose, 5 mM HEPES, 1 mM EDTA, pH 7.4) at 4°C for 20 min, and the cells were resuspended to form a cell suspension. After treatment, the cell suspension was homogenized 30 times at 4°C to obtain a homogenized product. Subsequently, the homogenized product was filtered through a 100 μm filter and a 40 μm filter five times each to obtain a homogenized product rich in HL-60 cell line mitochondria. The filtered homogenized product was incubated with Mito Tracker Red CMX Ros staining solution (working concentration 10 μm) at 37°C in the dark for 30 min. The cell pellet was then resuspended in PBS and centrifuged three times at 4°C for 5 min. Prepare labeled mitochondrial suspensions for later use.

[0059] The aforementioned receptor cells include p1 0 Cells and phoenix cells treated with autophagy inducers 0 Cells (removal of damaged mitochondria) 0 (Cells). The cell preparation methods for the two methods described above are described in Experiment 1 and Experiment 2. The autophagy inducer used was 100 ng / mL antimycin A. When Mito Tracker Red CMX Ros-labeled mitochondria and recipient cells were mixed, the concentrations (based on protein concentration) of Mito Tracker Red CMX Ros-labeled mitochondria were 6.25, 12.5, 25, and 50 μg / mL, respectively, and the concentration of recipient cells was 1 × 10⁻⁶. 6 The mitochondrial suspension was at a density of 1:1 (number of cells / mL) and the volume ratio of mitochondrial suspension to recipient cell suspension was 1:1. After centrifugation, the precipitate was collected to obtain the cell line infused with exogenous mitochondria. The cell lines infused with exogenous mitochondria were analyzed, and the percentage of cells containing the label (mean ± SD) was calculated.

[0060] For detailed experimental results, please refer to Figure 6 Mitochondria derived from the HL-60 cell line (human promyelocytic leukemia cells) were labeled, and then different concentrations of labeled mitochondrial suspensions were mixed with untreated p-cells containing autophagy inducers. 0 The cells were mixed with recipient cells treated with an autophagy inducer. After the storage system was established, flow cytometry was used to statistically analyze the fluorescence intensity of mitochondrial markers in the cells. The results showed that recipient cells treated with a mitophagy inducer could more efficiently absorb exogenous mitochondria, forming a hybrid cell line.

[0061] Experimental Example 4: p-type mitochondria after introduction of exogenous mitochondria 0 Cell proliferation efficiency study The survival efficiency of exogenous mitochondria after introduction into cells is affected by culture conditions. Mitochondrial antioxidants can reduce mitochondrial ROS levels, thereby preventing mitochondrial oxidative stress, maintaining mitochondrial homeostasis, improving cell proliferation efficiency, and simultaneously increasing mitochondrial division efficiency to obtain a greater number of mitochondria. Different concentrations of exogenous mitochondria were incubated with hybrid cells under different culture conditions for 72 hours. After culture, cell proliferation efficiency was detected and statistically analyzed. The specific procedure was as follows: mitochondria from the HL-60 cell line (filtered homogenized cell product) were obtained using the method in Example 3. ρm cells with damaged mitochondria removed were obtained using the method in Example 3. 0 Cells (HeLa cell line). Mitochondria from the HL-60 cell line were suspended in mitochondrial suspensions at concentrations of 6.25, 12.5, 25, 50, 100, and 200 μg / mL and then mixed with 1×10⁻⁶ cells. 6 The recipient cell suspension was mixed at a concentration of mitochondrial cells / mL, with a volume ratio of 1:1. After centrifugation, the pellet was collected to obtain the cell line infused with exogenous mitochondria. This cell line was then cultured for 72 hours in media containing and without oxidants. The oxidant-free medium was DMEM high-glucose medium (containing 10% FBS), while the oxidant-containing medium consisted of 100 ng / mL MitoQ antioxidant added to the basal medium.

[0062] The results showed that cells in culture medium supplemented with antioxidants exhibited faster proliferation efficiency. Figure 7 Subsequently, the number of mitochondria after 72 hours of culture was statistically analyzed, showing that cells cultured under antioxidant conditions yielded more mitochondria. Figure 8 ).

[0063] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing a mitochondrial storage system, characterized in that, This includes the processing of recipient cells, the acquisition of mitochondria, and the steps of mitochondrial introduction and culture. Treatment of recipient cells: Recipient cells were cultured using conditioned medium to obtain p-cells. 0 Cells; then cultured in a second conditioned medium. 0 Cells, obtaining p-type cells with damaged mitochondria removed 0 cell; The first conditioned medium contains a mitochondrial DNA decomposition agent; the second conditioned medium contains a first mitochondrial autophagy inducer or a mitochondrial depolarizer. Mitochondrial acquisition: Extracting mitochondria from cells or tissues; Mitochondrial introduction and culture: Introducing mitochondria with p-type mitochondria after removing damaged mitochondria. 0 Cells were mixed and centrifuged to obtain cells with introduced exogenous mitochondria; Cells in which exogenous mitochondria have been introduced were cultured using a third-conditioning medium to obtain a mitochondrial storage system; the third-conditioning medium contained a second mitophagy inducer and / or mitochondrial antioxidant.

2. The method for preparing a mitochondrial storage system according to claim 1, characterized in that, In the recipient cell treatment step, the mitochondrial DNA decomposition agent includes at least one of ethidium bromide, ammonium chloride, chloramphenicol, 4',6-diamidinyl-2-phenylindole, and FCCP; The first mitophagy inducer includes at least one of MTK458, rapamycin, niclosamide, urolithiasis A, MFI8, CCCP, ATP, MFI8, and antimycin A; The mitochondrial depolarizer includes at least one of ddC, antimycin A, FCCP, valproic acid, oligomycin, and nigrain.

3. The method for preparing a mitochondrial storage system according to claim 2, characterized in that, In the recipient cell treatment step, the concentration of mitochondrial DNA decomposition agent in the first conditioned medium was 1-1 × 10⁻⁶. 5 ng / mL; the concentration of the first mitophagy inducer or mitochondrial depolarizer in the second conditioned medium is 10 ng / mL-1000 ng / mL; Preferably, the content of the mitochondrial DNA decomposition agent is 100 ng / mL, and the content of the first mitophagy inducer or mitochondrial depolarizer is 100 ng / mL. The recipient cells were cultured in the first conditioned medium for 1-2400 hours; the ρ cells were cultured in the second conditioned medium. 0 The cell duration is 12-96 hours; Preferably, the recipient cells are cultured in the first-conditioning medium for 7-14 days; p is cultured in the second-conditioning medium. 0 The cell duration is 48-72 hours.

4. The method for preparing a mitochondrial storage system according to claim 1, characterized in that, In the mitochondrial introduction and culture step, the second mitophagy inducer includes at least one of ATP, CCCP, rapamycin, AICAR, Mdivi-1, and urolithiasis A; The mitochondrial antioxidants include CoQ10, α-tocopherol, ascorbic acid, MitoQ, resveratrol, Mito-TEMPO, and MitoVit-E.

5. A method for preparing a mitochondrial storage system according to claim 4, characterized in that, In the mitochondrial introduction and culture step, the ratio of mitochondria to cells was 1 × 10⁻⁶. 5 -1×10 7 One cell; 10-500 μg of mitochondria as protein content.

6. A method for preparing a mitochondrial storage system according to claim 5, characterized in that, In the mitochondrial introduction and culture steps, the concentration of the second mitophagy inducer in the third conditioned medium was 10-500 ng / mL; the concentration of the mitochondrial antioxidant in the third conditioned medium was 10-1000 ng / mL; and the culture time of the cells with introduced exogenous mitochondria in the third conditioned medium was 1-240 h.

7. A method for preparing a mitochondrial storage system according to claim 6, characterized in that, In the mitochondrial introduction and culture step, mitochondria are combined with p-type mitochondria after the removal of damaged mitochondria. 0 The cells were mixed and centrifuged at 100-5000g for 5-30 minutes to obtain cells with introduced exogenous mitochondria.

8. The method for preparing a mitochondrial storage system according to claim 1, characterized in that: In the mitochondrial acquisition process, mitochondria are derived from cells or tissues, including platelets and other cells; other cells include HeLa cells, 143B cells, and A549 cells. When extracting mitochondria from tissues and other cells, first obtain a single-cell suspension of cells or tissues, then perform lysis and homogenization, and finally filter the suspension through a 40-100μm filter sieve and a 10-40μm filter sieve to obtain mitochondria. When extracting mitochondria from platelets, the platelets are first homogenized, and then filtered sequentially through a 40-100 μm filter sieve and a 10-40 μm filter sieve to obtain mitochondria.

9. A mitochondrial storage system obtained by a method for preparing a mitochondrial storage system according to any one of claims 1-8.

10. The use of the mitochondrial storage system according to claim 9 in the preparation of a medicament for treating mitochondrial defect-related diseases.

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