CN114097768A - A kind of protective solution for bone marrow stem cells and preparation method and application thereof - Google Patents
A kind of protective solution for bone marrow stem cells and preparation method and application thereof Download PDFInfo
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/10—Preservation of living parts
- A01N1/12—Chemical aspects of preservation
- A01N1/122—Preservation or perfusion media
- A01N1/126—Physiologically active agents, e.g. antioxidants or nutrients
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a protective solution for bone marrow stem cells, which consists of the following raw materials: 0.9% sodium chloride injection, spina date seed saponin A, meglumine, cordycepin, sodium hyaluronate, and vitamin B2. The protective solution for the bone marrow stem cells provided by the invention is added with the synergistic effect of the spina date seed saponin A, the meglumine and the cordycepin, so that the bone marrow stem cells are preserved for 72 hours at 4-6 ℃, the cell viability is more than 89%, the use requirements of scientific research and clinic can be met, and the temporary preservation and transportation of the bone marrow stem cells are facilitated. The proliferation capacity of the bone marrow stem cells preserved by the protective solution is not affected, and the preserved cells can be continuously used for proliferation differentiation and the like. The invention also provides a preparation method of the bone marrow stem cell protective solution, which has simple process, easy operation and convenient product commercialization. The invention also provides the application of the protective solution, which is safe and nontoxic when used for preserving the bone marrow stem cells, is convenient to preserve and transport, and provides guarantee for clinical application of the bone marrow stem cells.
Description
Technical Field
The invention relates to the field of stem cells, in particular to a protective solution for bone marrow stem cells and a preparation method and application thereof.
Background
Mesenchymal Stem Cells (MSCs) are a class of adult stem cells of mesodermal origin with high self-renewal capacity and multipotentiality. The MSCs are widely present in tissues or organs such as bone marrow, fat, muscle, skin, amniotic fluid, umbilical cord and the like, have strong proliferation capacity and differentiation capacity, are low in immunogenicity, and have wide application prospects.
Bone marrow mesenchymal stem cells (BMSCs) are multipotent stem cells in bone marrow, have all commonalities of stem cells, namely self-renewal and multidirectional induced differentiation capacity, have the characteristics of wide sources, simple acquisition, easy culture, strong in vitro proliferation capacity, no rejection reaction of autologous transplantation and the like, are widely applied to the fields of tissue engineering and regenerative medicine, and are generally recognized as seed cells most commonly used in bone tissue engineering.
In clinical application, the prepared mesenchymal stem cells cannot be immediately applied for various reasons, and need to be kept in a protective solution temporarily. The cell activity can be ensured by cryopreservation in liquid nitrogen, but the cryopreservation operation is complex, and a great deal of inconvenience is brought to clinical application. The cost of handling is high for cells that need only be stored for a short period of time. The bone marrow stem cells with transportation requirements are preserved at ultra-low temperature by adopting liquid nitrogen, so that the requirements on transportation equipment are better, and the rapid transportation of the bone marrow stem cells is not easy to realize. In recent years, various preservation solutions capable of preserving bone marrow stem cells at low temperatures have been developed, but various problems such as short preservation time and low cell survival rate have been common, and thus the application of bone marrow stem cells has been limited.
Therefore, it is necessary to research a new preservation solution to preserve the bone marrow stem cells at 4-6 ℃, ensure the survival rate of the cells, and facilitate the short-term preservation and transportation of the bone marrow stem cells.
Disclosure of Invention
In order to overcome the defects of the prior art, one of the objectives of the present invention is to provide a protective solution for bone marrow stem cells, which does not require liquid nitrogen to be frozen, can ensure the activity of the bone marrow stem cells in a short time, and provides convenience for temporary storage and transportation of the bone marrow stem cells.
The second purpose of the invention is to provide a preparation method of the protective solution for the bone marrow stem cells.
The invention also aims to provide application of the bone marrow stem cell protective solution.
One of the purposes of the invention is realized by adopting the following technical scheme:
a protective solution for bone marrow stem cells is composed of the following raw materials: 0.9% sodium chloride injection, spina date seed saponin A, meglumine, cordycepin, sodium hyaluronate, and vitamin B2.
Further, the weight parts of the using amounts of the components are as follows: 75-85 parts of 0.9% sodium chloride injection, 1-2.5 parts of spina date seed saponin A, 2-4 parts of meglumine, 1-5 parts of cordycepin, 5-8 parts of sodium hyaluronate and 20.5-0.8 part of vitamin B.
Further, the weight parts of the using amounts of the components are as follows: 80 parts of 0.9% sodium chloride injection, 1.5 parts of spina date seed saponin A, 3 parts of meglumine, 2 parts of cordycepin, 6 parts of sodium hyaluronate and 20.7 parts of vitamin B.
The second purpose of the invention is realized by adopting the following technical scheme:
the preparation method of the protective solution for the bone marrow stem cells comprises the following steps: adding spina date seed saponin A, meglumine, cordycepin, sodium hyaluronate and vitamin B2 into 0.9% sodium chloride injection, mixing uniformly, and filtering with 0.22 mu m filter membrane to obtain the final product.
The third purpose of the invention is realized by adopting the following technical scheme:
the application of the protective solution is to use the protective solution in the preservation of bone marrow stem cells.
Further, the application comprises pre-cooling the protective solution to 4-6 deg.C, adding bone marrow stem cells, and storing at 4-6 deg.C.
Further, the concentration of the bone marrow stem cells in the protective solution is 1-5X 106one/mL.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention provides a protective solution for bone marrow stem cells, which is added with the synergistic effect of spina date seed saponin A, meglumine and cordycepin, so that the bone marrow stem cells are preserved for 72 hours at 4-6 ℃, the cell viability is more than 89%, the use requirements of scientific research and clinic can be met, and the temporary preservation and transportation of the bone marrow stem cells are facilitated. The proliferation capacity of the bone marrow stem cells preserved by the protective solution is not affected, and the preserved cells can be continuously used for proliferation differentiation and the like.
2. The protective solution provided by the invention has simple components and stable performance, does not add components with potential safety hazards such as a culture medium with complex components and DMSO (dimethyl sulfoxide), effectively protects the activity of the bone marrow stem cells, and avoids apoptosis.
3. The invention also provides a preparation method of the bone marrow stem cell protective solution, which has simple process, easy operation and convenient product commercialization.
4. The invention also provides the application of the protective solution, which is safe and nontoxic when used for preserving the bone marrow stem cells, is convenient to preserve and transport, and provides guarantee for clinical application of the bone marrow stem cells.
Drawings
FIG. 1 is a graph showing the proliferation curves of bone marrow stem cells preserved for 72h in example 1 of the present invention and bone marrow stem cells not preserved in a control group.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.
The procedure for preparing bone marrow stem cells used in examples 1 to 3 was as follows: killing SD rat of 6-8 weeks by cervical dislocation, taking femur and tibia, flushing marrow cavity with PBS, collecting marrow liquid, filtering, centrifuging at 1200r/min for 5min, discarding supernatant, adding 10ml erythrocyte lysate, lysing for 5min, adding equal amount of PBS1000r/min, centrifuging for 10min, removing supernatant, centrifuging at 1 × 106Inoculating to complete culture medium at a density of/mL, culturing in a 5% CO2 saturated humidity incubator at 37 deg.C, changing the culture solution after 2d, and taking P3 generation bone marrow stem cells for subsequent experiments.
Example 1
The protective solution for the bone marrow stem cells is characterized by comprising the following raw materials in parts by weight: 80 parts of 0.9% sodium chloride injection, 1.5 parts of spina date seed saponin A, 3 parts of meglumine, 2 parts of cordycepin, 6 parts of sodium hyaluronate and 20.7 parts of vitamin B.
The preparation method of the protective solution for the bone marrow stem cells comprises the following steps: adding spina date seed saponin A, meglumine, cordycepin, sodium hyaluronate and vitamin B2 into 0.9% sodium chloride injection, mixing uniformly, and filtering with 0.22 mu m filter membrane to obtain the final product.
The application of the bone marrow stem cell protective solution comprises subpackaging the protective solution in a sterile tube, pre-cooling in a refrigerator until the temperature is reducedAdding P3 generation bone marrow stem cells at 4 deg.C, with cell density of 1 × 106And (4) keeping the seeds/mL for 72h in a refrigerator at 4 ℃.
Example 2
The protective solution for the bone marrow stem cells is characterized by comprising the following raw materials in parts by weight: 75 parts of 0.9% sodium chloride injection, 1 part of spina date seed saponin A, 2 parts of meglumine, 1 part of cordycepin, 5 parts of sodium hyaluronate and 20.5 parts of vitamin B.
The preparation method of the protective solution for the bone marrow stem cells comprises the following steps: adding spina date seed saponin A, meglumine, cordycepin, sodium hyaluronate and vitamin B2 into 0.9% sodium chloride injection, mixing uniformly, and filtering with 0.22 mu m filter membrane to obtain the final product.
The application of the protective solution for the bone marrow stem cells comprises subpackaging the protective solution in a sterile tube, pre-cooling to 6 deg.C in a refrigerator, adding P3 substitute bone marrow stem cells with cell density of 3 × 106The cells/mL were kept in a refrigerator at 6 ℃ for 72 hours.
Example 3
The protective solution for the bone marrow stem cells is characterized by comprising the following raw materials in parts by weight: 85 parts of 0.9% sodium chloride injection, 2.5 parts of spina date seed saponin A, 4 parts of meglumine, 5 parts of cordycepin, 8 parts of sodium hyaluronate and 20.8 parts of vitamin B.
The preparation method of the protective solution for the bone marrow stem cells comprises the following steps: adding spina date seed saponin A, meglumine, cordycepin, sodium hyaluronate and vitamin B2 into 0.9% sodium chloride injection, mixing uniformly, and filtering with 0.22 mu m filter membrane to obtain the final product.
The application of the protective solution for the bone marrow stem cells comprises subpackaging the protective solution in a sterile tube, pre-cooling to 6 deg.C in a refrigerator, adding P3 substitute bone marrow stem cells with cell density of 5 × 106And (4) keeping the seeds/mL for 72h in a refrigerator at 4 ℃.
Comparative example 1
Comparative example 1 provides a bone marrow stem cell protective solution, which is different from example 1 in that: the same procedure as in example 1 was repeated except that the wild jujube seed saponin A was omitted.
Comparative example 2
Comparative example 2 provides a bone marrow stem cell protective solution, which is different from example 1 in that: the amount of meglumine was adjusted to 4.5 parts by omitting wild jujube seed saponin A, and the rest was the same as in example 1.
Comparative example 3
Comparative example 3 provides a bone marrow stem cell protective solution, which is different from example 1 in that: meglumine is omitted, the amount of jujuboside A is adjusted to 4.5 parts, and the rest is the same as example 1.
Comparative example 4
Comparative example 4 provides a bone marrow stem cell protective solution, which is different from example 1 in that: meglumine was replaced with sorbitol, and the rest was the same as in example 1.
Comparative example 5
Comparative example 5 provides a bone marrow stem cell protective solution, which is different from example 1 in that: cordycepin was omitted and the procedure was as in example 1.
Comparative example 6
Comparative example 6 provides a bone marrow stem cell protective solution, which is different from example 1 in that: cordycepin was replaced with adenosine, and the rest was the same as in example 1.
In the preservation of bone marrow stem cells in examples 1 to 3 and comparative examples 1 to 6, samples were taken at 24h, 48h and 72h of preservation, respectively, and 0.4% trypan blue was added for staining, and the number of live cells and dead cells was counted using a cell counter to calculate the survival rate of the cells, and the results are shown in table 1.
TABLE 1
| Sample (I) | Cell survival rate after 24h preservation | Cell survival rate of 48h | The survival rate of the cells is kept for 72h |
| Example 1 | 98.45% | 94.03% | 90.43% |
| Example 2 | 97.67% | 93.92% | 89.25% |
| Example 3 | 97.70% | 93.88% | 89.32% |
| Comparative example 1 | 78.92% | 52.59% | 35.49% |
| Comparative example 2 | 85.74% | 68.49% | 52.37% |
| Comparative example 3 | 88.93% | 75.49% | 61.01% |
| Comparative example 4 | 86.79% | 71.28% | 54.32% |
| Comparative example 5 | 82.43% | 62.91% | 40.29% |
| Comparative example 6 | 89.75% | 77.59% | 62.21% |
As can be seen from table 1: in examples 1 to 3, the survival rate of the bone marrow stem cells is slightly reduced along with the prolonging of the preservation time, and the survival rate of the bone marrow stem cells is more than 89% after 72 hours of preservation, thus meeting the requirements of experiments and clinics. The components of the protective solution were adjusted in comparative examples 1 to 6, and the cell survival rate after storage was significantly reduced.
In comparative example 1, spina date seed saponin A is omitted, and the prepared protection solution is used for preserving bone marrow stem cells, the survival rate of the cells is obviously reduced along with the prolonging of the preservation time, and the survival rate of the cells is only 35.49% after being preserved for 72h, which shows that the spina date seed saponin A in the protection solution plays an important role in maintaining the activity of the cells and avoiding the death of the cells.
In comparative example 2, spina date seed saponin A is omitted, the dosage of meglumine is adjusted, in comparative example 3, the dosage of spina date seed saponin A is omitted, in comparative example 4, the meglumine is replaced by sorbitol, and the survival rate of cells in the preservation solution is still lower compared with that in example 1, which shows that the meglumine can play a role in cooperation with the spina date seed saponin A, provide a stable external environment for the preservation of the cells, and ensure the survival rate of the cells.
In comparative example 5, cordycepin is omitted, in comparative example 6, cordycepin is replaced by adenosine, and the survival rate of the cells in comparative example 6 is improved to a certain extent compared with that in comparative example 5, but the survival rate of the cells in comparative example 6 is reduced to 62.21 percent after the cells are stored for 72 hours, so that the use requirement of the bone marrow stem cells cannot be met.
In conclusion, the protective solution disclosed by the invention realizes the preservation of the bone marrow stem cells at 4-6 ℃ for 72 hours by adding the spinasaponin A, the meglumine and the cordycepin under the synergistic action, the cell viability is more than 89%, the requirements of scientific research and clinical use are met, and the temporary preservation and transportation of the bone marrow stem cells are facilitated.
The bone marrow stem cells after 72 hours of storage in example 1 were used as a control group, and the non-stored P3-substituted bone marrow stem cells were inoculated into DMEM/F12 medium containing 10% FBS and 1% double antibody at a cell density of 1X 104cells/mL, the 12-well plate inoculated with cells was placed at 37 ℃ in 5% CO2The number of bone marrow stem cells was counted every day and a cell growth curve was plotted, and the results are shown in FIG. 1.
As can be seen from FIG. 1, the proliferation rate and proliferation amount of the bone marrow stem cells preserved by the protective solution of the present invention are not much different from those of bone marrow stem cells not preserved, which indicates that the protective solution provided by the present invention can effectively maintain the proliferation activity of the bone marrow stem cells and fully meet the application requirements of the bone marrow stem cells.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
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| CN107668024A (en) * | 2016-08-01 | 2018-02-09 | 北京世纪劲得生物技术有限公司 | A kind of stem cell protection liquid and preparation method thereof |
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| US20190343892A1 (en) * | 2013-08-19 | 2019-11-14 | National Cerebral And Cardiovascular Center | Method for producing amniotic mesenchymal stromal cell composition, method for cryopreserving the same, and therapeutic agent |
| CN113398140A (en) * | 2021-07-08 | 2021-09-17 | 辽宁中医药大学 | Application of spina date seed saponin in inhibiting oxidative damage of mesenchymal stem cells and enhancing immunoregulatory capacity of mesenchymal stem cells |
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| US20190343892A1 (en) * | 2013-08-19 | 2019-11-14 | National Cerebral And Cardiovascular Center | Method for producing amniotic mesenchymal stromal cell composition, method for cryopreserving the same, and therapeutic agent |
| CN107668024A (en) * | 2016-08-01 | 2018-02-09 | 北京世纪劲得生物技术有限公司 | A kind of stem cell protection liquid and preparation method thereof |
| CN110012898A (en) * | 2019-03-21 | 2019-07-16 | 广州赛莱拉干细胞科技股份有限公司 | Composition and its application as stem cell cryopreservation solution |
| CN113398140A (en) * | 2021-07-08 | 2021-09-17 | 辽宁中医药大学 | Application of spina date seed saponin in inhibiting oxidative damage of mesenchymal stem cells and enhancing immunoregulatory capacity of mesenchymal stem cells |
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