A method for storing isolated mitochondria in vitro

Abstract

The present invention relates to the field of biological technology. In order to solve the problems of poor in vitro preservation effect and short preservation time of mitochondria, an innovative method of using mitochondria-free cells as carriers to provide mitochondria with storage conditions closest to the intracellular environment to protect mitochondrial function and activity is used to solve the above problems. The present invention provides an in vitro storage method for isolated mitochondria, which mainly stores isolated mitochondria in receptor cells without mitochondria, greatly protects mitochondrial function and activity, and prolongs the in vitro preservation time of mitochondria. It can also more easily release mitochondria contained in red blood cells, without the need for complicated separation steps and separation reagents, thereby increasing the operability of the storage system.

Description

Technical Field

[0001] The invention relates to the field of biological technology, and in particular to an in vitro storage method for isolated mitochondria. Background Art

[0002] Mitochondria are involved in the occurrence and development of a variety of pathological and physiological processes, and the study of mitochondria has attracted more and more attention from researchers. Among them, maintaining the activity and function of mitochondria in vitro is a necessary condition for conducting mitochondrial research.

[0003] At present, the main means of preserving isolated mitochondria in clinical and experimental settings is mitochondrial storage solution or 0.01M PBS (Phosphate-Buffered Saline) buffer. These two storage methods mainly maintain the activity of isolated mitochondria by providing them with appropriate osmotic pressure and a neutral pH environment. However, since these two storage solutions are mainly composed of inorganic salts and lack antioxidant active substances, they have limited ability to protect the important enzyme activities in mitochondria and cannot meet the needs of long-term in vitro storage of mitochondria, limiting mitochondrial-related research and applications.

[0004] Existing technologies can achieve the introduction of mitochondria into nucleated cells containing complete organelle units, such as white blood cells, lymphocytes, etc. This type of technology mostly introduces mitochondria into cells with intact structure but impaired function. Its essential purpose is to repair the damaged mitochondrial function of the recipient cells through mitochondrial transplantation, and ultimately repair or enhance the function of the recipient cells. The purpose is not to preserve mitochondria in vitro. In addition, mitochondria perform biosynthesis and energy supply functions in target cells, and their respiratory pattern is regulated by the target cell environment, so that the function and type of mitochondria are reprogrammed in the recipient cells, losing the original characteristics of the mitochondria. This type of mitochondrial cell transplantation and preservation cannot meet the maintenance and protection of the original characteristics of the mitochondria. Summary of the invention

[0005] The present invention aims to solve the problems of poor in vitro preservation and short preservation time of mitochondria by innovatively using mitochondrial-free cells as carriers to provide mitochondria with storage conditions closest to the intracellular environment to protect mitochondrial function and activity.

[0006] The inventors found that for nucleated cells, if the recipient cell's own mitochondria are not cleared, the heterogeneous mitochondria will eventually be cleared by the cells, and the exogenous mitochondria cannot be preserved for a long time. At the same time, such nucleated cells contain complex cell components and organelles, which increases the difficulty of re-extracting mitochondria, and the purity of mitochondria is also reduced.

[0007] Specifically, the inventors used cells without mitochondria as carriers (preferably, mature red blood cells, which naturally lack nuclei and organelles, were selected as carriers), providing storage conditions for mitochondria that are closest to the intracellular environment, greatly protecting mitochondrial function and activity, and extending the in vitro storage time of mitochondria. At the same time, benefiting from the simple composition and structure of red blood cells, mitochondria, as a sensitive organelle, are not easily stimulated by intracellular components to undergo respiratory reprogramming. Moreover, the method for recollecting mitochondria in red blood cells is simple, that is, it is relatively convenient to release the mitochondria contained in red blood cells, and does not require complicated separation steps and separation reagents, which increases the operability of this storage system.

[0008] The embodiments of the present invention are implemented by the following technical solutions:

[0009] An in vitro storage method for isolated mitochondria mainly stores the isolated mitochondria in a recipient cell without mitochondria. Preferably, the recipient cell also does not have a nucleus, which is more conducive to the entry of exogenous cell bodies. Specifically, the method comprises the following steps:

[0010] S1. extracting red blood cells and mitochondria from the same or different subjects, and preparing red blood cell suspensions and mitochondrial suspensions, respectively;

[0011] S2. Introducing mitochondria into red blood cells: The mitochondrial suspension obtained in S1 is mixed with the red blood cell suspension, and the supernatant is removed by centrifugation to obtain a red blood cell precipitate containing mitochondria, and the cells are then dispersed in a red blood cell preservation solution.

[0012] Furthermore, the following steps are included:

[0013] 1.1 Red blood cell extraction

[0014] Mice (nude, Balb / C, C57BL / 6, Kunming mice) were anesthetized by intraperitoneal injection of 1-3 mL of 4%-10% anesthetic (sodium phenobarbital, sodium pentobarbital, sodium thiopental, ethyl carbamate, chloral hydrate), and 0.1-1.5 mL of peripheral blood was collected from the mice. The blood was centrifuged at 100-2000×g for 1-20 minutes at 4°C-25°C. After the centrifugation, the supernatant was removed to obtain the red blood cell pellet, and the red blood cells were resuspended in 1-1000 mL PBS for later use.

[0015] 1.2 Mitochondrial extraction

[0016] Mice (nude, Balb / C, C57BL / 6) were killed by cervical dislocation, and then mouse tissues (heart, muscle, spleen, kidney, liver) or peripheral blood leukocytes were isolated, and the tissues were digested with (0.05%-0.5%) pancreatic enzyme at 4℃-37℃ for 15-120 minutes. After mechanical grinding, the tissue was filtered into a single cell suspension using a 50-200 mesh cell sieve, and centrifuged at 100-2000×g for 1-20 minutes at 4℃-25℃. After centrifugation, the supernatant was removed to obtain a cell pellet. The cells were treated with 1-100 times the volume of the cell pellet and mitochondrial separation solution and mitochondrial lysis solution at 4℃-25℃ for 5-120 minutes, and then centrifuged at 100-2000g for 1-20 minutes at 4℃-25℃. After centrifugation, the supernatant was removed to obtain a mitochondrial pellet, and the mitochondria were resuspended in 1-1000mL PBS for use;

[0017] 1.3 Importing mitochondria into red blood cells

[0018] The mitochondrial suspension and red blood cell suspension obtained in 1.1 and 1.2 are mixed in a ratio of 20:1-1:20, and centrifuged at 100-2000g for 1-20 minutes at 4°C-25°C. After centrifugation, the supernatant is removed to obtain the red blood cell pellet containing mitochondria, and then the cells are dispersed in 1-100 mL of red blood cell preservation solution.

[0019] The technical solution of the embodiment of the present invention has at least the following advantages and beneficial effects:

[0020] In order to solve the problems of poor in vitro preservation and storage period of mitochondria, the present invention innovatively uses cells that naturally lack nuclei and organelles as carriers, preferably mature red blood cells, to provide storage conditions closest to the intracellular environment for mitochondria, greatly protecting mitochondrial function and activity, and extending the in vitro storage time of mitochondria. At the same time, benefiting from the simple composition and structure of red blood cells, mitochondria, as a sensitive organelle, are not easily stimulated by intracellular components to undergo respiratory mode reprogramming. Moreover, the method for recollecting mitochondria in red blood cells is simple, which can more easily release the mitochondria contained in the red blood cells, without the need for complicated separation steps and separation reagents, which increases the operability of this storage system. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0022] Figure 1A schematic diagram of the co-localization of red blood cells and mitochondria provided in Experimental Example 1 of the present invention (Mito Tracker: mitochondrial marker; DiO: cell membrane marker);

[0023] Figure 2 A schematic diagram of encapsulation efficiency at different mitochondria / red blood cell mixing ratios provided in Experimental Example 2 of the present invention;

[0024] Figure 3 This is a schematic diagram of the protective effect of red blood cells on mitochondrial activity provided in Experimental Example 3 of the present invention;

[0025] Figure 4 A schematic diagram of the repairing ability of mitochondria to damaged myocardial cells under different storage methods provided in Experimental Example 4 of the present invention; DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0027] Example 1

[0028] An in vitro storage method for isolated mitochondria comprises the following steps:

[0029] C57BL / 6 mice were anesthetized by intraperitoneal injection of 1.5 mL of 4% chloral hydrate, and 0.5 mL of peripheral blood was collected from the mice and centrifuged at 800 × g for 10 min at 4°C. After the centrifugation, the supernatant was removed to obtain the red blood cell pellet;

[0030] Then, the mouse heart tissue was separated and digested with 0.25% pancreatic enzyme at 37°C for 60 minutes. After mechanical grinding, the tissue was filtered into a single cell suspension using a 70-mesh cell sieve and centrifuged at 300×g for 20 minutes at 25°C. After the centrifugation, the supernatant was removed to obtain a cell pellet, and the red blood cells were resuspended in 1mL PBS for later use; the cells were treated with 10 times the volume of the cell pellet and mitochondrial separation solution and mitochondrial lysis solution at 4°C for 10 minutes, and then centrifuged at 800g for 5 minutes at 25°C. After the centrifugation, the supernatant was removed to obtain a mitochondrial pellet, and the mitochondria were resuspended in 1mL PBS for later use; the mitochondrial suspension and the red blood cell suspension were mixed in a 1:1 ratio, centrifuged at 800g for 20 minutes at 4°C, and the supernatant was removed after the centrifugation to obtain a red blood cell pellet containing mitochondria, and then the cells were dispersed in 1mL red blood cell preservation solution PBS.

[0031] Example 2

[0032] (2) Kunming mice were anesthetized by intraperitoneal injection of 1.5 mL of 5% sodium phenobarbital, and 1.5 mL of mouse peripheral blood was collected and centrifuged at 800 g for 5 minutes at 25°C. After the centrifugation, the supernatant was removed to obtain the red blood cell precipitate; then, the mouse heart tissue was isolated and digested with 0.20% pancreatic enzyme at 25°C for 120 minutes. After mechanical grinding, the tissue was filtered into a single cell suspension using a 40-mesh cell sieve and centrifuged at 800 g for 15 minutes at 25°C. The supernatant was removed after centrifugation to obtain a cell pellet, and the red blood cells were resuspended in 2 mL PBS for later use; the cells were treated with 10 times the volume of the cell pellet and mitochondrial separation solution at 25°C for 5 minutes, followed by centrifugation at 800 g for 20 minutes at 25°C. The supernatant was removed after centrifugation to obtain a mitochondrial pellet, and the mitochondria were resuspended in 5 mL PBS for later use; the mitochondrial suspension and the red blood cell suspension were mixed in a ratio of 2:1, and centrifuged at 800 g for 20 minutes at 25°C. The supernatant was removed after centrifugation to obtain a red blood cell pellet containing mitochondria, and the cells were then dispersed in 2 mL of red blood cell preservation solution PBS.

[0033] Example 3

[0034] Balb / C mice were anesthetized by intraperitoneal injection of 1 mL of 8% chloral hydrate, and 1 mL of peripheral blood was collected from the mice and centrifuged at 800 g for 10 minutes at 4°C. After the centrifugation, the supernatant was removed to obtain the red blood cell pellet; then the mouse heart tissue was separated and digested with 0.5% pancreatic enzyme at 37°C for 60 minutes. After mechanical grinding, the tissue was filtered into a single-cell suspension using a 70-mesh cell sieve and centrifuged at 400 g for 20 minutes at 4°C. The supernatant was removed after centrifugation to obtain a cell pellet, and the red blood cells were resuspended in 1 mL PBS for later use; the cells were treated with 10 times the volume of the cell pellet in mitochondrial separation solution and mitochondrial lysis solution at 25°C for 4 minutes, followed by centrifugation at 600 g for 20 minutes at 25°C. The supernatant was removed after centrifugation to obtain a mitochondrial pellet, and the mitochondria were resuspended in 10 mL PBS for later use; the mitochondrial suspension and the red blood cell suspension were mixed in a ratio of 1:5, and centrifuged at 800 g for 5 minutes at 20°C. The supernatant was removed after centrifugation to obtain a red blood cell pellet containing mitochondria, and the cells were then dispersed in 5 mL of red blood cell preservation solution PBS.

[0035] Example 4

[0036] C57BL / 6 mice were anesthetized by intraperitoneal injection of 0.5 mL of 10% sodium pentobarbital, and 0.5 mL of mouse peripheral blood was collected and centrifuged at 800 g for 10 minutes at 4°C. After the centrifugation, the supernatant was removed to obtain the red blood cell pellet; then the mouse heart tissue was separated and digested with 0.25% pancreatic enzyme at 37°C for 60 minutes. After mechanical grinding, the tissue was filtered into a single-cell suspension using a 700-mesh cell sieve and centrifuged at 300 g for 20 minutes at 25°C. The supernatant was removed after centrifugation to obtain a cell pellet, and the red blood cells were resuspended in 1 mL PBS for later use; the cells were treated with 10 times the volume of the cell pellet and mitochondrial separation solution at 4°C for 20 minutes, followed by centrifugation at 400 g for 25 minutes at 25°C. The supernatant was removed after centrifugation to obtain a mitochondrial pellet, and the mitochondria were resuspended in 10 mL PBS for later use; the mitochondrial suspension and the red blood cell suspension were mixed in a ratio of 5:1, and centrifuged at 800 g for 20 minutes at 4°C. The supernatant was removed after centrifugation to obtain a red blood cell pellet containing mitochondria, and the cells were then dispersed in 1 mL of red blood cell preservation solution Saline.

[0037] Experimental Example 1- Construction and verification of the mitochondrial red blood cell storage system

[0038] The red blood cell membrane was labeled with the cell membrane green fluorescent probe DiO, and the Mito RedCMX Ros was used to label mitochondria, and then the labeled mitochondrial suspension and red blood cell suspension were divided into the following categories according to protein content: number of cells = 80 μg: 10 × 10 6 The mixture was mixed in a ratio of , centrifuged at 800g for 20 minutes at 25°C, and the supernatant was removed after the centrifugation to obtain a red blood cell pellet containing mitochondria, and the cells were then dispersed in 2mL of red blood cell preservation solution. After the storage system was established, a small amount of sample was carefully added to a glass slide, and then the co-localization of mitochondria and red blood cells was analyzed under a fluorescence confocal microscope. The results showed that the fluorescence of the mitochondrial marker and the fluorescence of the red blood cell membrane marker were highly co-localized, indicating that the method of the present invention can successfully introduce mitochondria into red blood cells. The experimental results are shown in Figure 1 .

[0039] Experimental Example 2 - Encapsulation efficiency of mitochondrial red blood cell storage system

[0040] Using Mito Red CMX Ros was used to label mitochondria, and then 10 μg, 20 μg, 40 μg, 80 μg, 150 μg or 200 μg of labeled mitochondrial suspension was mixed with 10×10 6The red blood cells were mixed and centrifuged at 800g for 20 minutes at 25°C. After the centrifugation, the supernatant was removed to obtain the red blood cell pellet containing mitochondria, and then the cells were dispersed in 2mL of red blood cell preservation solution. After the storage system with different mixing ratios was established, the fluorescence intensity of mitochondrial markers in red blood cells was statistically analyzed by flow cytometry. The results showed that the mitochondrial suspension and red blood cell suspension were in accordance with the protein content: cell number = 200μg: 10×10 6 The highest mitochondrial encapsulation rate can be obtained by mixing the ratio of Figure 2 .

[0041] Experimental Example 3-Protective effect of red blood cells on mitochondrial activity

[0042] The mitochondria were stored in red blood cells, PBS or commercial mitochondrial storage solution, and then the mitochondrial ATP synthesis capacity and membrane potential levels were compared after being stored in vitro at 4°C for 24 hours using different storage methods. The results showed that compared with freshly extracted mitochondria (only transfer storage in the storage solution), the ATP content of mitochondria stored in red blood cells decreased less, while the ATP content of mitochondria stored in mitochondrial storage solution decreased significantly; the degree of decrease in mitochondrial membrane potential of mitochondria stored in red blood cells was significantly lower than that of the other two storage methods. In summary, red blood cell encapsulation has a good protective function on mitochondrial activity. Experimental results are shown in Figure 3 .

[0043] Experimental Example 4 - The repair ability of mitochondria on damaged myocardial cells under different storage methods

[0044] After establishing the isoproterenol ISO-induced cardiomyocyte injury model, mitochondria stored in vitro at 4°C for 24 hours using different storage methods were transplanted into cardiomyocytes for treatment. The results showed that cardiomyocytes transplanted with mitochondria stored in red blood cells showed higher ATP content and membrane potential, indicating that mitochondria stored in red blood cells still have transplantation therapeutic potential. The experimental results are shown in Figure 4 .

[0045] In summary, the present invention utilizes mitochondrial-free and nucleated red blood cells as carriers, providing storage conditions for mitochondria that are closest to the intracellular environment, greatly protecting mitochondrial function and activity, and extending the in vitro preservation time of mitochondria; at the same time, it also avoids the reprogramming of mitochondrial function and the elimination of heterogeneous mitochondria regulated by the cell nucleus, and is a stable means of preserving mitochondria in vitro.

[0046] In addition, the ability of PBS and mitochondrial storage solution to protect the activity of mitochondria is very limited. The activity of mitochondria decreased significantly after 24 hours of in vitro storage. However, mitochondria are stored in red blood cells and can be preserved in vitro for 35 to 42 days at (4±2)°C, which can provide a long-term protective environment for mitochondria and prolong the in vitro storage time of mitochondria.

[0047] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for storing isolated mitochondria in vitro, characterized in that: storing isolated mitochondria in recipient cells that do not have mitochondria; The recipient cell is a mature red blood cell in the blood without a cell nucleus; the isolated mitochondria are mitochondria with complete functions; Specifically, the steps include: S1. extracting red blood cells and mitochondria from the same or different subjects, and preparing red blood cell suspensions and mitochondrial suspensions, respectively; S2. Introducing mitochondria into red blood cells: The mitochondrial suspension obtained in S1 and the red blood cell suspension are mixed in a ratio of 20:1-1:20, and the supernatant is removed by centrifugation to obtain a red blood cell precipitate containing mitochondria, and the cells are then dispersed in a red blood cell preservation solution.

2. The in vitro storage method of isolated mitochondria according to claim 1, characterized in that: The isolated mitochondria are isolated mitochondria isolated from donor cells, and the donor cells and the recipient cells are obtained from the same or different subjects.

3. The in vitro storage method of isolated mitochondria according to claim 1, characterized in that: Step S1 includes S11. Red blood cell extraction: peripheral blood of the subject is collected and centrifuged for a period of time. After the centrifugation, the supernatant is removed to obtain the red blood cell precipitate, and the red blood cell suspension is obtained after resuspending.

4. The in vitro storage method of isolated mitochondria according to claim 1, characterized in that: Step S1 also includes S12. Mitochondrial extraction: taking the subject's tissue, digesting the tissue with pancreatic enzyme for a period of time, grinding and sieving the tissue to filter it into a single cell suspension; then centrifuging to obtain a cell precipitate, processing the cell precipitate, and then centrifuging to obtain a mitochondrial precipitate, and resuspending to obtain a mitochondrial suspension.

5. The in vitro storage method of isolated mitochondria according to claim 1, characterized in that: Step S1 also includes S13. Mitochondrial extraction: taking peripheral blood from a subject, and removing red blood cells using a red blood cell lysis solution; The cell pellet was centrifuged again to obtain a cell precipitate, the cell precipitate was treated, and then centrifuged to obtain a mitochondrial precipitate, which was then resuspended to obtain a mitochondrial suspension.

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