Kit for in-vitro extraction of cell mitochondria and method

Abstract

A kit for in-vitro extraction of cell mitochondria and a method. The kit comprises an extraction liquid A, an extraction liquid B, an extraction liquid C and a preservation liquid, the extraction liquid A comprising EDTA, sodium lactate and sodium chloride, the extraction liquid B being a heparin solution, the extraction liquid C comprising mannitol, EDTA, sodium lactate and sodium chloride, and the preservation liquid comprising mannitol, EDTA, sodium lactate, succinic acid, sodium pyruvate and sodium chloride. Using the extraction liquids and the preservation liquid can better maintain the functions of mitochondria in vitro, thus prolonging the time span from extracting mitochondria to applying same to the bodies of patients, and helping to clinical application of mitochondrial transplantation.

Description

A kit and method for extracting cell mitochondria in vitro Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a kit and method for extracting cell mitochondria in vitro. Background Art

[0002] Mitochondria have been considered a therapeutic target in medicine for the past three decades. A wealth of research has been conducted on targeting mitochondrial defects, suggesting their enormous potential. In recent years, mitochondrial transplantation for tissue restoration has garnered significant interest. To date, the application of mitochondrial transplantation has expanded to various disease models, including ischemia-reperfusion injury, neurodegenerative diseases, renal injury, and acute respiratory distress syndrome (ARDS).

[0003] In vitro mitochondrial extraction and preparation is the first step in the clinical application of mitochondrial transplantation. The main steps of in vitro mitochondrial extraction and preparation are: 1) cell lysis to isolate mitochondrial components, and 2) mitochondrial purification. Mitochondrial lysis methods mainly include: 1) mechanical lysis, including homogenization, which involves passing cells and tissues through a narrow passage using a specific grinding instrument (such as a Dounce or Potter-Elvehjem homogenizer), resulting in the fragmentation and release of cellular components; sonication, which involves the use of low-frequency ultrasound to disrupt cells; and 2) chemical lysis, including the use of digitonin or heparin to disrupt the cell outer membrane and release cellular components. Mitochondrial purification methods mainly include: 1) differential centrifugation, which involves at least two procedures with different centrifugation speeds: low-speed centrifugation is intended to discard large organelles such as the nucleus, while high-speed centrifugation is used to pellet mitochondria; 2) density gradient centrifugation, which uses a discontinuous density gradient medium (such as Ficoll) to centrifuge particles of equal density in the cell lysate to the same density layer, thereby purifying mitochondria; and 3) magnetic bead separation, which uses magnetic microbeads as an affinity material to capture mitochondria through antibody binding.

[0004] The extracted mitochondria need to be stored in the corresponding mitochondrial storage solution, and the next step needs to be carried out as soon as possible to prevent mitochondrial damage.

[0005] Mitochondrial transplantation requires the isolation of functional mitochondria with intact membrane potential and ATP production capacity. However, there is a delicate trade-off between the purity, yield, and function of isolated mitochondria. Functionally intact mitochondria are easily contaminated by non-mitochondrial components, and the removal of these non-mitochondrial components (such as the endoplasmic reticulum) may in turn impair mitochondrial function. For cell lysis, grinding and homogenization was the earliest method used in scientific research. However, this method requires high precision and specialized grinding equipment, making it unsuitable for direct clinical use. Furthermore, the grinding technique and frequency can lead to differences in the degree of cell lysis and mitochondrial viability. The ultrasonication process generates additional heat, and prolonged processing can affect mitochondrial viability. Therefore, chemical lysis is a preferred method for clinical mitochondrial extraction because it does not require additional equipment and the concentration can be controlled. However, currently, the only commercially available mitochondrial extraction kit using chemical lysis does not disclose the composition of the extraction reagents, and no effective domestic alternatives have emerged. Furthermore, it is unclear what composition and concentration of extracted mitochondria are most suitable for mitochondrial transplantation.

[0006] The internal formula components of currently available mitochondrial extraction and storage solution kits are undisclosed. According to literature reports, commonly used mitochondrial storage solutions also mainly include the following ingredients: osmotic pressure regulating solutions (such as mannitol, sorbitol, sucrose), ion buffers (such as Tris-HCl, HEPES), and calcium ion chelators (such as EGTA, EDTA). However, mitochondria preserved using current formulas lose membrane potential and respiratory function in a short period of time, and due to their complex composition, they cannot be used clinically in the short term.

[0007] Summary of the Invention

[0008] In order to overcome the defects in the prior art, the present invention provides a kit and method for extracting cell mitochondria in vitro.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] The first aspect of the present invention is to provide a kit for extracting cell mitochondria in vitro, comprising an extracting solution A, an extracting solution B, an extracting solution C, and a storage solution;

[0011] Among them, the above-mentioned extract A liquid includes EDTA, sodium lactate and sodium chloride, the above-mentioned extract B liquid is a heparin solution, the above-mentioned extract C liquid includes mannitol, EDTA, sodium lactate and sodium chloride, and the above-mentioned storage solution includes mannitol, EDTA, sodium lactate, succinic acid, sodium pyruvate and sodium chloride.

[0012] Furthermore, the extract A is prepared from EDTA, sodium lactate and sodium chloride, the extract C is prepared from mannitol, EDTA, sodium lactate and sodium chloride, and the storage solution is prepared from mannitol, EDTA, sodium lactate, succinic acid, sodium pyruvate and sodium chloride.

[0013] Furthermore, based on 100 ml of 0.6% sodium chloride solution, the extract solution A is prepared by the following method: 60-90 mg EDTA and 200-450 mg sodium lactate are dissolved in 100 ml of 0.6% sodium chloride and filtered to prepare solution A; preferably, 87.6 mg EDTA and 310 mg sodium lactate are dissolved in 100 ml of 0.6% sodium chloride solution and filtered to prepare solution A.

[0014] Furthermore, the mass volume ratio of the heparin solution is 1-10%, preferably 4%.

[0015] Furthermore, based on 100 ml of 0.9% sodium chloride solution, the extract C is prepared by the following method: 2-6 g mannitol, 60-90 mg EDTA, and 200-450 mg sodium lactate are dissolved in 100 ml of 0.9% sodium chloride solution and filtered to prepare liquid C; preferably, 4.55 g mannitol, 87.6 mg EDTA, and 310 mg sodium lactate are dissolved in 100 ml of 0.9% sodium chloride solution and filtered to prepare liquid C.

[0016] Furthermore, based on 100 ml of 0.9% sodium chloride solution, the above-mentioned stock solution is prepared by the following method: 2-6 g mannitol, 60-90 mg EDTA, 200-450 mg sodium lactate, 100-150 mg succinic acid, and 40-60 mg sodium pyruvate are dissolved in 100 ml of 0.9% sodium chloride solution and filtered to prepare a stock solution; preferably, 4.55 g mannitol, 87.6 mg EDTA, 310 mg sodium lactate, 135 mg succinic acid, and 55 mg sodium pyruvate are dissolved in 100 ml of 0.9% sodium chloride solution and filtered to prepare a stock solution.

[0017] The second aspect of the present invention is to provide a method for extracting cell mitochondria in vitro using the above kit, which comprises the following steps:

[0018] Step 1: Add the digested cells to Extraction Solution A, vortex and place on ice;

[0019] Step 2: Add Extraction Solution B and place on ice, vortexing periodically.

[0020] Step 3: Add Extraction Solution C, mix well, centrifuge, take the supernatant, centrifuge again, discard the supernatant, and precipitate mitochondria;

[0021] Step 4: Resuspend the pellet in storage solution and place on ice for later use.

[0022] Furthermore, in step 2, the mixture was placed on ice for 5 min and vortexed for 5 s every 1 min.

[0023] Furthermore, in step three, the centrifugation parameters are 700 g and centrifugation for 10 minutes.

[0024] Furthermore, in step three, the centrifugation parameters are 10,000 g and centrifugation for 15 minutes.

[0025] The present invention adopts the above technical solution, which has the following technical effects compared with the prior art:

[0026] The present invention utilizes the extract and storage solution to better maintain the function of mitochondria in vitro, prolongs the interval from mitochondrial extraction to application to patients, and provides favorable assistance for the clinical application of mitochondrial transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 shows that the proliferation capacity of bEnd.3 cells treated with mitochondria is significantly increased; Figures A and B show the EdU staining results of bEnd.3 cells treated with mitochondria and the corresponding statistical graphs, respectively;

[0028] Figure 2 shows the confocal microscopy imaging results after mitochondrial extraction. DETAILED DESCRIPTION

[0029] The present invention will be described in detail and specifically below through specific embodiments and drawings to provide a better understanding of the present invention. However, the following embodiments do not limit the scope of the present invention.

[0030] In the examples, conventional methods were used unless otherwise specified, and reagents used were conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified.

[0031] Example 1

[0032] This embodiment provides a kit for extracting cell mitochondria in vitro, which includes extraction solution A, extraction solution B, extraction solution C and storage solution.

[0033] The extract A was prepared by dissolving 87.6 mg EDTA and 310 mg sodium lactate in 100 ml 0.6% sodium chloride solution, and then filtering through a 0.22 μm filter to prepare solution A.

[0034] Extract solution B was prepared as follows: 200 mg of heparin was dissolved in 5 ml of deionized water and then filtered through a 0.22 μm filter to prepare solution B;

[0035] Extract solution C was prepared as follows: 4.55 g mannitol, 87.6 mg EDTA, and 310 mg sodium lactate were dissolved in 100 ml 0.9% sodium chloride solution, and then filtered through a 0.22 μm filter to prepare solution C;

[0036] The stock solution was prepared as follows: 4.55 g of mannitol, 87.6 mg of EDTA, 310 mg of sodium lactate, 135 mg of succinic acid, and 55 mg of sodium pyruvate were dissolved in 100 ml of 0.9% sodium chloride solution, and then filtered through a 0.22 μm filter to prepare the stock solution.

[0037] Example 2

[0038] This embodiment provides a method for extracting cell mitochondria in vitro using the kit provided in Example 1, which comprises the following steps:

[0039] Step 1: Add the digested cells to 10 ml of Extraction Solution A, vortex for 10 seconds, and place on ice for 2 minutes;

[0040] Step 2: Add 1 ml of solution B, place on ice for 5 min, and vortex for 5 s every 1 minute;

[0041] Step 3: Add 10 ml of solution C, mix thoroughly by inversion, and centrifuge at 700 g for 10 min. Transfer the supernatant to a new centrifuge tube and centrifuge at 10,000 g for 15 min. Discard the supernatant to precipitate mitochondria.

[0042] Step 4: Resuspend the pellet in storage solution and place on ice for later use.

[0043] Example 3

[0044] This example uses extracted bone cell mitochondria to stimulate bEnd.3 cells. The specific experimental steps and results are as follows:

[0045] We cultured MLO-Y4 cells in vitro, digested them, added 10 ml of solution A, vortexed for 10 seconds, and placed on ice for 2 minutes. Subsequently, 1 ml of solution B was added, placed on ice for 5 minutes, and vortexed for 5 seconds every minute. Subsequently, 10 ml of solution C was added, mixed by inversion, and centrifuged at 700g for 10 minutes. The supernatant was transferred to a new centrifuge tube and centrifuged at 10,000g for 15 minutes. The supernatant was discarded and the mitochondria were precipitated. The extracted mitochondria were resuspended in mitochondrial storage solution and placed on ice until use. The extracted mitochondria were transplanted into the mouse vascular endothelial cell line bEnd.3 at a donor / recipient cell ratio of 10:1 (i.e., mitochondria extracted from 10 donor cells were transplanted into 1 recipient cell) and their proliferation ability was detected by EdU staining.

[0046] The results are shown in FIG1 . The proportion of positive cells in the mitochondria-treated bEnd.3 cells increased significantly, indicating that the mitochondria extracted and stored by the method provided in Example 2 upregulated the proliferation capacity of bEnd.3.

[0047] Example 4

[0048] This example verifies that the kit provided in Example 1 can enable the extracted mitochondria to maintain membrane potential and respiratory function for a long time. The specific experimental steps and results are as follows:

[0049] 20 ml of human whole blood was collected, and the supernatant was taken after centrifugation at 400g for 10 minutes. Human platelets were obtained after centrifugation at 1000g for 10 minutes. 2 ml of solution A was added, vortexed for 10 seconds, and placed on ice for 2 minutes. Subsequently, 0.2 ml of solution B was added, placed on ice for 5 minutes, and vortexed for 5 seconds every minute. Subsequently, 2 ml of solution C was added, inverted to mix, and centrifuged at 700g for 10 minutes. The supernatant was transferred to a new centrifuge tube, centrifuged at 10000g for 15 minutes, and the supernatant was discarded to obtain platelet-derived mitochondria. We resuspended the extracted mitochondria in physiological saline and storage solution, respectively, and let them stand on ice for 2 hours. Subsequently, we used MitoTracker TM Green (ThermoM7514, a membrane potential-independent mitochondrial dye) and MitoTracker TM The extracted mitochondria were co-stained with Thermo Red (Thermo M22425, a membrane potential-dependent mitochondrial dye) and the mitochondrial membrane potential level was observed using a confocal microscope.

[0050] The mitochondrial staining results are shown in Figure 2. It can be seen that the mitochondria resuspended in the storage solution have a darker MitoTracker Red staining, indicating that the mitochondrial membrane potential is well preserved.

[0051] While the specific embodiments of the present invention have been described in detail above, these are intended to be exemplary only, and the present invention is not limited thereto. It will be apparent to those skilled in the art that any equivalent modifications and substitutions to the present invention fall within the scope of the present invention. Therefore, any equivalent changes and modifications made without departing from the spirit and scope of the present invention are intended to fall within the scope of the present invention.

Claims

1. A kit for extracting cell mitochondria in vitro, characterized in that: It includes extraction solution A, extraction solution B, extraction solution C and storage solution; Among them, the extracting solution A includes EDTA, sodium lactate and sodium chloride, the extracting solution B is a heparin solution, the extracting solution C includes mannitol, EDTA, sodium lactate and sodium chloride, and the storage solution includes mannitol, EDTA, sodium lactate, succinic acid, sodium pyruvate and sodium chloride.

2. The kit according to claim 1, characterized in that The extracting solution A is prepared from EDTA, sodium lactate and sodium chloride, the extracting solution C is prepared from mannitol, EDTA, sodium lactate and sodium chloride, and the storage solution is prepared from mannitol, EDTA, sodium lactate, succinic acid, sodium pyruvate and sodium chloride.

3. The kit according to claim 2, characterized in that Based on 100 ml of 0.6% sodium chloride solution, the extract A solution is prepared by the following method: 60-90 mg EDTA and 200-450 mg sodium lactate are dissolved in 100 ml of 0.6% sodium chloride solution, and filtered to prepare A solution; preferably, 87.6 mg EDTA and 310 mg sodium lactate are dissolved in 100 ml of 0.6% sodium chloride solution, and filtered to prepare A solution.

4. The kit according to claim 2, characterized in that The mass volume ratio of the heparin solution is 1-10%, preferably 4%.

5. The kit according to claim 2, characterized in that Based on 100 ml of 0.9% sodium chloride solution, the extract C solution is prepared by the following method: 2-6 g mannitol, 60-90 mg EDTA, and 200-450 mg sodium lactate are dissolved in 100 ml of 0.9% sodium chloride solution, and filtered to prepare C solution; preferably, 4.55 g mannitol, 87.6 mg EDTA, and 310 mg sodium lactate are dissolved in 100 ml of 0.9% sodium chloride solution, and filtered to prepare C solution.

6. The kit according to claim 2, characterized in that Based on 100 ml of 0.9% sodium chloride solution, the storage solution is prepared by the following method: 2-6 g mannitol, 60-90 mg EDTA, 200-450 mg sodium lactate, 100-150 mg succinic acid, and 40-60 mg sodium pyruvate are dissolved in 100 ml of 0.9% sodium chloride solution, and filtered to prepare a storage solution; preferably, 4.55 g mannitol, 87.6 mg EDTA, 310 mg sodium lactate, 135 mg succinic acid, and 55 mg sodium pyruvate are dissolved in 100 ml of 0.9% sodium chloride solution, and filtered to prepare a storage solution.

7. A method for extracting cell mitochondria in vitro using the kit according to any one of claims 1 to 6, characterized in that: The steps include: Step 1, adding the digested cells to the extract solution A, vortexing and placing on ice; Step 2, adding the extract B solution, placing on ice, and vortexing at intervals; Step 3, adding the extract C, mixing and centrifuging, taking the supernatant, centrifuging again, discarding the supernatant, and precipitating mitochondria; Step 4: resuspend the precipitate with the storage solution and place it on ice for later use.

8. The method according to claim 7, characterized in that In step 2, place on ice for 5 min and vortex for 5 s every 1 min.

9. The method according to claim 7, characterized in that: In step 3, the centrifugation parameters are 700g and centrifugation for 10 minutes.

10. The method according to claim 7, characterized in that In step 3, the centrifugation parameters are 10000g and centrifugation for 15 minutes.

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