Rapid and efficient extraction method of extracellular vesicles rich in mitochondria

Abstract

The invention discloses a rapid and efficient extraction method of extracellular vesicles rich in mitochondria. The extraction method comprises the following steps: (1) culturing mitochondrial donor cells in vitro; (2) treating the mitochondrial donor cells with cytochalasin D; (3) collecting the cells; (4) oscillating a cell suspension; according to the method disclosed by the invention, by combining cytochalasin D treatment with a subsequent gradient centrifugal separation step, a sample of extracellular vesicles rich in mitochondria can be efficiently extracted, meanwhile, the defects existing in a traditional mitochondrial transplantation technology are overcome, and a new thought and method are provided for clinical application of mitochondrial transplantation.

Description

Technical Field

[0001] The present invention relates to a method for rapidly and efficiently extracting extracellular vesicles rich in mitochondria, belonging to the technical field of mitochondrial extraction. Background Art

[0002] Mitochondria are important organelles in eukaryotic cells, and their abnormal functions are closely related to various diseases. As an emerging treatment strategy, mitochondrial transplantation involves transplanting functional mitochondria into damaged tissues or cells to replace or repair damaged mitochondria, thereby restoring cell function. However, current mitochondrial transplantation techniques mainly rely on extracting naked mitochondria from cells or tissues for transplantation, and this method has many defects: 1) Low activity: The activity of naked mitochondria rapidly decreases in the extracellular environment and it is difficult to maintain their functions for a long time. 2) Immune clearance: Naked mitochondria are easily recognized as foreign substances by the immune system and phagocytosed by immune cells such as macrophages, resulting in transplantation failure. 3) Difficult cell uptake: Naked mitochondria are difficult to efficiently enter recipient cells and exert their effects, limiting their therapeutic effects. Therefore, developing an alternative solution for mitochondrial extraction that can overcome the above defects and be used for transplantation is of great significance for improving the efficiency of mitochondrial transplantation and expanding its clinical application prospects. Summary of the Invention

[0003] The object of the present invention is: in view of the deficiencies of the prior art, the present invention provides a method for rapidly and efficiently extracting extracellular vesicles rich in mitochondria; the method of the present invention can efficiently extract samples of extracellular vesicles rich in mitochondria through treatment with cytochalasin D combined with subsequent gradient centrifugation separation steps, and at the same time overcomes the defects existing in traditional mitochondrial transplantation techniques, providing new ideas and methods for the clinical application of mitochondrial transplantation.

[0004] To achieve the above object, the present invention provides a method for extracting extracellular vesicles rich in mitochondria, comprising the following steps:

[0005] Step 1) Culturing mitochondrial donor cells in vitro;

[0006] Step 2) Treating the mitochondrial donor cells cultured in Step 1) with cytochalasin D;

[0007] Step 3) Collecting cell suspension: Gently scraping and collecting the cell suspension with a cell scraper;

[0008] Step 4) Oscillating the cell suspension: Placing the cell suspension in a vortex mixer to oscillate to separate mitochondrial vesicles from cells;

[0009] Step 5) Gradient centrifugation: Centrifuge at a low speed of 100 - 300 g to collect the supernatant; centrifuge the collected supernatant at a high speed of 1500 - 2500 g to collect the precipitate, namely, extracellular vesicles rich in mitochondria are extracted.

[0010] Preferably, the mitochondrial donor cells in step 1 include any one or more of cardiomyocytes, human umbilical vein endothelial cells, human umbilical cord-derived mesenchymal stem cells, human adipose tissue-derived mesenchymal stem cells, bone marrow mesenchymal stem cells, and placenta-derived mesenchymal stem cells.

[0011] Preferably, in step 2, the concentration of cytochalasin D is 0.8 - 1.2 μg / ml, the treatment time is 20 - 40 minutes, and the treatment conditions are: 37°C, 5% CO2.

[0012] Preferably, in step 4, the time for low-speed centrifugation is 5 - 10 minutes, and the number of times is 1 - 3 times.

[0013] Preferably, in step 4, the time for high-speed centrifugation is 15 - 25 minutes.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] (1) In the method of the present invention, cytochalasin D is used to treat mitochondrial donor cells. Cytochalasin D is an actin polymerization inhibitor that can disrupt the microfilament structure in the cytoskeleton and induce cytoskeleton remodeling, thereby promoting the formation and release of mitochondrial vesicles.

[0016] (2) The mitochondrial vesicles extracted by the present invention have a lipid bilayer structure, which can effectively prevent mitochondria from directly contacting the extracellular environment, thereby preventing mitochondria from being damaged in the extracellular environment; at the same time, this structure can also prevent mitochondria from being recognized and phagocytosed by the immune system; in addition, the lipid bilayer structure of mitochondrial vesicles enables them to fuse with the receptor cell membrane, and then efficiently deliver mitochondrial components into the receptor cells; compared with naked mitochondria, mitochondrial vesicles are more easily taken up by receptor cells and play functions within the cells, thus significantly improving the effect of mitochondrial transplantation.

[0017] (3) The present invention adopts a gradient centrifugation step, which can effectively remove cell debris and other impurities, and finally enrich and purify the supernatant rich in mitochondrial vesicles; the present invention not only improves the extraction efficiency of mitochondrial vesicles through gradient centrifugation, but also ensures the purity of the extract. Description of the Drawings

[0018] Figure 1 It is a schematic diagram of the extraction process of extracellular vesicles rich in mitochondria of the present invention;

[0019] Figure 2Video screenshots taken by a live cell imager prove the dynamic formation process of extracellular vesicles rich in mitochondria after cytochalasin D intervention;

[0020] Figure 3 Scanning electron microscope and transmission electron microscope images of extracellular vesicles rich in mitochondria extracted in the examples;

[0021] Figure 4 Flow cytometry was used to detect the number of functional mitochondria contained in the extracted vesicles, and MitoTracker DeepRed was used to label functional mitochondria;

[0022] Figure 5 Confocal detection was used to detect the ability of recipient cells to take up extracellular vesicles rich in mitochondria. Detailed implementation manners

[0023] To make the present invention more obvious and understandable, preferred embodiments are provided below in conjunction with the accompanying drawings for detailed description.

[0024] Embodiment

[0025] This embodiment provides a method for rapidly and efficiently extracting extracellular vesicles rich in mitochondria. In this embodiment, the extraction of mitochondria from H9C2 cardiomyocytes is taken as an example to list the implementation process of the method of the present invention. The cytochalasin D reagent used in this method was purchased from MedChemExpress (MCE) company (product number: HY-N6682). The extraction process is as Figure 1 shown, and the specific steps are as follows:

[0026] 1) In vitro culture of mitochondrial donor cells: Select a suitable cell line as the mitochondrial donor cell (including but not limited to cardiomyocytes, human umbilical vein endothelial cells, human umbilical cord-derived mesenchymal stem cells, human adipose-derived mesenchymal stem cells, etc.), and culture it in vitro until the density reaches 70%-90%.

[0027] 2) Cytochalasin D treatment: Treat the mitochondrial donor cells with 1 μg / ml cytochalasin D for 30 minutes (37°C, 5% CO2). A live cell imager was used to take videos to prove the dynamic formation process of extracellular vesicles rich in mitochondria after cytochalasin D intervention. The video screenshots are as Figure 2 shown. As can be seen from the figure, after cytochalasin D intervention, the cells shrink and produce many extracellular vesicles rich in mitochondria.

[0028] 3) Collect cells: Gently scrape and collect the cell suspension with a cell scraper.

[0029] 4) Shake the cell suspension: Place the collected cell suspension in a vortex mixer and shake it for 30 seconds to separate the mitochondrial vesicles from the cells.

[0030] 5) Gradient centrifugation: Centrifuge at a speed of 200 g for 5 minutes (4 °C), and collect the supernatant. Repeat once and collect the supernatant. Centrifuge the supernatant at a speed of 2000 g for 20 minutes (4 °C). The precipitate is the required mitochondrial-rich vesicles.

[0031] Confirm the presence of mitochondria in the above-extracted vesicles by scanning electron microscopy and transmission electron microscopy, as Figure 3 shown. It can be seen in the figure that the vesicles contain mitochondria, and their morphology is plump and complete, with dense and regularly arranged cristae, indicating that the extracted mitochondria are intact;

[0032] Detect the number of functional mitochondria contained in the extracted vesicles by flow cytometry. The results are as Figure 4 shown. MitoTracker DeepRed is used to label functional mitochondria in the figure. As can be seen from the figure, the highest proportion of functional mitochondria in the vesicles can reach 55.1%, indicating that the vesicles extracted by this method contain abundant functional mitochondria;

[0033] Detect the ability of receptor cells to uptake mitochondria-rich extracellular vesicles by confocal microscopy. The results are as Figure 5 shown. As can be seen from the figure, after co-incubating the mitochondria-rich extracellular vesicles (red) of donor cells with receptor cells for 12 hours, mitochondrial fluorescence co-localizes, indicating that the mitochondria-rich extracellular vesicles extracted by this method can be successfully uptaken by receptor cells and integrated into the mitochondrial network of receptor cells.

[0034] As described above, it is only a preferred embodiment of the present invention, and there is no restriction on the present invention in any form and substance. It should be pointed out that for those of ordinary skill in the art, without departing from the premise of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A method for extracting extracellular vesicles rich in mitochondria, characterized in that, Comprising the following steps: Step 1) Culturing mitochondrial donor cells in vitro; Step 2) Treating the mitochondrial donor cells cultured in Step 1) with cytochalasin D; Step 3) Collecting cell suspension: Gently scraping and collecting the cell suspension with a cell scraper; Step 4) Oscillating the cell suspension: Placing the cell suspension in a vortex for oscillation to separate mitochondrial vesicles from cells; Step 5) Gradient centrifugation: Centrifuging at a low speed of 100 - 300 g to collect the supernatant; Centrifuging the collected supernatant at a high speed of 1500 - 2500 g to collect the precipitate, namely, extracellular vesicles rich in mitochondria are extracted.

2. The extraction method according to claim 1, wherein The mitochondrial donor cells in Step 1) include any one or more of cardiomyocytes, human umbilical vein endothelial cells, human umbilical cord-derived mesenchymal stem cells, human adipose tissue-derived mesenchymal stem cells, bone marrow mesenchymal stem cells, and placenta-derived mesenchymal stem cells.

3. The extraction method according to claim 1, wherein In Step 2), the concentration of cytochalasin D is 0.8 - 1.2 μg / ml, the treatment time is 20 - 40 minutes, and the treatment conditions are: 37°C, 5% CO2.

4. The extraction method according to claim 1, characterized in that, In Step 4), the time for low-speed centrifugation is 5 - 10 minutes, and the number of times is 1 - 3 times.

5. The extraction method according to claim 1, characterized in that, In Step 4), the time for high-speed centrifugation is 15 - 25 minutes.

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