A culture medium and method for promoting mitochondrial proliferation

Abstract

The present application provides a culture medium and method for promoting mitochondrial proliferation, which belongs to the field of cell culture technology. The culture medium includes: DMEM / F12 culture medium, fetal bovine serum, double antibody and SFRP-1. The addition of SFRP-1 in the culture medium can improve the proliferation efficiency of mitochondria, promote the regeneration and development of mitochondria, keep the mitochondria in the cells in a better morphology, and significantly increase the number.

Description

Technical Field

[0001] The invention belongs to the technical field of stem cells and relates to a culture medium and a method for promoting mitochondrial proliferation. 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. 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 the injection of functional exogenous mitochondria into damaged organs to directly supplement the mitochondria in cells, promote the production of ATP (adenosine triphosphate), and help cells repair themselves. Therefore, in recent years, mitochondrial transplantation has attracted more and more attention as an innovative strategy for treating mitochondrial diseases. Specifically, the isolated and purified mitochondria are directly co-cultured with cells or directly injected into the damaged parts of the target tissues and organs. The exogenous mitochondria enter the cells through actin-dependent endocytosis or pinocytosis to form endosomes or macropinosomes, and fuse with endogenous mitochondria through MFN1 / 2 and OPA1 proteins to achieve the effect of treating the disease.

[0004] Mitochondrial transplantation has made important research progress in the fields of tumor treatment, myocardial injury, nerve damage, bone injury, ophthalmic diseases, assisted reproduction, etc., which has greatly increased the demand for mitochondria. However, there is still a lack of effective methods for the proliferation of mitochondria in China, resulting in the inability of mitochondrial production to meet application needs. Summary of the invention

[0005] The purpose of the present invention is to provide a culture medium and method for promoting mitochondrial proliferation, so as to solve the problem of slow mitochondrial proliferation in the prior art.

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

[0007] The present application provides a culture medium for promoting mitochondrial proliferation, the culture medium comprising DMEM / F12 culture medium, fetal bovine serum, double antibody and SFRP-1. Among them, DMEM / F12 culture medium is a commonly used mesenchymal stem cell culture medium. Fetal bovine serum contains abundant nutrients necessary for cell growth, is mainly used for in vitro culture of animal cells, and is also a commonly used natural culture medium for mesenchymal stem cells. The double antibody is penicillin-streptomycin.

[0008] SFRP-1, also known as Secreted frizzled related protein 1 or SARP-2, FrzA, is a soluble protein with specific biological activity. SFRP-1 has a wide spectrum of target genes, including multiple target genes related to regulating Wnt signaling and maintaining hematopoietic stem cell function. Many studies have shown that SFRP-1 is involved in biological effects such as cell generation, angiogenesis, energy metabolism of nucleosides, amino acids, and sugars, cell survival, apoptosis, and activity, as well as drug resistance, to maintain the stability of the internal environment of tissues and cells under hypoxic conditions. SFRP-1 can promote the decarboxylation of glutamate, thereby increasing the production of γ-aminobutyric acid, which is an important inhibitory neurotransmitter. SFRP-1 can promote transamination of transaminases, thereby increasing the content of dopamine in the body. Dopamine plays an important role in the nervous system and is closely related to emotions, behavior, and motor control.

[0009] In the present application, SFRP-1 can improve the proliferation efficiency of mitochondria, promote the regeneration and development of mitochondria, maintain a good morphology of mitochondria in cells, and significantly increase the number.

[0010] In the present application, the culture medium for promoting mitochondrial proliferation includes 84-94% DMEM / F12 culture medium, 5-15% fetal bovine serum and 0.5-1.5% double antibody by mass fraction, wherein the concentration of SFRP-1 in the culture medium is 0.1-0.5ng / ml. Preferably, the culture medium for promoting mitochondrial proliferation includes 89% DMEM / F12 culture medium, 10% fetal bovine serum and 1% double antibody by mass fraction, wherein the concentration of SFRP-1 in the culture medium is 0.3ng / ml.

[0011] In addition, the present application also provides a method for promoting mitochondrial proliferation, the method comprising:

[0012] S01: MSCs were cultured in the above culture medium without SFRP-1 to P3;

[0013] S02: When the mesenchymal stem cells grow to a coverage area of ​​55-65% in the culture bottle, SFRP-1 is added to the culture medium and the culture is continued for 36-48 hours;

[0014] S03: After the culture is completed, extracting mitochondria from the mesenchymal stem cells.

[0015] The present invention has the following beneficial effects:

[0016] In the present application, after mesenchymal stem cells are normally cultured to P3, SFRP-1 is added to the culture medium. The SFRP-1 can promote the mesenchymal stem cells to express more mitochondria, increase the content of mitochondria, and achieve the purpose of proliferating mitochondria. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a TEM image of the mitochondria in Comparative Example 1 when it is magnified 2000 times;

[0018] Figure 2 This is a TEM image of the mitochondria in Comparative Example 1 when it is magnified 10,000 times;

[0019] Figure 3 This is a TEM image of the mitochondria in Example 1 when it is magnified 2000 times;

[0020] Figure 4 This is a TEM image of the mitochondria in Example 1 when it is magnified 10,000 times;

[0021] Figure 5 The mitochondria extracted from Example 1 and Comparative Example 1 are 2 Figure 2. Mitochondrial number in the mitochondria. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0023] Example 1

[0024] The embodiment of the present application provides a culture medium for promoting mitochondrial proliferation, wherein the culture medium comprises 89% DMEM / F12 culture medium, 10% fetal bovine serum and 1% penicillin-streptomycin in terms of mass fraction, wherein the concentration of SFRP-1 is 0.3 ng / ml.

[0025] The method for promoting mitochondrial proliferation provided in the embodiments of the present application includes:

[0026] S101: MSCs were cultured in a medium without SFRP-1 until P3;

[0027] S102: When the mesenchymal stem cells grow to 55-65%, SFRP-1 is added to the culture medium and culture is continued for 36-48 hours;

[0028] S103: After the culture is completed, mitochondria in the mesenchymal stem cells are extracted normally according to the steps in the mitochondrial extraction kit.

[0029] Example 2

[0030] The embodiment of the present application provides a culture medium for promoting mitochondrial proliferation, wherein the culture medium comprises 84.5% DMEM / F12 culture medium, 15% fetal bovine serum and 0.5% penicillin-streptomycin in terms of mass fraction, wherein the concentration of SFRP-1 is 0.1 ng / ml.

[0031] The method for promoting mitochondrial proliferation provided in the present embodiment is the same as that in Example 1.

[0032] Example 3

[0033] An embodiment of the present application provides a culture medium for promoting mitochondrial proliferation, which includes 93.5% DMEM / F12 culture medium, 5% fetal bovine serum, 1.5% double antibody and SFRP-1 in mass fractions, wherein the concentration of SFRP-1 is 0.2 ng / ml.

[0034] The method for promoting mitochondrial proliferation provided in the present embodiment is the same as that in Example 1.

[0035] Example 4

[0036] The embodiment of the present application provides a culture medium for promoting mitochondrial proliferation, wherein the culture medium comprises 84% ​​DMEM / F12 culture medium, 14.5% fetal bovine serum and 1.5% penicillin-streptomycin in terms of mass fraction, wherein the concentration of SFRP-1 is 0.4 ng / ml.

[0037] The method for promoting mitochondrial proliferation provided in the present embodiment is the same as that in Example 1.

[0038] Example 5

[0039] The embodiment of the present application provides a culture medium for promoting mitochondrial proliferation, wherein the culture medium comprises 94% DMEM / F12 culture medium, 5.5% fetal bovine serum and 0.5% penicillin-streptomycin in terms of mass fraction, wherein the concentration of SFRP-1 is 0.5 ng / ml.

[0040] The method for promoting mitochondrial proliferation provided in the present embodiment is the same as that in Example 1.

[0041] Comparison group 1

[0042] The comparative example of the present application provides a culture medium for promoting mitochondrial proliferation, which comprises 89% DMEM / F12 culture medium, 10% fetal bovine serum and 1% penicillin-streptomycin in terms of mass fraction.

[0043] The method for promoting mitochondrial proliferation provided in Comparative Example 1 of the present application comprises:

[0044] D101: culture mesenchymal stem cells in the above culture medium to the P3 generation and continue culturing for 36-48 hours;

[0045] D102: After the culture is completed, mitochondria in mesenchymal stem cells are extracted normally according to the steps in the mitochondrial extraction kit.

[0046] The mitochondria extracted from Example 1 and Comparative Example 1 were subjected to transmission electron microscope examination (TEM) to observe the number and morphological structure of mitochondria in the microstructure of mesenchymal stem cells. When the magnification was 2000 times, the attached Figure 1 The TEM image of mitochondria in Comparative Example 1 and the attached Figure 3 TEM images of mitochondria in Example 1 shown in FIG. When the magnification is 10,000 times, the attached Figure 2 The TEM image of mitochondria in Comparative Example 1 and the attached Figure 4 The TEM image of the mitochondria in Example 1 is shown in FIG. At the same time, a statistical image of the mitochondria is drawn based on the TEM detection results to obtain the attached Figure 5 The number of mitochondria is calculated based on the number of 2 Calculation of mitochondrial numbers.

[0047] By the attached Figure 1 and attached Figure 3 It can be seen that when the mitochondria in Example 1 and Comparative Example 1 were magnified 2000 times, the number of mitochondria in Example 1 increased significantly, and the morphology was the same as that in Comparative Example 1. Figure 2 and attached Figure 4 It can be seen that when the mitochondria in Example 1 and Comparative Example 1 were magnified 10,000 times respectively, the number of mitochondria in Example 1 increased significantly. This indicates that the addition of SFRP-1 to the culture medium can proliferate mitochondria without destroying the structural morphology of mitochondria.

[0048] By the attached Figure 5 It can be seen that in Comparative Example 1, every 100 μm 2 The number of mitochondria in the sample is 6.63, and in Example 1, every 100 μm 2 The number of mitochondria in the culture medium was 14.81, which was significantly higher than that in Comparative Example 1. This indicates that the addition of SFRP-1 in the culture medium can make mesenchymal stem cells express more mitochondria, and the number of mitochondria is significantly increased, thereby achieving the purpose of mitochondrial proliferation.

[0049] 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 culture medium for promoting mitochondrial proliferation, characterized in that: include: DMEM / F12 culture medium, fetal bovine serum, double antibody and SFRP-1, wherein the mass fractions of DMEM / F12 culture medium, fetal bovine serum and double antibody are 84-94%, 5-15% and 0.5-1.5% respectively, and the concentration of SFRP-1 in the culture medium is 0.1-0.5 ng / ml.

2. The culture medium for promoting mitochondrial proliferation according to claim 1, characterized in that The culture medium includes 89% DMEM / F12 culture medium, 10% fetal bovine serum and 1% double antibody in terms of mass fraction, and the concentration of SFRP-1 in the culture medium is 0.3 ng / ml.

3. The culture medium for promoting mitochondrial proliferation according to claim 1, characterized in that The dual antibody is penicillin-streptomycin.

4. A method for promoting mitochondrial proliferation, characterized in that: include: Cultivating mesenchymal stem cells in the culture medium of any one of claims 1 to 3 without adding SFRP-1 to the P3 generation; When the mesenchymal stem cells grow to a coverage area of ​​55-65% in the culture bottle, SFRP-1 is added to the culture medium and the culture is continued for 36-48 hours; After the culture is completed, mitochondria in the mesenchymal stem cells are extracted.

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