A cryoprotectant for cell mitochondria and its usage method

Abstract

The present application provides a cryoprotectant for cell mitochondria and a method of use. The cryoprotectant includes glycerol, trehalose, SCF, and phosphate buffer. The addition of SCF can inhibit the apoptosis of cell mitochondria, ensure the survival of mitochondria, and can also regulate the mitochondrial membrane potential and metabolic activity, stabilize the cell energy supply, 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 its apoptosis rate.

Description

Technical Field

[0001] The present invention belongs to the technical field of cryopreservation, and relates to a cryoprotectant for cell mitochondria and a usage method thereof. Background Art

[0002] Mitochondria are organelles enclosed by two membranes that exist in most eukaryotic cells. They are the structures in cells that produce energy and are also the main sites for aerobic respiration in cells, known as the "power house". Except for a few prokaryotes, most eukaryotic cells have more or less mitochondria, but the number, size, appearance, etc. of the mitochondria they possess are different. Mitochondria have their own genetic material and genetic system, but the size of their genomes is limited. It is a semi-autonomous organelle. In addition to supplying energy to cells, mitochondria also participate in processes such as cell differentiation, cell information transmission, and cell apoptosis, and have the ability to regulate cell growth and the cell cycle.

[0003] Mitochondrial transplantation is to directly co-culture functional exogenous mitochondria with cells or directly inject them into the damaged parts of target tissues and organs to supplement intracellular mitochondria, thereby promoting the production of ATP (English name: Adenosinetriphosphate; Chinese name: Adenosine triphosphate), and helping cells self-repair. 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 mediated by MFN1 / 2 and OPA1 proteins to achieve the effect of treating diseases. Important research progress has been made in the fields of tumor treatment, myocardial injury, nerve injury, bone injury, ophthalmic diseases, assisted reproduction, etc.

[0004] Currently, in China, mitochondria are usually preserved in cryoprotectants. Common cryoprotectants include permeating cryoprotectants and non-permeating cryoprotectants, both of which achieve the purpose of cryopreservation by reducing ice crystal damage and solution damage. However, existing cryoprotectants cause greater damage to mitochondria, and after cryopreservation and resuscitation, the mitochondria not only suffer serious losses in activity, morphology, and quantity, but also undergo significant changes, which is not conducive to mitochondrial transplantation. Summary of the Invention

[0005] The purpose of the present invention is to provide a cryoprotectant for cell mitochondria and a usage method thereof to solve the problem that existing cryoprotectants cause greater damage to mitochondria.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] This application provides a cryoprotectant for cell mitochondria. The cryoprotectant includes glycerol, trehalose, SCF, and phosphate buffer solution, where the phosphate buffer solution is the solvent.

[0008] Glycerol can reduce the formation of ice crystals and mitochondrial damage. During cryopreservation, the water inside and outside the mitochondria forms 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.

[0009] Trehalose is a natural disaccharide. As a non-permeating cryoprotectant in mitochondrial cryopreservation, trehalose can promote the formation of an amorphous glassy state of the solution at low temperatures, avoid mechanical damage to mitochondria by ice crystals, and this glassy state can maintain molecular fluidity and reduce the harm caused by the increase in solute concentration during the freezing process.

[0010] 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 at the same time inhibits the activity of the pro-apoptotic protein Bad, ensuring 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 cell energy supply and reduces the accumulation of reactive oxygen species (ROS) and oxidative stress damage by regulating mitochondrial membrane potential and metabolic activity. In tissue homeostasis maintenance, SCF also enhances the interaction between cells and the microenvironment by promoting the expression of intercellular adhesion molecules, providing necessary survival signals for cells and organelles. 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.

[0011] In the cryoprotectant for cell mitochondria provided by this application, based on the addition amount of the phosphate buffer solution, 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. More preferably, based on the addition amount of the phosphate buffer solution, the final concentrations of glycerol, trehalose, and SCF are 20%, 0.5 mol / L, and 0.25 μg / mL, respectively.

[0012] In addition, this application provides a method for using the cryoprotectant for cell mitochondria. The method includes resuspending the mitochondria in the above-mentioned cryoprotectant for cell mitochondria, storing it at 4°C for 1 h, then cooling it to -20°C and storing it for 1 h, and finally cooling it to -80°C and storing it for 1 h, and finally storing it in liquid nitrogen. Among them, the mass-volume ratio of mitochondria to the cryoprotectant for cell mitochondria is 3:1.

[0013] In this application, the mitochondria are umbilical cord mesenchymal stem cell mitochondria, and the extraction method of the mitochondria includes:

[0014] S01: Collect umbilical cord mesenchymal stem cells that are stably expanded in vitro. After centrifugation, collect the cells, and add 1 mL of mitochondrial isolation reagent to the collected cells at a ratio of 2×10 7 . Gently suspend the cells and then let them stand on ice for 15 min to form a cell suspension; among them, the mitochondrial isolation reagent is a commercially available mitochondrial isolation reagent.

[0015] S02: Transfer the cell suspension to a glass homogenizer, homogenize about 15 times, and then perform trypan blue staining. When the positive ratio of live cells in the cell suspension exceeds 50%, centrifuge the cell suspension at 4°C and a centrifugal force of 600 g for 10 min to obtain a supernatant.

[0016] S03: Transfer the supernatant to a new EP tube, centrifuge at 4°C and a centrifugal force of 11,000 g for 10 min, discard the supernatant, and the obtained precipitate is umbilical cord mesenchymal stem cell mitochondria.

[0017] The present invention has the following beneficial effects:

[0018] The cryoprotectant in this application includes glycerol, trehalose, SCF, and phosphate buffer. The addition of SCF can inhibit the apoptosis of cell mitochondria, ensure the survival of mitochondria; it can also regulate the mitochondrial membrane potential and metabolic activity, stabilize the cell energy supply, reduce the accumulation of reactive oxygen species, reduce oxidative stress damage, and improve the activity of mitochondria after cryopreservation and resuscitation. The cryoprotectant added with SCF can effectively improve the cryopreservation survival rate of mitochondria and reduce its apoptosis rate. Description of the Drawings

[0019] Figure 1 Detection chart of the apoptosis rate of mitochondria in the cryoprotectant prepared for Control Group 1;

[0020] Figure 2 Detection chart of the apoptosis rate of mitochondria in the cryoprotectant prepared for Example 1. Detailed Embodiments

[0021] The mitochondria in the embodiments of this application are umbilical cord mesenchymal stem cell mitochondria, and the extraction method of the mitochondria includes:

[0022] S01: Collect umbilical cord mesenchymal stem cells that are stably expanded in vitro. After centrifugation, collect the cells, and add 1 mL of mitochondrial isolation reagent to the collected cells at a ratio of 2×10 7 . Gently suspend the cells and then let them stand on ice for 15 min to form a cell suspension; among them, the mitochondrial isolation reagent is a commercially available mitochondrial isolation reagent.

[0023] S02: Transfer the cell suspension to a glass homogenizer, homogenize about 15 times, and then perform trypan blue staining. When the positive ratio of viable cells in the cell suspension exceeds 50%, centrifuge the cell suspension at 4°C and a centrifugal force of 600g for 10 min to obtain the supernatant.

[0024] S03: Transfer the supernatant to a new EP tube, centrifuge at 4°C and a centrifugal force of 11,000g for 10 min, discard the supernatant, and the obtained precipitate is umbilical cord mesenchymal stem cell mitochondria.

[0025] The technical solution of the present invention will be further explained and illustrated below through specific examples, and the mitochondria used in the following examples are all extracted by the above method.

[0026] Example 1

[0027] The embodiment of the present application provides a cryoprotectant for cell mitochondria. Based on the addition amount of phosphate buffer solution, the cryoprotectant includes glycerol, trehalose and SCF with final concentrations of 20%, 0.5 mol / L and 0.25 μg / mL respectively.

[0028] Example 2

[0029] The embodiment of the present application provides a cryoprotectant for cell mitochondria. Based on the addition amount of phosphate buffer solution, the cryoprotectant includes glycerol, trehalose and SCF with final concentrations of 10%, 0.75 mol / L and 0.1 μg / mL respectively.

[0030] Example 3

[0031] The embodiment of the present application provides a cryoprotectant for cell mitochondria. Based on the addition amount of phosphate buffer solution, the cryoprotectant includes glycerol, trehalose and SCF with final concentrations of 30%, 0.25 mol / L and 0.5 μg / mL respectively.

[0032] Example 4

[0033] The embodiment of the present application provides a cryoprotectant for cell mitochondria. Based on the addition amount of phosphate buffer solution, the cryoprotectant includes glycerol, trehalose and SCF with final concentrations of 25%, 0.4 mol / L and 0.4 μg / mL respectively.

[0034] Control Group 1

[0035] The control group of the present application provides a cryoprotectant for cell mitochondria. Based on the addition amount of phosphate buffer solution, the cryoprotectant includes glycerol and trehalose with final concentrations of 20% and 0.5 mol / L respectively.

[0036] Equal amounts of mitochondria were added to the cryoprotectants prepared in Example 1 and Control Group 1 according to a mass-to-volume ratio of 3:1, and the protein concentration was adjusted to 0.1 mg / mL. Six parallel samples were set in each group. After cooling to -80°C according to the program of storing at 4°C for 1 h, cooling to -20°C for 1 h, and then cooling to -80°C for 1 h, they were transferred to a liquid nitrogen tank for long-term cryopreservation.

[0037] The mitochondrial samples after cryopreserving Example 1 and Control Group 1 for 60 days and 180 days were rapidly rewarmed and thawed respectively. The membrane potential of the thawed mitochondria was detected using a commercially available kit to obtain Table 1. At the same time, the apoptosis rate of the mitochondria cryopreserved for 180 days and then thawed was detected by flow cytometry to obtain Table 2 and Figure 1 、 2 。

[0038] Table 1: Survival rate of mitochondria in the cryoprotectants prepared in Example 1 and Control Group 1

[0039]

[0040] Table 2: Apoptosis rate of mitochondria in the cryoprotectants prepared in Example 1 and Control Group 1

[0041]

[0042] As can be seen from Table 1, after cryopreserving for 60 days and 180 days, 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 Control Group 1 were about 60.53% and about 52.37% respectively. This indicates that the cryoprotectant prepared in the embodiment of the present application can effectively protect mitochondria at low temperature and improve the survival rate of mitochondria.

[0043] As can be seen from Table 1, by detecting the membrane potential of the isolated mitochondria, the addition of SCF can significantly improve the survival rate of mitochondria after cryopreservation and resuscitation.

[0044] From Appendix 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 cryopreservation effect of mitochondria.

[0045] The above are only the preferred embodiments of the present application and are not used to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. 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: It comprises glycerol, trehalose, SCF and phosphate buffer; based on the amount of the phosphate buffer added, 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; The method for using the cryoprotectant comprises: Resuspend mitochondria in cell mitochondrial cryoprotectant; The mixture of mitochondria and cell mitochondrial cryoprotectant was stored at 4°C for 1 h, cooled to -20°C for 1 h, and finally cooled to -80°C for 1 h and stored in liquid nitrogen.

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 were 20%, 0.5 mol / L and 0.25 μg / mL, respectively.

3. The cell mitochondrial cryoprotectant according to claim 1, characterized in that: The mitochondria are umbilical cord mesenchymal stem cell mitochondria.

4. The cell mitochondrial cryoprotectant according to claim 3, 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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