Cell mitochondria cryoprotectant and use method thereof

Abstract

The invention provides a cell mitochondria cryoprotectant and a use method thereof. The cryoprotectant comprises glycerin, trehalose, SCF and a phosphate buffer solution. By adding the SCF, apoptosis of cell mitochondria can be inhibited, and mitochondria survival is guaranteed; in addition, by regulating and controlling mitochondrial membrane potential and metabolic activity, cell energy supply can be stabilized, active oxygen accumulation can be reduced, oxidative stress injury can be reduced, and activity of mitochondria after cryopreservation and resuscitation can be improved. The cryoprotectant added with the SCF can effectively improve the cryopreservation survival rate of mitochondria and reduce the apoptosis rate of the mitochondria.

Description

Technical Field

[0001] The invention belongs to the technical field of low temperature protection and relates to a cell mitochondrial cryoprotectant and a use method thereof. Background Art

[0002] Mitochondria are a type of organelles covered by two membranes that exist in most eukaryotic cells. They are structures that produce energy in cells and are the main place for cells to perform aerobic respiration. They are called "power houses". Except for a few prokaryotes, most eukaryotic cells have mitochondria to a greater or lesser extent, but the number, size, and appearance of their mitochondria vary. Mitochondria have their own genetic material and genetic system, but their genome size is limited. They are semi-autonomous organelles. In addition to providing energy for cells, mitochondria are also involved in processes such as cell differentiation, cell information transmission, and cell apoptosis, and have the ability to regulate cell growth and cell cycle.

[0003] Mitochondrial transplantation is to co-culture normal exogenous mitochondria with cells or directly inject them into damaged parts of target tissues and organs to replenish intracellular mitochondria, thereby promoting the production of ATP (English name: Adenosinetriphosphate; Chinese name: adenosine triphosphate) and helping cells to repair themselves. It is an innovative strategy for treating mitochondrial diseases. In addition, during mitochondrial transplantation, exogenous mitochondria enter cells through actin-dependent endocytosis or pinocytosis to form endosomes or macropinosomes, and then fuse with endogenous mitochondria through MFN1 / 2 and OPA1 proteins to achieve the effect of treating the disease. Mitochondrial transplantation has made important research progress in the fields of tumor treatment, myocardial injury, nerve injury, bone injury, ophthalmic diseases, and assisted reproduction.

[0004] At present, mitochondria are usually preserved in cryoprotectants in China. Common cryoprotectants include permeable cryoprotectants and non-permeable cryoprotectants, both of which achieve the purpose of cryoprotection by reducing ice crystal damage and solution damage. However, existing cryoprotectants cause great damage to mitochondria, and the activity, morphology and quantity of mitochondria after cryopreservation and resuscitation are seriously lost, and they also undergo major changes, which is not conducive to mitochondrial transplantation. Summary of the invention

[0005] The purpose of the present invention is to provide a cell mitochondrial cryoprotectant and a method of use, so as to solve the problem that existing cryoprotectants cause significant damage to mitochondria.

[0006] To achieve the above object, the present invention adopts the following technical solutions: The present application provides a cell mitochondrial cryoprotectant, which comprises glycerol, trehalose, SCF and phosphate buffer, wherein the phosphate buffer is a solvent.

[0007] Glycerol can reduce the formation of ice crystals and mitochondrial damage. When frozen, the water inside and outside the mitochondria will form sharp ice crystals that pierce the mitochondrial membrane. Glycerol can penetrate into the mitochondria, lower the freezing temperature of the water inside the mitochondria, and reduce the formation of ice crystals.

[0008] Trehalose is a natural disaccharide. As a non-permeable cryoprotectant in mitochondrial cryopreservation, trehalose can promote the solution to form an amorphous glass state at low temperatures, avoiding mechanical damage to mitochondria by ice crystals. This glass state can maintain molecular fluidity and reduce the harm of increased solute concentration during the freezing process.

[0009] SCF (English name: Stem cell factor; Chinese name: stem cell factor) is also known as mast cell growth factor (MGF). SCF significantly inhibits apoptosis through the PI3K / AKT pathway, upregulates the expression of anti-apoptotic proteins Bcl-2 and Mcl-1, and inhibits the activity of pro-apoptotic protein Bad to ensure mitochondrial survival. The JAK / STAT pathway activated by SCF can maintain the self-renewal ability of hematopoietic stem cells and progenitor cells and prevent premature differentiation. In addition, SCF stabilizes cellular energy supply by regulating mitochondrial membrane potential and metabolic activity, reduces the accumulation of reactive oxygen species (ROS), and reduces oxidative stress damage. In maintaining tissue homeostasis, SCF also promotes the expression of intercellular adhesion molecules, enhances the interaction between cells and the microenvironment, and provides cells and organelles with necessary survival signals. These mechanisms together ensure the long-term stability of mitochondria under different conditions. Therefore, adding SCF to the cryoprotectant can improve the activity of mitochondria after cryopreservation and recovery.

[0010] In the cell mitochondrial cryoprotectant provided in the present application, the final concentrations of glycerol, trehalose and SCF are 10-30%, 0.25-0.75 mol / L and 0.1-0.5 μg / mL, respectively, based on the amount of phosphate buffer added. Preferably, the final concentrations of glycerol, trehalose and SCF are 20%, 0.5 mol / L and 0.25 μg / mL, respectively, based on the amount of phosphate buffer added.

[0011] In addition, the present application provides a method for using a cell mitochondrial cryoprotectant, which comprises resuspending mitochondria in the cell mitochondrial cryoprotectant, storing at 4°C for 1 hour, cooling to -20°C for 1 hour, and finally cooling to -80°C for 1 hour, and finally storing in liquid nitrogen. The mass volume ratio of mitochondria to cell mitochondrial cryoprotectant is 3:1.

[0012] In the present application, the mitochondria are umbilical cord mesenchymal stem cell mitochondria, and the method for extracting the mitochondria includes: S01: Collect umbilical cord mesenchymal stem cells that have been stably expanded in vitro, collect the cells after centrifugation, and calculate the cell volume as 2×10 7 1 mL of mitochondrial isolation reagent was added to the collected cells at a ratio of , the cells were lightly suspended and then placed in an ice bath for 15 minutes to form a cell suspension; wherein, the mitochondrial isolation reagent used was a commercially available mitochondrial isolation reagent.

[0013] S02: The cell suspension was transferred to a glass homogenizer and homogenized for about 15 times before staining with trypan blue. When the positive ratio of live cells in the cell suspension exceeded 50%, the cell suspension was centrifuged at 4°C and 600 g for 10 min to obtain the supernatant.

[0014] S03: The supernatant was transferred to a new EP tube, and centrifuged at 4°C and 11000 g for 10 min. The supernatant was discarded, and the obtained precipitate was the umbilical cord mesenchymal stem cell mitochondria.

[0015] The present invention has the following beneficial effects: The cryoprotectants in this application include glycerol, trehalose, SCF and phosphate buffer. The addition of SCF can inhibit the apoptosis of cell mitochondria and ensure the survival of mitochondria; it can also stabilize the energy supply of cells by regulating the mitochondrial membrane potential and metabolic activity, reduce the accumulation of reactive oxygen species, reduce oxidative stress damage, and improve the activity of mitochondria after cryopreservation and recovery. The cryoprotectant added with SCF can effectively improve the cryopreservation survival rate of mitochondria and reduce their apoptosis rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a graph showing the apoptosis rate of mitochondria in the cryoprotectant prepared for control group 1; Figure 2 This is a graph showing the apoptosis rate of mitochondria in the cryoprotectant prepared in Example 1. DETAILED DESCRIPTION

[0017] The mitochondria in the embodiment of the present application are umbilical cord mesenchymal stem cell mitochondria, and the extraction method of the mitochondria includes: S01: Collect umbilical cord mesenchymal stem cells that have been stably expanded in vitro, collect the cells after centrifugation, and calculate the cell volume as 2×10 7 1 mL of mitochondrial isolation reagent was added to the collected cells at a ratio of , the cells were lightly suspended and then placed in an ice bath for 15 minutes to form a cell suspension; wherein, the mitochondrial isolation reagent used was a commercially available mitochondrial isolation reagent.

[0018] S02: The cell suspension was transferred to a glass homogenizer and homogenized for about 15 times before staining with trypan blue. When the positive ratio of live cells in the cell suspension exceeded 50%, the cell suspension was centrifuged at 4°C and 600 g for 10 min to obtain the supernatant.

[0019] S03: The supernatant was transferred to a new EP tube, and centrifuged at 4°C and 11000 g for 10 min. The supernatant was discarded, and the obtained precipitate was the umbilical cord mesenchymal stem cell mitochondria.

[0020] The technical solution of the present invention is further explained and illustrated by specific examples below, and the mitochondria used in the following examples are all extracted according to the above method.

[0021] Example 1 The embodiment of the present application provides a cell mitochondrial cryoprotectant, which includes glycerol, trehalose and SCF with final concentrations of 20%, 0.5 mol / L and 0.25 μg / mL respectively, based on the addition amount of phosphate buffer.

[0022] Example 2 The embodiment of the present application provides a cell mitochondrial cryoprotectant, which includes glycerol, trehalose and SCF with final concentrations of 10%, 0.75 mol / L and 0.1 μg / mL respectively, based on the addition amount of phosphate buffer.

[0023] Example 3 The embodiment of the present application provides a cell mitochondrial cryoprotectant, which includes glycerol, trehalose and SCF with final concentrations of 30%, 0.25 mol / L and 0.5 μg / mL respectively, based on the addition amount of phosphate buffer.

[0024] Example 4 The embodiment of the present application provides a cell mitochondrial cryoprotectant, which includes glycerol, trehalose and SCF with final concentrations of 25%, 0.4 mol / L and 0.4 μg / mL respectively, based on the addition amount of phosphate buffer.

[0025] Control group 1 The control group of the present application provides a cell mitochondrial cryoprotectant, which includes glycerol and trehalose with final concentrations of 20% and 0.5 mol / L respectively, based on the addition amount of phosphate buffer.

[0026] According to the mass-to-volume ratio of 3:1, equal amounts of mitochondria were added to the cryoprotectants prepared in Example 1 and Control Group 1, respectively, and the protein concentration was adjusted to 0.1 mg / mL. Six parallel samples were set up in each group, and the samples were stored at 4°C for 1 hour, cooled to -20°C for 1 hour, and cooled to -80°C for 1 hour. After cooling to -80°C, they were transferred to a liquid nitrogen tank for long-term cryopreservation.

[0027] The mitochondrial samples of Example 1 and Control Group 1 that had been frozen and stored for 60 days and 180 days were quickly thawed, and the membrane potential of the thawed mitochondria was detected using a commercially available kit, and the results were shown in Table 1. At the same time, the apoptosis rate of the mitochondria that had been frozen and stored for 180 days and then thawed was detected using flow cytometry, and the results were shown in Table 2 and Attached. Figure 1 , 2 .

[0028] Table 1: Survival rate of mitochondria in cryoprotectants prepared in Example 1 and Control Group 1 Table 2: Mitochondrial apoptosis rate in cryoprotectants prepared in Example 1 and Control Group 1 As can be seen from Table 1, after 60 days and 180 days of cryopreservation, the survival rates of mitochondria in the cryoprotectant prepared in Example 1 were about 80.10% and about 74.17%, respectively; while the survival rates of mitochondria in the cryoprotectant prepared in the control group 1 were about 60.53% and about 52.37%, respectively, which indicates that the cryoprotectant prepared in the examples of the present application can effectively protect mitochondria at low temperatures and improve the survival rate of mitochondria.

[0029] As can be seen from Table 1, by detecting the membrane potential of isolated mitochondria, the addition of SCF can significantly improve the survival rate of mitochondria after cryopreservation and recovery.

[0030] By Schedule 2 and Figure 1 , 2 It can be seen that by detecting the apoptosis rate of mitochondria, the addition of SCF can significantly reduce the apoptosis rate of mitochondria and improve the freezing effect of mitochondria.

[0031] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A cell mitochondrial cryoprotectant, characterized in that: Includes glycerol, trehalose, SCF and phosphate buffer.

2. The cell mitochondrial cryoprotectant according to claim 1, characterized in that: Based on the amount of the phosphate buffer added, the final concentrations of the glycerol, trehalose and SCF are 10-30%, 0.25-0.75 mol / L and 0.1-0.5 μg / mL, respectively.

3. The cell mitochondrial cryoprotectant according to claim 1, characterized in that: Based on the amount of the phosphate buffer added, the final concentrations of the glycerol, trehalose and SCF were 20%, 0.5 mol / L and 0.25 μg / mL, respectively.

4. A method for using a cell mitochondrial cryoprotectant, characterized in that: include: The mitochondria are resuspended in the cell mitochondrial cryoprotectant according to any one of claims 1 to 3, cooled to -80°C, and then stored in liquid nitrogen.

5. The method for using the cell mitochondrial cryoprotectant according to claim 4, characterized in that: The mass volume ratio of the mitochondria to the cell mitochondrial cryoprotectant is 3:

1.

6. The method for using the cell mitochondrial cryoprotectant according to claim 4, characterized in that: The process of cooling to -80°C includes: storing the mixture of mitochondria and cell mitochondrial cryoprotectant at 4°C for 1 hour, cooling to -20°C for 1 hour, and finally cooling to -80°C for 1 hour.

7. The method for using the cell mitochondrial cryoprotectant according to claim 4, characterized in that: The mitochondria are umbilical cord mesenchymal stem cell mitochondria.

8. The method for using the cell mitochondrial cryoprotectant according to claim 7, characterized in that: The method for extracting mitochondria of umbilical cord mesenchymal stem cells comprises: The umbilical cord mesenchymal stem cells were collected after centrifugation, and mitochondrial separation reagent was added to the collected cells. The cells were lightly suspended and then placed in an ice bath for 15 minutes to form a cell suspension; The cell suspension is homogenized and then stained with trypan blue; when the positive ratio of live cells in the cell suspension exceeds 50%, the cell suspension is centrifuged at 4° C. and a centrifugal force of 600 g for 10 minutes to obtain a supernatant; The supernatant was centrifuged at 4°C and a centrifugal force of 11,000 g for 10 min to obtain mitochondria.

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