Composition for extracting mitochondria as well as preparation method and application of composition

Abstract

The invention discloses a composition for mitochondria extraction and a preparation method and application thereof, and belongs to the technical field of cell therapy. The composition comprises mannitol, magnesium ions, sulfate ions, L-arginine, L-histidine, adenosine, sodium ions, potassium ions, hydrogen phosphate ions, citrate ions and dihydrogen phosphate ions. The stem cells are pre-cultured by using the composition before mitochondria extraction; the mitochondria extracted from the pre-cultured stem cells is transplanted to the immune cells by co-incubation with the immune cells, and compared with immune cells containing mitochondria of stem cells which are not pre-cultured, the survival rate and activity of the immune cells are further improved.

Description

Technical Field

[0001] The present application belongs to the field of cell therapy technology, and specifically relates to a composition for mitochondrial extraction, a preparation method, and an application thereof. Background Art

[0002] Mitochondria are the main organelles that provide energy to cells, powering multiple life processes such as proliferation, migration, and differentiation. They are also involved in regulating apoptosis, necrosis, autophagy, etc. Therefore, the health of mitochondria is crucial to maintaining the integrity and function of cells.

[0003] In the fight against cancer, the suppressive tumor microenvironment depletes the mitochondrial activity of immune cells such as T cells and NK cells, leading to immune cell exhaustion. This phenomenon hinders adoptive cell therapy (cell therapy in which healthy tumor-targeting immune cells are infused into cancer patients).

[0004] In related technologies, a paper titled "Intercellular nanotube-mediated mitochondrial transfer enhances T-cell metabolic fitness and antitumor efficacy" published in Cell describes a method of "charging" T cells by transplanting extra mitochondria from bone marrow stromal cells (BMSC) into T cells via intercellular nanotubes. In preclinical cancer models, these "supercharged" T cells showed higher anti-tumor activity and reduced signs of exhaustion, indicating that this technology can help improve existing cancer immunotherapy and open up new avenues for the next generation of cell therapy.

[0005] For example, Chinese invention patent publication number CN109983120A describes natural killer cells containing exogenous mitochondria and pharmaceutical compositions containing the same. The invention involves delivering mitochondria from normal human liver cells (WRL-68) (CRL1458, ATCC) and umbilical cord-derived mesenchymal stem cells (UC-MSC) to NK cells. The introduction of exogenous mitochondria into NK cells not only increases cytotoxicity, leading to an increase in cancer-specific killing effects, but also has no side effects on the presence of immune cells in the body. In this patent application, the generation of NK cells with the introduction of mitochondria from umbilical cord-derived mesenchymal stem cells includes:

[0006] Placenta-derived mesenchymal stem cells were seeded in α-minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum, 100 μg / ml streptomycin, and 100 U / ml ampicillin and cultured for 72 hours. After the culture was completed, the cells were washed twice with Dulbecco's phosphate buffered saline. The washed cells were treated with 0.25% trypsin-EDTA to obtain cells. For the obtained cells, the cell number was measured using a hemocytometer to extract mitochondria, and approximately 2×10 7 cells / ml; performing a preliminary centrifugation of the cell line at 350×g for 10 minutes at a temperature of about 4° C.; collecting the resulting precipitate, resuspending it, and homogenizing it in a buffer solution for 10 to 15 minutes; performing a second centrifugation of the composition containing the precipitate at 1,100×g for 3 minutes at a temperature of about 4° C. to obtain a supernatant; and then performing a third centrifugation of the supernatant at 12,000×g for 15 minutes at a temperature of about 4° C. to separate mitochondria from the cell line;

[0007] The isolated mitochondria were divided into 1 × 10 5 Weights of 0.3, 1, 3, 5, and 10 μg of individual recipient cells were injected into a test tube containing human NK cells (NK92mi) (CRL2408, ATCC) alone and centrifuged at 2,500 × g for 15 minutes at approximately 4°C; after removing the supernatant, the cells were washed with PBS and centrifuged at approximately 4°C for 5 minutes; and washed twice under the same conditions.

[0008] In their research, the applicant found that simply extracting mitochondria from bone marrow mesenchymal stem cells and incubating them with NK cells to obtain NK cells containing exogenous mitochondria had a very limited effect on the survival rate of NK cells when used, especially in solid tumors. This may be due to the influence of low oxygen and high lactate in the immunosuppressive microenvironment of solid tumors. Summary of the Invention

[0009] 1. Technical problems to be solved

[0010] This application addresses the technical problem that the survival rate of immune cells containing exogenous mitochondria prepared by the preparation methods in the prior art is limited, and provides a composition for mitochondrial extraction, its preparation method and application. Before mitochondrial extraction, stem cells are pre-cultured using the composition of the present application, and the mitochondria extracted from the pre-cultured stem cells are transplanted into the immune cells by co-incubation with immune cells. Compared with immune cells containing mitochondria from stem cells that have not been pre-cultured, the survival rate and activity of the immune cells are further improved.

[0011] 2. Technical solution

[0012] In order to solve the above technical problems, the technical solutions adopted in this application are as follows:

[0013] The present application provides a composition for mitochondrial extraction, comprising:

[0014] Mannitol, 23-37 g / L, to maintain osmotic pressure;

[0015] Magnesium ion, 1~6mmol / L, Mg 2+ Used to maintain cell activity;

[0016] Sulfate ion, 1~6mmol / L, SO4 2- Used to regulate osmotic pressure, maintain ion balance, resist oxidative damage and protect cell structure;

[0017] L-arginine, 1.3-2.7 mmol / L, is used for antioxidant and cell energy supply;

[0018] L-histidine, 20-50 mmol / L, is used for antioxidant and cell energy supply;

[0019] Adenosine, 3-6 mmol / L, is used to provide cellular energy;

[0020] Sodium ion: 53.7~121.5mmol / L;

[0021] Potassium ion: 9-20mmol / L;

[0022] Hydrogen phosphate ion: 23.7~51.3mol / L;

[0023] Citrate ion: 2.1~6.3mmol / L;

[0024] Dihydrogen phosphate ion: 9-20mmol / L, Na + , K + 、 Used to maintain osmotic pressure and adjust pH,

[0025] The solvent is water.

[0026] Furthermore, the above composition for mitochondrial extraction, wherein:

[0027] Mannitol, 30 g / L;

[0028] Magnesium ion, 4mmol / L;

[0029] Sulfate ion, 4 mmol / L;

[0030] L-arginine, 2 mmol / L;

[0031] L-histidine, 30 mmol / L;

[0032] Adenosine, 5 mmol / L;

[0033] Sodium ion: 90mmol / L;

[0034] Potassium ion: 15mmol / L;

[0035] Hydrogen phosphate ion: 42.5 mol / L;

[0036] Citrate ion: 5mmol / L;

[0037] Dihydrogen phosphate ion: 15mmol / L.

[0038] The present application also provides a method for preparing the above-mentioned composition for mitochondrial extraction, which comprises:

[0039] Mannitol, magnesium sulfate, L-arginine, L-histidine, adenosine, disodium hydrogen phosphate, sodium citrate and potassium dihydrogen phosphate are prepared separately and dissolved in water to obtain a composition for mitochondrial extraction.

[0040] Furthermore, the water is sterile double-distilled water.

[0041] The present application also provides the use of the above composition for mitochondrial extraction in mitochondrial extraction, which includes:

[0042] Before extracting the mitochondria of the cells, the cells are pre-cultured with the composition, the pre-culture temperature is 3-7° C., and the time is 4-12 hours.

[0043] Furthermore, the above-mentioned pre-culture is carried out in a closed environment.

[0044] Furthermore, the pre-culture temperature is 3-5°C. Furthermore, the pre-culture temperature is 4°C.

[0045] Furthermore, the pre-culture time is 4 to 10 hours. Still further, the pre-culture time is 6 hours.

[0046] Furthermore, the initial amount of cells in the pre-culture is (1-5)×10 7 indivual.

[0047] Furthermore, the initial amount of cells in the pre-culture is 3×10 7 indivual.

[0048] Furthermore, before the pre-culture, the cells are cultured in a culture medium until the confluence reaches 80-90%.

[0049] Furthermore, before the pre-culture, the cells were cultured to a confluence of 80-90% and then washed with phosphate buffered saline (PBS).

[0050] Furthermore, the above cells include somatic cells or stem cells.

[0051] Furthermore, the above-mentioned stem cells include bone marrow mesenchymal stem cells.

[0052] Furthermore, the above-mentioned stem cells are bone marrow mesenchymal stem cells.

[0053] Furthermore, the bone marrow mesenchymal stem cells are human or mouse bone marrow mesenchymal stem cells.

[0054] Furthermore, the above-mentioned bone marrow mesenchymal stem cells are mouse bone marrow mesenchymal stem cells.

[0055] Furthermore, the above-mentioned bone marrow mesenchymal stem cell culture medium includes: basal culture medium + 10% FBS.

[0056] Furthermore, the above-mentioned basal culture medium includes DMEM / F12.

[0057] The present application also provides a mitochondrial extraction method, which comprises the following steps:

[0058] The cells were cultured in culture medium until the confluence reached 80-90%;

[0059] Pre-culturing the cells using the above composition in a closed environment at a temperature of 3 to 7° C. for 4 to 12 hours;

[0060] Extracting cell mitochondria; As a further explanation of the present application, the present application does not limit the method for extracting mitochondria. The method for extracting mitochondria from cells is a conventional technical means in the art, such as using a mitochondrial extraction kit.

[0061] Furthermore, the above-mentioned pre-culture is carried out in a closed environment.

[0062] Furthermore, the pre-culture temperature is 3-5°C. Furthermore, the pre-culture temperature is 4°C.

[0063] Furthermore, the pre-culture time is 4 to 10 hours. Still further, the pre-culture time is 6 hours.

[0064] Furthermore, the initial amount of cells in the pre-culture is (1-5)×10 7 indivual.

[0065] Furthermore, the initial amount of cells in the pre-culture is 3×10 7 indivual.

[0066] Furthermore, before the pre-culture, the cells were cultured to a confluence of 80-90% and then washed with phosphate buffered saline (PBS).

[0067] Furthermore, the above cells include somatic cells or stem cells.

[0068] Furthermore, the above-mentioned stem cells include bone marrow mesenchymal stem cells.

[0069] Furthermore, the bone marrow mesenchymal stem cells can be immortalized bone marrow mesenchymal stem cells, which have a significantly lower cost compared to primary ones.

[0070] Furthermore, the above-mentioned stem cells are bone marrow mesenchymal stem cells.

[0071] Furthermore, the bone marrow mesenchymal stem cells are human or mouse bone marrow mesenchymal stem cells.

[0072] Furthermore, the above-mentioned bone marrow mesenchymal stem cells are mouse bone marrow mesenchymal stem cells.

[0073] Furthermore, the above-mentioned bone marrow mesenchymal stem cell culture medium includes: basal culture medium + 10% FBS.

[0074] Furthermore, the above-mentioned basal culture medium includes DMEM / F12.

[0075] The present application also provides mitochondria extracted by the above mitochondrial extraction method.

[0076] The present application also provides the above-mentioned composition for mitochondrial extraction, the above-mentioned mitochondrial extraction method or the use of the above-mentioned mitochondria in preparing immune cells transplanted with exogenous mitochondria.

[0077] Furthermore, the above application includes: co-incubating mitochondria with therapeutic immune cells to obtain immune cells transplanted with exogenous mitochondria.

[0078] Furthermore, the above-mentioned therapeutic immune cells include any one or more of T cells, NK cells, macrophages, DC cells, etc.

[0079] Furthermore, the therapeutic immune cells are NK cells. As a further illustration of the present application, the present application is not limited to specific NK cells. As an example, the NK cells in the present application are commercially available NK92MI cells purchased from Zhejiang Meisen Cell Technology Co., Ltd., which can autosecrete IL2 activating factor.

[0080] Furthermore, in the above co-incubation, every 1×10 5For each therapeutic immune cell, mitochondria are added according to the total mitochondrial protein content of 5 to 30 μg. As a further explanation of this application, the total mitochondrial protein content is detected by directly adding a common protein lysis buffer to the mitochondria after extraction, and then detecting it with a common BCA protein concentration detection kit.

[0081] Furthermore, in the above co-incubation, the co-incubation temperature is 37±1° C. and the time is 20 to 40 minutes.

[0082] Furthermore, in the above co-incubation, the co-incubation time is 30 minutes.

[0083] Furthermore, in the above co-incubation, the immune cells transplanted with exogenous mitochondria are obtained by centrifuging at 4°C, 500-1500 rpm for 4-6 minutes after the co-incubation is completed, and discarding the supernatant to obtain the immune cells transplanted with exogenous mitochondria.

[0084] Furthermore, after the above co-incubation was completed, centrifugation was performed at 1000 rpm for 5 minutes.

[0085] The present application also provides a method for preparing immune cells transplanted with exogenous mitochondria, the method comprising:

[0086] Mitochondria are extracted using the above-mentioned mitochondrial extraction method, and the mitochondria are co-incubated with therapeutic immune cells to obtain immune cells transplanted with exogenous mitochondria.

[0087] Furthermore, the above-mentioned therapeutic immune cells include any one or more of T cells, NK cells, macrophages, DC cells, etc.

[0088] Furthermore, the therapeutic immune cells are NK cells. As a further illustration of the present application, the present application is not limited to specific NK cells. As an example, the NK cells in the present application are commercially available NK92MI cells purchased from Zhejiang Meisen Cell Technology Co., Ltd., which can autosecrete IL2 activating factor.

[0089] Furthermore, in the above co-incubation, every 1×10 5 For each therapeutic immune cell, mitochondria are added according to the total mitochondrial protein content of 5 to 30 μg. As a further explanation of this application, the total mitochondrial protein content is detected by directly adding a common protein lysis buffer to the mitochondria after extraction, and then detecting it with a common BCA protein concentration detection kit.

[0090] Furthermore, in the above co-incubation, the co-incubation temperature is 37±1° C. and the time is 20 to 40 minutes.

[0091] Furthermore, in the above co-incubation, the co-incubation time is 30 minutes.

[0092] Furthermore, in the above co-incubation, the immune cells transplanted with exogenous mitochondria are obtained by centrifuging at 4°C, 500-1500 rpm for 4-6 minutes after the co-incubation is completed, and discarding the supernatant to obtain the immune cells transplanted with exogenous mitochondria.

[0093] Furthermore, after the above co-incubation was completed, centrifugation was performed at 1000 rpm for 5 minutes.

[0094] The present application also provides immune cells transplanted with exogenous mitochondria prepared by the above-mentioned method for preparing immune cells transplanted with exogenous mitochondria.

[0095] The present application also provides the use of the above-mentioned mitochondria and / or immune cells transplanted with exogenous mitochondria in the preparation of drugs for treating cancer.

[0096] Furthermore, the above-mentioned cancers include solid tumors.

[0097] Furthermore, the solid tumor is selected from one or more of glioma, nasopharyngeal carcinoma, lung cancer, breast cancer, gastric cancer, liver cancer, colorectal cancer, kidney cancer, prostate cancer, ovarian cancer, osteosarcoma, and soft tissue sarcoma.

[0098] Furthermore, the above-mentioned solid tumor is prostate cancer.

[0099] Furthermore, the mitochondria are mitochondria extracted from bone marrow mesenchymal stem cells, and the immune cells transplanted with exogenous mitochondria are NK cells transplanted with mitochondria extracted from bone marrow mesenchymal stem cells.

[0100] The present application also provides a pharmaceutical composition for treating cancer, which comprises the above-mentioned immune cells transplanted with exogenous mitochondria.

[0101] Furthermore, the above-mentioned cancers include solid tumors.

[0102] Furthermore, the solid tumor is selected from one or more of glioma, nasopharyngeal carcinoma, lung cancer, breast cancer, gastric cancer, liver cancer, colorectal cancer, kidney cancer, prostate cancer, ovarian cancer, osteosarcoma, and soft tissue sarcoma.

[0103] Furthermore, the above-mentioned solid tumor is prostate cancer.

[0104] Furthermore, the immune cells transplanted with exogenous mitochondria are NK cells transplanted with mitochondria extracted from bone marrow mesenchymal stem cells.

[0105] 3. Beneficial effects

[0106] Compared with the prior art, the present application has the following advantages:

[0107] (1) The composition for mitochondrial extraction provided in this application, as well as its preparation method and application, can be used to pre-culture cells, especially bone marrow mesenchymal stem cells, using the composition to quickly, conveniently and effectively activate mitochondria in bone marrow mesenchymal stem cells, extract mitochondria from the pre-cultured cells, co-incubate the mitochondria with therapeutic immune cells, especially NK cells, and absorb the mitochondria by the therapeutic immune cells to obtain transplanted exogenous mitochondrial immune cells. The transplanted exogenous mitochondrial immune cells can better adapt to the hypoxic and high lactic acid microenvironment of the tumor, increase the survival rate in the tumor microenvironment, improve activity, and enhance their killing phenotype, thereby reducing the number of NK cells and the number of activators used in tumor treatment, reducing treatment costs while improving treatment effects.

[0108] (2) The present application provides a composition for mitochondrial extraction, a preparation method thereof, and an application thereof. The composition comprises mannitol, magnesium ions, sulfate ions, L-arginine, L-histidine, adenosine, sodium ions, potassium ions, therapeutic hydrogen phosphate, therapeutic citrate, and therapeutic dihydrogen phosphate. The reagents are readily available and relatively low in cost. The composition is used to pre-culture cells, especially bone marrow mesenchymal stem cells. The pre-culture method is simple and easy to operate. The composition can simply, economically, and efficiently improve the survival rate and activity of NK cells in the tumor microenvironment.

[0109] (3) The composition for mitochondrial extraction provided in this application, as well as its preparation method and application, first pre-treats bone marrow mesenchymal stem cells, then extracts their mitochondria and co-incubates them with NK cells. Compared with the co-culture of bone marrow mesenchymal stem cells and immune cells, the latter requires long-term culture of bone marrow mesenchymal stem cells and immune cells, which is not conducive to rapid preparation; it is necessary to consider the optimization and selection of culture medium when stem cells and NK cells are co-cultured; in the co-culture system, the secretory factors of bone marrow mesenchymal stem cells will also affect immune cells, increasing the workload and complexity of quality control; subsequent treatment requires the removal of bone marrow mesenchymal stem cells, which increases the cost of re-purification of NK cells. However, this application does not need to consider the co-culture system, and the purified mitochondria are easier to remove than cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] Figure 1 This is a statistical bar chart of the changes in ROS levels in NK cells detected by flow cytometry. *** represents P < 0.001, vs Normal; ### represents P < 0.001, vs 1% O2 + 10 mM lactate.

[0111] Figure 2 The expression levels of mitochondrial function-related factors in NK cells before and after hypoxia and high lactate treatment were detected by qRT-PCR. *** represents P < 0.001, vs Normal.

[0112] Figure 3 The expression of mitochondria-related factors in NK cells and NK cells transplanted with HmBMSC mitochondria was detected by qRT-PCR in a hypoxic and high-lactate environment, * represents P < 0.05, ** represents P < 0.01, vs 1% O2 + 10mM lactate.

[0113] Figure 4 Figure 2 is the relative content of mouse mitochondrial DNA relative to human mitochondrial DNA in NK cells transplanted with HmBMSC mitochondria detected by qRT-PCR. ** represents P < 0.01 vs 24 h; ### represents P < 0.001 vs 48 h.

[0114] Figure 5 Figure 3: Flow cytometry analysis of apoptosis of NK cells and BMSC-mito-transplanted NK cells in a hypoxic, high-lactate environment. The figure shows the proportion of viable cells (viable cell proportion = 100% - apoptotic cell proportion). A is a flow cytometer graph, and B is a bar graph. ** indicates P < 0.01, vs. 1% O2 + 10 mM lactate.

[0115] Figure 6 Figure 3. Flow cytometry analysis of apoptosis of NK cells and NK cells transplanted with HBMSC-mito in a hypoxic, high-lactate environment. The figure shows the proportion of viable cells (viable cell proportion = 100% - apoptotic cell proportion). A is a flow cytometer graph, and B is a bar graph. *** indicates P < 0.001, vs 1% O2 + 10 mM lactate.

[0116] Figure 7 Figure 2 qRT-PCR analysis of the changes in the expression of NK anti-tumor activity factors in a hypoxic and high-lactate environment after HmBMSC mitochondrial transplantation. * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001 vs 1% O2 + 10 mM lactate.

[0117] Figure 8 Flow cytometry analysis of PC3 and NK cell apoptosis in a hypoxic, high-lactate environment. The bar graph shows the proportion of live cells (live cell proportion = 100% - apoptotic cell proportion). * represents P < 0.05, ** represents P < 0.01, * represents P < 0.05 vs 1% O2 + 10mM lactate, ## represents P < 0.01 vs 1% O2 + 10mM lactate + mBMSC-mito.

[0118] Figure 9Figure 3 is a flow cytometric analysis of NK cell mitochondrial uptake by PC3 cells under a hypoxic and high lactate environment; A is a flow cytometric graph, and B is a bar graph. *** represents P < 0.001, vs ET = 0.1.

[0119] Figure 10 The figure shows the apoptosis of PC3 cells that have taken up NK cell mitochondria by flow cytometry. The figure shows the proportion of apoptotic cells. * represents P < 0.05, * represents P < 0.05, vs ET = 0.2; ## represents P < 0.01, ### represents P < 0.001 vs NK-mito + PC3.

[0120] Figure 11 The expression of PDL-1 in PC3 cells detected by flow cytometry after NK cell mitochondria uptake. *** represents P < 0.001 vs vs ET = 0.2; ### represents P < 0.001 vs NK-mito + PC3.

[0121] Figure 12 The flow cytometry test was used to detect the uptake of NK cell mitochondria by PC3 cells under a hypoxic and high lactate environment. DETAILED DESCRIPTION

[0122] The present application is further described below with reference to specific embodiments.

[0123] It should be noted that, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application belongs; the term "and / or" used herein includes any and all combinations of one or more of the relevant listed items.

[0124] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0125] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement, or value. One skilled in the art can readily determine the degree of flexibility for a particular variable.

[0126] As used herein, the term "at least one of" is intended to be synonymous with "one or more of." For example, "at least one of A, B, and C" explicitly includes only A, only B, only C, and combinations of each thereof.

[0127] Concentration, amount and other numerical data can be presented in range format in this article.Should be understood that such range format is only used for convenience and brevity, and should be flexibly interpreted as not only including the numerical value clearly described as range limit, but also including all independent numerical values ​​or subranges encompassed within the scope, just as each numerical value and subrange are clearly described.For example, the numerical range of about 1 to about 4.5 should be interpreted as not only including the limit value of 1 to about 4.5 clearly described, but also including independent numerals (such as 2,3,4) and subranges (such as 1 to 3,2 to 4 etc.).The same principle is applicable to the scope of only narrating a numerical value, such as "less than about 4.5", which should be interpreted as including all above-mentioned values ​​and scopes.In addition, no matter how the breadth of described scope or feature is, this explanation should be applicable.

[0128] As used herein, phosphate-buffered saline (PBS) is a commonly used buffer in biochemical research. Its main components include potassium dihydrogen phosphate (KH2PO4), disodium hydrogen phosphate (Na2HPO4), sodium chloride (NaCl), and potassium chloride (KCl). These components are dissolved and brought to volume with distilled water. The phosphate ratio is adjusted to maintain a specific pH, typically between 7.2 and 7.4, which is close to the pH of physiological environments such as human blood. For example, a 1L PBS solution typically contains 8g NaCl, 0.2g KCl, 1.42g Na2HPO4, and 0.27g KH2PO4.

[0129] Example 1

[0130] This embodiment provides a composition for mitochondrial extraction and a preparation method thereof.

[0131] In this embodiment, the composition for mitochondrial extraction includes:

[0132] Mannitol, 30 g / L; magnesium ion, 4 mmol / L; sulfate ion, 4 mmol / L; L-arginine, 2 mmol / L; L-histidine, 30 mmol / L; adenosine, 5 mmol / L; sodium ion: 90 mmol / L; potassium ion: 15 mmol / L; hydrogen phosphate ion: 42.5 mol / L; citrate ion: 5 mmol / L; dihydrogen phosphate ion: 15 mmol / L; the solvent is sterile double-distilled water.

[0133] Its preparation method is:

[0134] 30 g of mannitol, 4 mmol of magnesium sulfate, 2 mmol of L-arginine, 30 mmol of L-histidine, 5 mmol of adenosine, 42.5 mmol of disodium hydrogen phosphate, 5 mmol of sodium citrate, and 15 mmol of potassium dihydrogen phosphate were weighed and dissolved in 1 L of sterile double-distilled water to obtain a composition for mitochondrial extraction. The composition comprises:

[0135] Mg 2+ The concentration is 4mmol / L, The concentration is 4mmol / L, Na + The concentration is 90mmol / L, K + The concentration is 15mmol / L, The concentration is 42.5mmol / L, (Citrate) concentration is 5mmol / L, and H2PO-4 concentration is 15mmol / L.

[0136] Example 2

[0137] This embodiment provides a composition for mitochondrial extraction and a preparation method thereof.

[0138] In this embodiment, the composition for mitochondrial extraction includes:

[0139] Mannitol, 23 g / L; magnesium ion, 1 mmol / L; sulfate ion, 1 mmol / L; L-arginine, 1.3 mmol / L; L-histidine, 20 mmol / L; adenosine, 3 mmol / L; sodium ion: 53.7 mmol / L; potassium ion: 9 mmol / L; hydrogen phosphate ion: 23.7 mol / L; citrate ion: 2.1 mmol / L; dihydrogen phosphate ion: 9 mmol / L; the solvent is sterile double-distilled water.

[0140] Example 3

[0141] This embodiment provides a composition for mitochondrial extraction and a preparation method thereof.

[0142] In this embodiment, the composition for mitochondrial extraction includes:

[0143] Mannitol, 37 g / L; magnesium ion, 6 mmol / L; sulfate ion, 6 mmol / L; L-arginine, 2.7 mmol / L; L-histidine, 50 mmol / L; adenosine, 6 mmol / L; sodium ion: 121.5 mmol / L; potassium ion: 20 mmol / L; hydrogen phosphate ion: 51.3 mol / L; citrate ion: 6.3 mmol / L; dihydrogen phosphate ion: 20 mmol / L; the solvent is sterile double-distilled water.

[0144] Example 4

[0145] This embodiment provides a method for preparing immune cells transplanted with exogenous mitochondria and the prepared immune cells transplanted with exogenous mitochondria.

[0146] In this embodiment, the mitochondria are mitochondria extracted from bone marrow mesenchymal stem cells, and the bone marrow mesenchymal stem cells are mouse immortalized bone marrow mesenchymal stem cells (mBMSC), purchased from Zhejiang Ruyao Biotechnology Co., Ltd.; the immune cells are NK cells (NK92MI cells), purchased from Zhejiang Meisen Cell Technology Co., Ltd., and the immune cells transplanted with exogenous mitochondria are NK cells transplanted with mitochondria extracted from bone marrow mesenchymal stem cells.

[0147] The preparation method comprises the following steps:

[0148] Primary cell culture: Immortalized mouse bone marrow mesenchymal stem cells (mBMSCs) were cultured in 10 cm dishes in a 37°C incubator until the confluence reached 80-90%.

[0149] Pre-culture of the composition: discard the culture medium, wash twice with phosphate buffered saline, add 8 mL of the composition prepared in Example 1, and the initial cell volume is 3×10 7 , pre-cultured in a closed environment at 4 °C for 6 h;

[0150] Mitochondrial extraction: remove the composition at low temperature (on ice), quickly scrape the bone marrow mesenchymal stem cells with a cell scraper, collect them into a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes, and discard the supernatant; use the mitochondrial extraction kit (purchased from Beijing Solebow Technology Co., Ltd., catalog number: SM0020) to extract mitochondria according to the instructions, specifically: add 1.0 mL of ice-cold Lysis Buffer to resuspend the cells, transfer the cell suspension to a small-capacity glass homogenizer, grind it in an ice bath at 0°C for 30 to 40 times, take out the homogenate and centrifuge it at 4°C, 1000g for 5 minutes, take the supernatant, transfer it to a new centrifuge tube, centrifuge it again at 4°C, 1000g for 5 minutes, take the supernatant, transfer it to a new centrifuge tube, centrifuge it at 4°C, 12000g for 10 minutes, take the precipitate, add 0.5 mL Resuspend the mitochondrial pellet in WashBuffer, centrifuge at 1000g for 5 minutes at 4°C, collect the supernatant, transfer it to a new centrifuge tube, and centrifuge at 12000g for 10 minutes at 4°C. Discard the supernatant, and highly pure mitochondria will be precipitated at the bottom of the tube. This mitochondria is named HmBMSC mitochondria.

[0151] Preparation of immune cells transplanted with exogenous mitochondria: Resuspend the extracted mitochondria in 100 μL NK culture medium (purchased from Zhejiang Meisen Cell Technology Co., Ltd.) and co-incubate with NK cells at a rate of 1×10 5Mitochondria were added to NK cells with a total mitochondrial protein content of 5 to 30 μg, and the cells were incubated in a 37°C incubator for 30 minutes. The cells were blown evenly and aspirated into a 15 mL centrifuge tube. The tube was centrifuged at 4°C and 1000 rpm for 5 minutes, and the supernatant was discarded to obtain NK cells transplanted with the mitochondria extracted from bone marrow mesenchymal stem cells mentioned above, which were named HmBMSC-NK cells.

[0152] Comparative Example 1

[0153] This comparative example provides a method for preparing immune cells transplanted with exogenous mitochondria and the prepared immune cells transplanted with exogenous mitochondria, but the mitochondria are extracted without pre-culture of the composition.

[0154] Specifically refer to Example 4, which differs in that the step of "pre-culture of the composition" is missing, and the rest is the same as Example 4. The prepared mitochondria are named mBMSC mitochondria, and the NK cells transplanted with mBMSC mitochondria are named mBMSC-NK cells.

[0155] Example 5

[0156] This example provides a study on the ROS levels and the expression of mitochondrial function-related factors in HmBMSC-NK cells.

[0157] (1) Flow cytometry detection of NK cell ROS levels

[0158] The cells were divided into three treatment groups: NK cells were cultured under normal conditions using NK culture medium (Normal), NK cells were cultured under 1% O2+10mM lactate conditions (1% O2+10mM lactate culture group), and NK cells transplanted with HmBMSC-mito were cultured under 1% O2+10mM lactate conditions (1% O2+10mM lactate+HmBMSC-mito culture group). After 24 hours of culture, NK cells or HmBMSC-NK cells were cultured with reactive oxygen species (ROS) detection staining solution prepared in NK cell culture medium (purchased from Shanghai Maokang Biotechnology Co., Ltd., product number C10422, i.e., the reactive oxygen species probe was diluted 1:1000 with NK culture medium) and incubated in a 37°C incubator for 30 minutes. The cells were collected in a centrifuge tube, centrifuged at 1200 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in PBS and detected by flow cytometry.

[0159] (2) qRT-PCR detection of the expression of mitochondrial function-related factors in NK cells before and after hypoxia and high lactate treatment

[0160] NK cells (without mitochondrial transplantation) were cultured in a normal culture environment (Normal), a 1% O2+10mM lactate environment (1% O2+10mM lactate), and NK cells transplanted with HmBMSC-Mito (HmBMSC-NK cells) were cultured in a 1% O2+10mM lactate environment (1% O2+10mM lactate+HmBMSC-Mito) for 24 hours. RNA was extracted using the Trizol method, specifically: the culture medium was aspirated, the cells were washed once with 4°C pre-cooled PBS, and 1 mL of PBS was added to each well. After gently pipetting Trizol to mix, let it stand at room temperature for 10 minutes; transfer the suspension into EP tubes (1.5 mL), add 0.2 mL of chloroform to each tube, shake vigorously first, and then let it stand at room temperature for 5 minutes; then centrifuge at 4°C and 12000 rpm for 15 minutes, carefully transfer the upper colorless and transparent aqueous phase (approximately 400 μL) to a new EP tube; add an equal amount of isopropyl alcohol (IPA), mix thoroughly by inverting, and centrifuge at 12000 rpm and 4°C for 15 minutes. min centrifugation, a white precipitate can be observed at the bottom of the tube; the supernatant is carefully removed, 1 mL of ethanol (75%) is added to each tube, and the tube is thoroughly mixed by inversion. The tube is centrifuged at 12000 rpm and 4°C for 10 min, the supernatant is carefully removed, and the tube is dried at room temperature for 5-15 min. 0.02-0.03 mL of DPEC water is then added, and the tube is allowed to stand at room temperature for 5-15 min to fully dissolve the precipitate. The RNA concentration is then measured, and the samples are stored at -80°C.

[0161] The expression of function-related factors was quantitatively analyzed using qRT-PCR. The qRT-PCR kit was purchased from Nanjing Novozymes Biotechnology Co., Ltd. The reverse transcription kit was HiScriptⅡQ RT SuperMix for qPCR, product number R123-01, and the PCR kit was ChamQ SYBR qPCR Master Mix, product number Q341-02.

[0162] (1) Genomic DNA removal

[0163] The reaction system is shown in Table 1. Mix gently by pipetting and incubate at 42°C for 2 min to obtain the reaction solution.

[0164] Table 1

[0165] <![CDATA[RNase-free ddH2O]]> to 16μL 4×gDNA wiper mix 4μL Total RNA 0.5 μg Total 16μL

[0166] (2) Reverse transcription reaction

[0167] The reaction system is shown in Table 2:

[0168] Table 2

[0169] The reaction solution in step (1) 16μL 5×HiScriptⅡqRT SuperMixⅡ 4 μL Total 20 μL

[0170] The reaction conditions are shown in Table 3:

[0171] Table 3

[0172] temperature time Number of cycles 37℃ 15min 1 85℃ 5sec 1 4℃ - -

[0173] (3) PCR reaction

[0174] The reaction system is shown in Table 4, where the q-RTPCR results are statistically analyzed using 2 －ΔΔCt Calculation; primer sequences are shown in Table 5; reaction conditions are shown in Table 6:

[0175] Table 4

[0176] 2x ChamQ SYBR qPCR Master Mix(Low ROX Premixed) 10.0μL Primer 1 (10 μL) 0.4μL Primer 2 (10 μL) 0.4μL Template DNA / cDNA 1 μL ddH2O 8.2μL Total 20 μL

[0177] Table 5

[0178]

[0179] Table 6

[0180] temperature time Number of cycles 95℃ 30sec 1 95℃ 10sec 40 60℃ 30sec 40 95℃ 15sec 1 60℃ 60sec 1 95℃ 15sec 1

[0181] Result analysis:

[0182] Compared with normal culture, the ROS level of NK cells increased significantly under 1% O2 + 10mM lactate culture conditions ( Figure 1 ), the expression of PGC-1α and Mfn2 was significantly downregulated ( Figure 2 ); however, after HmBMSC mitochondria were transplanted, the ROS of HmBMSC-NK cells decreased significantly ( Figure 1 ), and the expression of PGC-1α and Mfn2 was significantly restored ( Figure 3 ), PGC-1α is the main regulatory factor of mitochondrial biogenesis, which plays an important role in maintaining mitochondrial dynamic balance and metabolic stability; Mfn2 is one of the key proteins mediating mitochondrial fusion, and is involved in regulating mitochondrial dynamic balance and autophagy. The above results show that transplantation of HmBMSC mitochondria helps NK cells maintain mitochondrial homeostasis in a 1% O2+10mM lactate environment.

[0183] Example 6

[0184] This example provides the changes in the relative proportion of mouse mitochondrial DNA (mtDNA) in HmBMSC-NK cells.

[0185] HmBMSC-NK cells were cultured in NK medium in a 1% O2 + 10mM lactic acid environment for 72 hours. Cells were collected at 24 hours, 48 ​​hours, and 72 hours, and DNA was extracted. The extracted DNA was used as a template to detect the relative contents of human (homo) and mouse (mouse) mitochondrial DNA, namely hDLoop and mDLoop, respectively, by qRT-PCR.

[0186] DNA was extracted using a DNA extraction kit (purchased from Kangwei Century Biotechnology Co., Ltd., catalog number CW3064S). The operation was referred to the kit instructions. As an example, the operation was as follows:

[0187] The collected cell suspension was centrifuged at 1200 rpm, the supernatant was discarded, and the cells were resuspended in 200 μL PBS. 20 μL Proteinase K, 200 μL sample, 200 μL lysis buffer, and 300 μL isopropanol were added to a 1.5 mL centrifuge tube and vortexed for 5 seconds. The tube was then shaken at 1200 rpm and room temperature (25°C) for 20 minutes using a thermomixer.

[0188] The resulting solution was transferred to an adsorption column and centrifuged at 12,000 rpm for 1 min to remove the liquid from the collection tube. 0.5 mL of rinse solution 1 was added to the adsorption column and centrifuged at 12,000 rpm for 1 min to remove the waste liquid from the collection tube. 0.5 mL of rinse solution 2 was then added to the adsorption column and centrifuged at 12,000 rpm for 1 min to remove the waste liquid. The adsorption column was then centrifuged at 12,000 rpm for another 2 min to remove the waste liquid. The column was then transferred to room temperature to air dry for 2 min.

[0189] Then move the adsorption column to a new centrifuge tube (1.5 mL), carefully add 40 μL of DNase-free water to the membrane, let it stand at room temperature for 2 minutes to promote its dissolution, then centrifuge at 12,000 rpm for 1 minute. The extracted DNA is in the collection tube, the concentration is measured, and then stored at -20°C.

[0190] The relative content of hDLoop and mDLoop was detected using a kit (purchased from Nanjing Novozymes Biotechnology Co., Ltd., ChamQ SYBR qPCR Master Mix, Catalog No. Q331). The above-extracted DNA was used as a template, and the human nuclear DNA gene hnβactin was used as an internal reference. The qRT-PCR reaction conditions were based on those in Example 5. The qRT-PCR results were statistically calculated as follows: the content of mouse mitochondrial DNA relative to human mitochondrial DNA = [CT value (mDLoop) / CT value (hnβactin)] / [CT value (hDLoop) / CT value (hnβactin)]; the primers used in this test are as follows:

[0191] Table 7

[0192]

[0193] Result analysis:

[0194] The mBMSC mitochondrial DNA content in NK cells transplanted with HmBMSC mitochondria was approximately 0.5% to 1% of that in NK cell mitochondria (the mouse mDLOOP CT value divided by the human mDLOOP value refers to the relative content), and within 24h to 48h after transplantation, the HmBMSC mitochondrial DNA content increased significantly, but then decreased, but at 72h it was still higher than the HmBMSC mitochondrial content at 24h ( Figure 4 ), suggesting that HmBMSC mitochondria can stably exist in NK cells for a period of time and will not continue to proliferate in large quantities, but can be maintained at a certain level.

[0195] Example 7

[0196] This example provides a study on the activity of HmBMSC-NK cells in a hypoxic and high lactate environment.

[0197] In this example, HmBMSC-NK cells (NK + HmBMSC-mito1% + 10mM Lactate), mBMSC-NK cells (NK + mBMSC-mito1% + 10mM Lactate), and NK cells (NK1% + 10mM Lactate) were resuspended in NK culture medium containing 10mM lactate and cultured in a 1% O2 cell culture incubator for 24h. The cells were collected in a centrifuge tube, centrifuged at 1200rpm for 5min, the supernatant was discarded, and the cells were washed once with PBS. The 5× buffer and ddH2O in the apoptosis kit (purchased from Shanghai Biyuntian Biotechnology Co., Ltd., product number C1062S) were mixed in a ratio of 1:4 to prepare a working buffer. 250 μL was added to each tube, and then 2.5 μL Annexin V-FITC and 5 μL PI were added in sequence. After blowing evenly, the cells were incubated in the dark for 5min and detected by upflow cytometry.

[0198] Result analysis:

[0199] The results are as follows Figure 5 and Figure 6 As shown in the results, the survival rate of NK cells transplanted with HmBMSC mitochondria (HmBMSC-NK cells) was significantly higher than that of NK cells transplanted with only mBMSC mitochondria (mBMSC-NK cells), and the activity of NK cells transplanted with mBMSC cell mitochondria was also significantly higher than that of the non-transplanted group.

[0200] (2) qRT-PCR was used to detect the expression of active factors in HmBMSC-NK cells in a 1% O2 + 10 mM lactic acid environment. The qRT-PCR detection method was similar to that in Example 5, and the primer sequences used are shown in Table 8.

[0201] Table 8

[0202]

[0203]

[0204] Result analysis:

[0205] Compared with the non-transplanted group, the expression of Nkp46, IL12 and Perforin in NK cells transplanted with HmBMSC mitochondria was significantly increased ( Figure 7 ), Nkp46 is mainly involved in recognizing and killing virus-infected cells and tumor cells; Perforin is a key molecule for NK cells to exert cytotoxicity, and IL-12 activates Th1 immune response, enhances the activity of NK cells and T cells, and promotes the killing of tumor cells. The upregulation of these factors suggests that the immune regulation and cytotoxicity of NK cells are enhanced; these results indicate that HmBMSC mitochondrial transplantation activates the immune surveillance and killing function of NK cells.

[0206] Example 8

[0207] This example provides the effect of HmBMSC-NK cells on tumor killing ability.

[0208] (1) Flow cytometry was used to detect the killing ability of transplanted HmBMSC mitochondrial NK cells (HmBMSC-NK cells) on prostate cancer PC3 cells in a 1% O2 + 10mM lactic acid culture environment

[0209] PC3 cells were incubated with CFSE staining solution (purchased from Shanghai Maokang Biotechnology Co., Ltd., product number MX3009) in 24-well plates for 15 minutes, the supernatant was discarded, the cells were washed three times with PBS, 500 μL of serum-containing F12K culture medium (purchased from Zhejiang Meisen Biological Co., Ltd.) was added, and the cells were incubated for 5 minutes. The supernatant was discarded, and the cells were added with the required number of NK cells at ET = 0.2. The cells were cultured with NK cell-specific culture medium (purchased from Zhejiang Meisen Cell Technology Co., Ltd.) and cultured in a 1% O2 + 10 mM lactic acid environment for 24 hours. All cells were collected, centrifuged at 1200 rpm for 5 minutes, resuspended in 500 μL PBS, centrifuged at 1200 rpm for 5 minutes, the supernatant was discarded, 250 μL of the diluted buffer in the apoptosis kit was added, and the cells were blown evenly. 2.5 μL of Annexin V-FITC and 5 μL of PI dye were added to each sample, incubated in the dark for 5 minutes, and then 250 μL of PBS was used to detect the apoptotic ratio of CFSE-positive area (PC3 cells) and the apoptotic ratio of CFSE-negative area (NK cells) on flow cytometry.

[0210] Results: In the 1% O2+10mM lactate environment, compared with the non-transplanted group (1% O2+10mM lactate) and the group transplanted with mBMSC-mito only (1% O2+10mM lactate+mBMSC-Mito), the apoptosis rate of PC3 cells co-cultured with NK cells transplanted with HmBMSC mitochondria was significantly increased; and the apoptosis rate of NK cells transplanted with HmBMSC mitochondria was significantly reduced in the environment co-cultured with PC3 ( Figure 8). It was found that in a low-oxygen, high-lactic acid environment, when ET = 0.5, after NK cells were co-cultured with PC3, NK cells showed obvious apoptotic damage. After pre-transplantation of mBMSC-mito, NK cell activity increased, but very limited, consistent with the results in Example 7 above, and PC3 cell activity decreased. However, after transplantation of HmBMSC mitochondria, NK cell activity was significantly improved, and the killing efficacy against PC3 cells was even more significant. Prostate cancer is a recognized cold tumor with low immunogenicity and low levels of effector immune cell infiltration. This field seeks to enhance NK cell activity and killing function as much as possible under the condition of low NK cell infiltration, so a low ET ratio was used in this study.

[0211] Example 9

[0212] This example provides a study on the uptake of mitochondria by prostate cancer cells in NK cells and HmBMSC-NK cells.

[0213] (1) Study on PC3 cell uptake of NK cell mitochondria

[0214] (a) PC3 cells uptake NK cell mitochondria

[0215] Experimental method: The ET ratio indicates the ratio of the initial cell numbers of NK cells and PC3 cells. PC3 cells were stained with CFSE, and NK cell mitochondria were stained with Mito tracker Deep Red. Then, the cells were co-cultured for 24 hours according to the ET ratio. The supernatant was discarded, the adherent cells were trypsinized, and the cells were collected in flow cytometry tubes for detection. The proportion of cells in the CFSE and Mito tracker Deep Red double-positive area was the PC3 cells that took up NK cell mitochondria.

[0216] (b) Anti-apoptosis ability of PC3 cells that have taken up NK cell mitochondria

[0217] Experimental method: PC3 cells were stained with CFSE, NK cell mitochondria were stained with Mito tracker Deep Red, and then co-cultured for 24 hours according to the ET ratio. The supernatant was discarded, the adherent cells were trypsinized, collected into flow tubes, centrifuged at 1200 rpm for 5 minutes, the supernatant was discarded, resuspended in PBS, centrifuged at 1200 rpm for 5 minutes, and the above-mentioned apoptosis detection kit was used. Each sample was resuspended in 250 μL buffer, 5 μL PI dye was added, incubated in the dark for 5 minutes, and then 250 μL PBS was added to mix. The cells were tested on the machine, and the proportion of PI-positive cells in the Mitotracker Deep Red and CFSE double-positive areas was counted ( Figure 10 green dots) and the proportion of PI-positive cells in the Mitotracker Deep Red negative and CFSE-positive areas ( Figure 10 middle red point), where: * represents vs ET=0.2; # represents vs NK-mito - PC3.

[0218] (c) Immune escape ability of PC3 cells that ingest NK cell mitochondria

[0219] Experimental method: PC3 cells were stained with CFSE, NK cell mitochondria were stained with Mito tracker Deep Red, and then co-cultured for 24 hours according to the ET ratio. The supernatant was discarded, the adherent cells were trypsinized, collected into flow tubes, centrifuged at 1200 rpm for 5 minutes, the supernatant was discarded, resuspended in PBS, centrifuged at 1200 rpm for 5 minutes, each sample was resuspended in 100 μL PBS, 0.3 μL PDL-1-PE flow cytometry antibody (purchased from Invitrogen, product number 12-5982-81) was added, incubated in the dark for 30 minutes, and then 400 μL PBS was added to mix. The cells were detected by the machine, and the average fluorescence intensity of PDL-1 in the Mito tracker Deep Red and CFSE double-positive areas was calculated ( Figure 11 MFI of the middle green dot) and the mean fluorescence intensity of PDL-1 in the Mito tracker Deep Red negative and CFSE positive areas ( Figure 11 Middle red dot MFI), where: * represents vs ET=0.2; # represents vs NK-mito - PC3.

[0220] Result analysis:

[0221] The results showed that as the ET ratio increased, the proportion of PC3 cells that took up NK cell mitochondria also increased ( Figure 9 ), which significantly enhances the anti-apoptosis of PC3 cells after uptake ( Figure 10 ) and immune escape ability ( Figure 11 ).

[0222] (2) Study on PC3 cells taking up HmBMSC mitochondria in HmBMSC-NK cells

[0223] HmBMSC cell mitochondria were stained with Mito tracker Deep Red and transplanted to NK cells as described above. The cells were then co-cultured with PC3 cells at ET = 0.5. The culture conditions were normal culture, 1% O2, and 1% O2 + 10 mM lactate. The supernatant was discarded after 24 h (PC3 cells grow adherently, while NK cells grow in suspension, so only the PC3 cells can be collected by aspirating the supernatant). The adherent cells were trypsinized and collected into flow tubes. The proportion of Mito tracker Deep Red-positive areas to total cells was detected by flow cytometry.

[0224] The results showed that PC3 cells did not take up mitochondria of HmBMSC cells in NK cells under normal culture, 1% O2 and 1% O2 + 10mM lactate culture conditions. Figure 12 ), suggesting that the transplanted HmBMSC mitochondria would not be quickly lost due to the mitochondrial uptake of NK cells by PC3 cells.

Claims

1. A composition for mitochondrial extraction, characterized in that The composition comprises: Mannitol, 23-37 g / L; Magnesium ion, 1-6 mmol / L; Sulfate ion, 1-6 mmol / L; L-arginine, 1.3-2.7 mmol / L; L-histidine, 20-50 mmol / L; Adenosine, 3-6 mmol / L; Sodium ion: 53.7~121.5mmol / L; Potassium ion: 9-20mmol / L; Hydrogen phosphate ion: 23.7~51.3mol / L; Citrate ion: 2.1~6.3mmol / L; Dihydrogen phosphate ion: 9-20 mmol / L; The solvent is water.

2. The composition for mitochondrial extraction according to claim 1, characterized in that The composition comprises: Mannitol, 30 g / L; Magnesium ion, 4mmol / L; Sulfate ion, 4 mmol / L; L-arginine, 2 mmol / L; L-histidine, 30 mmol / L; Adenosine, 5 mmol / L; Sodium ion: 90mmol / L; Potassium ion: 15mmol / L; Hydrogen phosphate ion: 42.5 mol / L; Citrate ion: 5mmol / L; Dihydrogen phosphate ion: 15mmol / L.

3. The method for preparing the composition for mitochondrial extraction according to claim 1 or 2, characterized in that: The method comprises: Mannitol, magnesium sulfate, L-arginine, L-histidine, adenosine, disodium hydrogen phosphate, sodium citrate and potassium dihydrogen phosphate are prepared separately and dissolved in water to obtain a composition for mitochondrial extraction.

4. Use of the composition for mitochondrial extraction according to claim 1 or 2 in mitochondrial extraction, characterized in that: The applications include: Before extracting the mitochondria of the cells, the cells are pre-cultured using the composition, the pre-culture temperature is 3 to 7° C., and the time is 4 to 12 hours.

5. A mitochondrial extraction method, characterized in that: The method comprises the following steps: The cells were cultured in culture medium until the confluence reached 80-90%; Pre-culturing cells with the composition for mitochondrial extraction according to claim 1 or 2, wherein the pre-culturing temperature is 3 to 7° C. and the pre-culturing time is 4 to 12 hours; Extract cell mitochondria.

6. A mitochondrial extraction method according to claim 5, characterized in that: The cells include somatic cells or stem cells; preferably bone marrow mesenchymal stem cells; more preferably human or mouse bone marrow mesenchymal stem cells.

7. Mitochondria extracted by the mitochondrial extraction method according to claim 5 or 6.

8. Use of the composition for mitochondrial extraction according to claim 1 or 2, the method for mitochondrial extraction according to claim 5 or 6, or the mitochondria according to claim 7 in preparing immune cells transplanted with exogenous mitochondria.

9. The use according to claim 8, characterized in that The application includes: co-incubating mitochondria with therapeutic immune cells to obtain immune cells transplanted with exogenous mitochondria.

10. The use according to claim 9, characterized in that The therapeutic immune cells include any one or more of T cells, NK cells, macrophages, and DC cells; preferably NK cells.

11. A method for preparing immune cells transplanted with exogenous mitochondria, characterized in that: The method comprises: The cells were cultured in culture medium until the confluence reached 80-90%; Pre-culturing cells with the composition for mitochondrial extraction according to claim 1 or 2, wherein the pre-culturing temperature is 3 to 7° C. and the pre-culturing time is 4 to 12 hours; Extract cell mitochondria; Mitochondria are co-incubated with therapeutic immune cells to obtain immune cells transplanted with exogenous mitochondria.

12. The method for preparing immune cells transplanted with exogenous mitochondria according to claim 11, characterized in that: The therapeutic immune cells include any one or more of T cells, NK cells, macrophages, and DC cells; preferably NK cells.

13. The immune cells transplanted with exogenous mitochondria prepared by the method for preparing immune cells transplanted with exogenous mitochondria according to claim 11 or 12.

14. Use of the mitochondria according to claim 7 or the immune cells transplanted with exogenous mitochondria according to claim 13 in the preparation of a drug for treating cancer.

15. The use according to claim 14, characterized in that The cancer includes solid tumors.

16. A pharmaceutical composition for treating cancer, characterized in that: The composition comprises the immune cells transplanted with exogenous mitochondria according to claim 13.

Images