TW202244276A - Protective solution for isolating mitochondria from cells and protecting mitochondria and use of protective solution for isolating mitochondria from cells and maintaining mitochondria activity - Google Patents

Abstract

The present disclosure provides a protective solution for isolating mitochondria from cells and protecting mitochondria, wherein the osmolarity of the protective solution is greater than 0 and less than or equal to 220 mOsm/L. Using the protective solution to isolate mitochondria from cells can obtain mitochondria with high efficiency and maintain the function of mitochondria.

Description

自細胞分離並保護粒線體用之萃取保護液及萃取保護液用於自細胞分離粒線體並維持粒線體活性的用途Extraction protection solution for separating and protecting mitochondria from cells and use of extraction protection solution for separating mitochondria from cells and maintaining mitochondrial activity

本發明係關於自細胞分離並保護粒線體用之萃取保護液。The present invention relates to an extraction and protection solution for separating and protecting mitochondria from cells.

粒線體(Mitochondrion)是細胞內重要的胞器之一。粒線體除了是能量(ATP)的提供者外，更是氧化壓力、細胞凋亡、細胞間溝通與訊號傳遞的重要調控者。近年許多國際期刊証實粒線體跟老化與疾病形成有著密不可分的關係(Braticet al., 2013)。更有國際研究團隊指出可以透過移植粒線體的方式來修補細胞或組織的損傷(Pacak et al., 2015; Cowanet al., 2017)。Mitochondrion is one of the important organelles in cells. In addition to providing energy (ATP), mitochondria are also important regulators of oxidative stress, apoptosis, intercellular communication and signal transmission. In recent years, many international journals have confirmed that mitochondria are closely related to aging and disease formation (Bratic et al., 2013). Some international research teams have pointed out that cell or tissue damage can be repaired by transplanting mitochondria (Pacak et al., 2015; Cowan et al., 2017).

因此，如何快速獲取粒線體並維持粒線體的功能及活性為目前的發展方向之一。Therefore, how to quickly obtain mitochondria and maintain the function and activity of mitochondria is one of the current development directions.

本發明實施例提供自細胞分離粒線體用之萃取保護液，可快速獲取粒線體並維持粒線體的功能及活性。The embodiment of the present invention provides an extraction protection solution for separating mitochondria from cells, which can quickly obtain mitochondria and maintain the function and activity of mitochondria.

本發明實施例提供自細胞分離並保護粒線體用之萃取保護液，萃取保護液的滲透度為大於0且小於等於220 mOsm/L。An embodiment of the present invention provides an extraction and protection solution for separating and protecting mitochondria from cells. The osmolarity of the extraction and protection solution is greater than 0 and less than or equal to 220 mOsm/L.

本發明實施例提供一種萃取保護液用於自細胞分離粒線體並維持粒線體活性之用途，其中萃取保護液為低張溶液，且該萃取保護液包含氯化鈉、葡萄糖、磷酸二氫鈉或甘露醇。An embodiment of the present invention provides an extraction and protection solution for separating mitochondria from cells and maintaining mitochondrial activity, wherein the extraction and protection solution is a hypotonic solution, and the extraction and protection solution contains sodium chloride, glucose, dihydrogen phosphate sodium or mannitol.

本發明實施例提供自細胞分離並保護粒線體用之萃取保護液。在破壞細胞膜時，例如以長針來回抽吸使細胞膜因摩擦而受損時，藉由此萃取保護液的滲透度為大於0且小於等於220 mOsm/L，可使細胞在萃取保護液之滲透度的幫助下容易破壞細胞膜進而釋出粒線體。這種輔助破壞細胞膜的方式較不會損及粒線體，故可以簡單便利的方式高效率地自細胞分離出粒線體，更重要的是，分離出的粒線體能夠維持良好的功能。An embodiment of the present invention provides an extraction and protection solution for separating and protecting mitochondria from cells. When destroying the cell membrane, for example, when the cell membrane is damaged due to friction by pumping back and forth with a long needle, the permeability of the extraction protection solution is greater than 0 and less than or equal to 220 mOsm/L. With the help of the cell membrane, it is easy to destroy the cell membrane and release the mitochondria. This method of assisting in the destruction of the cell membrane will not damage the mitochondria, so the mitochondria can be isolated from the cells in a simple and convenient way, and more importantly, the isolated mitochondria can maintain good functions.

於以下實施方式中詳細敘述本發明之詳細特徵及優點，其內容足以使任何熟習相關技藝者了解本發明之技術內容並據以實施，且根據本說明書所揭露的內容、申請專利範圍及圖式，任何熟習相關技藝者可輕易理解本發明相關之目的及優點。以下實施例係進一步詳細說明本發明之觀點，但非以任何觀點限制本發明之範疇。The detailed features and advantages of the present invention are described in detail in the following embodiments, the content of which is sufficient to enable anyone familiar with the relevant art to understand the technical content of the present invention and implement it accordingly, and according to the content disclosed in this specification, the scope of the patent application and the drawings , anyone skilled in the art can easily understand the purpose and advantages of the present invention. The following examples are to further describe the concept of the present invention in detail, but not to limit the scope of the present invention in any way.

本發明實施方式與實施例提供用以分離粒線體的套組、分離粒線體的方法以及自細胞分離並保護粒線體用之萃取保護液。The embodiments and examples of the present invention provide a kit for isolating mitochondria, a method for isolating mitochondria, and an extraction protection solution for isolating and protecting mitochondria from cells.

首先說明本發明第一實施方式之用以分離粒線體的套組。請參考圖1，圖1為本發明第一實施方式之用以分離粒線體的套組的示意圖。本發明第一實施方式之用以分離粒線體的套組1包含萃取用管體11、萃取保護液12及抽吸用針頭13。Firstly, the kit for isolating mitochondria according to the first embodiment of the present invention will be described. Please refer to FIG. 1 , which is a schematic diagram of a kit for isolating mitochondria according to a first embodiment of the present invention. The kit 1 for separating mitochondria according to the first embodiment of the present invention includes an extraction tube body 11 , an extraction protection solution 12 and a suction needle 13 .

萃取用管體11通常為圓管體。萃取用管體11具有封閉的底端和開口，並用以盛裝細胞以及各種溶液。細胞為具有粒線體的細胞。在本實施方式中，萃取用管體11為圓管體，但不以此為限。在其他實施方式中，萃取用管體可為任何形狀的管體，只要為離心機可接受之管體即可。The extraction tube body 11 is usually a round tube body. The extraction tube body 11 has a closed bottom end and an opening, and is used for containing cells and various solutions. A cell is a cell having mitochondria. In this embodiment, the tube body 11 for extraction is a round tube body, but it is not limited thereto. In other embodiments, the tube for extraction can be of any shape, as long as it is acceptable for a centrifuge.

萃取保護液12用以在萃取用管體11中與細胞混合，形成混合溶液。在本實施方式中，萃取保護液12的滲透度(osmolarity)為大於0且小於等於220 mOsm/L，但不以此為限。在其他實施方式中，萃取保護液可為常見之可保存胞器或維持胞器活性的緩衝液。在本實施方式中，萃取保護液12盛裝在不同於萃取用管體11的容器中，但不以此為限。在其他實施方式中，萃取保護液亦可盛裝在萃取用管體中。藉此，在分離粒線體的操作過程中，可直接將細胞加入萃取用管體中的萃取保護液中以形成混合溶液。The extraction protection solution 12 is used to mix with the cells in the extraction tube body 11 to form a mixed solution. In this embodiment, the osmolarity of the extraction protection solution 12 is greater than 0 and less than or equal to 220 mOsm/L, but it is not limited thereto. In other embodiments, the extraction protection solution can be a common buffer that can preserve organelles or maintain the activity of organelles. In this embodiment, the extraction protection solution 12 is contained in a container different from the extraction tube body 11, but it is not limited thereto. In other embodiments, the extraction protection solution can also be contained in the extraction tube. Thereby, during the operation of isolating mitochondria, the cells can be directly added to the extraction protection solution in the extraction tube body to form a mixed solution.

抽吸用針頭13具有連接端131和尖端132。連接端131用以連接到針筒。尖端132用以吸取或注射溶液。藉由抽吸用針頭13來回抽吸萃取用管體11中包含細胞與萃取保護液12的混合溶液，混合溶液中的細胞會在抽吸用針頭13中與針頭的內壁來回摩擦而使細胞膜破裂，進而釋放出粒線體。在本實施方式中，抽吸用針頭13的長度配合萃取用管體11的長度，為70毫米，但不以此為限。在其他實施方式中，抽吸用針頭的長度亦可為15毫米。在本實施方式中，抽吸用針頭13的內徑為0.337毫米，但不以此為限。在其他實施方式中，抽吸用針頭13的內徑亦可為0.318毫米至0.356毫米之間。The suction needle 13 has a connection end 131 and a tip 132 . The connection end 131 is used to connect to the syringe. Tip 132 is used to aspirate or inject solutions. The mixed solution containing the cells and the extraction protection solution 12 in the extraction tube body 11 is sucked back and forth by the suction needle 13, the cells in the mixed solution will rub back and forth with the inner wall of the needle in the suction needle 13 to make the cell membrane rupture, releasing the mitochondria. In this embodiment, the length of the suction needle 13 matches the length of the extraction tube 11 and is 70 mm, but it is not limited thereto. In other embodiments, the length of the suction needle can also be 15 mm. In this embodiment, the inner diameter of the suction needle 13 is 0.337 mm, but it is not limited thereto. In other embodiments, the inner diameter of the suction needle 13 may also be between 0.318 mm and 0.356 mm.

在本實施方式中，萃取用管體11、萃取保護液12及抽吸用針頭13皆為無菌狀態，但不以此為限。在其他實施方式中，亦可於使用前再進行滅菌處理。In this embodiment, the extraction tube body 11 , the extraction protection solution 12 and the suction needle 13 are all sterile, but not limited thereto. In other embodiments, sterilization can also be performed before use.

接下來說明使用本發明第一實施方式之套組分離粒線體的方法。請參考圖2與圖3。圖2為使用本發明第一實施方式之套組分離粒線體的方法的流程圖。圖3為本發明第一實施方式之套組的使用示意圖。Next, the method for isolating mitochondria using the kit according to the first embodiment of the present invention will be described. Please refer to Figure 2 and Figure 3. Fig. 2 is a flowchart of a method for isolating mitochondria using the kit according to the first embodiment of the present invention. Fig. 3 is a schematic view of the use of the kit according to the first embodiment of the present invention.

首先，在萃取用管體11中混合複數個細胞與萃取保護液形成混合溶液，萃取保護液的滲透度為大於0且小於等於220 mOsm/L(S11)。詳細來說，可使用其他針頭、移液管或以傾倒的方式將細胞與萃取保護液12加入萃取用管體11中以形成混合溶液，加入細胞及萃取保護液12的順序並無限制。細胞可為周邊血的單核球細胞或幹細胞。周邊血的單核球細胞可以任何習知的方式將周邊血分層，並取出所需之單核球細胞。在本實施方式中，萃取保護液12為低張溶液，滲透度為大於0且小於等於220 mOsm/L，且萃取保護液12每1毫升可處理約1×10 ⁶至5×10 ⁶個細胞，但不以此為限。在本實施方式中，細胞與萃取保護液12在萃取用管體中混合，但不以此為限。在其他實施方式中，細胞與萃取保護液亦可在萃取用管體以外的容器中混合後再加入至萃取用管體。 Firstly, a plurality of cells are mixed with the extraction protection solution in the extraction tube body 11 to form a mixed solution, and the osmolarity of the extraction protection solution is greater than 0 and less than or equal to 220 mOsm/L (S11). In detail, other needles, pipettes or pouring methods can be used to add the cells and extraction protection solution 12 into the extraction tube body 11 to form a mixed solution, and the order of adding the cells and extraction protection solution 12 is not limited. The cells may be monocytes or stem cells from peripheral blood. Mononuclear Cells from Peripheral Blood Peripheral blood can be stratified and desired mononuclear cells removed in any known manner. In this embodiment, the extraction protection solution 12 is a hypotonic solution, the osmolarity is greater than 0 and less than or equal to 220 mOsm/L, and the extraction protection solution 12 can treat about 1×10 ⁶ to 5×10 ⁶ cells per 1 ml , but not limited to this. In this embodiment, the cells and the extraction protection solution 12 are mixed in the extraction tube, but it is not limited thereto. In other embodiments, the cells and the extraction protection solution may also be mixed in a container other than the extraction tube and then added to the extraction tube.

接著，摩擦混合溶液中的細胞，使細胞的細胞膜受損，以幫助萃取保護液進入細胞並破壞細胞的細胞膜(S12)。詳細來說，使用抽吸用針頭13搭配針筒S來回抽吸萃取用管體11中的混合溶液。如圖3所示，使用抽吸用針頭13來回抽吸數次萃取用管體11中的混合溶液，例如來回抽吸至少五次。藉此，混合溶液中的細胞會在抽吸用針頭13中與針頭的內壁來回摩擦而使細胞膜受損，讓萃取保護液由細胞膜受損處進入細胞中而破壞細胞膜，進而釋放出粒線體。Next, the cells in the mixed solution are rubbed to damage the cell membranes of the cells, so as to help extract the protective solution into the cells and destroy the cell membranes of the cells (S12). In detail, the mixed solution in the extraction tube 11 is sucked back and forth by using the suction needle 13 and the syringe S. As shown in FIG. 3 , use the suction needle 13 to suck back and forth the mixed solution in the extraction tube 11 several times, for example, back and forth at least five times. In this way, the cells in the mixed solution will rub back and forth against the inner wall of the needle in the suction needle 13 to damage the cell membrane, allowing the extraction protection solution to enter the cell from the damaged part of the cell membrane to destroy the cell membrane, and then release the granule body.

接著，將混合溶液離心(S13)。詳細來說，將經過抽吸用針頭13來回抽吸後的混合溶液以1500至2500 rpm離心5至15分鐘。透過離心的方式將細胞碎片與含有粒線體的上清液分層。Next, the mixed solution is centrifuged (S13). Specifically, the mixed solution that has been sucked back and forth by the suction needle 13 is centrifuged at 1500 to 2500 rpm for 5 to 15 minutes. Cell debris was separated from the mitochondria-containing supernatant by centrifugation.

最後，採集混合溶液經離心後所得到含有粒線體的上清液(S14)。詳細來說，可使用針頭、移液管或傾倒的方式收集含有粒線體的上清液，以此達到分離出粒線體的目的。Finally, the supernatant containing mitochondria obtained after the mixed solution is collected and centrifuged (S14). In detail, the supernatant containing mitochondria can be collected by needle, pipette or pouring, so as to achieve the purpose of isolating mitochondria.

接下來說明本發明第二實施方式之用以分離粒線體的套組。請參考圖4，圖4為本發明第二實施方式之用以分離粒線體的套組的示意圖。本發明第二實施方式與第一實施方式相似，以下僅對差異處說明。本發明第二實施方式之用以分離粒線體的套組2除了包含萃取用管體21、萃取保護液22、抽吸用針頭23以外，還更包含塞子24、平衡管25、採取細胞用針頭26及採集粒線體用針頭27。第二實施方式之萃取用管體21、萃取保護液22及抽吸用針頭23的描述，請參考第一實施方式之萃取用管體11、萃取保護液12及抽吸用針頭13的描述，於此不再贅述。Next, the kit for isolating mitochondria according to the second embodiment of the present invention will be described. Please refer to FIG. 4 , which is a schematic diagram of a set for isolating mitochondria according to a second embodiment of the present invention. The second embodiment of the present invention is similar to the first embodiment, and only the differences will be described below. The kit 2 for isolating mitochondria according to the second embodiment of the present invention not only includes the extraction tube body 21, the extraction protection solution 22, and the suction needle 23, but also includes a plug 24, a balance tube 25, and a cell collection tube. A needle 26 and a needle 27 for collecting mitochondria. For the description of the extraction tube body 21, extraction protection solution 22 and suction needle 23 of the second embodiment, please refer to the description of the extraction tube body 11, extraction protection solution 12 and suction needle 13 of the first embodiment, No more details here.

塞子24的大小對應萃取用管體21之開口的大小，塞子24用以密封萃取用管體21，可防止萃取用管體21內的溶液受到外界的污染。在本實施方式中，塞子24為橡膠塞，在以針頭穿刺後仍可維持萃取用管體21的密封性，但不以此為限。在其他實施方式中，可不具有塞子，而是以蓋子來封閉萃取用管體。The size of the plug 24 corresponds to the size of the opening of the extraction tube 21, and the plug 24 is used to seal the extraction tube 21 to prevent the solution in the extraction tube 21 from being polluted by the outside. In this embodiment, the stopper 24 is a rubber stopper, which can still maintain the tightness of the extraction tube body 21 after being punctured by a needle, but it is not limited thereto. In other embodiments, there may be no stopper, but a cap to close the extraction tube.

平衡管25的重量與萃取用管體21的重量相等。平衡管25用以在離心萃取用管體21時維持平衡。詳細來說，當萃取用管體21以塞子24密封時，平衡管25亦可具有塞子或與塞子24等重的蓋子，以在離心時維持平衡。當萃取用管體21內裝有溶液時，亦可在平衡管25內加入等重的溶液以在離心時維持平衡。在其他實施方式中，平衡管可為與萃取用管體同樣的管體，亦可不具有平衡管，只要在離心時使離心機中的轉盤保持平衡即可。The weight of the balance tube 25 is equal to the weight of the tube body 21 for extraction. The balance tube 25 is used to maintain balance when the tube body 21 for extraction is centrifuged. In detail, when the extraction tube body 21 is sealed with a stopper 24, the balance tube 25 may also have a stopper or a cover with the same weight as the stopper 24 to maintain balance during centrifugation. When the extraction tube body 21 is equipped with a solution, an equal weight solution can also be added into the balance tube 25 to maintain balance during centrifugation. In other embodiments, the balance tube may be the same tube body as the extraction tube body, or may not have a balance tube, as long as the turntable in the centrifuge is kept balanced during centrifugation.

採取細胞用針頭26具有連接端261和尖端262。連接端261用以連接到針筒。尖端262用以吸取或注射溶液。採取細胞用針頭26用以將含有細胞的溶液注入萃取用管體21。採取細胞用針頭26的長度與孔徑之規格可與抽吸用針頭23的長度與孔徑之規格相同，亦可相異。在其他實施方式中，可不具有採取細胞用針頭，而是可使用移液管將溶液注入萃取用管體。The needle 26 for collecting cells has a connection end 261 and a tip 262 . The connection end 261 is used to connect to the syringe. Tip 262 is used to aspirate or inject solutions. The needle 26 for collecting cells is used to inject the solution containing cells into the tube body 21 for extraction. The specifications of the length and aperture of the needle 26 for taking cells can be the same as those of the suction needle 23 or different. In other embodiments, instead of having a needle for collecting cells, a pipette may be used to inject the solution into the extraction tube.

採集粒線體用針頭27具有連接端271和尖端272。連接端271用以連接到針筒。尖端272用以吸取或注射溶液。採集粒線體用針頭27用以採集含有粒線體的上清液。採集粒線體用針頭27的長度與孔徑之規格可與抽吸用針頭23的長度與孔徑之規格相同，亦可相異。在其他實施方式中，可不具有採集粒線體用針頭，而是可使用移液管採集上清液。The needle 27 for collecting mitochondria has a connection end 271 and a tip 272 . The connection end 271 is used to connect to the syringe. Tip 272 is used to aspirate or inject solutions. The needle 27 for collecting mitochondria is used to collect the supernatant containing mitochondria. The specifications of the length and aperture of the needle 27 for collecting mitochondria can be the same as those of the suction needle 23 or different. In other embodiments, instead of having a needle for collecting mitochondria, a pipette can be used to collect the supernatant.

接下來說明使用本發明第二實施方式之套組分離粒線體的方法。請參考圖5。圖5為使用本發明第二實施方式之套組分離粒線體的方法的流程圖。Next, a method for isolating mitochondria using the kit according to the second embodiment of the present invention will be described. Please refer to Figure 5. Fig. 5 is a flowchart of a method for isolating mitochondria using the kit according to the second embodiment of the present invention.

首先，使用採取細胞用針頭26將含有細胞的溶液注入萃取用管體21中(S21)。詳細來說，使用採取細胞用針頭26將含有細胞的溶液注入蓋有塞子24的萃取用管體21中。細胞可為周邊血的單核球細胞或幹細胞。周邊血的單核球細胞可以任何習知的方式將周邊血分層，並以採取細胞用針頭26取出所需之單核球細胞。於此，不使用抽吸用針頭23，而是使用採取細胞用針頭26來注入含有細胞的溶液，可避免與抽吸用針頭23接觸的萃取保護液22受到汙染。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 covered with the stopper 24 using the cell collection needle 26 . The cells may be monocytes or stem cells from peripheral blood. The mononuclear cells of the peripheral blood can be stratified by any known method, and the required mononuclear cells can be taken out with the needle 26 for collecting cells. Here, instead of the suction needle 23, the cell collection needle 26 is used to inject the cell-containing solution, so that the extraction protection solution 22 in contact with the suction needle 23 can be prevented from being contaminated.

接著，將溶液離心(S22)。詳細來說，在平衡管25中加入液體以使其與含有細胞之萃取用管體21等重，將含有細胞溶液的萃取用管體21與平衡管25在離心機中以1000至1500 rpm離心5至10分鐘，使細胞溶液分層為不含細胞的上清液與含有細胞的沉澱物。Next, the solution is centrifuged (S22). Specifically, liquid is added to the balance tube 25 so that it has the same weight as the extraction tube body 21 containing the cells, and the extraction tube body 21 containing the cell solution and the balance tube 25 are centrifuged in a centrifuge at 1000 to 1500 rpm. For 5 to 10 minutes, the cell solution was stratified into a cell-free supernatant and a cell-containing pellet.

接者，移除溶液經離心後不含細胞的上清液，留下細胞(S23)。詳細來說，使用採取細胞用針頭26移除不含細胞的上清液，以將細胞留在萃取用管體21中。進一步移除不含細胞的上清液可使萃取用管體11中的細胞濃度提升，同時避免後續加入的萃取保護液12被其他液體稀釋而降低萃取效率。於此，不使用抽吸用針頭23，而是使用採取細胞用針頭26來移除上清液，可避免與抽吸用針頭23接觸的萃取保護液22受到汙染。Next, the cell-free supernatant of the centrifuged solution is removed, leaving the cells (S23). Specifically, the cell-free supernatant is removed using the cell collection needle 26 to leave the cells in the extraction tube 21 . Further removing the cell-free supernatant can increase the cell concentration in the extraction tube body 11 , and at the same time prevent the subsequently added extraction protection solution 12 from being diluted by other liquids to reduce the extraction efficiency. Here, instead of the suction needle 23, the cell collection needle 26 is used to remove the supernatant, so that the extraction protection solution 22 in contact with the suction needle 23 can be prevented from being polluted.

接著，在萃取用管體21中混合細胞與萃取保護液22形成混合溶液，萃取保護液的滲透度為大於0且小於等於220 mOsm/L(S24)。詳細來說，使用抽吸用針頭23吸取適量萃取保護液22加至含有細胞的萃取用管體21，以形成混合溶液。在本實施方式中，萃取保護液22為低張溶液，滲透度為大於0且小於等於220 mOsm/L，但不以此為限。在其他實施方式中，萃取保護液可為常見之可保存胞器或維持胞器活性的緩衝液。在本實施方式中，萃取保護液22每1毫升可處理約1×10 ⁶至5×10 ⁶個細胞。 Next, the cells are mixed with the extraction protection solution 22 in the extraction tube body 21 to form a mixed solution, and the osmolarity of the extraction protection solution is greater than 0 and less than or equal to 220 mOsm/L (S24). In detail, use the suction needle 23 to draw a proper amount of extraction protection solution 22 and add it to the extraction tube 21 containing the cells to form a mixed solution. In this embodiment, the extraction protection solution 22 is a hypotonic solution, and the osmolarity is greater than 0 and less than or equal to 220 mOsm/L, but it is not limited thereto. In other embodiments, the extraction protection solution can be a common buffer that can preserve organelles or maintain the activity of organelles. In this embodiment, the extraction protection solution 22 can treat about 1×10 ⁶ to 5×10 ⁶ cells per 1 ml.

接著，使用抽吸用針頭23搭配針筒S來回抽吸萃取用管體21中的混合溶液(S25)。詳細來說，如圖3所示，使用抽吸用針頭23來回抽吸數次萃取用管體21中的混合溶液，例如來回抽吸至少五次。藉此，混合溶液中的細胞會在抽吸用針頭23中與針頭的內壁來回摩擦而使細胞膜受損，讓萃取保護液由細胞膜受損處進入細胞中而破壞細胞膜，進而釋放出粒線體。Next, use the suction needle 23 and the syringe S to suck back and forth the mixed solution in the extraction tube 21 (S25). In detail, as shown in FIG. 3 , use the suction needle 23 to suck back and forth the mixed solution in the extraction tube 21 several times, for example, at least five times. In this way, the cells in the mixed solution will rub back and forth against the inner wall of the needle in the suction needle 23 to damage the cell membrane, allowing the extraction protection solution to enter the cell from the damaged part of the cell membrane to destroy the cell membrane, and then release the granule body.

接者，將混合溶液靜置至少五分鐘(S26)。詳細來說，將混合溶液靜置於平穩處，靜置時間為至少五分鐘，但不以此為限。靜置使低張之萃取保護液有足夠的時間擴散而幫助受損的細胞破裂，進而幫助釋放粒線體。Next, the mixed solution is left to stand for at least five minutes (S26). Specifically, the mixed solution is placed in a stable place for at least five minutes, but not limited thereto. Standing still allows the hypotonic extraction protection solution to have enough time to diffuse and help the damaged cells to rupture, thereby helping to release the mitochondria.

接著，將混合溶液離心(S27)。詳細來說，在平衡管25中加入液體以使其與含有混合溶液之萃取用管體21等重，再將靜置後含有混合溶液的萃取用管體21與平衡管25放入離心機中以1500至2500 rpm離心5至15分鐘。透過離心的方式將細胞碎片與含有粒線體的上清液分層。Next, the mixed solution is centrifuged (S27). In detail, liquid is added to the balance tube 25 so that it has the same weight as the extraction tube body 21 containing the mixed solution, and then the extraction tube body 21 and the balance tube 25 containing the mixed solution after standing still are put into the centrifuge Centrifuge at 1500 to 2500 rpm for 5 to 15 minutes. Cell debris was separated from the mitochondria-containing supernatant by centrifugation.

最後，使用採集粒線體用針頭27採集混合溶液經離心後所得到含有粒線體的上清液(S28)。詳細來說，使用採集粒線體用針頭27採集混合溶液經離心後所得到含有粒線體的上清液，藉以自混合溶液中分離粒線體與細胞碎片。於此，不使用抽吸用針頭23，而是使用採集粒線體用針頭27來採集含有粒線體的上清液，可避免已接觸過萃取保護液及混合溶液的抽吸用針頭23再次接觸到含有粒線體的上清液而造成汙染。Finally, the mitochondria-collecting needle 27 is used to collect the mixed solution and centrifuge to obtain a supernatant containing mitochondria (S28). Specifically, the mitochondrial-containing supernatant obtained after centrifuging the mixed solution with the needle 27 for collecting mitochondria is used to separate mitochondria and cell debris from the mixed solution. Here, instead of using the needle 23 for aspiration, the needle 27 for collecting mitochondria is used to collect the supernatant containing mitochondria, which can prevent the needle 23 for aspiration that has been in contact with the extraction protection solution and the mixed solution from being exposed again. Contamination by contact with the supernatant containing mitochondria.

以下實驗一至實驗五說明使用本發明第二實施方式之套組並依照本發明之分離粒線體的方法來分離粒線體之粒線體萃取效率分析，以及自細胞萃取粒線體的結果。The following experiments 1 to 5 illustrate the analysis of mitochondrial extraction efficiency and the results of extracting mitochondria from cells using the kit according to the second embodiment of the present invention and the method for isolating mitochondria of the present invention.

具體而言，使用採取細胞用針頭採取單核球細胞層的細胞或脂肪幹細胞，並將細胞移至萃取用管體，其中單核球細胞層係透過自靜脈抽取周邊血8至20毫升並以2000 rpm離心10分鐘使血液分層來取得。將萃取用管體以1000 rpm離心5分鐘，使細胞沉澱並使用採取細胞用針頭移除上清液。使用抽吸用針頭將1至2毫升的萃取保護液加入至含有細胞的萃取用管體，在萃取用管體中形成混合溶液，並使用抽吸用針頭來回抽吸數次。將混合溶液靜置至少五分鐘，再將混合溶液以2000 rpm離心10分鐘使混合溶液中的粒線體與細胞碎片分層。使用採集粒線體用針頭採集混合溶液經離心後所得到含有粒線體的上清液。在實驗中使用的萃取保護液為滲透度為42.8 mOsm/L的氯化鈉溶液。Specifically, the cells or adipose stem cells of the mononuclear cell layer are collected using a needle for collecting cells, and the cells are moved to the tube for extraction. The blood was obtained by centrifugation at 2000 rpm for 10 minutes. Centrifuge the extraction tube at 1000 rpm for 5 minutes to pellet the cells and remove the supernatant using a cell collection needle. Use a suction needle to add 1 to 2 ml of extraction protection solution to the extraction tube containing cells to form a mixed solution in the extraction tube, and use the suction needle to pump back and forth several times. The mixed solution was allowed to stand for at least five minutes, and then the mixed solution was centrifuged at 2000 rpm for 10 minutes to separate the mitochondria and cell debris in the mixed solution. The supernatant containing mitochondria was obtained by centrifuging the mixed solution collected with a needle for collecting mitochondria. The extraction protection solution used in the experiment was sodium chloride solution with an osmolarity of 42.8 mOsm/L.

粒線體萃取效率分析係透過細胞影像計數儀求得。細胞被破壞就會釋出粒線體，因此利用細胞影像計數儀來計算受到破壞的細胞數，再計算受到破壞的細胞數與原本總細胞數的比例，以此比例定義為粒線體萃取效率。The analysis of mitochondrial extraction efficiency was obtained by cell image counter. When cells are destroyed, mitochondria will be released. Therefore, the number of damaged cells is calculated using a cell image counter, and then the ratio of the number of damaged cells to the original total number of cells is calculated. This ratio is defined as the mitochondrial extraction efficiency. .

粒線體功能分析係透過測量粒線體的膜電位改變來判斷粒線體功能的好壞。TMRE (tetramethylrhodamine ethyl ester)是一種帶正電的螢光染劑，會聚集在具有活性的粒線體上，故可使用TMRE來標記健康的粒線體。當粒線體活性較低或呈現去極化現象時粒線體的膜電位會降低，造成TMRE無法保留於粒線體上。FCCP (Carbonylcyanide 4-(trifluoromethoxy) phenylhydrazone)為可跨越粒線體內膜的離子載體，會與質子結合來破壞ATP的合成造成膜電位改變。FCCP可用來消除粒線體的膜電位，因此常被用於粒線體去活性或去極化的對照組。藉由TMRE與FCCP染色處理，分析螢光強度以得知粒線體的膜電位變化，以此作為判斷粒線體功能的依據。透過流式細胞儀偵測以TMRE處理後的上清液，可根據TMRE標記分析有功能之粒線體佔上清液中所有粒子的比例。Mitochondrial function analysis is to judge the quality of mitochondrial function by measuring the change of mitochondrial membrane potential. TMRE (tetramethylrhodamine ethyl ester) is a positively charged fluorescent dye that accumulates on active mitochondria, so TMRE can be used to label healthy mitochondria. When the mitochondrial activity is low or depolarized, the membrane potential of the mitochondria will decrease, resulting in the inability of TMRE to remain on the mitochondria. FCCP (Carbonylcyanide 4-(trifluoromethoxy) phenylhydrazone) is an ionophore that can cross the inner mitochondrial membrane. It will bind to protons to disrupt the synthesis of ATP and change the membrane potential. FCCP can be used to eliminate the membrane potential of mitochondria, so it is often used as a control group for mitochondrial inactivation or depolarization. Through TMRE and FCCP staining, the fluorescence intensity was analyzed to obtain the change of mitochondrial membrane potential, which was used as the basis for judging mitochondrial function. The supernatant treated with TMRE is detected by flow cytometry, and the ratio of functional mitochondria to all particles in the supernatant can be analyzed according to the TMRE marker.

粒線體質量檢測係使用Pierce™ BCA Protein Assay Kit進行檢測。詳細操作方式請參考此套組的使用指南。本量測係以牛血清白蛋白(BSA)作為標準品，BSA原液濃度為2微克/毫升(µg/mL)；將Pierce™ BCA Protein Assay Kit的試劑A(無色)及試劑B(藍色)以50：1的比例配製成工作試劑。標準品的配製請參考表1。使用分光光度計在波長562奈米進行量測，依據標準曲線推算樣品中粒線體的質量。表1中，空白試驗用於扣除背景值；含粒線體的樣品為經粒線體萃取後含有粒線體的上清液。The mitochondrial quality detection system uses Pierce™ BCA Protein Assay Kit for detection. For detailed operation methods, please refer to the user guide of this kit. This measurement system uses bovine serum albumin (BSA) as a standard, and the concentration of BSA stock solution is 2 micrograms/milliliter (µg/mL); reagent A (colorless) and reagent B (blue) of Pierce™ BCA Protein Assay Kit Prepare the working reagent at a ratio of 50:1. Please refer to Table 1 for the preparation of standard products. Use a spectrophotometer to measure at a wavelength of 562 nm, and calculate the mass of mitochondria in the sample based on the standard curve. In Table 1, the blank test is used to subtract the background value; the sample containing mitochondria is the supernatant containing mitochondria after mitochondrial extraction.

表1 BSA濃度(µg/mL)   - 空白試驗 - 198µL工作試劑+ 2µL含粒線體的樣品 0 200µL工作試劑 40 198µL工作試劑+2µL BSA 80 196µL工作試劑+4µL BSA 120 194µL工作試劑+6µL BSA 160 192µL工作試劑+8µL BSA 200 190µL工作試劑+10µL BSA Table 1 BSA concentration (µg/mL) - blank test - 198 µL working reagent + 2 µL sample containing mitochondria 0 200 µL working reagent 40 198µL Working Reagent + 2µL BSA 80 196µL Working Reagent + 4µL BSA 120 194µL Working Reagent + 6µL BSA 160 192µL Working Reagent + 8µL BSA 200 190µL Working Reagent + 10µL BSA

〔實驗一〕〔experiment one〕

實驗一係使用不同長度之抽吸用針頭對混合溶液進行不同次數之來回抽吸的粒線體萃取效率分析。在依本發明第二實施方式之套組分離粒線體的方法進行的本實驗中，各實驗組分別含有1×10 ⁶個單核球細胞；萃取保護液體積為1毫升；靜置時間為5分鐘。此外，長針為23G(內徑0.337毫米)、長度70毫米之針頭；短針為23G(內徑0.337毫米)、長度15毫米之針頭；來回抽吸次數分別為0、5、10、15及20次。實驗結果揭示於圖6，圖6為不同針頭長度與不同抽吸次數的粒線體萃取效率分析。 Experiment 1 was an analysis of the mitochondrial extraction efficiency of the mixed solution by using different lengths of suction needles to pump back and forth for different times. In this experiment carried out according to the method for separating mitochondria of sets of sets according to the second embodiment of the present invention, each experimental group contained 1× ¹⁰ monocytes respectively; the volume of the extraction protection solution was 1 milliliter; the standing time was 5 minutes. In addition, the long needle is 23G (0.337mm inner diameter) and 70mm in length; the short needle is 23G (0.337mm inner diameter) and 15mm in length; the number of back and forth suctions is 0, 5, 10, 15 and 20 times respectively . The experimental results are shown in Figure 6, which shows the analysis of mitochondrial extraction efficiency with different needle lengths and different pumping times.

由於混合溶液中的細胞會與針頭的內壁來回摩擦、碰撞而使細胞膜受損進而釋放粒線體，故抽吸用針頭的長度愈長、抽吸次數愈多，表示細胞受到摩擦的路徑也愈長、受到的摩擦與碰撞也愈多，可幫助提高破壞細胞膜的效率。因此，如圖6所示，過少的抽吸次數會造成粒線體萃取效率不佳，增加抽吸次數可提高粒線體萃取效率，但過多的抽吸次數對粒線體萃取效率沒有太大的幫助。並且，在相同抽吸次數下，長針的萃取效率優於短針的萃取效率。依據本實驗結果，即使使用短針、來回抽吸次數為5次，仍可獲得約50%的粒線體萃取效率；使用長針、來回抽吸次數為15次，即可獲得接近100%的粒線體萃取效率。Since the cells in the mixed solution will rub and collide back and forth with the inner wall of the needle, the cell membrane will be damaged and the mitochondria will be released. Therefore, the longer the suction needle and the more suction times, it means that the path for the cells to be rubbed is also different. The longer it is, the more friction and collision it receives, which can help improve the efficiency of destroying cell membranes. Therefore, as shown in Figure 6, too few pumping times will result in poor mitochondrial extraction efficiency, increasing the pumping times can improve the mitochondrial extraction efficiency, but too many pumping times will not affect the mitochondrial extraction efficiency too much s help. Moreover, under the same pumping times, the extraction efficiency of the long needle is better than that of the short needle. According to the results of this experiment, even if a short needle is used and the number of pumping back and forth is 5 times, the extraction efficiency of mitochondria can still be about 50%; using a long needle and the number of pumping back and forth is 15 times, nearly 100% of the mitochondria can be obtained body extraction efficiency.

〔實驗二〕[Experiment 2]

實驗二係使用不同體積的萃取保護液對不同細胞數的粒線體萃取效率分析。在依本發明第二實施方式之套組分離粒線體的方法進行的本實驗中，使用23G(內徑0.337毫米)、長度70毫米的抽吸用針頭；來回抽吸次數為15次；萃取保護液體積為1毫升；靜置時間為5分鐘。此外，各實驗組分別含有1×10 ⁶或1×10 ⁷個單核球細胞；萃取保護液體積分別為0.5、1、1.5、2毫升。實驗結果揭示於圖7，圖7為不同細胞數與不同萃取保護液體積的粒線體萃取效率分析。 In the second experiment, different volumes of extraction protection solution were used to analyze the extraction efficiency of mitochondria with different cell numbers. In this experiment carried out according to the method for separating mitochondria of the set of the second embodiment of the present invention, a suction needle of 23G (inner diameter 0.337 millimeters) and 70 millimeters in length was used; the number of times of pumping back and forth was 15 times; The volume of the protection solution is 1 ml; the standing time is 5 minutes. In addition, each experimental group contained 1×10 ⁶ or 1×10 ⁷ mononuclear cells; the volumes of the extraction protection solution were 0.5, 1, 1.5, and 2 ml, respectively. The experimental results are shown in Figure 7, which is an analysis of the mitochondrial extraction efficiency of different cell numbers and different extraction protection solution volumes.

由圖7可知，在細胞數為1×10 ⁶個的情況下，萃取保護液體積為1毫升時具有較佳的粒線體萃取效率；在細胞數為1×10 ⁷個的情況下，萃取保護液體積為2毫升時具有較佳的粒線體萃取效率。依據本實驗結果，滲透度為42.8 mOsm/L之氯化鈉的萃取保護液每1毫升可處理約1×10 ⁶至5×10 ⁶個細胞，在此範圍內皆可達到80%以上的粒線體萃取效率。 It can be seen from Figure 7 that when the number of cells is 1×10 ⁶ , when the volume of the extraction protection solution is 1 ml, the extraction efficiency of mitochondria is better; when the number of cells is 1×10 ⁷ , the extraction When the volume of protection solution is 2 ml, the extraction efficiency of mitochondria is better. According to the results of this experiment, the extraction and protection solution of sodium chloride with an osmolarity of 42.8 mOsm/L can treat about 1×10 ⁶ to 5×10 ⁶ cells per 1 ml, and it can reach more than 80% of the cells within this range. Line body extraction efficiency.

〔實驗三〕[Experiment 3]

實驗三係來回抽吸後靜置不同平衡時間的粒線體萃取效率分析。在依本發明第二實施方式之套組分離粒線體的方法進行的本實驗中，各實驗組分別含有1×10 ⁶個單核球細胞；使用23G(內徑0.337毫米)、長度70毫米的抽吸用針頭；來回抽吸次數為15次；萃取保護液體積為1毫升。此外，靜置的平衡時間分別為0、5、10、15、30分鐘。實驗結果揭示於圖8，圖8為不同靜置時間的粒線體萃取效率分析。 The analysis of the mitochondrial extraction efficiency of the three series of experiments after reciprocating pumping and resting for different equilibration time. In this experiment carried out according to the method for isolating mitochondria in sets of sets according to the second embodiment of the present invention, each experimental group contains 1×10 ⁶ monocytes; The needle used for suction; the number of back and forth pumping is 15 times; the volume of extraction protection solution is 1 ml. In addition, the equilibrium time of standing was 0, 5, 10, 15, 30 minutes, respectively. The experimental results are shown in Figure 8, which is the analysis of mitochondrial extraction efficiency at different resting times.

靜置使低張之萃取保護液有足夠的時間擴散而幫助受損的細胞破裂，故可幫助提高破壞細胞膜的效率。如圖8所示，在來回抽吸混合溶液之後，靜置數分鐘的混合溶液較未靜置的混合溶液具有較佳的粒線體萃取效率。依據本實驗結果，將混合溶液靜置至少五分鐘，即可獲得80%以上的粒線體萃取效率，靜置更長的時間則不會有明顯變化。Standing still allows the hypotonic extraction protection solution to have enough time to diffuse and help the damaged cells to rupture, so it can help improve the efficiency of destroying the cell membrane. As shown in FIG. 8 , after pumping the mixed solution back and forth, the mixed solution that has been left to stand for several minutes has better mitochondrial extraction efficiency than the mixed solution that has not been left to stand. According to the results of this experiment, the mitochondrial extraction efficiency of more than 80% can be obtained by standing the mixed solution for at least five minutes, and there will be no significant change after standing for a longer time.

〔實驗四〕[Experiment 4]

實驗四係自周邊血的單核球細胞中分離出粒線體。血液中單核球細胞的細胞數約為2.5×10 ⁶個(相當於約8毫升的周邊血)；萃取保護液為滲透度為42.8 mOsm/L之氯化鈉溶液，體積為1毫升；抽吸用針頭為23G(內徑0.337毫米)、長度70毫米的長針；來回抽吸次數為15次；靜置時間為5分鐘。實驗結果如表2所示，自2.5×10 ⁶個周邊血單核球細胞中可分離出8.30微克的粒線體；有功能之粒線體佔上清液中所有粒子的比例(在表中簡稱為純度)為48.55%。 Experiment 4. Mitochondria were isolated from peripheral blood mononuclear cells. The number of mononuclear cells in the blood is about 2.5×10 ⁶ (equivalent to about 8 ml of peripheral blood); the extraction protection solution is a sodium chloride solution with an osmolarity of 42.8 mOsm/L, and the volume is 1 ml; The suction needle is a 23G (inner diameter 0.337 mm) long needle with a length of 70 mm; the number of pumping times is 15 times; the standing time is 5 minutes. The experimental results are shown in Table 2. From 2.5×10 ⁶ peripheral blood mononuclear cells, 8.30 micrograms of mitochondria can be isolated; the ratio of functional mitochondria to all particles in the supernatant (in the table Abbreviated as purity) is 48.55%.

〔實驗五〕[Experiment 5]

實驗五係自脂肪幹細胞中分離出粒線體。脂肪幹細胞的細胞數約為5×10 ⁶個；萃取保護液為滲透度為42.8 mOsm/L 之氯化鈉溶液，體積為1毫升；抽吸用針頭為23G(內徑0.337毫米)、長度70毫米的長針；來回抽吸次數為15次；靜置時間為5分鐘。實驗結果如表2所示，自5×10 ⁶個脂肪幹細胞中可分離出10.97微克的粒線體；有功能之粒線體佔上清液中所有粒子的比例(在表中簡稱為純度)為39.68%。 In experiment five, mitochondria were isolated from adipose stem cells. The cell number of adipose stem cells is about 5×10 ⁶ ; the extraction protection solution is sodium chloride solution with an osmolarity of 42.8 mOsm/L, and the volume is 1 ml; mm long needle; 15 back and forth pumping times; resting time is 5 minutes. The experimental results are shown in Table 2, 10.97 micrograms of mitochondria can be isolated from 5×10 ⁶ adipose-derived stem cells; the ratio of functional mitochondria to all particles in the supernatant (abbreviated as purity in the table) It was 39.68%.

表2   實驗四 實驗五 細胞數(個) 2.5×10 ⁶ 5×10 ⁶ 粒線體質量(微克) 8.30 10.97 純度(%) 48.55 39.68 Table 2 Experiment four Experiment five number of cells 2.5×10 ⁶ 5×10 ⁶ Mitochondrial mass (micrograms) 8.30 10.97 purity(%) 48.55 39.68

本發明實施方式提供用以分離粒線體的套組及分離粒線體的方法，藉由此套組中的抽吸用針頭及萃取保護液，在細胞與抽吸用針頭摩擦的過程中以及在萃取保護液的幫助下使細胞膜受損，而使細胞膜受損的方式較不會損及粒線體，故可以簡單便利的方式高效率地自細胞分離出粒線體，且分離出的粒線體能維持良好的功能。Embodiments of the present invention provide a set for isolating mitochondria and a method for isolating mitochondria. With the aspiration needle and extraction protection solution in the set, during the process of friction between cells and the aspiration needle and With the help of the extraction protection solution, the cell membrane is damaged in a way that will not damage the mitochondria, so the mitochondria can be isolated from the cells in a simple and convenient way, and the isolated mitochondria The thread body can maintain good function.

接下來將進一步說明本發明實施方式之用以分離粒線體的套組中的萃取保護液。Next, the extraction protection solution in the kit for isolating mitochondria according to the embodiment of the present invention will be further described.

萃取保護液係一種自細胞分離並保護粒線體用之萃取保護液，萃取保護液的滲透度(osmolarity)可為大於0且小於等於220 mOsm/L。在部分實施例中，萃取保護液的滲透度可為42.8 mOsm/L至220 mOsm/L。在另一部分實施例中，萃取保護液的滲透度可為42.8 mOsm/L至113 mOsm/L。在部分實施例中，萃取保護液可包含氯化鈉、葡萄糖、磷酸二氫鈉或甘露醇。在部分實施例中，萃取保護液可包含氯化鈉及葡萄糖，其中氯化鈉及葡萄糖的重量比為1：0.06至1：2560。在另一部分實施例中，萃取保護液可包含氯化鈉及磷酸二氫鈉，其中氯化鈉及磷酸二氫鈉的重量比為1：0.015至1：133。在再一部分實施例中，萃取保護液可包含葡萄糖及磷酸二氫鈉，其中葡萄糖及磷酸二氫鈉的重量比為1：0.0007至1：22。在部分實施例中，萃取保護液的溶質僅由氯化鈉及葡萄糖組成，不添加其他溶質。在另一部分實施例中，萃取保護液的溶質僅由氯化鈉及磷酸二氫鈉組成，不添加其他溶質。在再一部分實施例中，萃取保護液的溶質僅由葡萄糖及磷酸二氫鈉組成，不添加其他溶質。The extraction and protection solution is an extraction and protection solution for separating and protecting mitochondria from cells. The osmolarity of the extraction and protection solution can be greater than 0 and less than or equal to 220 mOsm/L. In some embodiments, the osmolarity of the extraction protection solution may range from 42.8 mOsm/L to 220 mOsm/L. In another part of the embodiments, the osmolarity of the extraction protection solution may be 42.8 mOsm/L to 113 mOsm/L. In some embodiments, the extraction protection solution may contain sodium chloride, glucose, sodium dihydrogen phosphate or mannitol. In some embodiments, the extraction protection solution may include sodium chloride and glucose, wherein the weight ratio of sodium chloride and glucose is 1:0.06 to 1:2560. In another part of the embodiment, the extraction protection solution may include sodium chloride and sodium dihydrogen phosphate, wherein the weight ratio of sodium chloride and sodium dihydrogen phosphate is 1:0.015 to 1:133. In yet another embodiment, the extraction protection solution may comprise glucose and sodium dihydrogen phosphate, wherein the weight ratio of glucose and sodium dihydrogen phosphate is 1:0.0007 to 1:22. In some embodiments, the solute of the extraction protection solution is only composed of sodium chloride and glucose, and no other solutes are added. In another part of the embodiment, the solute of the extraction protection solution is only composed of sodium chloride and sodium dihydrogen phosphate, and no other solutes are added. In yet another part of the embodiments, the solute of the extraction protection solution is only composed of glucose and sodium dihydrogen phosphate, and no other solutes are added.

以下說明本發明實施例之萃取保護液及比較例之比較萃取液的成分與滲透度，請參考表3及表4。The following describes the composition and permeability of the extraction protection solution of the embodiment of the present invention and the comparative extraction solution of the comparative example, please refer to Table 3 and Table 4.

表3   成分 氯化鈉 葡萄糖 磷酸二氫鈉 甘露醇 滲透度(mOsm/L) 42.8 實施例一 實施例四 實施例七 實施例十 113 實施例二 實施例五 實施例八 實施例十一 220 實施例三 實施例六 實施例九 實施例十二 520 比較例一 比較例三 比較例五 比較例七 1025 比較例二 比較例四 比較例六 比較例八 table 3 Element Sodium chloride glucose Sodium dihydrogen phosphate Mannitol Permeability (mOsm/L) 42.8 Embodiment one Embodiment four Embodiment seven Embodiment ten 113 Embodiment two Embodiment five Embodiment eight Embodiment Eleven 220 Embodiment Three Embodiment six Embodiment nine Embodiment 12 520 Comparative example one Comparative example three Comparative example five Comparative example seven 1025 Comparative example two Comparative example four Comparative example six Comparative example eight

表4   成分 滲透度(mOsm/L) 實施例十三 氯化鈉、葡萄糖 42.8 實施例十四 氯化鈉、磷酸二氫鈉 42.8 實施例十五 葡萄糖、磷酸二氫鈉 42.8 Table 4 Element Permeability (mOsm/L) Embodiment Thirteen Sodium Chloride, Glucose 42.8 Embodiment Fourteen Sodium Chloride, Sodium Dihydrogen Phosphate 42.8 Embodiment 15 Glucose, sodium dihydrogen phosphate 42.8

以下實驗六至實驗八說明使用本發明實施例之萃取保護液萃取並保護粒線體。參照本發第二實施方式之用以分離粒線體的套組以及使用其分離粒線體的方法，分別使用本發明實施例之萃取保護液及比較例之比較萃取液來進行粒線體的分離，再進行粒線體功能分析以及粒線體萃取效率分析。The following experiments 6 to 8 illustrate the extraction and protection of mitochondria using the extraction and protection solution of the embodiment of the present invention. Referring to the kit for isolating mitochondria and the method for isolating mitochondria using it according to the second embodiment of the present invention, use the extraction protection solution of the embodiment of the present invention and the comparison extraction solution of the comparative example to carry out the isolation of mitochondria Separation, and then analysis of mitochondrial function and mitochondrial extraction efficiency.

具體而言，首先，混合多個細胞與萃取保護液以形成混合溶液。詳細來說，採取固定數量之細胞(1×10 ⁶個)，細胞來源為周邊血單核球細胞或脂肪幹細胞，但不以此為限，細胞來源亦可為任何具有粒線體的細胞。接著，將1毫升之本發明實施例之萃取保護液或比較例之比較萃取液與細胞混合形成混合溶液。 Specifically, first, a plurality of cells are mixed with an extraction protection solution to form a mixed solution. Specifically, a fixed number of cells (1×10 ⁶ ) is used, and the source of the cells is peripheral blood mononuclear cells or adipose stem cells, but not limited thereto, and the source of the cells can also be any cells with mitochondria. Next, 1 ml of the extraction protection solution of the embodiment of the present invention or the comparative extraction solution of the comparative example was mixed with the cells to form a mixed solution.

接著，摩擦混合溶液中的細胞，使細胞的細胞膜受損，以幫助萃取保護液進入細胞並破壞細胞的細胞膜。詳細來說，在混合細胞與萃取保護液的過程中，細胞的細胞膜因摩擦而受損。細胞膜受損可幫助萃取保護液進入細胞以加速細胞破裂。於此，使用尺寸23G、長度70毫米之長針來回抽吸混合溶液，使細胞膜受損，但不以此為限。本發明各實施例之萃取保護液在使用時，亦可配合實驗器材需求使用不同尺寸的長針，或是使用諸如研磨機等方式，藉以使細胞膜受損以幫助萃取保護液進入細胞進而使細胞破裂。Next, the cells in the mixed solution are rubbed to damage the cell membranes of the cells to help extract the protective solution into the cells and destroy the cell membranes of the cells. In detail, during the process of mixing the cells and extracting the protective solution, the cell membranes of the cells were damaged by friction. Damaged cell membranes can help extract protective fluid into cells to accelerate cell rupture. Here, a long needle with a size of 23G and a length of 70 mm was used to pump the mixed solution back and forth to damage the cell membrane, but not limited thereto. When using the extraction and protection solution of various embodiments of the present invention, you can also use long needles of different sizes according to the requirements of experimental equipment, or use a method such as a grinder, so as to damage the cell membrane to help the extraction and protection solution enter the cell and rupture the cell. .

接著，離心混合溶液，使混合溶液分層。詳細來說，將混合溶液靜置後離心，使混合溶液分成含有粒線體的上清液以及含有細胞碎片的沉澱物。Next, the mixed solution was centrifuged to separate the mixed solution. Specifically, the mixed solution was left to stand and then centrifuged to separate the mixed solution into a supernatant containing mitochondria and a precipitate containing cell debris.

接著，收集含有粒線體的上清液。詳細來說，可使用針頭、移液管或傾倒的方式收集含有粒線體的上清液。Next, the supernatant containing mitochondria was collected. In detail, the supernatant containing mitochondria can be collected using a needle, pipette or pouring.

最後，對收集到含有粒線體的上清液進行粒線體功能分析以及粒線體萃取效率分析。Finally, the collected supernatant containing mitochondria was analyzed for mitochondrial function and mitochondrial extraction efficiency.

〔實驗六〕[Experiment 6]

實驗六係使用包含不同滲透度之氯化鈉的萃取保護液，自周邊血單核球細胞中分離粒線體，進行粒線體功能分析以及粒線體萃取效率分析。請參考圖9、圖10及表5，圖9為使用不同滲透度之氯化鈉的萃取保護液自周邊血單核球細胞中分離粒線體的粒線體功能分析，圖10為使用不同滲透度之氯化鈉的萃取保護液自周邊血單核球細胞中分離粒線體的粒線體萃取效率分析。圖10中，#表示相較於滲透度為520 mOsm/L之比較例具有顯著差異(P＜0.05)，*表示相較於滲透度為1025 mOsm/L之比較例具有顯著差異(P＜0.05)。由圖9及表5可知，使用滲透度為大於0且小於等於220 mOsm/L之氯化鈉的萃取保護液來分離粒線體，可使粒線體維持良好的功能，粒線體功能皆大於10%。由圖10及表5可知，使用滲透度為大於0且小於等於220 mOsm/L之氯化鈉的萃取保護液來分離粒線體，可獲得良好的萃取效率，萃取效率皆大於50%。綜合來看，使用滲透度為大於0且小於等於220 mOsm/L之氯化鈉的萃取保護液自周邊血單核球細胞中分離粒線體，可在維持粒線體功能(大於10%)的前提下，同時獲得良好的萃取效率(大於50%)。In Experiment 6, using extraction protection solutions containing sodium chloride with different osmolarity, mitochondria were isolated from peripheral blood mononuclear cells, and mitochondrial function analysis and mitochondrial extraction efficiency analysis were performed. Please refer to Figure 9, Figure 10 and Table 5. Figure 9 shows the functional analysis of mitochondria isolated from peripheral blood mononuclear cells using sodium chloride extraction protection solution with different osmolarity. Analysis of mitochondrial extraction efficiency of mitochondria isolated from peripheral blood mononuclear cells by osmolarity of sodium chloride extraction protection solution. In Fig. 10, # indicates that there is a significant difference (P<0.05) compared with the comparative example with the permeability of 520 mOsm/L, and * indicates that there is a significant difference (P<0.05) compared with the comparative example with the permeability of 1025 mOsm/L ). It can be seen from Figure 9 and Table 5 that using the sodium chloride extraction protection solution with an osmolarity greater than 0 and less than or equal to 220 mOsm/L to separate mitochondria can maintain good mitochondrial function, and the mitochondrial function is stable. Greater than 10%. It can be seen from Figure 10 and Table 5 that using the sodium chloride extraction protection solution with an osmolarity greater than 0 and less than or equal to 220 mOsm/L to separate mitochondria can obtain good extraction efficiency, and the extraction efficiency is greater than 50%. Taken together, the isolation of mitochondria from peripheral blood mononuclear cells using sodium chloride extraction protection solution with an osmolarity greater than 0 and less than or equal to 220 mOsm/L can maintain mitochondrial function (greater than 10%) Under the premise, good extraction efficiency (greater than 50%) is obtained at the same time.

表5   滲透度(mOsm/L) 粒線體功能 (%) 粒線體萃取效率 (%) 實施例一 42.8 17.2±3.8 97.0±2.0 實施例二 113 15.4±5.2 72.7±4.2 實施例三 220 15.7±5.0 56.7±6.5 比較例一 520 10.4±3.8 47.6±5.9 比較例二 1025 9.2±5.1 40.6±7.9 細胞來源：單核球細胞 table 5 Permeability (mOsm/L) Mitochondrial function (%) Mitochondrial extraction efficiency (%) Embodiment one 42.8 17.2±3.8 97.0±2.0 Embodiment two 113 15.4±5.2 72.7±4.2 Embodiment three 220 15.7±5.0 56.7±6.5 Comparative example one 520 10.4±3.8 47.6±5.9 Comparative example two 1025 9.2±5.1 40.6±7.9 Cell source: monocytes

〔實驗七〕[Experiment 7]

實驗七係使用包含不同滲透度之氯化鈉、葡萄糖、磷酸二氫鈉或甘露醇的萃取保護液，自脂肪幹細胞中分離粒線體，進行粒線體功能分析。請參考圖11、圖12及表6，圖11為使用包含不同滲透度之氯化鈉、葡萄糖、磷酸二氫鈉或甘露醇的萃取保護液自脂肪幹細胞中分離粒線體的粒線體功能分析，圖12為使用包含不同滲透度之氯化鈉、葡萄糖、磷酸二氫鈉或甘露醇的萃取保護液自脂肪幹細胞中分離粒線體的粒線體萃取效率分析。圖11及圖12中，#表示相較於滲透度為520 mOsm/L之比較例具有顯著差異(P＜0.05)，*表示相較於滲透度為1025 mOsm/L之比較例具有顯著差異(P＜0.05)。由圖11及表6可知，使用滲透度為大於0且小於等於220 mOsm/L之氯化鈉、葡萄糖、磷酸二氫鈉或甘露醇的萃取保護液來分離粒線體，可使粒線體維持良好的功能，粒線體功能皆大於10%。由圖12及表6可知，使用滲透度為大於0且小於等於220 mOsm/L之氯化鈉的萃取保護液來分離粒線體，可獲得良好的萃取效率，萃取效率皆大於50%。綜合來看，使用滲透度為大於0且小於等於220 mOsm/L之氯化鈉、葡萄糖、磷酸二氫鈉或甘露醇的萃取保護液自脂肪幹細胞中分離粒線體，可在維持粒線體功能(大於10%)的前提下，同時獲得良好的萃取效率(大於50%)。In Experiment 7, mitochondria were isolated from adipose-derived stem cells using extraction protection solutions containing sodium chloride, glucose, sodium dihydrogen phosphate or mannitol with different osmolarity, and mitochondrial function analysis was performed. Please refer to Figure 11, Figure 12 and Table 6. Figure 11 shows the mitochondrial function of mitochondria isolated from adipose-derived stem cells using extraction protection solutions containing sodium chloride, glucose, sodium dihydrogen phosphate or mannitol with different osmolarity Analysis, FIG. 12 is an analysis of the mitochondrial extraction efficiency of mitochondria isolated from adipose stem cells using extraction protection solutions containing sodium chloride, glucose, sodium dihydrogen phosphate or mannitol with different osmolarity. In Fig. 11 and Fig. 12, # indicates that there is a significant difference (P<0.05) compared with the comparative example that the permeability is 520 mOsm/L, and * indicates that there is a significant difference compared with the comparative example that the permeability is 1025 mOsm/L ( P<0.05). From Figure 11 and Table 6, it can be seen that using the extraction protection solution of sodium chloride, glucose, sodium dihydrogen phosphate or mannitol with an osmolarity greater than 0 and less than or equal to 220 mOsm/L to separate mitochondria can make mitochondria To maintain good function, the mitochondrial function is greater than 10%. From Figure 12 and Table 6, it can be seen that using the sodium chloride extraction protection solution with an osmolarity greater than 0 and less than or equal to 220 mOsm/L to separate mitochondria can obtain good extraction efficiency, and the extraction efficiency is greater than 50%. Taken together, using the extraction and protection solution of sodium chloride, glucose, sodium dihydrogen phosphate or mannitol with an osmolarity greater than 0 and less than or equal to 220 mOsm/L to isolate mitochondria from adipose-derived stem cells can maintain mitochondria On the premise of the function (greater than 10%), good extraction efficiency (greater than 50%) is obtained at the same time.

表6   滲透度(mOsm/L) 粒線體功能 (%) 粒線體萃取效率 (%) 氯化鈉 實施例一 42.8 20.7±6.5 95.9±3.6 實施例二 113 21.9±5.4 76.9±20.1 實施例三 220 14.6±1.8 59.4±9.4 比較例一 520 2.0±1.0 52.6±3.8 比較例二 1025 2.0±0.2 41.5±2.4 葡萄糖 實施例四 42.8 12.4±1.8 99.5±0.9 實施例五 113 19.8±3.4 82.9±14.9 實施例六 220 13.1±2.6 73.9±9.8 比較例三 520 2.2±1.1 66.4±7.0 比較例四 1025 6.1±0.5 65.8±6.9 磷酸二氫鈉 實施例七 42.8 12.3±2.9 97.7±2.1 實施例八 113 11.6±0.9 75.5±12.7 實施例九 220 11.5±4.0 51.6±9.7 比較例五 520 4.5±2.0 42.7±6.7 比較例六 1025 2.4±2.1 35.9±7.8 甘露醇 實施例十 42.8 17.2±0.5 100±0 實施例十一 113 13.0±4.8 98.2±0.4 實施例十二 220 13.4±0.5 78.3±7.9 比較例七 520 7.5±5.1 64.8±9.5 比較例八 1025 5.0±0.2 55±12.0 細胞來源：脂肪幹細胞 Table 6 Permeability (mOsm/L) Mitochondrial function (%) Mitochondrial extraction efficiency (%) Sodium chloride Embodiment one 42.8 20.7±6.5 95.9±3.6 Embodiment two 113 21.9±5.4 76.9±20.1 Embodiment three 220 14.6±1.8 59.4±9.4 Comparative example one 520 2.0±1.0 52.6±3.8 Comparative example two 1025 2.0±0.2 41.5±2.4 glucose Embodiment four 42.8 12.4±1.8 99.5±0.9 Embodiment five 113 19.8±3.4 82.9±14.9 Embodiment six 220 13.1±2.6 73.9±9.8 Comparative example three 520 2.2±1.1 66.4±7.0 Comparative example four 1025 6.1±0.5 65.8±6.9 Sodium dihydrogen phosphate Embodiment seven 42.8 12.3±2.9 97.7±2.1 Embodiment Eight 113 11.6±0.9 75.5±12.7 Embodiment nine 220 11.5±4.0 51.6±9.7 Comparative example five 520 4.5±2.0 42.7±6.7 Comparative example six 1025 2.4±2.1 35.9±7.8 Mannitol Embodiment ten 42.8 17.2±0.5 100±0 Embodiment Eleven 113 13.0±4.8 98.2±0.4 Embodiment 12 220 13.4±0.5 78.3±7.9 Comparative example seven 520 7.5±5.1 64.8±9.5 Comparative example eight 1025 5.0±0.2 55±12.0 Cell Source: Adipose Stem Cells

〔實驗八〕[Experiment 8]

實驗八係使用包含不同成分且滲透度皆為42.8 mOsm/L的萃取保護液，自脂肪幹細胞中分離粒線體，進行粒線體功能分析。請參考圖13及表7，圖13為使用包含不同成分且滲透度皆為42.8 mOsm/L的萃取保護液自脂肪幹細胞中分離粒線體的粒線體功能分析。由圖13及表7可知，在滲透度為42.8 mOsm/L的情況下，使用分別包含單一成分之氯化鈉、葡萄糖、磷酸二氫鈉之實施例一、實施例四、實施例七的萃取保護液自脂肪幹細胞中分離粒線體，可使粒線體維持良好的功能，粒線體功能皆大於10%。並且，使用氯化鈉、葡萄糖、磷酸二氫鈉其中兩者相互組合之實施例十三、實施例十四、實施例十五的萃取保護液自脂肪幹細胞中分離粒線體，可使粒線體維持更佳良好的功能，粒線體功能皆大於15%。In the eighth experiment, the extraction protection solution containing different components and the osmolarity of 42.8 mOsm/L was used to isolate mitochondria from adipose-derived stem cells and analyze the mitochondrial function. Please refer to FIG. 13 and Table 7. FIG. 13 shows the functional analysis of mitochondria isolated from adipose-derived stem cells using the extraction protection solution containing different components and the osmolarity of 42.8 mOsm/L. As can be seen from Figure 13 and Table 7, in the case of an osmolarity of 42.8 mOsm/L, the extractions of Example 1, Example 4, and Example 7 containing sodium chloride, glucose, and sodium dihydrogen phosphate respectively containing a single component The protective solution separates mitochondria from adipose stem cells, which can maintain good mitochondrial function, and the mitochondrial function is greater than 10%. And, using sodium chloride, glucose, and sodium dihydrogen phosphate in which the two are combined with each other to extract and protect the solution of Example 13, Example 14, and Example 15 to separate mitochondria from adipose stem cells can make the mitochondria The body maintains better and better function, and the mitochondrial function is greater than 15%.

表7   成分 粒線體功能 (%) 實施例一 氯化鈉 18.0±7.8 實施例四 葡萄糖 12.4±1.8 實施例七 磷酸二氫鈉 12.3±2.9 實施例十三 氯化鈉、 葡萄糖 22.5±5.8 實施例十四 氯化鈉、 磷酸二氫鈉 20.4±6.1 實施例十五 葡萄糖、 磷酸二氫鈉 17.3±2.7 細胞來源：脂肪幹細胞 Table 7 Element Mitochondrial function (%) Embodiment one Sodium chloride 18.0±7.8 Embodiment Four glucose 12.4±1.8 Embodiment seven Sodium dihydrogen phosphate 12.3±2.9 Embodiment Thirteen Sodium Chloride, Glucose 22.5±5.8 Embodiment Fourteen Sodium Chloride, Sodium Dihydrogen Phosphate 20.4±6.1 Embodiment 15 Glucose, sodium dihydrogen phosphate 17.3±2.7 Cell Source: Adipose Stem Cells

由上述實驗結果可知使用低張溶液會比使用高張溶液有更好的萃取效率。並且，由上述實驗證實使用滲透度為大於0且小於等於220 mOsm/L之萃取保護液來分離粒線體，可在維持粒線體功能的前提下，同時獲得良好的萃取效率。再者，由上述實驗證實在滲透度為42.8 mOsm/L的情況下，使用含有氯化鈉、葡萄糖、磷酸二氫鈉其中兩者相互組合的萃取保護液來分離粒線體，獲得的粒線體具有較佳的粒線體功能。在不同實驗中，數值的差異係源自實驗批次進行時的實驗誤差，上述數值的差異皆在本技術領域可接受的誤差範圍中。From the above experimental results, it can be seen that using hypotonic solution will have better extraction efficiency than using hypertonic solution. Moreover, the above experiments have confirmed that using an extraction protection solution with an osmolarity greater than 0 and less than or equal to 220 mOsm/L to separate mitochondria can achieve good extraction efficiency while maintaining mitochondrial function. Furthermore, it has been confirmed by the above experiments that when the osmotic degree is 42.8 mOsm/L, using the extraction protection solution containing sodium chloride, glucose, and sodium dihydrogen phosphate combined with each other to separate mitochondria, the obtained mitochondria The body has better mitochondrial function. In different experiments, the differences in numerical values are due to the experimental errors when the experimental batches are carried out, and the differences in the above numerical values are all within the acceptable error range in the art.

本發明實施例提供自細胞分離並保護粒線體用之萃取保護液。在破壞細胞膜時，例如以長針來回抽吸使細胞膜因摩擦而受損時，藉由此萃取保護液的滲透度為大於0且小於等於220 mOsm/L，可使細胞在萃取保護液之滲透度的幫助下容易破裂進而釋出粒線體。這種輔助破壞細胞膜的方式較不會損及粒線體，故可以簡單便利的方式高效率地自細胞分離出粒線體，更重要的是，分離出的粒線體能夠維持良好的功能。An embodiment of the present invention provides an extraction and protection solution for separating and protecting mitochondria from cells. When destroying the cell membrane, for example, when the cell membrane is damaged due to friction by pumping back and forth with a long needle, the permeability of the extraction protection solution is greater than 0 and less than or equal to 220 mOsm/L. It is easy to rupture with the help of the mitochondria and release the mitochondria. This method of assisting in the destruction of the cell membrane will not damage the mitochondria, so the mitochondria can be isolated from the cells in a simple and convenient way, and more importantly, the isolated mitochondria can maintain good functions.

雖然本發明以前述之實施例揭露如上，然其並非用以限定本發明。在不脫離本發明之精神和範圍內，所為之更動與潤飾，均屬本發明之專利保護範圍。關於本發明所界定之保護範圍請參考所附之申請專利範圍。Although the present invention is disclosed by the aforementioned embodiments, they are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, all changes and modifications are within the scope of patent protection of the present invention. For the scope of protection defined by the present invention, please refer to the appended scope of patent application.

1,2:分離粒線體之套組 11,21:萃取管 12,22:萃取保護液 13,23:抽吸用針頭 24:塞子 25:平衡管 26:採取細胞用針頭 27:採集粒線體用針頭 131,231,261,271:連接端 132,232,262,272:尖端 S:針筒 S11~S14,S21~S28:步驟 1,2: Kit for isolating mitochondria 11,21: extraction tube 12,22: Extraction protection solution 13,23: Needle for aspiration 24: stopper 25: Balance tube 26: Collect cells with a needle 27: Needle for collection of mitochondria 131,231,261,271: connection end 132, 232, 262, 272: cutting edge S: Syringe S11~S14, S21~S28: steps

圖1為本發明第一實施方式之用以分離粒線體的套組的示意圖。 圖2為使用本發明第一實施方式之套組分離粒線體的方法的流程圖。 圖3為本發明第一實施方式之套組的使用示意圖。 圖4為本發明第二實施方式之用以分離粒線體的套組的示意圖。 圖5為使用本發明第二實施方式之套組分離粒線體的方法的流程圖。 圖6為不同針頭長度與不同抽吸次數的粒線體萃取效率分析。 圖7為不同細胞數與不同萃取保護液體積的粒線體萃取效率分析。 圖8為不同靜置時間的粒線體萃取效率分析。 圖9為使用不同滲透度之氯化鈉的萃取保護液自周邊血單核球細胞中分離粒線體的粒線體功能分析。 圖10為使用不同滲透度之氯化鈉的萃取保護液自周邊血單核球細胞中分離粒線體的粒線體萃取效率分析。 圖11為使用包含不同滲透度之氯化鈉、葡萄糖、磷酸二氫鈉或甘露醇的萃取保護液自脂肪幹細胞中分離粒線體的粒線體功能分析。 圖12為使用包含不同滲透度之氯化鈉、葡萄糖、磷酸二氫鈉或甘露醇的萃取保護液自脂肪幹細胞中分離粒線體的粒線體萃取效率分析。 圖13為使用包含不同成分且滲透度皆為42.8 mOsm/L的萃取保護液自脂肪幹細胞中分離粒線體的粒線體功能分析。 FIG. 1 is a schematic diagram of a kit for isolating mitochondria according to a first embodiment of the present invention. Fig. 2 is a flowchart of a method for isolating mitochondria using the kit according to the first embodiment of the present invention. Fig. 3 is a schematic view of the use of the kit according to the first embodiment of the present invention. FIG. 4 is a schematic diagram of a kit for isolating mitochondria according to a second embodiment of the present invention. Fig. 5 is a flowchart of a method for isolating mitochondria using the kit according to the second embodiment of the present invention. Figure 6 is the analysis of mitochondrial extraction efficiency with different needle lengths and different pumping times. Figure 7 is an analysis of the mitochondrial extraction efficiency of different cell numbers and different extraction protection solution volumes. Figure 8 is the analysis of mitochondrial extraction efficiency at different resting times. FIG. 9 is a functional analysis of mitochondria isolated from peripheral blood mononuclear cells using sodium chloride extraction protection solution with different osmolarity. FIG. 10 is an analysis of the mitochondrial extraction efficiency of isolated mitochondria from peripheral blood mononuclear cells using sodium chloride extraction protection solution with different osmolarity. Figure 11 is a functional analysis of mitochondria isolated from adipose-derived stem cells using extraction protection solutions containing sodium chloride, glucose, sodium dihydrogen phosphate or mannitol with different osmolarity. Figure 12 is an analysis of the mitochondrial extraction efficiency of mitochondria isolated from adipose stem cells using extraction protection solutions containing sodium chloride, glucose, sodium dihydrogen phosphate or mannitol with different osmolarity. Figure 13 is the mitochondrial function analysis of mitochondria isolated from adipose-derived stem cells using extraction protection solutions containing different components and an osmolarity of 42.8 mOsm/L.

Claims (10)

一種自細胞分離並保護粒線體用之萃取保護液，其中該萃取保護液為低張溶液。An extraction and protection solution for separating and protecting mitochondria from cells, wherein the extraction and protection solution is a hypotonic solution. 如請求項1所述之萃取保護液，其中該萃取保護液的滲透度為大於0且小於等於220 mOsm/L。The extraction protection solution as claimed in claim 1, wherein the osmolarity of the extraction protection solution is greater than 0 and less than or equal to 220 mOsm/L. 如請求項1所述之萃取保護液，其中該萃取保護液的滲透度為42.8 mOsm/L至220 mOsm/L。The extraction protection solution as described in Claim 1, wherein the osmotic degree of the extraction protection solution is 42.8 mOsm/L to 220 mOsm/L. 如請求項1所述之萃取保護液，包含氯化鈉、葡萄糖、磷酸二氫鈉或甘露醇。The extraction protection solution as described in claim 1, comprising sodium chloride, glucose, sodium dihydrogen phosphate or mannitol. 如請求項1所述之萃取保護液，包含氯化鈉及甘露醇。The extraction protection solution as described in claim 1, comprising sodium chloride and mannitol. 如請求項1所述之萃取保護液，包含甘露醇及磷酸二氫鈉。The extraction protection solution as described in claim 1, comprising mannitol and sodium dihydrogen phosphate. 如請求項1所述之萃取保護液，包含葡萄糖及甘露醇。The extraction protection solution as described in claim 1, comprising glucose and mannitol. 一種如請求項1至7之任一項所述之萃取保護液用於自細胞分離粒線體並維持粒線體活性之用途。A use of the extraction protection solution as described in any one of Claims 1 to 7 for isolating mitochondria from cells and maintaining mitochondrial activity. 如請求項8所述之用途，其中該萃取保護液的體積與所處理之細胞的細胞數的比例為每毫升1×10 ⁷個細胞以下。 The use as described in claim 8, wherein the ratio of the volume of the extraction protection solution to the number of cells to be treated is less than 1×10 ⁷ cells per milliliter. 如請求項8所述之用途，其中該萃取保護液的體積與所處理之細胞的細胞數的比例為每毫升5×10 ⁶個細胞以下。 The use as described in claim 8, wherein the ratio of the volume of the extraction protection solution to the number of cells to be treated is below 5×10 ⁶ cells per milliliter.

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