Neural stem cell low-temperature preservation solution

Abstract

The invention discloses a low-temperature preservation solution for neural stem cells, which is prepared by organically combining polysaccharide, ginsenoside Rg3 and baicalin, and can well maintain the motility rate of the neural stem cells cryopreserved for a long time in the absence of dimethyl sulfoxide, and experiments show that the low-temperature preservation solution for the neural stem cells has the advantages that the survival rate of the neural stem cells cryopreserved for a long time can be well maintained; the ginseng polysaccharide and the astragalus polysaccharide are compounded, so that compared with single ginseng polysaccharide or astragalus polysaccharide, the compound has better capability of maintaining the motility rate of the neural stem cells cryopreserved for a long time.

Description

Technical Field

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

[0002] Neural stem cells, as cells with self-renewal ability and multidirectional differentiation potential, are derived from neural tissue and can generate neural tissue. Under appropriate conditions, they can differentiate into neurons, oligodendrocytes and astrocytes. Neural stem cells provide a new way for functional reconstruction and neural regeneration of central nervous system diseases, and have broad clinical application prospects. Neural stem cells, namely adult neural stem cells, are adult stem cells, and generally need to be stored at low temperatures, that is, dispersed in a preservation solution and cryopreserved under the protection of a preservation solution. The preservation solution suitable for long-term preservation is a cryopreservation solution, which is used to freeze cells at extremely low temperatures to prolong their survival time and maintain their functions. The current cell cryopreservation solution usually contains about 5% to 10% dimethyl sulfoxide (DMSO) by volume, but dimethyl sulfoxide itself is damaging to cells, and long-term freezing will enhance this damaging effect; on the other hand, after being preserved in the existing preservation solution, the stem cell damage will occur during the recovery process, affecting the survival rate. Currently, developing DMSO-free cell cryopreservation fluid is one of the hot research directions. Different cells have different biological characteristics and environmental tolerance, but currently there are few or even no technical solutions for dimethyl sulfoxide-free cryopreservation fluid for ultra-low temperature preservation of neural stem cells. Summary of the invention

[0003] Therefore, based on the above background, the present invention provides a neural stem cell cryopreservation solution, which is free of dimethyl sulfoxide and can preserve neural stem cells for a long time under freezing conditions, and maintain a high cell survival rate after recovery.

[0004] The technical solution of the present invention is: A neural stem cell cryopreservation solution, which 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 remainder DMEM / F-12 culture medium.

[0005] Furthermore, the polysaccharide includes at least one of ginseng polysaccharide and astragalus polysaccharide.

[0006] Furthermore, the polysaccharide is composed of ginseng polysaccharide and astragalus polysaccharide, and the mass ratio of ginseng polysaccharide to astragalus polysaccharide is (1-6):1.

[0007] Furthermore, the mass ratio of ginseng polysaccharide to astragalus polysaccharide is 3:1.

[0008] Furthermore, the serum is serum from species from which neural stem cells are derived.

[0009] Based on the same inventive concept, the present invention provides a method for preparing a neural stem cell cryopreservation solution, comprising the following steps: 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.

[0010] Based on the same inventive concept, the present invention provides a method for freezing neural stem cells, which comprises the following steps: 1) A neural stem cell cryopreservation solution prepared as described above; 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.

[0011] The beneficial effects achieved by the technical solution of the present invention are: The invention discloses a neural stem cell cryopreservation solution. The cryopreservation solution for neural stem cell cryopreservation is prepared by organically combining polysaccharides, ginsenoside Rg3 and baicalin. The cryopreservation solution can well maintain the viability of neural stem cells cryopreserved for a long time in the absence of dimethyl sulfoxide. Experiments have shown that, compared with single use of ginseng polysaccharide or astragalus polysaccharide, compounding ginseng polysaccharide and astragalus polysaccharide has better ability to maintain the viability of neural stem cells cryopreserved for a long time. DETAILED DESCRIPTION

[0012] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is 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.

[0013] The technical solution of the present invention: A neural stem cell cryopreservation solution, which is made of human 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 remainder DMEM / F-12 culture medium.

[0014] The serum is the serum of the species from which the neural stem cells originate. For example, if the neural stem cells are derived from humans, the serum is human serum.

[0015] Preferably, the polysaccharide is composed of ginseng polysaccharide and astragalus polysaccharide, and the mass ratio of ginseng polysaccharide to astragalus polysaccharide is (1-6):1. More preferably, the mass ratio of ginseng polysaccharide to astragalus polysaccharide is 3:1.

[0016] The method for preparing the neural stem cell cryopreservation solution comprises the following steps: 1) adding polysaccharide and human serum into 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.

[0017] The main materials and instruments used in the following examples are as follows: Carbon dioxide incubator (SANYO, Japan); ordinary refrigerator (Haier, Qingdao); cell counter (Rayward); Neural stem cells were derived from the human neural stem cell line of the Pediatric Laboratory of the Sixth Medical Center of PLA General Hospital; Human serum (Hengyuan Biotechnology, product number HY-10069); DMEM / F-12 culture medium (Guanglan Biotechnology, catalog number GS-3301); Ginsenoside Rg3 (Solebol, CAS No.: 14197-60-5); Ginsenoside Rg1 (Yunnan Xili Biological, CAS No.: 22427-39-0) Baicalin (Yunnan Xili Biological, CAS No.: 21967-41-9); Astragalus polysaccharide (Shanghai Aladdin Biochemical Technology, CAS No.: 89250-26-0); Ginseng polysaccharide (Fufeng Ciyuan Biological, No. Ksm104) Example 1: A neural stem cell cryopreservation solution, wherein each 100 ml of the neural stem cell cryopreservation solution contains: Human serum 15ml, propylene glycol 8ml, polysaccharide 0.4g, ginsenoside Rg3 0.4mg, baicalin 2mg, and the balance DMEM / F-12 culture medium.

[0018] The polysaccharide consists of ginseng polysaccharide and astragalus polysaccharide, and the mass ratio of the ginseng polysaccharide to the astragalus polysaccharide is 3:1.

[0019] Example 2: A neural stem cell cryopreservation solution, wherein each 100 ml of the neural stem cell cryopreservation solution contains: Human serum 15ml, propylene glycol 8ml, ginseng polysaccharide 0.4g, ginsenoside Rg3 0.4mg, baicalin 2mg, and the balance DMEM / F-12 culture medium.

[0020] Example 3: A neural stem cell cryopreservation solution, wherein each 100 ml of the neural stem cell cryopreservation solution contains: Human serum 15ml, propylene glycol 8ml, astragalus polysaccharide 0.4g, ginsenoside Rg3 0.4mg, baicalin 2mg, and the balance DMEM / F-12 culture medium.

[0021] Comparative Example 1: A neural stem cell cryopreservation solution, wherein each 100 ml of the neural stem cell cryopreservation solution contains: 15 ml human serum, 8 ml propylene glycol, 0.3 g ginseng polysaccharide, 0.1 g astragalus polysaccharide, 0.4 mg ginsenoside Rg3, and the balance DMEM / F-12 culture medium.

[0022] Comparative Example 2: A neural stem cell cryopreservation solution, wherein each 100 ml of the neural stem cell cryopreservation solution contains: A neural stem cell cryopreservation solution, wherein each 100 ml of the neural stem cell cryopreservation solution contains: 15 ml human serum, 8 ml propylene glycol, 0.4 mg ginsenoside Rg3, 2 mg baicalin, and the balance DMEM / F-12 culture medium.

[0023] Comparative Example 3: A neural stem cell cryopreservation solution, wherein each 100 ml of the neural stem cell cryopreservation solution contains: Each 100 ml of the neural stem cell cryopreservation solution contains: 15 ml human serum, 8 ml propylene glycol, 0.4 g polysaccharide, 2 mg baicalin, and the remainder DMEM / F-12 culture medium.

[0024] The polysaccharide consists of ginseng polysaccharide and astragalus polysaccharide, and the mass ratio of the ginseng polysaccharide to the astragalus polysaccharide is 3:1.

[0025] Comparative Example 4: A neural stem cell cryopreservation solution, wherein each 100 ml of the neural stem cell cryopreservation solution contains: 15 ml human serum, 8 ml propylene glycol, and the balance DMEM / F-12 culture medium.

[0026] Comparative Example 5: A neural stem cell cryopreservation solution, wherein each 100 ml of the neural stem cell cryopreservation solution contains: Each 100 ml of the neural stem cell cryopreservation solution contains: Human serum 15ml, propylene glycol 8ml, polysaccharide 0.4g, ginsenoside Rg1 0.4mg, baicalin 2mg, and the balance DMEM / F-12 culture medium.

[0027] The polysaccharide consists of ginseng polysaccharide and astragalus polysaccharide, and the mass ratio of the ginseng polysaccharide to the astragalus polysaccharide is 3:1.

[0028] The composition of each 100 ml of the neural stem cell cryopreservation solution in the above embodiments and comparative examples is specifically shown in Table 1.

[0029] Table 1: Composition of the preservation solution of each embodiment and comparative example (per 100 ml of neural stem cell cryopreservation solution)

[0030] According to the preservation solution composition in Table 1, a neural stem cell preservation solution was prepared according to the technical solution of the present invention for cryopreservation experiments. The subsequent cryopreservation and resuscitation experiments were all conducted in the Pediatric Laboratory of the Sixth Medical Center of the PLA General Hospital.

[0031] Cryopreservation experiment: Take the stem cells in the logarithmic growth phase, digest them, count them, and divide the cells into 24 equal parts, with 1×10 cells in each part. 6 After centrifugation at 2 000 r / min for 5 min, discard the supernatant, flick the cell pellet to mix, then add it to the cryopreservation solution prepared above and resuspend it. Then put it into sterile cell freezing tubes. The cryopreservation solution for each group was repeated three times.

[0032] Cryopreservation operation: First place it at 4℃ for 50min, then pre-freeze it at -20℃ for 1.5h, then cool it down to -80℃ at 1℃ / min and keep it for 8h, and then transfer it to liquid nitrogen for storage.

[0033] Then, after being frozen for 1 month, a resuscitation experiment was performed.

[0034] After the cryovials were taken out of liquid nitrogen, they were revived in a 37°C water bath, and the cell viability was counted using trypan blue staining and a cell counter.

[0035] Cell viability (%) = total number of live cells*100 / (total number of live cells+total number of dead cells).

[0036] The cell viability of each group of cryopreserved fluid was taken as the average value of three replicate groups, and the results are shown in Table 2.

[0037] Table 2: Cell viability of neural stem cells after resuscitation in each group

[0038] As shown in Table 1, the cryopreservation solution prepared by organically combining polysaccharides, ginsenoside Rg3 and baicalin based on DMEM / F-12 culture medium can make neural stem cells have a good cell viability after long-term cryopreservation, and the effect of compounding ginseng polysaccharide and astragalus polysaccharide in a ratio of 3:1 is better than that of using ginseng polysaccharide or astragalus polysaccharide alone, which shows that the two have a certain synergistic effect. In addition, for the cell viability of neural stem cells after long-term cryopreservation, ginsenoside Rg3 has a better effect than ginsenoside Rg1.

[0039] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection 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 remainder DMEM / F-12 culture medium.

2. A neural stem cell cryopreservation solution according to claim 1, characterized in that: The polysaccharide includes at least one of ginseng polysaccharide and astragalus polysaccharide.

3. A neural stem cell cryopreservation solution according to claim 2, characterized in that: 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.

4. A neural stem cell cryopreservation solution according to claim 3, characterized in that: The mass ratio of the ginseng polysaccharide to the astragalus polysaccharide is 3:

1.

5. 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.

6. The method for preparing a neural stem cell cryopreservation solution according to any one of claims 1 to 5, 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.

7. 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 6; 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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