A cryopreservation solution for neural stem cells

Abstract

The present invention discloses a cryopreservation solution for neural stem cells. The cryopreservation solution for neural stem cells prepared by organically combining polysaccharides, ginsenoside Rg3 and baicalin can well maintain the viability of neural stem cells cryopreserved for a long time in the absence of dimethyl sulfoxide. And through experiments, it is found that by compounding ginseng polysaccharide and astragalus polysaccharide, compared with using only ginseng polysaccharide or astragalus polysaccharide alone, its ability to maintain the viability of neural stem cells cryopreserved for a long time is better.

Description

Technical Field

[0001] The present invention relates to the technical field of neural stem cells, and in particular to a cryopreservation solution for neural stem cells. Background Art

[0002] As a kind of cells with self-renewal ability and multi-directional differentiation potential, neural stem cells are derived from neural tissues and can generate neural tissues, and can differentiate into neurons, oligodendrocytes and astrocytes under appropriate conditions. Neural stem cells provide a new way for the functional reconstruction and nerve regeneration of central nervous system diseases, and have broad clinical application prospects. Neural stem cells, that is, adult neural stem cells, are adult stem cells and generally need to be cryopreserved, that is, dispersed in a preservation solution and cryopreserved at low temperature under the protection of the preservation solution. The preservation solution suitable for long-term preservation is a cryopreservation solution, which is used to freeze and preserve cells at extremely low temperatures to extend their survival time and maintain their functions. Currently, the commonly used cell cryopreservation solution usually contains about 5% - 10% (volume fraction) of dimethyl sulfoxide (DMSO). However, DMSO itself has damage to cells, and this damage effect will be enhanced during long-term cryopreservation. On the other hand, after the existing preservation solution is used for preservation, it will cause damage to stem cells during the resuscitation process, affecting the survival rate. Currently, developing a DMSO-free cell cryopreservation solution is one of the hot research directions. Different cells have different biological characteristics and environmental tolerances, but currently, there are few or no technical solutions for DMSO-free cryopreservation solutions for cryopreserving neural stem cells at extremely low temperatures. Summary of the Invention

[0003] Therefore, based on the above background, the present invention provides a cryopreservation solution for neural stem cells without DMSO, which can cryopreserve neural stem cells for a long time under cryopreservation conditions and has a high cell survival rate after resuscitation.

[0004] The technical solution of the present invention is as follows:

[0005] A cryopreservation solution for neural stem cells is made up of serum, DMEM / F-12 culture medium, polysaccharide, ginsenoside Rg3, baicalin, and propylene glycol.

[0006] Each 100 ml of the cryopreservation solution for neural stem cells contains:

[0007] 10 - 40 ml of serum, 5 - 10 ml of propylene glycol, 0.2 - 0.5 g of polysaccharide, 0.2 - 1 mg of ginsenoside Rg3, 0.5 - 4 mg of baicalin, and the balance of DMEM / F-12 culture medium.

[0008] Further, the polysaccharide includes at least one of ginseng polysaccharide and astragalus polysaccharide.

[0009] Further, the polysaccharide is composed of ginseng polysaccharide and astragalus polysaccharide, and the mass ratio of the ginseng polysaccharide to the astragalus polysaccharide is (1 - 6):1.

[0010] Further, the mass ratio of the ginseng polysaccharide to the astragalus polysaccharide is 3:1.

[0011] Further, the serum is the serum of the species from which the neural stem cells are derived.

[0012] Based on the same inventive concept, the present invention provides a method for preparing a cryopreservation solution for neural stem cells, comprising the following steps:

[0013] 1) Take the polysaccharide and the serum and add them to the DMEM / F-12 culture medium respectively, mix them evenly to obtain a first mixture;

[0014] 2) Take ginsenoside Rg3 and baicalin, add them to propylene glycol, stir and dissolve them, then add them to the first mixture in step 1), stir and mix evenly, and filter and sterilize to obtain the cryopreservation solution for neural stem cells.

[0015] Based on the same inventive concept, the present invention provides a method for cryopreserving neural stem cells, which comprises the following steps:

[0016] 1) Prepare the cryopreservation solution for neural stem cells according to the above formulation;

[0017] 2) Select neural stem cells in the logarithmic growth phase and inoculate them into the cryopreservation solution for neural stem cells prepared in step 1);

[0018] 3) Then place it at 0 - 4°C for 30 - 70 min first, pre-freeze it at -15 - -20°C for 0.5 - 3 h, then cool it to -80°C at a rate of 1 - 5°C / min and maintain it for 5 - 10 h, and finally transfer it to liquid nitrogen for storage.

[0019] The beneficial effects achieved by adopting the technical solution of the present invention are as follows:

[0020] The present invention discloses a cryopreservation solution for neural stem cells. The cryopreservation solution for neural stem cell cryopreservation prepared by organically combining polysaccharide, ginsenoside Rg3 and baicalin can well maintain the viability of neural stem cells cryopreserved for a long time in the absence of dimethyl sulfoxide. And through experiments, it is found that by compounding ginseng polysaccharide and astragalus polysaccharide, compared with using ginseng polysaccharide or astragalus polysaccharide alone, its ability to maintain the viability of neural stem cells cryopreserved for a long time is better. Detailed implementation mode

[0021] In order to make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below in conjunction with its embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0022] Technical solution of the present invention: A cryopreservation solution for neural stem cells, which is made of human serum, DMEM / F-12 culture medium, polysaccharide, ginsenoside Rg3, baicalin, and propylene glycol;

[0023] Each 100 ml of the cryopreservation solution for neural stem cells contains:

[0024] 10 - 40 ml of serum, 5 - 10 ml of propylene glycol, 0.2 - 0.5 g of polysaccharide, 0.2 - 1 mg of ginsenoside Rg3, 0.5 - 4 mg of baicalin, and the balance of DMEM / F-12 culture medium.

[0025] The serum is the serum of the species from which the neural stem cells are derived. For example, if the neural stem cells are derived from humans, the serum is human serum.

[0026] Preferably, the polysaccharide is composed of ginseng polysaccharide and astragalus polysaccharide, and the mass ratio of the ginseng polysaccharide to the astragalus polysaccharide is (1 - 6):1. Further preferably, the mass ratio of the ginseng polysaccharide to the astragalus polysaccharide is 3:1.

[0027] The preparation method of the above-mentioned cryopreservation solution for neural stem cells includes the following steps:

[0028] 1) Take the polysaccharide and human serum and add them to the DMEM / F-12 culture medium respectively, mix them evenly to obtain a first mixture;

[0029] 2) Take ginsenoside Rg3 and baicalin, add them to propylene glycol and stir to dissolve, then add them to the first mixture in step 1), stir and mix evenly, and filter and sterilize to obtain the cryopreservation solution for neural stem cells.

[0030] The main materials and instruments used in the following experimental examples are as follows:

[0031] Carbon dioxide incubator (SANYO, Japan); ordinary refrigerator (Haier, Qingdao); cell counter (Rewod);

[0032] The neural stem cells are from the human neural stem cell line in the Pediatric Laboratory of the Sixth Medical Center of the Chinese PLA General Hospital;

[0033] Human serum (Hengyuan Bio, product number HY-10069);

[0034] DMEM / F-12 culture medium (Guanglan Bio, product number GS-3301);

[0035] Ginsenoside Rg3 (Solarbio, CAS No.: 14197-60-5);

[0036] Ginsenoside Rg1 (Yunnan XiLi Biotechnology, CAS No.: 22427-39-0)

[0037] Baicalin (Yunnan XiLi Biotechnology, CAS No.: 21967-41-9);

[0038] Astragalus polysaccharide (Shanghai Aladdin Biochemical Technology, CAS No.: 89250-26-0);

[0039] Ginseng polysaccharide (Fufeng Ciyuan Biotechnology, No., Ksm104)

[0040] Example 1: A cryopreservation solution for neural stem cells. In every 100 ml of the cryopreservation solution for neural stem cells, there is:

[0041] 15 ml of human serum, 8 ml of propylene glycol, 0.4 g of polysaccharide, 0.4 mg of ginsenoside Rg3, 2 mg of baicalin, and the balance is DMEM / F-12 culture medium.

[0042] The polysaccharide consists of ginseng polysaccharide and astragalus polysaccharide, and the mass ratio of ginseng polysaccharide to astragalus polysaccharide is 3:1.

[0043] Example 2: A cryopreservation solution for neural stem cells. In every 100 ml of the cryopreservation solution for neural stem cells, there is:

[0044] 15 ml of human serum, 8 ml of propylene glycol, 0.4 g of ginseng polysaccharide, 0.4 mg of ginsenoside Rg3, 2 mg of baicalin, and the balance is DMEM / F-12 culture medium.

[0045] Example 3: A cryopreservation solution for neural stem cells. In every 100 ml of the cryopreservation solution for neural stem cells, there is:

[0046] 15 ml of human serum, 8 ml of propylene glycol, 0.4 g of astragalus polysaccharide, 0.4 mg of ginsenoside Rg3, 2 mg of baicalin, and the balance is DMEM / F-12 culture medium.

[0047] Comparative Example 1: A cryopreservation solution for neural stem cells. In every 100 ml of the cryopreservation solution for neural stem cells, there is:

[0048] 15 ml of human serum, 8 ml of propylene glycol, 0.3 g of ginseng polysaccharide, 0.1 g of astragalus polysaccharide, 0.4 mg of ginsenoside Rg3, and the balance is DMEM / F-12 culture medium.

[0049] Comparative Example 2: A cryopreservation solution for neural stem cells. In every 100 ml of the cryopreservation solution for neural stem cells, there is:

[0050] A cryopreservation solution for neural stem cells, in every 100 ml of the cryopreservation solution for neural stem cells, there is:

[0051] 15 ml of human serum, 8 ml of propylene glycol, 0.4 mg of ginsenoside Rg3, 2 mg of baicalin, and the balance is DMEM / F-12 culture medium.

[0052] Comparative Example 3: A cryopreservation solution for neural stem cells, in every 100 ml of the cryopreservation solution for neural stem cells, there is:

[0053] In every 100 ml of the cryopreservation solution for neural stem cells, there is:

[0054] 15 ml of human serum, 8 ml of propylene glycol, 0.4 g of polysaccharide, 2 mg of baicalin, and the balance is DMEM / F-12 culture medium.

[0055] The polysaccharide is composed of ginseng polysaccharide and astragalus polysaccharide, and the mass ratio of the ginseng polysaccharide to the astragalus polysaccharide is 3:1.

[0056] Comparative Example 4: A cryopreservation solution for neural stem cells, in every 100 ml of the cryopreservation solution for neural stem cells, there is:

[0057] 15 ml of human serum, 8 ml of propylene glycol, and the balance is DMEM / F-12 culture medium.

[0058] Comparative Example 5: A cryopreservation solution for neural stem cells, in every 100 ml of the cryopreservation solution for neural stem cells, there is:

[0059] In every 100 ml of the cryopreservation solution for neural stem cells, there is:

[0060] 15 ml of human serum, 8 ml of propylene glycol, 0.4 g of polysaccharide, 0.4 mg of ginsenoside Rg1, 2 mg of baicalin, and the balance is DMEM / F-12 culture medium.

[0061] The polysaccharide is composed of ginseng polysaccharide and astragalus polysaccharide, and the mass ratio of the ginseng polysaccharide to the astragalus polysaccharide is 3:1.

[0062] The compositions of every 100 ml of the cryopreservation solution for neural stem cells in the above examples and comparative examples are shown in Table 1 specifically.

[0063] Table 1: Compositions of the preservation solutions in the examples and comparative examples (every 100 ml of the cryopreservation solution for neural stem cells)

[0064]

[0065] Prepare the nerve stem cell cryopreservation solution according to the technical solution of the present invention with the cryopreservation solution composition shown in Table 1 for the cryopreservation experiment. The subsequent cryopreservation and resuscitation experiments will all be carried out in the pediatric laboratory of the Sixth Medical Center of the Chinese PLA General Hospital.

[0066] Cryopreservation experiment:

[0067] Take the nerve stem cells in the logarithmic growth phase, digest them, count them, and divide the cells into 24 equal parts, with the number of cells in each part being 1×10 6 , after centrifuging at 2,000 r / min for 5 min, discard the supernatant, gently flick to mix the cell pellet, then add it to the prepared cryopreservation solution above to resuspend, and transfer it into a sterile cell cryopreservation tube. Each cryopreservation solution in each group has three replicates.

[0068] Cryopreservation operation:

[0069] First, place it at 4°C for 50 min, then pre-freeze it at -20°C for 1.5 h, then cool it to -80°C at a rate of 1°C / min and hold for 8 h, and then transfer it to liquid nitrogen for storage.

[0070] Then, after 1 month of cryopreservation, conduct the resuscitation experiment.

[0071] After taking out the cryopreservation tube from liquid nitrogen, resuscitate it in a 37°C water bath, and use the trypan blue staining method and a cell counter to count the cell viability.

[0072] Cell viability (%) = total number of live cells * 100 / (total number of live cells + total number of dead cells).

[0073] The cell viability of each cryopreservation solution group takes the average value of three replicate groups, and the results are shown in Table 2.

[0074] Table 2: Cell viability of nerve stem cells after resuscitation in each group

[0075]

[0076] As can be seen from the results in Table 1, based on DMEM / F-12 culture medium, the cryopreservation solution prepared by the organic combination of polysaccharides, ginsenoside Rg3, and baicalin can enable nerve stem cells to have a good cell viability after long-term cryopreservation. And the effect of the compounding of ginseng polysaccharide and astragalus polysaccharide in a ratio of 3:1 is better than that of using ginseng polysaccharide or astragalus polysaccharide alone, indicating that the two have a certain synergistic effect. And for the cell viability of nerve stem cells after long-term cryopreservation, ginsenoside Rg3 has a better effect than ginsenoside Rg1.

[0077] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A neural stem cell cryopreservation solution, characterized in that: It is made of serum, DMEM / F-12 culture medium, polysaccharide, ginsenoside Rg3, baicalin, and propylene glycol; Each 100 ml of the neural stem cell cryopreservation solution contains: Serum 10-40ml, propylene glycol 5-10ml, polysaccharide 0.2-0.5g, ginsenoside Rg3 0.2-1mg, baicalin 0.5-4mg, and the rest DMEM / F-12 culture medium; The polysaccharide consists of ginseng polysaccharide and astragalus polysaccharide, and the mass ratio of the ginseng polysaccharide to the astragalus polysaccharide is 3:

1.

2. A neural stem cell cryopreservation solution according to claim 1, characterized in that: The serum is serum from species where neural stem cells are derived.

3. The method for preparing a neural stem cell cryopreservation solution according to claim 1 or 2, characterized in that: The steps include: 1) adding polysaccharide and serum to DMEM / F-12 culture medium respectively and mixing evenly to obtain a first mixture; 2) Ginsenoside Rg3 and baicalin are added to propylene glycol and stirred to dissolve, and then added to the first mixture of step 1), stirred and mixed, and filtered and sterilized to obtain the neural stem cell cryopreservation solution.

4. A method for freezing neural stem cells, characterized in that: It includes the following steps: 1) preparing a neural stem cell cryopreservation solution according to claim 3; 2) Selecting neural stem cells in the logarithmic growth phase and inoculating them into the neural stem cell cryopreservation solution prepared in step 1); 3) Place it at 0-4℃ for 30-70min, pre-freeze it at -15-20℃ for 0.5-3h, cool it down to -80℃ at 1-5℃ / min and keep it for 5-10h. Finally, transfer it to liquid nitrogen for storage.

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