Protective solution for isolating and protecting mitochondria, and method for isolating mitochondria

Abstract

To provide a method for efficiently isolating mitochondria from cells, and a protective solution for isolating and protecting mitochondria.SOLUTION: A method for isolating mitochondria comprises: mixing animal cells having mitochondria with a protective solution containing sodium chloride, glucose, sodium dihydrogen phosphate, or mannitol, the osmolarity of which is greater than 0 and less than or equal to 220 mOsm / L, to form a mixture solution; rubbing the animal cells in the mixture solution to damage the animal cell membrane so as to promote destruction of the animal cell membrane by penetration of the protective solution into the animal cells; centrifuging the mixture solution; and collecting a mitochondria-containing supernatant obtained after centrifugation.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] This application claims the benefit of priority from Application No. 109123093, filed in Taiwan on July 8, 2022, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to a protection solution for isolating and protecting mitochondria and a method for isolating mitochondria. [Background technology]

[0003] Mitochondria are one of the most important organelles in cells. In addition to providing cells with energy (adenosine triphosphate, ATP), mitochondria are also involved in regulating oxidative stress, apoptosis, cell-cell communication, and signal transduction. In recent years, many studies have reported that mitochondria are closely related to aging and disease formation (Bratic et al., 2013). Furthermore, other studies have shown that transplanting mitochondria can repair cell and tissue damage (Pacak et al., 2015; Cowan et al., 2017). Summary of the Invention [Problem to be solved by the invention]

[0004] Therefore, one of the development goals is to efficiently obtain mitochondria and maintain their function and activity. [Means for solving the problem]

[0005] According to an embodiment of the present disclosure, there are provided a protection solution for isolating mitochondria, a kit including the same, and a method for isolating mitochondria, which allow mitochondria to be efficiently isolated from cells in a simple and convenient manner, while maintaining the function and activity of the isolated mitochondria.

[0006] According to one embodiment of the present disclosure, a kit for isolating mitochondria includes an extraction tube for holding cells, a protective liquid for mixing with the cells in the extraction tube to form a mixed solution, and an inhalation needle for repeatedly inhaling the mixed solution.

[0007] According to one embodiment of the present disclosure, a method for isolating mitochondria includes: mixing cells with a protective solution having an osmolality concentration greater than 0 and less than or equal to 220 mOsm / L to form a mixed solution; rubbing the cells in the mixed solution to damage the cell membranes of the cells so as to promote the penetration of the protective solution into the cells and disrupt the cell membranes of the cells; centrifuging the mixed solution; and collecting a mitochondria-containing supernatant obtained after the mixed solution is centrifuged.

[0008] According to one embodiment of the present disclosure, there is provided a protection solution for isolating and protecting mitochondria from cells, the protection solution having an osmolality concentration of greater than 0 and not greater than 220 mOsm / L.

[0009] According to one embodiment of the present disclosure, there is provided the use of a protective solution, which is a hypotonic solution and includes sodium chloride, glucose, sodium dihydrogen phosphate, or mannitol, for isolating mitochondria from cells and maintaining mitochondrial activity.

[0010] Based on the above, the present disclosure provides a protective solution for isolating mitochondria, a kit for isolating mitochondria containing the same, and a method for isolating mitochondria. The cell membrane of cells is disrupted by friction of the cells within the inhalation needle using the protective solution, using the inhalation needle and protective solution in the kit. This method of disrupting the cell membrane can avoid damaging the mitochondria. Furthermore, by using the protective solution with an osmotic pressure of greater than 0 and less than or equal to 220 mOsm / L, the cell membrane is disrupted by the hypotonicity of the protective solution, allowing the mitochondria to be released. Therefore, mitochondria can be efficiently isolated from cells in a simple and convenient manner, and the isolated mitochondria can have excellent functionality. [Brief explanation of the drawings]

[0011] The present disclosure will become more fully understood from the following detailed description and the accompanying drawings, which are given by way of example only and are not intended to limit the disclosure.

[0012] [Figure 1] FIG. 1 is a diagram schematically illustrating a kit for isolating mitochondria according to a first embodiment of the present disclosure. [Figure 2] 1 is a flow chart showing a method for isolating mitochondria using the kit of the first embodiment of the present disclosure. [Figure 3] FIG. 1 is a diagram schematically illustrating the use of the kit of the first embodiment of the present disclosure. [Figure 4] FIG. 1 is a schematic diagram showing a kit for isolating mitochondria according to a second embodiment of the present disclosure. [Figure 5] 1 is a flow chart showing a method for isolating mitochondria using a kit according to a second embodiment of the present disclosure. [Figure 6] FIG. 10 shows the extraction efficiency at different needle lengths and different puff times. [Figure 7] FIG. 1 shows extraction efficiency at different cell numbers and different volumes of protective liquid. [Figure 8] FIG. 1 shows the extraction efficiency at different settling times. [Figure 9] FIG. 1 shows the function of mitochondria isolated from peripheral blood mononuclear cells in protective solutions containing sodium chloride at different osmolality concentrations. [Figure 10] FIG. 1 shows the extraction efficiency of mitochondria isolated from peripheral blood mononuclear cells using protective solutions containing sodium chloride at different osmotic concentrations. [Figure 11] FIG. 1 shows the function of mitochondria isolated from adipose-derived stem cells in a protective solution containing sodium chloride, glucose, sodium dihydrogen phosphate, or mannitol at different osmolality concentrations. [Figure 12]FIG. 1 shows the extraction efficiency of mitochondria isolated from adipose-derived stem cells using a protective solution containing sodium chloride, glucose, sodium dihydrogen phosphate, or mannitol at different osmotic concentrations. [Figure 13] FIG. 1 shows the function of mitochondria isolated from adipose-derived stem cells in protective solutions containing different components, all with an osmotic concentration of 42.8 mOsm / L. DETAILED DESCRIPTION OF THE INVENTION

[0013] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments. However, it will be apparent that one or more embodiments may be practiced without such specific details. In other instances, well-known structures and devices are shown in schematic form to simplify the drawings.

[0014] Embodiments according to the present disclosure provide kits for isolating mitochondria, methods for isolating mitochondria, and protective solutions for protecting mitochondria after separation from cells.

[0015] A kit for isolating mitochondria according to a first embodiment of the present disclosure will now be described. Referring to Fig. 1, Fig. 1 is a schematic diagram of the kit for isolating mitochondria according to the first embodiment of the present disclosure. The kit 1 for isolating mitochondria according to the first embodiment of the present disclosure includes an extraction tube 11, a protection solution 12, and an inhalation needle 13.

[0016] The extraction tube 11 is typically cylindrical. The extraction tube 11 has a closed bottom and an opening. The extraction tube is used to hold cells and a solution. The cells are cells that have mitochondria. In this embodiment, the extraction tube 11 is a circular tube, but the present disclosure is not limited thereto. In other embodiments, the extraction tube may be a tube having any shape as long as it can be accommodated in a centrifuge.

[0017] The protection liquid 12 is mixed with the cells in the extraction tube 11 to form a mixed solution. In the first embodiment, the osmolarity of the protection liquid 12 is greater than 0 and less than 220 mOsm / L, but the present disclosure is not limited thereto. In other embodiments, the protection liquid may be a buffer capable of preserving general organelles and maintaining organelle activity. In the first embodiment, the protection liquid 12 is held in a container separate from the extraction tube 11, but the present disclosure is not limited thereto. In other embodiments, the protection liquid is held in the extraction tube, so that the cells can be added directly to the extraction tube and mixed with the protection liquid held in the extraction tube to form a mixed solution.

[0018] The suction needle 13 has a connecting end 131 and a tip end 132. The connecting end 131 is used for connecting to a syringe. The tip end 132 is used for sucking up or injecting a solution. The suction needle 13 repeatedly aspirates a mixed solution containing cells and the protection solution 12 in the extraction tube 11, causing the cells in the mixed solution to rub against the inner wall of the suction needle 13. The friction damages the cell membrane, releasing mitochondria. In the first embodiment, the length of the suction needle 13 is 70 mm, matching the length of the extraction tube 11, but the present disclosure is not limited to this. In other embodiments, the length of the suction needle 13 may be 15 mm. In the first embodiment, the inner diameter of the suction needle 13 is 0.337 mm, but the present disclosure is not limited to this. In other embodiments, the inner diameter of the suction needle 13 may be 0.318 mm to 0.356 mm.

[0019] In the first embodiment, the extraction tube 11, the protection liquid 12, and the aspiration needle 13 are sterile, but the present disclosure is not limited thereto. In other embodiments, they may be sterilized before use.

[0020] A method for isolating mitochondria using the kit of the first embodiment of the present disclosure will be described below. Referring to Figures 2 and 3, Figure 2 is a flowchart showing the method for isolating mitochondria using the kit of the first embodiment of the present disclosure, and Figure 3 is a diagram schematically showing the use of the kit of the first embodiment of the present disclosure.

[0021] First, cells are mixed with a protective solution (12) having an osmolality of greater than 0 and less than or equal to 220 mOsm / L in an extraction tube 11 to form a mixed solution (S11). Specifically, the cells and protective solution 12 can be added to the extraction tube 11 using a needle, a pipette, or by pouring to form the mixed solution. The procedure for adding the cells and protective solution 12 is not limited. The cells may be any cells that have mitochondria, such as peripheral blood mononuclear cells, platelets, somatic stem cells, adipose-derived stem cells, embryonic stem cells, mesenchymal stem cells, hematopoietic stem cells, amniotic stem cells, amniotic fluid stem cells, neural stem cells, hair follicle stem cells, olfactory ensheathing stem cells, CD34+ stem cells, bone marrow stem cells, skeletal muscle cells, hepatocytes, kidney cells, fibroblasts, endothelial cells, oral epithelial cells, cardiac muscle cells, neurons, keratinocytes, and epithelial cells. Mononuclear cells and platelets from peripheral blood can be obtained by separating peripheral blood by any known method and collecting the desired mononuclear cells and platelets. In this embodiment, the protection solution 12 is a hypotonic solution with an osmotic pressure concentration of greater than 0 and less than or equal to 220 mOsm / L. The protection solution 12 has a concentration of 1×10 6 ~5×10 6 Although 1 mL per cell can be processed, the present disclosure is not limited thereto. In this embodiment, the cells and the protection solution 12 are mixed in the extraction tube 11, but the present disclosure is not limited thereto. In other embodiments, the cells and the protection solution 12 may be mixed in a container other than the extraction tube 11 and then added to the extraction tube 11.

[0022] Next, the cells in the mixed solution are rubbed and the cell membranes of the cells are damaged so that the penetration of the protection liquid 12 into the cells promotes destruction of the cell membranes of the cells (S12). Specifically, the mixed solution in the extraction tube 11 is repeatedly aspirated using the suction needle 13 and the syringe S. As shown in FIG. 3, the mixed solution in the extraction tube 11 is repeatedly aspirated several times (e.g., five times) using the suction needle 13 and the syringe S. This causes the cells in the mixed solution to rub back and forth against the inner wall of the suction needle 13. The cell membranes are damaged by the friction, and the protection liquid 12 enters the cells through the damaged cell membrane, destroying the cell membrane and releasing mitochondria.

[0023] Next, the mixed solution is centrifuged (S13). Specifically, the mixed solution after repeated inhalation through the inhalation needle 13 is centrifuged at 1500 to 2500 rpm for 5 to 15 minutes. By centrifugation, a pellet containing cell debris and a supernatant containing mitochondria are separated.

[0024] Finally, the mitochondria-containing supernatant obtained after centrifugation is collected (S14). Specifically, the mitochondria-containing supernatant can be collected using a needle, a pipette, or by pouring, thereby achieving the goal of isolating mitochondria.

[0025] A kit for isolating mitochondria according to a second embodiment of the present disclosure will now be described. Referring to FIG. 4, FIG. 4 is a schematic diagram of a kit for isolating mitochondria according to a second embodiment of the present disclosure. Because the second embodiment is similar to the first embodiment, only the differences will be described below. The kit 2 for isolating mitochondria according to the second embodiment includes an extraction tube 21, a protection solution 22, and an aspiration needle 23, as well as a stopper 24, an equilibration tube 25, a cell collection needle 26, and a mitochondrial collection needle 27. The extraction tube 21, the protection solution 22, and the aspiration needle 23 according to the second embodiment are similar to the extraction tube 11, the protection solution 12, and the aspiration needle 13 according to the first embodiment. Therefore, for details of these components, please refer to the description of the first embodiment and a detailed description will be omitted here.

[0026] The size of the stopper 24 corresponds to the size of the opening of the extraction tube 21. The stopper 24 is used to seal the extraction tube 21 to prevent the solution in the extraction tube 21 from being contaminated from the outside. In this embodiment, the stopper 24 is a rubber stopper that can maintain the sealing of the extraction tube 21 even when pierced with a needle, but the present disclosure is not limited thereto. In other embodiments, the extraction tube can be sealed with a cap or lid without a stopper.

[0027] The weight of the equilibrium tube 25 is similar to the weight of the extraction tube 21. The equilibrium tube 25 is used to maintain balance when the extraction tube 21 is centrifuged. Specifically, if the extraction tube 21 is sealed with a stopper 24, the equilibrium tube 25 may have a stopper, cap, or lid having the same weight as the stopper 24 to maintain balance during centrifugation. If a solution is held in the extraction tube 21, the same weight of the solution may be added to the equilibrium tube 25 to maintain balance during centrifugation. In other embodiments, the equilibrium tube may be a tube similar to the extraction tube, and may not have a balance tube as long as the centrifuge's rotating plate can maintain balance during centrifugation.

[0028] The cell collection needle 26 has a connecting end 261 and a tip end 262. The connecting end 261 is used for connecting to a syringe. The tip end 262 is used for sucking up or injecting a solution. The cell collection needle 26 is used to suck up the cell-containing solution and inject it into the extraction tube 21. The length and inner diameter of the cell collection needle 26 may be the same as or different from the length and inner diameter of the suction needle 23. In other embodiments, a cell collection needle may not be provided, and a pipette may be used to inject the cell-containing solution into the extraction tube.

[0029] The mitochondrial collection needle 27 has a connecting end 271 and a tip end 272. The connecting end 271 is used for connecting to a syringe. The tip end 272 is used for sucking up or injecting a solution. The mitochondrial collection needle 27 is used to collect the mitochondria-containing supernatant. The length and pore diameter of the mitochondrial collection needle 27 may be the same as or different from the length and pore diameter of the suction needle 23. In other embodiments, the mitochondrial collection needle may not be provided, and the mitochondria-containing supernatant may be collected using a pipette.

[0030] Hereinafter, a method for isolating mitochondria using the kit of the second embodiment of the present disclosure will be described. Referring to Figure 5, Figure 5 is a flow chart showing a method for isolating mitochondria using the kit of the second embodiment of the present disclosure.

[0031] First, a cell-containing solution is injected into the extraction tube 21 using the cell collection needle 26 (S21). Specifically, the cell-containing solution is injected into the extraction tube 21 sealed with the stopper 24 using the cell collection needle 26. The cells may be any cells that have mitochondria, such as peripheral blood mononuclear cells, platelets, somatic stem cells, adipose-derived stem cells, embryonic stem cells, mesenchymal stem cells, hematopoietic stem cells, amniotic stem cells, amniotic fluid stem cells, neural stem cells, hair follicle stem cells, olfactory nerve stem cells, CD34+ stem cells, bone marrow stem cells, skeletal muscle cells, hepatocytes, kidney cells, fibroblasts, endothelial cells, oral epithelial cells, cardiac muscle cells, neurons, keratinocytes, and epithelial cells. Peripheral blood mononuclear cells and platelets can be obtained by separating the peripheral blood using any known method, and then using the cell collection needle 26 to obtain the desired mononuclear cells and platelets. Here, by injecting the cell-containing solution using the cell collection needle 26 instead of the suction needle 23, contamination of the protection liquid 22 that comes into contact with the suction needle 23 can be avoided.

[0032] The solution is then centrifuged (S22). Specifically, a liquid is added to the equilibration tube 25 so that it has the same weight as the extraction tube 21 containing the cell solution. The extraction tube 21 containing the cell solution and the equilibration tube 25 are centrifuged at 1000 to 1500 rpm for 5 to 10 minutes, and the cell solution is separated into a pellet containing cells and a supernatant containing no cells.

[0033] Next, after centrifugation, the supernatant not containing cells is removed, leaving the cells in the extraction tube 21 (S23). Specifically, the cell-free supernatant is removed using the cell collection needle 26, leaving the cells in the extraction tube 21. By further removing the cell-free supernatant, the cell concentration in the extraction tube 21 is improved, while preventing the protection liquid 22 added thereafter from being diluted with other liquids, which would reduce the extraction efficiency. Here, by removing the supernatant using the cell collection needle 26 rather than the suction needle 23, contamination of the protection liquid 22 that comes into contact with the suction needle 23 can be avoided.

[0034] Next, the cells are mixed with a protection liquid 22 having an osmolality greater than 0 and less than or equal to 220 mOsm / L in the extraction tube 21 to form a mixed solution (S24). Specifically, an appropriate amount of the protection liquid 22 is added to the extraction tube 21 containing the cells using a suction needle 23 to form a mixed solution. In this embodiment, the protection liquid 22 is a hypotonic solution having an osmolality greater than 0 and less than or equal to 220 mOsm / L, but the present disclosure is not limited thereto. In other embodiments, the protection liquid may be a general buffer capable of preserving organelles and maintaining organelle activity. In a second embodiment, the protection liquid 22 is about 1×10 6 ~5×10 6 1 mL of cells can be processed.

[0035] Next, the mixed solution in the extraction tube 21 is repeatedly aspirated using the suction needle 23 and the syringe S (S25). Specifically, as shown in FIG. 3, the mixed solution in the extraction tube 21 is repeatedly aspirated several times (e.g., five times) using the suction needle 23 and the syringe S. This causes the cells in the mixed solution to rub back and forth against the inner wall of the suction needle 23. The cell membrane is damaged by the friction, and the protective liquid 22 enters the cells through the damaged cell membrane, destroying the cell membrane and releasing mitochondria.

[0036] The mixed solution is then allowed to stand for at least 5 minutes (S26). Specifically, the mixed solution is placed in a stable location and allowed to stand for at least 5 minutes, but the present disclosure is not limited thereto. Allowing the mixed solution to stand allows sufficient time for the hypotonic protective solution to spread, promoting the rupture of damaged cells and the release of mitochondria.

[0037] Next, the mixed solution is centrifuged (S27). Specifically, a liquid is added to the equilibration tube 25 so that it has the same weight as the extraction tube 21 containing the mixed solution. The extraction tube 21 containing the stationary mixed solution and the equilibration tube 25 are centrifuged at 1500 to 2500 rpm for 5 to 15 minutes. The centrifugation separates the pellet containing cell debris from the supernatant containing mitochondria.

[0038] Finally, the mitochondria-containing supernatant obtained after centrifugation is collected using the mitochondria collection needle 27 (S28). Specifically, mitochondria are separated from cell debris in the mixed solution by collecting the mitochondria-containing supernatant using the mitochondria collection needle 27. Here, by collecting the mitochondria-containing supernatant using the mitochondria collection needle 27 rather than the suction needle 23, contamination of the mitochondria-containing supernatant by the suction needle 23, which comes into contact with the protective solution and the mixed solution, can be avoided.

[0039] Experiments 1 to 5 demonstrate the isolation of mitochondria according to the method using the kit of the second embodiment of the present disclosure, and demonstrate the extraction efficiency and the function of the isolated mitochondria.

[0040] Specifically, peripheral blood mononuclear cells or adipose-derived stem cells are collected using a cell collection needle and injected into an extraction tube. Peripheral blood mononuclear cells are collected by collecting 8–20 mL of peripheral blood via vein and centrifuging the blood at 2000 rpm for 10 minutes to separate the cells, and the peripheral blood mononuclear cell layer is collected. The cell-containing extraction tube is centrifuged at 1000 rpm for 5 minutes to precipitate the cells, and the supernatant is removed using a cell collection needle. Then, 1–2 mL of protective solution is added to the cell-containing extraction tube to form a mixed solution. The mixed solution is inhaled several times using an inhalation needle. The mixed solution is then allowed to stand for at least 5 minutes, and then centrifuged at 2000 rpm for 10 minutes to separate the mixed solution, forming a mitochondria-containing supernatant and a cell debris-containing pellet. The mitochondria-containing supernatant obtained after centrifugation is collected using a mitochondria collection needle. The protective solution used in the experiment is a sodium chloride solution with an osmolality of 42.8 mOsm / L.

[0041] The mitochondrial extraction efficiency is obtained by a cell image counter. When cells are disrupted, mitochondria are released from the cells. The cell image counter calculates the number of disrupted cells and the total number of cells. The ratio of disrupted cells to total cells is defined as the extraction efficiency.

[0042] Mitochondrial function is assessed by measuring the membrane potential. Tetramethylrhodamine ethyl ester (TMRE) is a positively charged fluorescent dye. TMRE accumulates on active mitochondria and is therefore used to label healthy mitochondria. When mitochondria become less active or depolarized, the membrane potential decreases, and TMRE is no longer able to be retained on mitochondria. Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) is an ionophore that can cross the inner mitochondrial membrane. FCCP binds protons to disrupt ATP synthesis, causing a change in membrane potential. FCCP is commonly used to inactivate or depolarize mitochondria because it can eliminate the membrane potential. Changes in membrane potential are analyzed by fluorescence analysis following treatment with TMRE and FCCP to determine mitochondrial function. TMRE-treated cells are detected using a flow cytometer, and the proportion of functional mitochondria relative to the total number of cells can be analyzed based on TMRE analysis.

[0043] Mitochondrial mass was measured using the Pierce™ Protein Assay Kit. For the procedure, refer to the kit's guidelines. For this measurement, bovine serum albumin (BSA) was used as the standard. The BSA stock solution was 2 μg / mL. The working reagent was prepared by mixing Reagent A (colorless) and Reagent B (blue) from the Pierce™ Protein Assay Kit in a 50:1 ratio. The preparation of the standard is shown in Table 1. Samples were measured at 562 nm using a spectrophotometer, and the mitochondrial mass in the sample was calculated using the calibration curve. In Table 1, the blank was used to correct for background values, and the sample was the mitochondria-containing supernatant after extraction.

[0044] [Table 1]

[0045] [Experiment 1] In Experiment 1, the extraction efficiency was investigated by repeatedly inhaling the mixed solution for different times and using inhalation needles of different lengths. This experiment was conducted according to the method using the kit of the second embodiment of the present disclosure. In this experiment, each group was assigned to 1 x 10 peripheral blood mononuclear cells. 6 The sample contained 1 mL of protective solution, and the incubation time was 5 minutes. The long needle was a 23G (inner diameter 0.337 mm) needle with a length of 70 mm, and the short needle was a 23G (inner diameter 0.337 mm) needle with a length of 15 mm. The number of repeated inhalations was 0, 5, 10, 15, and 20, respectively. The results are shown in Figure 6, which shows the extraction efficiency for different needle lengths and different inhalation numbers.

[0046] The cells in the mixed solution rub against and collide with the inner wall of the needle, damaging the cell membrane and releasing mitochondria. Therefore, the longer the needle and the more repeated inhalations, the longer the path the cells have to rub, and the more friction and collisions there are, which promotes the efficiency of damaging the cell membrane. Therefore, as shown in Figure 6, the fewer the repeated inhalations, the lower the extraction efficiency. As the number of repeated inhalations increases, the extraction efficiency improves, but excessive inhalations do not contribute to the efficiency. Furthermore, the extraction efficiency of the long needle is superior to that of the short needle at the same number of repeated inhalations. The experimental results showed that the short needle achieved an extraction efficiency of approximately 50% even with five repeated inhalations. The long needle achieved an extraction efficiency of nearly 100% with 15 repeated inhalations.

[0047] [Experiment 2] Experiment 2 investigated the extraction efficiency with different cell numbers and different volumes of protective liquid. This experiment was conducted according to the method using the kit of the second embodiment of the present disclosure. In this experiment, the inhalation needle was 23G (inner diameter 0.337 mm) and 70 mm long, the number of repeated inhalations was 15, and the resting time was 5 minutes. Each group was treated with 1 x 10 peripheral blood mononuclear cells. 6 or 1×10 7 The cells contained 0.5, 1, 1.5, or 2 mL of protection liquid. The results are shown in Figure 7, which shows the extraction efficiency at different cell numbers and different volumes of protection liquid.

[0048] As shown in Figure 7, the cell count was 1 × 10 6 When the number of cells is 1 x 10, 1 mL of protection solution has excellent extraction efficiency. 7 In this case, 2 mL of the protection solution has excellent extraction efficiency. According to the results of this experiment, the protection solution, which is a sodium chloride solution with an osmotic concentration of 42.8 mOsm / L, can extract approximately 1 × 10 6 ~5×10 6 1 mL of cells could be processed, and an extraction efficiency of over 80% was achieved within this range.

[0049] [Experiment 3] Experiment 3 investigated the extraction efficiency at different resting times. This experiment was conducted according to the method using the kit of the second embodiment of the present disclosure. In this experiment, each group received 1 × 10 peripheral blood mononuclear cells. 6 The sample contained 1000 pieces of the solution, the inhalation needle was 23G (inner diameter 0.337 mm) and 70 mm long, the number of repeated inhalations was 15, and the protective solution was 1 mL. The incubation times were 0, 5, 10, 15, and 30 minutes, respectively. The results are shown in Figure 8, which shows the extraction efficiency at different incubation times.

[0050] Allowing the mixed solution to stand allows sufficient time for the hypotonic protective solution to spread, promoting further rupture of damaged cell membranes, improving the efficiency of cell membrane destruction. As shown in Figure 8, the mixed solution that was allowed to stand for several minutes after repeated inhalation had a better extraction efficiency than the mixed solution that was not allowed to stand. The results of this experiment showed that when the mixed solution was allowed to stand for at least 5 minutes, an extraction efficiency of over 80% was achieved, and there was no significant change even with longer standing times.

[0051] [Experiment 4] In experiment 4, mitochondria were isolated from peripheral blood mononuclear cells (PBMCs). In this experiment, the number of PBMCs in the blood was approximately 2.5 × 10 6The number of samples was approximately 8 mL of peripheral blood, the protective solution was 1 mL of sodium chloride solution with an osmolality of 42.8 mOsm / L, the inhalation needle was 23 G (inner diameter 0.337 mm) and 70 mm long, the number of repeated inhalations was 15, and the resting time was 5 minutes. The results are shown in Table 2. 6 8.30 μg of mitochondria were isolated from each peripheral blood mononuclear cell, and the ratio of functional mitochondria to all particles in the supernatant, expressed as purity in Table 2, was 48.55%.

[0052] [Experiment 5] In experiment 5, mitochondria were isolated from adipose-derived stem cells. In this experiment, the number of adipose-derived stem cells was approximately 5 × 10 6 The protective solution was 1 mL of sodium chloride solution with an osmolality of 42.8 mOsm / L, the inhalation needle was 23 G (inner diameter 0.337 mm) and 70 mm long, the number of repeated inhalations was 15, and the resting time was 5 minutes. The results are shown in Table 2. 6 10.97 μg of mitochondria were isolated from each adipose-derived stem cell, and the ratio of functional mitochondria to all particles in the supernatant, represented as purity in Table 2, was 39.68%.

[0053] [Table 2] The present disclosure provides a kit and method for isolating mitochondria. The kit includes an inhalation needle and a protective solution. The cell membranes of cells are disrupted by friction between the cells and the inhalation needle, preventing damage to the mitochondria. Therefore, mitochondria can be efficiently isolated from cells in a simple and convenient manner, and the isolated mitochondria have excellent function and activity.

[0054] The protection solution in the kit for isolating mitochondria according to the present disclosure will be further described below.

[0055] The protection solution is a solution used to separate mitochondria from cells and protect the separated mitochondria. The osmolality of the protection solution may be greater than 0 and less than 220 mOsm / L. In some embodiments, the osmolality of the protection solution may be 42.8 mOsm / L to 220 mOsm / L. In some embodiments, the osmolality of the protection solution may be 42.8 mOsm / L to 113 mOsm / L. In some embodiments, the protection solution contains sodium chloride, glucose, sodium dihydrogen phosphate, or mannitol. In some embodiments, the protection solution contains sodium chloride and glucose, and the weight ratio of sodium chloride to glucose may be 1:0.06 to 1:2560. In some embodiments, the protection solution contains sodium chloride and sodium dihydrogen phosphate, and the weight ratio of sodium chloride to sodium dihydrogen phosphate may be 1:0.015 to 1:133. In some embodiments, the protection solution may include glucose and sodium dihydrogen phosphate, and the weight ratio of glucose to sodium dihydrogen phosphate may be 1:0.0007 to 1:22. In some embodiments, the protection solution may include only sodium chloride and glucose, with no other solutes. In some embodiments, the protection solution may include only sodium chloride and sodium dihydrogen phosphate, with no other solutes. In some embodiments, the protection solution may include only glucose and sodium dihydrogen phosphate, with no other solutes.

[0056] The components and osmotic concentrations of the protection solution of the example of the present disclosure and the extract solution of the comparative example are shown in Tables 3 and 4.

[0057] [Table 3]

[0058] [Table 4]

[0059] Experiments 6 to 8 demonstrate the isolation of mitochondria using a protection solution according to the present disclosure. Mitochondria were isolated using the protection solution according to the present disclosure and an extraction solution as a comparative example, using the kit and method of the second embodiment of the present disclosure, and the function and extraction efficiency of the isolated mitochondria were analyzed.

[0060] Specifically, cells and a protection solution are first mixed to form a mixed solution. 6 The cells are collected. The cells may be peripheral blood mononuclear cells or adipose-derived stem cells, but the present disclosure is not limited thereto. The cells may be any cells that contain mitochondria. The cells are then mixed with 1 mL of the protection solution of the example or 1 mL of the comparative extraction solution of the comparative example to form a mixed solution.

[0061] Next, the cells in the mixed solution are rubbed, damaging the cell membranes of the cells, to promote the penetration of the protective solution into the cells and disrupting their cell membranes. Specifically, during the process of mixing the cells with the protective solution, the cell membranes of the cells are damaged by friction. The damaged cell membranes promote the penetration of the protective solution into the cells, leading to further cell destruction. Here, a 23G, 70mm long needle is used to repeatedly aspirate the mixed solution and damage the cells, but the present disclosure is not limited thereto. When using the protective solution disclosed herein, needles of different sizes or machines such as a grinder can be used depending on the experimental equipment to further damage the cell membrane and promote the penetration of the protective solution into the cells.

[0062] The mixed solution is then centrifuged to separate the layers. Specifically, the mixed solution is allowed to stand and then centrifuged to separate the mixed solution into a supernatant containing mitochondria and a pellet containing cell debris.

[0063] The mitochondria-containing supernatant is then collected, specifically by using a needle, a pipette, or by pouring.

[0064] Finally, the mitochondria-containing supernatant is analyzed for extraction efficiency and mitochondrial function.

[0065] [Experiment 6] In Experiment 6, mitochondria were isolated from peripheral blood mononuclear cells (PBMCs) using protective solutions containing sodium chloride at different osmolality concentrations, and the extraction efficiency and mitochondrial function were analyzed. Referring to Figures 9 and 10, Figure 9 shows the function of mitochondria isolated from PBMCs using protective solutions containing sodium chloride at different osmolality concentrations, and Figure 10 shows the extraction efficiency of mitochondria isolated from PBMCs using protective solutions containing sodium chloride at different osmolality concentrations. In Figure 10, "#" indicates a significant difference (P<0.05) compared to the control (520 mOsm / L), and "*" indicates a significant difference (P<0.05) compared to the control (1025 mOsm / L).

[0066] Figure 9 and Table 5 show that when mitochondria are isolated using a protection solution containing sodium chloride at an osmolality of greater than 0 and less than 220 mOsm / L, the isolated mitochondria have excellent functionality, with the mitochondrial function exceeding 10%. Figure 10 and Table 5 show that when mitochondria are isolated using a protection solution containing sodium chloride at an osmolality of greater than 0 and less than 220 mOsm / L, excellent extraction efficiency is achieved, with the extraction efficiency exceeding 50%. In summary, isolating mitochondria from peripheral blood mononuclear cells using a protection solution containing sodium chloride at an osmolality of greater than 0 and less than 220 mOsm / L maintains mitochondrial functionality (more than 10%) while achieving excellent extraction efficiency (more than 50%).

[0067] [Table 5]

[0068] [Experiment 7] In Experiment 7, mitochondria were isolated from adipose-derived stem cells using a protective solution containing sodium chloride, glucose, sodium dihydrogen phosphate, or mannitol at different osmolality concentrations, and the extraction efficiency and mitochondrial function were analyzed. Referring to Figures 11 and 12, Figure 11 shows the function of mitochondria isolated from adipose-derived stem cells using a protective solution containing sodium chloride, glucose, sodium dihydrogen phosphate, or mannitol at different osmolality concentrations, and Figure 12 shows the extraction efficiency of mitochondria isolated from adipose-derived stem cells using a protective solution containing sodium chloride, glucose, sodium dihydrogen phosphate, or mannitol at different osmolality concentrations. In Figures 11 and 12, "#" indicates a significant difference (P<0.05) compared to the comparative example at 520 mOsm / L, and "*" indicates a significant difference (P<0.05) compared to the comparative example at 1025 mOsm / L.

[0069] Figure 11 and Table 6 show that when mitochondria are isolated using a protection solution with an osmolality greater than 0 and less than 220 mOsm / L and containing sodium chloride, glucose, sodium dihydrogen phosphate, or mannitol, the isolated mitochondria have excellent functionality, with mitochondrial function exceeding 10%. Figure 12 and Table 6 show that when mitochondria are isolated using a protection solution with an osmolality greater than 0 and less than 220 mOsm / L and containing sodium chloride, glucose, sodium dihydrogen phosphate, or mannitol, excellent extraction efficiency is achieved, with the extraction efficiency exceeding 50%. In summary, isolating mitochondria from adipose-derived stem cells using a protection solution with an osmolality greater than 0 and less than 220 mOsm / L and containing sodium chloride, glucose, sodium dihydrogen phosphate, or mannitol maintains mitochondrial function (more than 10%) while achieving excellent extraction efficiency (more than 50%).

[0070] [Table 6]

[0071] [Experiment 8] In Experiment 8, mitochondria were isolated from adipose-derived stem cells using a protection solution containing different components with an osmotic concentration of 42.8 mOsm / L, and the function of the mitochondria was analyzed. Referring to Figure 13 and Table 7, Figure 13 shows the function of mitochondria isolated from adipose-derived stem cells using a protection solution containing different components, all with an osmotic concentration of 42.8 mOsm / L.

[0072] 13 and Table 7 show that when mitochondria were isolated from adipose-derived stem cells using a protection solution containing a single component (Example 1: sodium chloride, Example 4: glucose, Example 7: sodium dihydrogen phosphate) at an osmolality of 42.8 mOsm / L, the isolated mitochondria had excellent functionality, with mitochondrial function exceeding 10%. Furthermore, when mitochondria were isolated from adipose-derived stem cells using a protection solution containing two of sodium chloride, glucose, and sodium dihydrogen phosphate (Example 13: sodium chloride and glucose, Example 14: sodium chloride and sodium dihydrogen phosphate, Example 15: glucose and sodium dihydrogen phosphate) at an osmolality of 42.8 mOsm / L, the isolated mitochondria had even better functionality, with mitochondrial function exceeding 15%.

[0073] [Table 7]

[0074] The above results demonstrate that the use of hypotonic solutions provides better extraction efficiency than the use of hypertonic solutions. Furthermore, the above experiments demonstrate that isolating mitochondria using a protection solution with an osmolality greater than 0 and less than 220 mOsm / L can achieve excellent extraction efficiency while maintaining mitochondrial function. Furthermore, the above experiments demonstrate that when the osmolality is 42.8 mOsm / L, isolating mitochondria using a protection solution containing two of sodium chloride, glucose, and sodium dihydrogen phosphate results in better isolated mitochondria with better function. The differences in values ​​between different experiments are due to experimental error when experiments are performed in batches, and are within the tolerances accepted in the art.

[0075] An embodiment of the present disclosure provides a protective solution for isolating mitochondria from cells and protecting the isolated mitochondria. When damaging the cell membrane, for example, by repeatedly suctioning with a long needle, the cell membrane is damaged by friction. The osmolality of the protective solution is greater than 0 and less than 220 mOsm / L, so that the cell membrane is destroyed using a hypotonic protective solution, releasing the mitochondria. This method of destroying the cell membrane can prevent mitochondrial damage in cells. Therefore, mitochondria can be efficiently isolated from cells in a simple and convenient way, and the isolated mitochondria have excellent function and activity.

[0076] Although the embodiments of the present disclosure have been disclosed as above, the present disclosure is not limited thereto. Those skilled in the art may make appropriate changes to the shape, structure, characteristics, and spirit of the present disclosure without departing from the spirit and scope of the present disclosure. The description in the specification belongs to the scope of patent protection. [Explanation of symbols]

[0077] 1, 2: Kit for isolating mitochondria 11, 21: Extraction tube 12, 22: Protective liquid 13, 23: Inhalation needle 24: Stopper 25: Balance tube 26: Cell collection needle 27: Mitochondria collection needle 131, 231, 261, 271: Connecting end 132, 232, 262, 272: Tip S:Syringe S11~S14, S21~S28: Step

Claims

1. Mixing animal cells having mitochondria with a protective solution containing sodium chloride, glucose, sodium dihydrogen phosphate, or mannitol and having an osmotic pressure concentration of greater than 0 and less than or equal to 220 mOsm / L to form a mixed solution; rubbing the animal cells in the mixed solution to damage the animal cell membranes of the animal cells so as to promote the penetration of the protection solution into the animal cells and the destruction of the animal cell membranes of the animal cells; centrifuging the mixed solution; collecting the mitochondria-containing supernatant obtained after centrifugation; 1. A method for isolating mitochondria, comprising:

2. The method according to claim 1, wherein the mixed solution is repeatedly inhaled using an inhalation needle during the rubbing of the animal cells.

3. Before mixing the animal cells with the protection solution, injecting a solution containing the animal cells into an extraction tube using an animal cell collection needle; centrifuging the solution; removing the supernatant containing no animal cells and leaving the animal cells; The method of claim 1.

4. 2. The method of claim 1, wherein the step of collecting the mitochondria-containing supernatant is performed using a mitochondria collection needle.

5. When mixing the animal cells with the protection solution, the ratio of the number of the animal cells to the volume of the protection solution is 5 × 10 per mL. ６ The method of claim 1 , wherein the number of the ions is equal to or less than 1.

6. 3. The method according to claim 2, wherein the length of the inhalation needle is 70 mm, and the number of repeated inhalations of the mixed solution is at least 5.

7. 3. The method of claim 2, further comprising allowing the mixed solution to stand for an equilibration period of at least 5 minutes after repeatedly inhaling the mixed solution using the inhalation needle.

8. A protection solution for isolating and protecting mitochondria from animal cells, which contains sodium chloride, glucose, sodium dihydrogen phosphate, or mannitol and has an osmolality concentration of greater than 0 and not greater than 220 mOsm / L.

9. The protection solution according to claim 8, wherein the osmotic concentration is 42.8 mOsm / L to 113 mOsm / L.

10. 9. The protection solution according to claim 8, comprising sodium chloride and glucose, wherein the weight ratio of sodium chloride to glucose is 1:0.06 to 1:2560.

11. 9. The protection solution according to claim 8, comprising sodium chloride and sodium dihydrogen phosphate, wherein the weight ratio of sodium chloride to sodium dihydrogen phosphate is 1:0.015 to 1:

133.

12. 9. The protection solution according to claim 8, comprising glucose and sodium dihydrogen phosphate, wherein the weight ratio of glucose to sodium dihydrogen phosphate is 1:0.0007 to 1:22.

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