Human neural stem cell preserving fluid and preparation method thereof

Abstract

The invention discloses a human neural stem cell preserving fluid and a preparation method thereof, and the preserving fluid comprises 30 mM of potassium ions, 30 mM of sodium ions, 0.5 mM of magnesium ions, 5 mM of dihydrogen phosphate ions, 5 mM of hydroxyl ions, 20 mM of acetate ions, 32 mM of chloride ions, 5 mM of glucose, 200 mM of sorbitol and pure water. The preparation method comprises the following steps: mixing potassium chloride, sodium hydroxide, sodium dihydrogen phosphate dihydrate, magnesium chloride hexahydrate, sodium pyruvate, glucose and sorbitol, uniformly stirring while adding pure water, fixing the volume, and filtering by using a 0.22 mu m filter to obtain the product. And centrifuging 500g of human neural stem cells for 3 minutes, discarding the supernatant, resuspending the cells by using the preserving fluid, subpackaging and transferring to 2-8 DEG C for preservation. The preserving fluid can prolong the survival rate maintaining time of the human neural stem cells, and the cell survival rate is not lower than 90% after the human neural stem cells are preserved for 72 hours.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and in particular relates to a human neural stem cell preservation solution and a preparation method thereof. Background Art

[0002] Neural stem cells (NSC) are important members of the stem cell family. They originate from the ectoderm in the early stages of development and have the potential for self-renewal and differentiation. They can partially replace lost and damaged nerve cells and repair damaged nervous systems. Although the detailed mechanism of neural stem cells in the treatment of neurodegenerative diseases such as Alzheimer's disease is still unclear, their clinical application has received increasing attention and has achieved certain results. They have now become the focus of widespread attention in the future fields of life sciences and medical research.

[0003] At present, there are few domestic institutions that are qualified to achieve high-throughput separation, culture, and expansion of human neural stem cells, but the clinical application of human neural stem cells is very wide. The cultivation of neural stem cells has become mature, and the application of neural stem cell therapy is also very in-depth. However, neural stem cell clinical projects and neural stem cell therapies with a wide range of audiences often involve long-distance transportation between cell preparation units and cell transplantation units. Living cells are the key active ingredients of cell products, and long-term transportation places high-quality requirements on the maintenance of cell viability. Therefore, there is an urgent need to develop a neural stem cell preservation solution that can maintain the activity of neural stem cells in this protective solution for a long time, meet the needs of transportation to most parts of the country and neighboring countries, and can be directly used in the human body, safe and non-toxic.

[0004] At present, domestic stem cell institutions usually use normal saline and cell culture medium as solution media for clinical stem cell applications. Normal saline is safe and convenient, and is often used as an infusion medium. However, the vitality of stem cells in normal saline decreases rapidly. The method of using only normal saline as a cell preservation fluid is only suitable for short-term preservation or immediate use of stem cell products. Cell culture medium is also often used as the main component of cell protection fluid, which provides the nutrients required for cell growth and metabolism, including amino acids, sugars, inorganic salts, vitamins, etc., and has a good effect on maintaining cell osmotic pressure and vitality. However, most of the current domestic cell culture media are scientific research-level applications and cannot be used in clinical research. At the same time, due to different manufacturers and production methods, the cost and quality of cell culture media are also different, and better quality cell culture media are more expensive. Summary of the invention

[0005] In view of the above problems existing in the prior art, the main purpose of the present invention is to provide a human neural stem cell preservation solution, which can maintain a high cell viability of human neural stem cells during transportation.

[0006] Another object of the present invention is to provide a method for preparing the human neural stem cell preservation solution.

[0007] Another object of the present invention is to provide a human neural stem cell suspension, the cell viability of which is not less than 90% after being stored for 72 hours.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] The invention provides a human neural stem cell preservation solution, which comprises: 30mM potassium ions, 30mM sodium ions, 0.5mM magnesium ions, 5mM dihydrogen phosphate ions, 5mM hydroxide ions, 20mM acetate ions, 32mM chloride ions, 5mM glucose, 200mM sorbitol and pure water.

[0010] Preferably, the glucose is a glucose solution approved for clinical use.

[0011] Preferably, the potassium ions come from potassium chloride;

[0012] The magnesium ions come from magnesium chloride hexahydrate;

[0013] The dihydrogen phosphate ion comes from sodium dihydrogen phosphate dihydrate;

[0014] The hydroxide ions come from sodium hydroxide;

[0015] The acetate ion comes from sodium pyruvate;

[0016] The sodium ions come from sodium hydroxide, sodium dihydrogen phosphate dihydrate and sodium pyruvate;

[0017] The chloride ions come from potassium chloride and magnesium chloride hexahydrate.

[0018] Preferably, the human neural stem cell preservation medium is a liquid medium free of bacteria, viruses, and mycoplasma contamination and with an endotoxin content of <5EU / mL.

[0019] The present invention also provides a method for preparing the human neural stem cell preservation solution, comprising: mixing potassium chloride, sodium hydroxide, sodium dihydrogen phosphate dihydrate, magnesium chloride hexahydrate, sodium pyruvate, glucose and sorbitol, adding pure water while stirring evenly, fixing the volume to 100 mL, and filtering with a 250 mL 0.22 μm filter to obtain the human neural stem cell preservation solution.

[0020] The present invention also provides a human neural stem cell suspension, the preparation method of which includes: centrifuging the human neural stem cells at 500g for 3 minutes, discarding the supernatant, resuspending the human neural stem cells in the obtained preservation solution, adjusting the concentration to 1-9×10^6 / mL, packaging the suspension, and transferring it to 2-8°C for storage.

[0021] Preferably, the concentration of human neural stem cells in the human neural stem cell suspension is 2-5×10^6 / mL.

[0022] Compared with the prior art, the beneficial effects of the present invention are: the human neural stem cell preservation solution of the present invention prolongs the viability maintenance time of neural stem cells, expands the physical application range of neural stem cells by utilizing existing transportation vehicles, reduces the preparation cost of the protective solution, has a wide source, is simple in method, and is safe and reliable, and can be directly applied clinically. After testing, after the neural stem cells are preserved in the protective solution for 72 hours, the cell viability is still not less than 90%. DETAILED DESCRIPTION

[0023] In order to more fully understand and demonstrate the technical solutions, purposes and advantages of the present invention, the technical effects produced by the present invention are further described in detail and completely in conjunction with specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all. It should be pointed out that for those of ordinary skill in the art, other embodiments obtained without departing from the concept of the present invention all belong to the protection scope of the present invention, and the present invention is specifically described in conjunction with the embodiments below.

[0024] Unless otherwise specified in the following examples, all reagents and materials used were commercially available.

[0025] Example 1

[0026] The steps for preserving 2×10^6 / mL human neural stem cells are as follows:

[0027] 1. Preparation of human neural stem cell preservation solution: Mix potassium chloride, sodium hydroxide, sodium dihydrogen phosphate dihydrate, magnesium chloride hexahydrate, sodium pyruvate, glucose and sorbitol, add purified water and stir evenly, make the volume to 100 mL, and filter with a 250 mL 0.22 μm filter.

[0028] 2. Preparation of human neural stem cell suspension: After centrifugation (500g, 3min), the supernatant was discarded, and the cells were resuspended in preservation solution to adjust the concentration to 2×10^6 / mL. The cell suspension of this concentration was divided into 5 tubes, 0.2mL / tube, and the cells resuspended in 5 tubes of preservation solution were transferred to 2-8°C. Samples were taken at 0h, 24h, 48h, 72h, and 168h, respectively, and stained with AO / PI dye. The cell survival rate was counted on a cell counter, as shown in Table 1.

[0029] Table 1

[0030] Storage time (h) Cell viability 0 92.53±0.13％ 24 91.01±0.38％ 48 90.79±1.07％ 72 85.03±0.61％ 168 79.32±1.41％

[0031] The results in Table 1 show that under 2-8°C conditions, the viability of cells with a total cell concentration of 2×10^6 / mL preserved in the preservation solution within 48 hours was higher than 90%; the viability of cells with a total cell concentration of 2×10^6 / mL preserved in the preservation solution for 72 hours was lower than 90%; and beyond 72 hours, the cell viability was significantly lower than 90%. The use of cell preservation solution can maintain the viability of low-concentration cells above 90% for at least 48 hours, which is used for long-term transportation and storage of transfused cells, meeting clinical transfusion requirements.

[0032] Example 2

[0033] The steps for preserving 5×10^6 / mL human neural stem cells are as follows:

[0034] 1. Preparation of human neural stem cell preservation solution: Mix potassium chloride, sodium hydroxide, sodium dihydrogen phosphate dihydrate, magnesium chloride hexahydrate, sodium pyruvate, glucose and sorbitol, add purified water and stir evenly, make the volume to 100 mL, and filter with a 250 mL 0.22 μm filter.

[0035] 2. Preparation of human neural stem cell suspension: After centrifugation (500g, 3min), the supernatant was discarded, and the cells were resuspended in preservation solution to adjust the concentration to 5×10^6 / mL. The cell suspension at this concentration was divided into 5 tubes, 0.2mL / tube, and the cells resuspended in 5 tubes of preservation solution were transferred to 2-8°C. Samples were taken at 0h, 24h, 48h, 72h, and 168h, respectively, and stained with AO / PI dye. The cell survival rate was counted on a cell counter, as shown in Table 2.

[0036] Table 2

[0037] Storage time (h) Cell viability 0 94.43±0.12％ 24 92.32±0.09％ 48 92.37±0.29％ 72 93.34±0.56％ 168 87.24±2.26％

[0038] The results in Table 2 show that under 2-8°C conditions, the viability of cells with a total cell concentration of 5×10^6 / mL preserved in preservation solution within 72 hours is higher than 90%, and can even reach 93%; after preservation in preservation solution for 168 hours, the viability of cells with a total cell concentration of 5×10^6 / mL can still be maintained above 80%. The cell preservation solution can maintain a high cell concentration of 5×10^6 / mL cell viability of more than 90% for at least 72 hours, which is used for long-term transportation and storage of transfused cells, meeting clinical transfusion requirements.

[0039] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A human neural stem cell preservation solution, characterized in that: Contains: 30mM potassium ion, 30mM sodium ion, 0.5mM magnesium ion, 5mM dihydrogen phosphate ion, 5mM hydroxide ion, 20mM acetate ion, 32mM chloride ion, 5mM glucose, 200mM sorbitol and purified water.

2. The human neural stem cell preservation solution according to claim 1, characterized in that: The glucose is a glucose solution approved for clinical use.

3. The human neural stem cell preservation solution according to claim 1, characterized in that: The potassium ions come from potassium chloride; The magnesium ions come from magnesium chloride hexahydrate; The dihydrogen phosphate ion comes from sodium dihydrogen phosphate dihydrate; The hydroxide ions come from sodium hydroxide; The acetate ion comes from sodium pyruvate; The sodium ions come from sodium hydroxide, sodium dihydrogen phosphate dihydrate and sodium pyruvate; The chloride ions are potassium chloride and magnesium chloride hexahydrate.

4. The human neural stem cell preservation solution according to claim 1, characterized in that: The human neural stem cell preservation medium is a liquid medium free of bacteria, viruses, and mycoplasma contamination and with an endotoxin content of <5 EU / mL.

5. The method for preparing the human neural stem cell preservation solution according to any one of claims 1 to 4, characterized in that: include: Mix potassium chloride, sodium hydroxide, sodium dihydrogen phosphate dihydrate, magnesium chloride hexahydrate, sodium pyruvate, glucose and sorbitol, add purified water while stirring evenly, make the volume to 100 mL, and filter with a 250 mL 0.22 μm filter to obtain the product.

6. A human neural stem cell suspension, characterized in that: It comprises the human neural stem cell preservation solution according to any one of claims 1 to 4, with a concentration of 1-9×10^6 / mL; The preparation method of the human neural stem cell suspension comprises: centrifuging the human neural stem cells at 500g for 3 minutes, discarding the supernatant, resuspending the human neural stem cells in the obtained preservation solution, adjusting the concentration to 1-9×10^6 / mL, aliquoting the suspension, and transferring to 2-8°C for storage.

7. The human neural stem cell suspension according to claim 6, characterized in that: The concentration of human neural stem cells in the human neural stem cell suspension is 2-5×10^6 / mL.