Patent Publication Number: US-2023143850-A1

Title: Protective solution for isolating mitochondria, use thereof, kit comprising the same, and method for isolating mitochondria

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation-in-part of International Application No. PCT/MY2021/050056, filed on Jul. 7, 2021, which claiming priority to Patent Application No(s). 100123093 filed in Taiwan (R.O.C.) on Jul. 8, 2020, the entire contents of which are hereby incorporated by reference. 
    
    
     BACKGROUND 
     1. Technical Field 
     This disclosure relates to a protective solution for isolating mitochondria, use thereof, a kit comprising the same, and a method for isolating mitochondria. 
     2. Related Art 
     A mitochondrion is one of the important organelles in a cell. Mitochondria not only supply energy (adenosine triphosphate, ATP) for cells but also involve the regulation of oxidative stress, apoptosis, communication between cells, and transportation of signals. Recently, many studies have reported that aging and the formation of diseases have a close relationship with mitochondria (Braticet al., 2013). Some studies have further shown that a technique of mitochondrial transplantation can repair the damages to cells and tissues (Pacak et al., 2015; Cowanet al., 2017). 
     Therefore, how to obtain mitochondria and maintain their function and activity in an efficient way is one of the aims of development. 
     SUMMARY 
     Accordingly, this disclosure provides a protective solution for isolating mitochondria, a kit comprising the same, and a method for isolating mitochondria. In this disclosure, mitochondria may be isolated from cells with high efficiency in a simple and convenient way, and the isolated mitochondria may maintain their function and activity. 
     According to one embodiment of the present disclosure, a kit for isolating mitochondria comprises an extraction tube for containing cells; a protective solution for forming a mixed solution with the cells in the extraction tube; and a sucking needle for sucking the mixed solution back and forth. 
     According to one embodiment of the present disclosure, a method for isolating mitochondria comprises mixing cells and a protective solution to form a mixed solution, with a osmolarity of the protective solution greater than 0 and less than or equal to 220 mOsm/L; rubbing the cells in the mixed solution to damage cell membranes of the cells to facilitate an entry of the protective solution into the cells and destroy the cell membranes of the cells; 
     centrifuging the mixed solution; and collecting supernatant comprising mitochondria obtained after centrifuging. 
     According to one embodiment of the present disclosure, a protective solution for isolating and protecting mitochondria from cells is provided, wherein a osmolarity of the protective solution is greater than 0 and less than or equal to 220 mOsm/L. 
     According to one embodiment of the present disclosure, a use of a protective solution in isolating mitochondria from cells and maintaining the activity of mitochondria is provided, wherein the protective solution is a hypotonic solution and comprises sodium chloride, glucose, sodium dihydrogen phosphate, or mannitol. 
     In view of the above description, the present disclosure provides a protective solution for isolating mitochondria, a kit comprising the same, and a method for isolating mitochondria. The cell membranes of the cells may be destroyed by the sucking needle and the protective solution in the kit through the friction of the cells in the sucking needle with the help of the protective solution, and this way of destroying the cell membranes may not damage the mitochondria in the cells. In addition, the osmolarity of the protective solution is greater than 0 and less than or equal to 220 mOsm/L so that the cell membranes of the cells may be destroyed with the help of the hypotonicity of the protective solution to release the mitochondria. Therefore, the mitochondria may be isolated from cells with high efficiency in a simple and convenient way, and the isolated mitochondria may have excellent function and activity. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The present disclosure will become more fully understood from the detailed description given hereinbelow and the accompanying drawings which are given by way of illustration only and thus are not limitative of the present disclosure and wherein: 
         FIG.  1    is a schematic view illustrating a kit for isolating mitochondria according to the first embodiment of the present disclosure. 
         FIG.  2    is a flow chart illustrating a method for isolating mitochondria by using the kit according to the first embodiment of the present disclosure. 
         FIG.  3    is a schematic view illustrating the implementation of the kit according to the first embodiment of the present disclosure. 
         FIG.  4    is a schematic view illustrating a kit for isolating mitochondria according to the second embodiment of the present disclosure. 
         FIG.  5    is a flow chart illustrating a method for isolating mitochondria by using the kit according to the second embodiment of the present disclosure. 
         FIG.  6    shows the extraction efficiency for different numbers of times for sucking back and forth and different lengths of the needle. 
         FIG.  7    shows the extraction efficiency for different numbers of the cells and different volumes of the protective solution. 
         FIG.  8    shows the extraction efficiency for different times for standing. 
         FIG.  9    shows the function of mitochondria isolated from the peripheral blood mononuclear cells by the protective solution comprising NaCl with different osmolarities. 
         FIG.  10    shows the extraction efficiency of mitochondria isolated from the peripheral blood mononuclear cells by the protective solution comprising NaCl with different osmolarities. 
         FIG.  11    shows the function of mitochondria isolated from the adipose-derived stem cells by the protective solution comprising NaCl, glucose, NaH 2 PO 4 , or mannitol with different osmolarities. 
         FIG.  12    shows the extraction efficiency of mitochondria isolated from the adipose-derived stem cells by the protective solution comprising NaCl, glucose, NaH 2 PO 4 , or mannitol with different osmolarities. 
         FIG.  13    shows the function of mitochondria isolated from the adipose-derived stem cells by the protective solution comprising different compositions with an osmolarity of 42.8 mOsm/L. 
     
    
    
     DETAILED DESCRIPTION 
     In the following detailed description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments. It will be apparent, however, that one or more embodiments may be practiced without these specific details. In other instances, well-known structures and devices are schematically shown in order to simplify the drawings. 
     The embodiments according to the present disclosure provide a kit, a method, and a protective solution for isolating mitochondria from cells and protecting the isolated mitochondria. The aforementioned cells may include any cells having mitochondria, for example, peripheral blood mononuclear cells, hematopoietic stem cells, embryonic stem cells, mesenchymal stem cells, adipose-derived mesenchymal stem cells, umbilical cord mesenchymal stem cells, CD34+ stem cells, amniotic fluid mesenchymal stem cells, bone marrow mesenchymal stem cells, induced pluripotent stem cells, neural stem cells, epithelial stem cells, skin stem cells, somatic stem cells, somatic cells, smooth muscle cells, liver cells, kidney cells, cardiomyocytes, monocytes, lymphocytes, keratinocytes, and hair follicle cells. 
     A kit for isolating mitochondria according to the first embodiment of the present disclosure will be described below. Please refer to  FIG.  1   , wherein  FIG.  1    is a schematic view illustrating a kit for isolating mitochondria according to the first embodiment of the present disclosure. The kit  1  for isolating mitochondria in the first embodiment of the present disclosure comprises an extraction tube  11 , a protective solution  12 , and a sucking needle  13 . 
     The extraction tube is usually a cylinder. The extraction tube  11  has a closed bottom and an opening. The extraction tube is used to contain cells and solutions. The cells are cells that have mitochondria. In this embodiment, the extraction tube  11  is a cylinder tube, but the present disclosure is not limited thereto. In other embodiments, the extraction tube may be a tube in any shape as long as acceptable to the centrifuge. 
     The protective solution  12  is used for mixing with the cells in the extraction tube  11  to form a mixed solution. In the first embodiment, the osmolarity of the protective solution  12  is greater than 0 and less than or equal to 220 mOsm/L, but the present disclosure is not limited thereto. In other embodiments, the protective solution may be any common buffer that can preserve organelles or maintain the activity of organelles. In the first embodiment, the protective solution  12  is contained in a container different from the extraction tube  11 , but the present disclosure is not limited thereto. In other embodiments, the protective solution may be contained in the extraction tube so that the cells may be directly added to the extraction tube and mixed with the protective solution contained in the extraction tube to form the mixed solution. 
     The sucking needle  13  has a connection end  131  and a tip end  132 . The connection end  131  is used to connect to a syringe. The tip end  132  is used to suck or inject solutions. The sucking needle  13  is used to suck back and forth the mixed solution comprising the cells and the protective solution  12  in the extraction tube  11 , and the cells in the mixed solution may be rubbed back and forth with the inner wall of the needle in the sucking needle  13 . The cell membranes are damaged due to the friction thereby releasing the mitochondria. In the first embodiment, the length of the sucking needle  13  corresponds to the length of the extraction tube  11  to be 70 mm, but the present disclosure is not limited thereto. In other embodiments, the length of the sucking needle may be 15 mm. In the first embodiment, the inner diameter of the sucking needle  13  is 0.337 mm, but the present disclosure is not limited thereto. In other embodiments, the inner diameter of the sucking needle may be 0.318 to 0.356 mm. 
     In the first embodiment, the extraction tube  11 , the protective solution  12 , and the sucking needle  13  are sterile, but the present disclosure is not limited thereto. In other embodiments, they may be sterilized before used. 
     A method for isolating mitochondria by using the kit according to the first embodiment of the present disclosure will be described below. Please refer to  FIGS.  2  and  3   , wherein  FIG.  2    is a flow chart illustrating a method for isolating mitochondria by using the kit according to the first embodiment of the present disclosure, and  FIG.  3    is a schematic view illustrating the implementation of the kit according to the first embodiment of the present disclosure. 
     Firstly, cells and a protective solution  12  are mixed in an extraction tube  11  to form a mixed solution, and the osmolarity of the protective solution is greater than 0 and less than or equal to 220 mOsm/L (S 11 ). In detail, the cells and the protective solution  12  may be added to the extraction tube  11  by using other needles or pipettes or by pouring, to form the mixed solution. The sequence of adding the cells and adding the protective solution  12  is not limited. The cells may be any cell having 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, hepatic cells, kidney cells, fibroblasts, endothelial cells, oral epithelial cells, myocardial cells, nerve cells, keratinocytes, epithelial cells, etc. The peripheral blood mononuclear cells or platelets may be obtained by stratifying the peripheral blood and collecting the desired mononuclear cells or platelets by any known method. In this embodiment, the protective solution  12  is a hypotonic solution, the osomolarity is greater than 0 and less than or equal to 220 mOsm/L, and each milliliter of the protective solution  12  may be used for treating 1×10 6  to 5×10 6  cells, but the present disclosure is not limited thereto. In this embodiment, the cells and the protective solution  12  are mixed in the extraction tube  11 , but the present disclosure is not limited thereto. In other embodiments, the cells and the protective solution  12  may be mixed in a container other than the extraction tube and then added to the extraction tube. 
     Subsequently, the cells in the mixed solution are rubbed to damage the cell membranes of the cells to facilitate the entry of the protective solution  12  into the cells and destroy the cell membranes of the cells (S 12 ). In detail, the mixed solution in the extraction tube  11  is sucked back and forth by using a sucking needle  13  and a syringe S. As shown in  FIG.  3   , the mixed solution in the extraction tube  11  is sucked back and forth several times (e.g., 5 times) by using the sucking needle  13  and the syringe S. Thereby, the cells in the mixed solution are rubbed back and forth with the inner wall of the needle in the sucking needle  13 . The cell membranes are damaged due to the friction so that the protective solution  12  enters into the cells through the damaged membranes to destroy the cell membrane, thereby releasing the mitochondria. 
     Subsequently, the mixed solution is centrifuged (S 13 ). In detail, the mixed solution after being sucked back and forth by the sucking needle  13  is centrifuged at 1500 to 2500 rpm for 5 to 15 minutes. The pellet comprising cell debris and the supernatant comprising the mitochondria are stratified by the centrifugation. 
     Finally, the supernatant comprising the mitochondria obtained after the centrifugation is collected (S 14 ). In detail, the supernatant comprising the mitochondria may be collected by using other needles or pipettes or by pouring so as to achieve the purpose of isolating the mitochondria. 
     A kit for isolating mitochondria according to the second embodiment of the present disclosure will be described below. Please refer to  FIG.  4   , wherein  FIG.  4    is a schematic view illustrating a kit for isolating mitochondria according to the second embodiment of the present disclosure. The second embodiment is similar to the first embodiment, and only differences will be described below. In addition to an extraction tube  21 , a protective solution  22 , and a sucking needle  23 , the kit  2  in the second embodiment of the present disclosure further comprises a stopper  24 , a balance tube  25 , a cell collection needle  26 , and a mitochondria collection needle  27 . The extraction tube  21 , the protective solution  22 , and the sucking needle  23  in the second embodiment are the same as the extraction tube  11 , the protective solution  12 , and the sucking needle  13  in the first embodiment. Therefore, please refer to the description of the first embodiment for the details of these components, and the details are not repeated here. 
     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 by the outside. In this embodiment, the stopper  24  is a rubber stopper, and the rubber stopper maintains the seal of the extraction tube  21  after being pierced by the needle, but the present disclosure is not limited thereto. In other embodiments, the extraction tube may be closed by a cap or a lid rather than a rubber stopper. 
     The weight of the balance tube  25  is equal to that of the extraction tube  21 . The balance tube  25  is used to keep balance with the extraction tube  21  during centrifugation. In detail, when the extraction tube  21  is sealed by the stopper  24 , the balance tube  25  may have a stopper, a cap, or a lid that has the same weight as the stopper  24  to keep balance during centrifugation. When the extraction tube  21  contains a solution, the balance tube  25  may be added with the same weight of the solution to keep balance during centrifugation. In other embodiments, the balance tube may be the same as the extraction tube, or there may be not a balance tube as long as the turntable of the centrifuge keeps balance during centrifugation. 
     The cell collection needle  26  has a connection end  261  and a tip end  262 . The connection end  261  is used to connect to a syringe. The tip end  262  is used to suck or inject solutions. The cell collection needle  26  is used to suck a solution comprising cells and inject the solution into the extraction tube  21 . The length and the inner diameter of the cell collection needle  26  may be the same as or different from those of the sucking needle  23 . In other embodiments, there may be not a cell collection needle, and the solution comprising the cells may be injected into the extraction tube by a pipette. 
     The mitochondria collection needle  27  has a connection end  271  and a tip end  272 . The connection end  271  is used to connect to a syringe. The tip end  272  is used to suck or inject solutions. The mitochondria collection needle  27  is used to collect the supernatant comprising mitochondria. The length and the inner diameter of the mitochondria collection needle  27  may be the same as or different from those of the sucking needle  23 . In other embodiments, there may be not a mitochondria collection needle, and the supernatant comprising the mitochondria may be collected by a pipette. 
     A method for isolating mitochondria by using the kit according to the second embodiment of the present disclosure will be described below. Please refer to  FIG.  5   , wherein  FIG.  5    is a flow chart illustrating a method for isolating mitochondria by using the kit according to the second embodiment of the present disclosure. 
     Firstly, a solution comprising cells is injected to an extraction tube  21  by using a cell collection needle  26  (S 21 ). In detail, the solution comprising the cells is injected into the extraction tube  21  sealed with a stopper  24  by using the cell collection needle  26 . The cells may be any cell having 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, hepatic cells, kidney cells, fibroblasts, endothelial cells, oral epithelial cells, myocardial cells, nerve cells, keratinocytes, epithelial cells, etc. The peripheral blood mononuclear cells or platelets may be obtained by stratifying the peripheral blood by any known method and collecting the desired mononuclear cells or platelets by using the cell collection needle  26 . Here, the solution comprising the cells is injected by the cell collection needle  26  rather than the sucking needle  23 , which may prevent the protective solution  22  contacted with the sucking needle  23  from being contaminated. 
     Subsequently, the solution is centrifuged (S 22 ). In detail, the balance tube  25  is added with liquid to have the same weight as the extraction tube  21  which contains the solution of the cells. The extraction tube  21  containing the solution of the cells and the balance tube  25  are centrifuged at 1000 to 1500 rpm for 5 to 10 minutes so that the solution of the cells is stratified to form the pellet comprising the cells and the supernatant without cells. 
     Subsequently, the supernatant without the cells after centrifugation is removed, and the cells are retained in the extraction tube  21  (S 23 ). In detail, the supernatant without the cells is removed by using the cell collection needle  26 , and the cells are retained in the extraction tube  21 . Removing the supernatant without the cells may increase the concentration of the cells in the extraction tube  21  and prevent the protective solution  22  subsequently added from being diluted by other liquid which decreases the extraction efficiency. Here, the supernatant is removed by the cell collection needle  26  rather than the sucking needle  23 , which may prevent the protective solution  22  contacted with the sucking needle  23  from being contaminated. 
     Subsequently, the cells and the protective solution  22  are mixed in the extraction tube  21  to form a mixed solution, and the osmolarity of the protective solution  22  is greater than 0 and less than or equal to 220 mOsm/L (S 24 ). In detail, a proper amount of the protective solution  22  is added to the extraction tube  21  containing the cells by the sucking needle  23 , to form the mixed solution. In this embodiment, the protective solution  22  is a hypotonic solution, and the osmolarity is greater than 0 and less than or equal to 220 mOsm/L, but the present disclosure is not limited thereto. In other embodiments, the protective solution may be any common buffer that can preserve organelles or maintain the activity of organelles. In the second embodiment, each milliliter of the protective solution  22  may be used for treating approximately 1×10 6  to 5×10 6  cells. 
     Subsequently, the mixed solution in the extraction tube  21  is sucked back and forth by the sucking needle  23  and a syringe S (S 25 ). In detail, as shown in  FIG.  3   , the mixed solution in the extraction tube  21  is sucked back and forth several times (e.g., 5 times) by using the sucking needle  23  and the syringe S. Thereby, the cells in the mixed solution are rubbed back and forth with the inner wall of the needle in the sucking needle  23 . The cell membranes are damaged due to the friction so that the protective solution  22  enters into the cells through the damaged membranes to destroy the cell membrane, thereby releasing the mitochondria. 
     Subsequently, the mixed solution stands for at least five minutes (S 26 ). In detail, the mixed solution is placed at a stable place, and the time for standing is at least five minutes, but the present disclosure is not limited thereto. Standing of the mixed solution allows the hypotonic protective solution has enough time to diffuse to facilitate the destruction of the damaged cell, thereby facilitating the release of the mitochondria. 
     Subsequently, the mixed solution is centrifuged (S 27 ). In detail, the balance tube  25  is added with liquid to have the same weight as the extraction tube  21  which contains the mixed solution. The extraction tube  21  containing the mixed solution after standing and the balance tube  25  are centrifuged at 1500 to 2500 rpm for 5 to 15 minutes. The pellet comprising cell debris and the supernatant comprising the mitochondria are stratified by the centrifugation. 
     Finally, the supernatant comprising the mitochondria obtained after the centrifugation is collected by using a mitochondria collection needle  27  (S 28 ). In detail, the supernatant comprising the mitochondria is collected by the mitochondria collection needle  27 , thereby isolating the mitochondria from the cell debris in the mixed solution. Here, the supernatant comprising the mitochondria is collected by the mitochondria collection needle  27  rather than the sucking needle  23 , which may prevent the supernatant comprising the mitochondria from being contaminated by the sucking needle  23  contacted with the protective solution and the mixed solution. 
     Experiments 1 to 5 demonstrate isolating mitochondria by the method using the kit according to the second embodiment of the present disclosure, and the extraction efficiency and the function of the isolated mitochondria will be shown. 
     Specifically, the peripheral blood mononuclear cells or the adipose-derived stem cells are collected by using the cell collection needle, and the cells are injected into the extraction tube. The peripheral blood mononuclear cells are obtained by drawing 8 to 20 mL of the peripheral blood from veins, centrifuging the blood at 2000 rpm for 10 min to stratify it, and collecting the layer of the peripheral blood mononuclear cells. The extraction tube containing the cells is centrifuged at 1000 rpm for 5 min to precipitate the cells, and the supernatant is removed by the cell collection needle. Then, 1 to 2 mL of the protective solution is added to the extraction tube containing the cells to form the mixed solution. The mixed solution is sucked back and forth several times by the sucking needle. Then, the mixed solution stands for at least 5 min and then centrifuged at 2000 rpm for 10 min, and the mixed solution is stratified to form the supernatant comprising mitochondria and the pellet comprising the cell debris. The supernatant comprising the mitochondria after centrifugation is collected by using the mitochondria collection needle. The protective solution used in the experiments is NaCl solution with the osmolarity of 42.8 mOsm/L. 
     The extraction efficiency of the mitochondria is obtained by a cell image counter. If the cells are damaged, the mitochondria will be released from the cells. The cell image counter counts the number of the total cells and the damaged cells. The ratio of the damaged cells to the total cells is defined as the extraction efficiency. 
     The function of the mitochondria is determined by measuring the membrane potential. Tetramethylrhodamine ethyl ester (TMRE) is a fluorescent dye with positive charge. TMRE can gather on the healthy mitochondria so that TMRE can be used to label the healthy mitochondria. When the mitochondria are less active or depolarized, the membrane potential decreases, and TMRE cannot be retained on the mitochondria. Carbonylcyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) is an ionophore that can cross the inner membranes of the mitochondria. FCCP combines protons to disrupt the synthesis of ATP and cause changes in the membrane potential. FCCP is able to eliminate the membrane potential and thus often used to deactivate or depolarize the mitochondria. The changes in the membrane potential are analyzed by fluorescent analysis through the treatment of TMRE and FCCP to determine the function of the mitochondria. The cells treated with TMRE are detected by a flow cytometer, and the ratio of the functional mitochondria to the total particles is analyzed based on TMRE analysis. 
     The mass of the mitochondria is measured by Pierce™ BCA Protein Assay Kit. For the operation procedure, please refer to the guideline of this kit. The bovine serum albumin (BSA) is used as the standard in this measurement, and the stock solution of BSA is 2 μg/mL. The reagent A (colorless) and the reagent B (blue) in Pierce™ BCA Protein Assay Kit are prepared in a ratio of 50:1 as the working reagent. The preparation of the standard solution is shown in Table 1. Samples are measured at 562 nm by the spectrophotometer, and the mitochondria mass in the samples is calculated based on the standard curve. In Table 1, the blank is used to correct the background, and the samples are the supernatant comprising mitochondria after extraction. 
     The purity of the mitochondria herein represents the percentage of the functional mitochondria to the total particles in the supernatant and is analyzed by fluorescent analysis through the treatment of only TMRE. 
     The content of the mitochondria herein represents the percentage of the all mitochondria to the total particles in the supernatant and is analyzed by 10-N-Nonyl acridine orange (NAO) staining. NAO is an acridine orange derivative and is generally used as a fluorescent marker of the inner mitochondrial membrane so that NAO can stain functional and non-functional mitochondria for analyzing the content of mitochondria. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 BSA (μg/mL) 
               
               
                   
                   
               
             
            
               
                   
                 — 
                 blank 
               
               
                   
                 — 
                     198 μL working reagent + 2 μL sample 
               
               
                   
                 0 
                 200 μL working reagent 
               
               
                   
                 40 
                 198 μL working reagent + 2 μL BSA 
               
               
                   
                 80 
                 196 μL working reagent + 4 μL BSA 
               
               
                   
                 120 
                 194 μL working reagent + 6 μL BSA 
               
               
                   
                 160 
                 192 μL working reagent + 8 μL BSA 
               
               
                   
                 200 
                  190 μL working reagent + 10 μL BSA 
               
               
                   
                   
               
            
           
         
       
     
     Experiment 1 
     In Experiment 1, the extraction efficiency for different numbers of times for sucking the mixed solution back and forth and different lengths of the sucking needle is studied. This experiment is conducted by the method using the kit according to the second embodiment of the present disclosure. In this experiment, each group contains 1×10 6  cells of the peripheral blood mononuclear cells, the protective solution is 1 mL, and the time for standing is 5 min. In addition, the long needle is a needle of 23 G (0.337 mm of the inner diameter) and 70 mm, the short needle is a needle of 23 G (0.337 mm of the inner diameter) and 15 mm, and the number of times for sucking back and forth is 0, 5, 10, 15, or 20 times. The result is shown in  FIG.  6   , and  FIG.  6    shows the extraction efficiency for different numbers of times for sucking back and forth and different lengths of the needle. 
     The cells in the mixed solution are rubbed and collided with the inner wall of the needle to damage the cell membrane, thereby releasing the mitochondria. Thus, the longer needle and the more times for sucking back and forth cause the longer path that the cells are subjected to friction and the more friction and collision, and this facilitates the increase of the efficiency for damaging the cell membrane. Therefore, as shown in  FIG.  6   , fewer times for sucking back and forth causes the lower extraction efficiency, the increasing times for sucking back and forth increases the extraction efficiency, but the excessive times for sucking back and forth do not have more help for the extraction efficiency. In addition, under the same times for sucking back and forth, the extraction efficiency of the long needle is superior to that of the short needle. According to the result of this experiment, even if the short needle is used and the times for sucking back and forth is 5 times, it still obtains approximately 50% of the extraction efficiency. When the long needle is used and the times for sucking back and forth is 15 times, it obtains close to 100% of the extraction efficiency. 
     Experiment 2 
     In Experiment 2, the cell numbers for per milliliter of the protective solution is studied, and the mitochondria are isolated from peripheral blood mononuclear cells. This experiment is conducted by the method using the kit according to the second embodiment of the present disclosure. In this experiment, the protective solution is NaCl solution with the osmolarity of 42.8 mOsm/L, the sucking needle is a needle of 23 G (0.337 mm of the inner diameter) and 70 mm in length, the number of times for sucking back and forth of the protective solution along with the peripheral blood mononuclear cells therein is 15 times, and the time for standing after sucking is 5 mins. For the first part, each group contains 1×10 6  or 1×10 7  cells of the peripheral blood mononuclear cells, and the protective solution is 0.5, 1, 1.5, or 2 mL. For the second part, 12 mL of the protective solution is used for 3×10 8  or 3×10 9  cells of the peripheral blood mononuclear cells, that is, 2.5×10 7  or 2.5×10 8  cells/mL for each group. The result of the first part is shown in  FIG.  7    and Table 2,  FIG.  7    shows the extraction efficiency for different numbers of the cells and different volumes of the protective solution. The result of the second part is shown in Table 2. 
     As shown in  FIG.  7    and Table 2, when the cell number is 133 10 6  cells, 1 mL of the protective solution has excellent extraction efficiency of 80% or more. When the cell number is 1×10 7  cells, 2 mL of the protective solution has excellent extraction efficiency of 80% or more. When the cell number is 1×10 6  to 2.5×10 8 cells/mL, the protective solution has good extraction efficiency of 70% or more. According to the result of this experiment, each milliliter of the protective solution that is NaCl solution with the osmolarity of 42.8 mOsm/L may be used for treating approximately 2.5×10 8  or less cells, preferably 1×10 6  to 2.5×10 8  cells. Preferably, each milliliter of the protective solution that is NaCl solution with the osmolarity of 42.8 mOsm/L may be used for treating approximately 2.5×10 8  or less cells of peripheral blood mononuclear cells, preferably 1×10 6  to 2.5×10 8  cells of peripheral blood mononuclear cells. 
     
       
         
           
               
               
             
               
                   
                 TABLE 2 
               
             
            
               
                   
                   
               
               
                   
                 Cell 
               
               
                   
                 Peripheral blood mononuclear cell 
               
               
                   
                 Cell number (cells/mL) 
               
            
           
           
               
               
               
               
               
            
               
                   
                 1 × 10 6   
                 5 × 10 6   
                 2.5 × 10 7   
                 2.5 × 10 8   
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 Extraction 
                 90.83 ± 3.03 
                  92.5 ± 7.23 
                 95.07 ± 3.77 
                 71.17 ± 6.14 
               
               
                 efficiency (%) 
               
               
                 Purity (%) 
                  15.7 ± 4.97 
                 36.15 ± 7.22 
                 49.41 ± 3.77 
                  47.3 ± 6.66 
               
               
                 Content (%) 
                 69.58 ± 9.61 
                 75.95 ± 6.19 
                 76.55 ± 7.11 
                 72.53 ± 5.95 
               
               
                   
               
            
           
         
       
     
     Experiment 3 
     In Experiment 3, the extraction efficiency for different times for standing is studied. 
     This experiment is conducted by the method using the kit according to the second embodiment of the present disclosure. In this experiment, each group contains 1×10 6  cells of the peripheral blood mononuclear cells, the sucking needle is a needle of 23 G (0.337 mm of the inner diameter) and 70 mm, the number of times for sucking back and forth is 15 times, and the protective solution is 1 mL. In addition, the time for standing is 0, 5, 10, 15, or 30 min. The result is shown in  FIG.  8   ,  FIG.  8    shows the extraction efficiency for different times for standing. 
     Standing of the mixed solution allows the hypotonic protective solution has enough time to diffuse to facilitate the further destruction of the damaged cell membranes, thereby increasing the efficiency for destroying the cell membrane. As shown in  FIG.  8   , after sucking the mixed solution back and forth, the mixed solution that stands for minutes has a better extraction efficiency than the mixed solution without standing. According to the result of this experiment, when the mixed solution stands for at least 5 min, it obtains above 80% of the extraction efficiency, and there is no significant change if being stood for longer time. 
     Experiment 4 
     In Experiment 4, the mitochondria are isolated from the peripheral blood mononuclear cells. In this experiment, the number of the peripheral blood mononuclear cells in the blood (about 8 mL of the peripheral blood) is approximately 2.5×10 6 , the protective solution is 1 mL of NaCl solution with the osmolarity of 42.8 mOsm/L, the sucking needle is a needle of 23 G (0.337 mm of the inner diameter) and 70 mm, the number of times for sucking back and forth is 15 times, and the time for standing is 5 min. The result is shown in Table 3. There are 8.30 μg of the mitochondria are isolated from 2.5×10 6  cells of the peripheral blood mononuclear cells, and the percentage of the functional mitochondria to the total particles in the supernatant, represented as purity in Table 3, is 48.55%. 
     Experiment 5 
     In Experiment 5, the mitochondria are isolated from the adipose-derived stem cells. In this experiment, the adipose-derived stem cells are approximately 5×10 6  cells, the protective solution is 1 mL of NaCl solution with the osmolarity of 42.8 mOsm/L, the sucking needle is a needle of 23 G (0.337 mm of the inner diameter) and 70 mm, the number of times for sucking back and forth is 15 times, and the time for standing is 5 min. The result is shown in Table 3. There are 10.97 μg of the mitochondria are isolated from 5×10 6  cells of the adipose-derived stem cells, and the percentage of the functional mitochondria to the total particles in the supernatant, represented as purity in Table 3, is 39.68%. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                 Experiment 4 
                 Experiment 5 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Cell 
                 peripheral blood 
                 adipose-derived 
               
               
                   
                   
                 mononuclear cell 
                 stem cell 
               
               
                   
                 Cell number 
                 2.5 × 10 6   
                 5 × 10 6   
               
               
                   
                 Mitochondria mass (μg) 
                  8.30 
                 10.97 
               
               
                   
                 Purity (%) 
                 48.55 
                 39.68 
               
               
                   
                   
               
            
           
         
       
     
     The embodiments according to the present disclosure provide a kit and a method for isolating mitochondria. The cell membranes of the cells may be destroyed by the sucking needle and the protective solution in the kit through the friction of the cells in the sucking needle with the help of the protective solution, and this way of destroying the cell membranes may not damage the mitochondria in the cells. Therefore, the mitochondria may be isolated from cells with high efficiency in a simple and convenient way, and the isolated mitochondria may have excellent function and activity. 
     The protective solution in the kit for isolating mitochondria according to the present disclosure will be further described below. 
     The protective solution is a solution used for isolating mitochondria from cells and protecting the isolated mitochondria. The osmolarity of the protective solution may be greater than 0 and less than or equal to 220 mOsm/L. In some embodiments, the osmolarity of the protective solution may be 42.8 mOsm/L to 220 mOsm/L. In some embodiments, the osmolarity of the protective solution may be 42.8 mOsm/L to 113 mOsm/L. In some embodiments, the protective solution may comprise NaCl, glucose, NaH 2 PO 4 , or mannitol. In some embodiments, the protective solution may comprise NaCl and glucose, and the weight ratio of NaCl to glucose may be 1:0.06 to 1:2560. In some embodiments, the protective solution may comprise NaCl and NaH 2 PO 4 , and the weight ratio of NaCl to NaH 2 PO 4  may be 1:0.015 to 1:133. In some embodiments, the protective solution may comprise glucose and NaH 2 PO 4 , and the weight ratio of glucose to NaH 2 PO 4  may be 1:0.0007 to 1:22. In some embodiments, the protective solution may only comprise NaCl and glucose without other solutes. In some embodiments, the protective solution may only comprise NaCl and NaH 2 PO 4  without other solutes. In some embodiments, the protective solution may only comprise glucose and NaH 2 PO 4  without other solutes. 
     The composition and the osmolarity of the examples (Ex.) of the protective solution according to the present disclosure and the comparative examples (Com.) of the extraction solution are shown in Table 4 and 5. 
     
       
         
           
               
               
             
               
                   
                 TABLE 4 
               
             
            
               
                   
                   
               
               
                   
                 Composition 
               
            
           
           
               
               
               
               
               
            
               
                   
                 NaCl 
                 Glucose 
                 NaH 2 PO 4   
                 Mannitol 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Osmolarity 
                 42.8 
                 Ex. 1 
                 Ex. 4 
                 Ex. 7 
                 Ex. 10 
               
               
                 (mOsm/L) 
                 113 
                 Ex. 2 
                 Ex. 5 
                 Ex. 8 
                 Ex. 11 
               
               
                   
                 220 
                 Ex. 3 
                 Ex. 6 
                 Ex. 9 
                 Ex. 12 
               
               
                   
                 520 
                 Com. 1 
                 Com. 3 
                 Com. 5 
                 Com. 7 
               
               
                   
                 1025 
                 Com. 2 
                 Com. 4 
                 Com. 6 
                 Com. 8 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                   
                 TABLE 5 
               
               
                   
                   
               
               
                   
                 Composition 
                 Osmolarity (mOsm/L) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Ex. 13 
                 NaCl + Glucose 
                 42.8 
               
               
                   
                 Ex. 14 
                     NaCl + NaH 2 PO 4   
                 42.8 
               
               
                   
                 Ex. 15 
                 Glucose + NaH 2 PO 4   
                 42.8 
               
               
                   
                   
               
            
           
         
       
     
     Experiments 6 to 8 demonstrate isolating mitochondria by the protective solution according to the present disclosure. The mitochondria are isolated by using the examples (Ex.) of the protective solution according to the present disclosure and the comparative examples (Com.) of the extraction solution, according to the method and the kit of the second embodiment of the present disclosure, and the function and the extraction efficiency of the isolated mitochondria are analyzed. 
     Specifically, firstly, the cells and the protective solution are mixed to form the mixed solution. In detail, a certain number of the cells (1×10 6  cells) are collected. The cells are the peripheral blood mononuclear cells or the adipose-derived stem cells, but the present disclosure is not limited thereto. The cells may be any cells that have mitochondria. Then, the cells and 1 mL of the protective solution of the examples or 1 mL of the extraction solution of the comparative examples are mixed to form the mixed solution. 
     Subsequently, the cells in the mixed solution are rubbed to damage the cell membranes of the cells to facilitate the entry of the protective solution into the cells and the destruction of the cell membranes of the cells. In detail, during mixing the cells and the protective solution, the cell membranes of the cells is damaged due to friction. The damaged cell membranes facilitate the entry of the protective solution into the cells, thereby further destroying the cell membrane. Here, a long needle of 23 G and 70 mm is used to suck the mixed solution back and forth to damage the cells, but the present disclosure is not limited thereto. When using the protective solution according to the present disclosure, a needle of different sizes may be used corresponding to the experimental equipment, or machines such a grinder may be used, thereby damaging the cell membranes to facilitate the entry of the protective solution into the cells so as to further destroy the cell membrane. 
     Subsequently, the mixed solution is centrifuged to be stratified. In detail, the mixed solution stands and then centrifuged, and the mixed solution is stratified to form the supernatant comprising the mitochondria and the pellet comprising cell debris. 
     Subsequently, the supernatant comprising the mitochondria is collected. In detail, the supernatant comprising the mitochondria may be collected by using needle or pipette or by pouring. 
     Finally, the supernatant comprising the mitochondria is analyzed for the extraction efficiency and the function of the mitochondria. 
     Experiment 6 
     In Experiment 6, the mitochondria are isolated from the peripheral blood mononuclear cells by using the protective solution comprising NaCl with different osmolarities, and the extraction efficiency and the function of the mitochondria are analyzed. Please refer to  FIGS.  9  and  10   , wherein  FIG.  9    shows the function of mitochondria isolated from the peripheral blood mononuclear cells by the protective solution comprising NaCl with different osmolarities, and  FIG.  10    shows the extraction efficiency of mitochondria isolated from the peripheral blood mononuclear cells by the protective solution comprising NaCl with different osmolarities. In  FIG.  10   , “#” indicates a significant difference (P&lt;0.05) compared with the comparative example of 520 mOsm/L, “*” indicates a significant difference (P&lt;0.05) compared with the comparative example of 1025 mOsm/L. 
     From  FIG.  9    and Table 6, it can be seen that by using the protective solution comprising NaCl with the osmolarity greater than 0 and less than or equal to 220 mOsm/L to isolate mitochondria, the isolated mitochondria have excellent function, and the function of the mitochondria is greater than 10%. From  FIG.  10    and Table 6, it can be seen that by using the protective solution comprising NaCl with the osmolarity greater than 0 and less than or equal to 220 mOsm/L to isolate mitochondria, excellent extraction efficiency is obtained, and the extraction efficiency is greater than 50%. Taking together, by using the protective solution comprising NaCl with the osmolarity greater than 0 and less than or equal to 220 mOsm/L to isolate mitochondria from the peripheral blood mononuclear cells, the mitochondria maintain their function (greater than 10%) while obtaining the excellent extraction efficiency (greater than 50%). 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 6 
               
               
                   
                   
               
               
                   
                 Osmolarity 
                   
                 Extraction 
               
               
                   
                 (mOsm/L) 
                 Function (%) 
                 efficiency (%) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Ex. 1 
                 42.8 
                 17.2 ± 3.8 
                 97.0 ± 2.0 
               
               
                   
                 Ex. 2 
                 113 
                 15.4 ± 5.2 
                 72.7 ± 4.2 
               
               
                   
                 Ex. 3 
                 220 
                 15.7 ± 5.0 
                 56.7 ± 6.5 
               
               
                   
                 Com. 1 
                 520 
                 10.4 ± 3.8 
                 47.6 ± 5.9 
               
               
                   
                 Com. 2 
                 1025 
                  9.2 ± 5.1 
                 40.6 ± 7.9 
               
               
                   
                   
               
               
                   
                 Cell: peripheral blood mononuclear cell 
               
            
           
         
       
     
     Experiment 7 
     In Experiment 7, the mitochondria are isolated from the adipose-derived stem cells by using the protective solution comprising NaCl, glucose, NaH 2 PO 4 , or mannitol with different osmolarities, and the extraction efficiency and the function of the mitochondria are analyzed. Please refer to  FIGS.  11  and  12   , wherein  FIG.  11    shows the function of mitochondria isolated from the adipose-derived stem cells by the protective solution comprising NaCl, glucose, NaH 2 PO 4 , or mannitol with different osmolarities, and  FIG.  12    shows the extraction efficiency of mitochondria isolated from the adipose-derived stem cells by the protective solution comprising NaCl, glucose, NaH 2 PO 4 , or mannitol with different osmolarities. In  FIGS.  11  and  12   , “#” indicates a significant difference (P&lt;0.05) compared with the comparative example of 520 mOsm/L, “*” indicates a significant difference (P&lt;0.05) compared with the comparative example of 1025 mOsm/L. 
     From  FIG.  11    and Table 7, it can be seen that by using the protective solution comprising NaCl, glucose, NaH 2 PO 4 , or mannitol with the osmolarity greater than 0 and less than or equal to 220 mOsm/L to isolate mitochondria, the isolated mitochondria have excellent function, and the function of the mitochondria is greater than 10%. From  FIG.  12    and Table 7, it can be seen that by using the protective solution comprising NaCl, glucose, NaH 2 PO 4 , or mannitol with the osmolarity greater than 0 and less than or equal to 220 mOsm/L to isolate mitochondria, excellent extraction efficiency is obtained, and the extraction efficiency is greater than 50%. Taking together, by using the protective solution comprising NaCl, glucose, NaH 2 PO 4 , or mannitol with the osmolarity greater than 0 and less than or equal to 220 mOsm/L to isolate mitochondria from the adipose-derived stem cells, the mitochondria maintain their function (greater than 10%) while obtaining the excellent extraction efficiency (greater than 50%). 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 7 
               
               
                   
                   
               
               
                   
                 Osmolarity 
                   
                 Extraction 
               
               
                   
                 (mOsm/L) 
                 Function (%) 
                 efficiency (%) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 NaCl 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Ex. 1 
                 42.8 
                 20.7 ± 6.5 
                 95.9 ± 3.6 
               
               
                   
                 Ex. 2 
                 113 
                 21.9 ± 5.4 
                  76.9 ± 20.1 
               
               
                   
                 Ex. 3 
                 220 
                 14.6 ± 1.8 
                 59.4 ± 9.4 
               
               
                   
                 Com. 1 
                 520 
                  2.0 ± 1.0 
                 52.6 ± 3.8 
               
               
                   
                 Com. 2 
                 1025 
                  2.0 ± 0.2 
                 41.5 ± 2.4 
               
            
           
           
               
            
               
                 Glucose 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Ex. 4 
                 42.8 
                 12.4 ± 1.8 
                 99.5 ± 0.9 
               
               
                   
                 Ex. 5 
                 113 
                 19.8 ± 3.4 
                  82.9 ± 14.9 
               
               
                   
                 Ex. 6 
                 220 
                 13.1 ± 2.6 
                 73.9 ± 9.8 
               
               
                   
                 Com. 3 
                 520 
                  2.2 ± 1.1 
                 66.4 ± 7.0 
               
               
                   
                 Com. 4 
                 1025 
                  6.1 ± 0.5 
                 65.8 ± 6.9 
               
            
           
           
               
            
               
                 NaH 2 PO 4   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Ex. 7 
                 42.8 
                 12.3 ± 2.9 
                 97.7 ± 2.1 
               
               
                   
                 Ex. 8 
                 113 
                 11.6 ± 0.9 
                  75.5 ± 12.7 
               
               
                   
                 Ex. 9 
                 220 
                 11.5 ± 4.0 
                 51.6 ± 9.7 
               
               
                   
                 Com. 5 
                 520 
                  4.5 ± 2.0 
                 42.7 ± 6.7 
               
               
                   
                 Com. 6 
                 1025 
                  2.4 ± 2.1 
                 35.9 ± 7.8 
               
            
           
           
               
            
               
                 Mannitol 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Ex. 10 
                 42.8 
                 17.2 ± 0.5 
                 100 ± 0  
               
               
                   
                 Ex. 11 
                 113 
                 13.0 ± 4.8 
                 98.2 ± 0.4 
               
               
                   
                 Ex. 12 
                 220 
                 13.4 ± 0.5 
                 78.3 ± 7.9 
               
               
                   
                 Com. 7 
                 520 
                  7.5 ± 5.1 
                 64.8 ± 9.5 
               
               
                   
                 Com. 8 
                 1025 
                  5.0 ± 0.2 
                   55 ± 12.0 
               
               
                   
                   
               
               
                   
                 Cell: adipose-derived stem cell 
               
            
           
         
       
     
     Experiment 8 
     In Experiment 8, the mitochondria are isolated from the adipose-derived stem cells by using the protective solution comprising different compositions with an osmolarity of 42.8 mOsm/L, and the function of the mitochondria is analyzed. Please refer to  FIG.  13    and Table 8, wherein  FIG.  13    shows the function of mitochondria isolated from the adipose-derived stem cells by the protective solution comprising different compositions with an osmolarity of 42.8 mOsm/L. 
     From  FIG.  13    and Table 8, it can be seen that under the osmolarity of 42.8 mOsm/L, by using the protective solution comprising single composition (Ex. 1, NaCl; Ex. 4, glucose; Ex. 7, NaH 2 PO 4 ) to isolate mitochondria from the adipose-derived stem cells, the isolated mitochondria have excellent function, and the function of the mitochondria is greater than 10%. In addition, under the osmolarity of 42.8 mOsm/L, by using the protective solution comprising two of NaCl, glucose, and NaH 2 PO 4  (Ex. 13, NaCl and glucose; Ex. 14, NaCl and NaH 2 PO 4 ; Ex. 15, Glucose+NaH 2 PO 4 ) to isolate mitochondria from the adipose-derived stem cells, the isolated mitochondria have more excellent function, and the function of the mitochondria is greater than 15%. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 8 
               
               
                   
                   
               
               
                   
                 Composition 
                 Function (%) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Ex. 1 
                 NaCl 
                 18.0 ± 7.8 
               
               
                   
                 Ex. 4 
                 Glucose 
                 12.4 ± 1.8 
               
               
                   
                 Ex. 7 
                 NaH 2 PO 4   
                 12.3 ± 2.9 
               
               
                   
                 Ex. 13 
                 NaCl + Glucose 
                 22.5 ± 5.8 
               
               
                   
                 Ex. 14 
                     NaCl + NaH 2 PO 4   
                 20.4 ± 6.1 
               
               
                   
                 Ex. 15 
                 Glucose + NaH 2 PO 4   
                 17.3 ± 2.7 
               
               
                   
                   
               
               
                   
                 Cell: adipose-derived stem cell 
               
            
           
         
       
     
     Experiment 9 
     In Experiment 9, the cell numbers for per milliliter of the protective solution is studied, and the mitochondria are isolated from adipose-derived stem cells. This experiment is conducted by the method using the kit according to the second embodiment of the present disclosure. In this experiment, the protective solution is NaCl solution with the osmolarity of 42.8 mOsm/L. The sucking needle is a needle of 23 G (0.337 mm of the inner diameter) and 70 mm in length, the number of times for sucking back and forth of the protective solution along with the adipose-derived stem cells therein is 15 times, and the time for standing after sucking is 5 mins. 12 mL of the protective solution is used for 3×10 8  or 3×10 9  cells of the adipose-derived stem cells, that is, 2.5×10 7  or 2.5×10 8  cells/mL for each group. The result is shown in Table 9. 
     From Table 9, it can be seen that when the protective solution is used at 2.5×10 7  or 2.5×10 8  cells/mL, a good extraction efficiency which is greater than 65% is obtained. The percentage of the functional mitochondria to the total particles in the supernatant, represented as purity in Table 9, is 50.22% and 52.6% for each group. The percentage of all mitochondria to the total particles in the supernatant, represented as content in Table 9, is 78.91% and 73.8% for each group. Further, when the protective solution is used at 2.5×10 7  cells/mL, an excellent extraction efficiency which is greater than 90% is obtained. 
     From Experiment 9, it can be seen that each milliliter of the protective solution may be used for treating approximately 2.5×10 8  or less cells, preferably 2.5×10 7  to 2.5×10 8  cells. Preferably, each milliliter of the protective solution that is NaCl solution with the osmolarity of 42.8 mOsm/L may be used for treating approximately 2.5×10 8  or less cells of adipose-derived stem cells, preferably 2.5×10 7  to 2.5×10 8  cells for adipose-derived stem cells. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 9 
               
             
            
               
                   
                   
               
               
                   
                 Cell 
                   
               
               
                   
                 Adipose-derived stem cell 
               
            
           
           
               
               
               
               
            
               
                   
                 Cell number (cells/mL) 
                 2.5 × 10 7   
                 2.5 × 10 8   
               
               
                   
                   
               
               
                   
                 Extraction efficiency (%) 
                 93.11 ± 2.10 
                 67.2 ± 3.80 
               
               
                   
                 Purity (%) 
                 50.22 ± 9.80 
                 52.6 ± 5.39 
               
               
                   
                 Content (%) 
                 78.91 ± 7.47 
                 73.8 ± 6.09 
               
               
                   
                   
               
            
           
         
       
     
     According to the above results, using a hypotonic solution has better extraction efficiency than using a hypertonic solution. In addition, the above experiments demonstrate that by using the protective solution of the osmolarity greater than 0 and less than or equal to 220 mOsm/L to isolate mitochondria, the mitochondria maintain their function while obtaining the excellent extraction efficiency. Further, the above experiments demonstrate that by using the protective solution comprising two of NaCl, glucose, and NaH 2 PO 4  to isolate mitochondria, the isolated mitochondria have more excellent function. In different experiments, the differences in numerical values may be derived from the experimental error in each batch of experiments, and these differences in numerical values are in the acceptable range in the art. 
     The embodiments according to the present disclosure provide a protective solution for isolating mitochondria from cells and protecting the isolated mitochondria. When damaging the cell membrane, for example, when using a long needle to suck back and forth to damage the cell membranes due to friction, the osmolarity of the protective solution is greater than 0 and less than or equal to 220 mOsm/L so that the cell membranes of the cells may be destroyed with the help of the hypotonicity of the protective solution to release the mitochondria, and this way of destroying the cell membranes may not damage the mitochondria in the cells. Therefore, the mitochondria may be isolated from cells with high efficiency in a simple and convenient way, and the isolated mitochondria may have excellent function and activity. 
     Though the embodiment according to the present disclosure is described above, the present disclosure is not limited thereto. Without departing from the spirit and scope of the present disclosure, any skilled person in the field can do some appropriate change in the shapes, structures, characteristics and spirits. The extent of patent protection subject to the claim in the specification.