Cell protection liquid, protection method and application

Abstract

The invention relates to the technical field of cell protection, and provides a cell protection solution, a protection method and application. The cell protection liquid comprises leonurine, human serum albumin and normal saline. The cell protection method comprises the following steps: S1, adding the human serum albumin into normal saline to prepare a cell preservation solution; s2, adding the leonurine into a cell preservation solution to prepare a cell protection solution; s3, resuspending and storing the collected mesenchymal stem cells or immune cells by using the cell protection liquid. The cell protection liquid is applied to mesenchymal stem cells and immune cells. According to the cell protection liquid, the protection method and the application provided by the invention, the cell activity can be maintained after mesenchymal stem cells and immune cells are prepared into a cell preparation, and the cell protection liquid has no toxic or side effect on the cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell protection, in particular to a cell protection solution, a protection method and application. BACKGROUND

[0002] Mesenchymal stem cells are a kind of multipotent stem cells, which have all the common properties of stem cells, i.e. self-renewal and multi-directional differentiation ability, and are most used in clinical application. Combined with hematopoietic stem cells, the success rate of transplantation can be improved and hematopoietic reconstruction can be accelerated. Immune cells refer to cells involved in or related to immune response, including lymphocytes, dendritic cells, monocyte / macrophage cells, granulocytes, mast cells, etc. Immune cells can be divided into many kinds, and various immune cells play an important role in the human body.

[0003] At present, mesenchymal stem cells and immune cells often face problems such as reduced cell activity and impaired function during the preservation and application process after being prepared into cell preparations. Existing cell protection agents or protection methods have limited effect, high cost, potential toxic side effects, etc. For example, some commonly used chemical protective agents may be toxic to cells or affect the biological properties of cells after long-term storage.

[0004] Therefore, it is an urgent problem to find a method that can maintain cell activity and reduce toxic side effects on cells after mesenchymal stem cells and immune cells are prepared into cell preparations. SUMMARY

[0005] In view of the defects in the prior art, the purpose of the present application is to provide a cell protection solution, a protection method and application, which can maintain cell activity and have no toxic side effects on cells after mesenchymal stem cells and immune cells are prepared into cell preparations.

[0006] The present application provides a cell protection solution, which comprises Leonurine, human serum albumin and physiological saline.

[0007] Further, the volume percentage of the human serum albumin is 2-5%.

[0008] Further, the concentration of the Leonurine is n μM, wherein 0 < n ≤ 100.

[0009] The present application provides a cell protection method, which comprises the following steps: S1, adding the human serum albumin into physiological saline to prepare a cell preservation solution; S2, adding the Leonurine into the cell preservation solution to prepare a cell protection solution; S3, resuspending and preserving the collected mesenchymal stem cells or immune cells with the cell protection solution.

[0010] Further, before step S2, the leonurine is dissolved in DMSO to prepare a leonurine solution.

[0011] Further, in step S2, the temperature of the cell protection solution is 4 DEG C, and the pH value of the cell protection solution ranges from 7.2 to 7.4.

[0012] Further, the leonurine is artificially synthesized.

[0013] The application provides application of the cell protection solution in a cell preparation.

[0014] Further, the cell protection solution prolongs the cell activity maintenance time of the cell preparation to more than 24 hours.

[0015] Further, the cell preparation is at least one of a stem cell preparation, an immune cell preparation and a somatic cell preparation.

[0016] Further, the stem cell preparation is a UCMSC cell preparation, and the immune cell preparation is an NK cell preparation.

[0017] The application provides a cell protection solution, a protection method and application, and leonurine can significantly improve the cell activity of mesenchymal stem cells and immune cells after the mesenchymal stem cells and the immune cells are prepared into a cell preparation, so that the survival rate of the mesenchymal stem cells and the immune cells is obviously higher than that of a control group without adding leonurine. Moreover, leonurine is a natural product or an artificial synthetic product, has better biocompatibility and lower risk of toxic side effects compared with some chemically synthesized cell protection agents, and is conducive to popularization and application in the field of clinic and the like. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description only show some embodiments of the application, and for those skilled in the art, other drawings can also be obtained according to the structures shown in the drawings without any creative effort.

[0019] Figure 1 A diagram for the influence of leonurine on the activity of mesenchymal stem cells; Figure 2 A diagram for the influence of leonurine on the activity of immune cells; Figure 3 A diagram for the influence of multiple groups of cell protection solutions on the activity of mesenchymal stem cells; Figure 4 A diagram for the influence of multiple groups of cell protection solutions on the activity of immune cells; Figure 5Figure 1 shows the effect of a multi-cell protective solution without human serum albumin on the viability of immune cells. DETAILED DESCRIPTION

[0020] It should be understood that the specific embodiments described herein are merely exemplary and are not intended to limit the present application.

[0021] The technical solutions in the embodiments of the present application will be clearly and completely described with reference to the drawings of the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0022] The composition of the cell protective solution includes leonuride, human serum albumin and physiological saline. The concentration of leonuride is n μM, where 0 < n ≤ 100, the volume percentage of human serum albumin is 2-5%, and the volume percentage of physiological saline is 95-98%.

[0023] Example 1, composition of the cell protective solution: The concentration of leonuride is 0 μM (i.e. no leonuride), the volume percentage of human serum albumin is 3%, and the volume percentage of physiological saline is 97%.

[0024] Example 2, composition of the cell protective solution: The concentration of leonuride is 5 μM, the volume percentage of human serum albumin is 3%, and the volume percentage of physiological saline is 97%.

[0025] Example 3, composition of the cell protective solution: The concentration of leonuride is 10 μM, the volume percentage of human serum albumin is 3%, and the volume percentage of physiological saline is 97%.

[0026] Example 4, composition of the cell protective solution: The concentration of leonuride is 25 μM, the volume percentage of human serum albumin is 3%, and the volume percentage of physiological saline is 97%.

[0027] Example 5, composition of the cell protective solution: The concentration of leonuride is 50 μM, the volume percentage of human serum albumin is 3%, and the volume percentage of physiological saline is 97%.

[0028] Example 6, composition of the cell protective solution: The concentration of leonuride is 100 μM, the volume percentage of human serum albumin is 3%, and the volume percentage of physiological saline is 97%.

[0029] The collected mesenchymal stem cells are added to the cell protection solution to detect the effect of Leonurine on the activity of mesenchymal stem cells. The effect of Leonurine on the activity of mesenchymal stem cells is detected by detecting the OD value of CCK8. In the CCK8 experiment, the CCK8 reagent will react with the metabolic products in the mesenchymal stem cells to form a soluble yellow product. The activity of mesenchymal stem cells is determined by measuring the absorbance of the yellow product.

[0030] As shown in Figure 1 , six groups of mesenchymal stem cell activity detection experiments were carried out at the same time. 96-well plates were used, and the same number of mesenchymal stem cells were inoculated in each group. After adhering, 0 μM, 5 μM, 10 μM, 20 μM, 50 μM and 100 μM concentrations of Leonurine were added respectively. After 24 h and 48 h of culture, CCK8 detection was performed, and it was found that different concentrations of Leonurine had no effect on the activity of mesenchymal stem cells, indicating that Leonurine had no toxic effect on the growth of mesenchymal stem cells.

[0031] As shown in Figure 2 , six groups of immune cell activity detection experiments were carried out at the same time. 96-well plates were used, and the same number of mesenchymal stem cells were inoculated in each group. After adhering, 0 μM, 5 μM, 10 μM, 20 μM, 50 μM and 100 μM concentrations of Leonurine were added respectively. After 24 h and 48 h of culture, CCK8 detection was performed, and it was found that most concentrations of Leonurine had no effect on the activity of immune cells, and only 100 μM Leonurine at 24 h and 10 μM at 48 h enhanced the activity of immune cells, indicating that Leonurine not only had no toxic effect on the growth of immune cells, but also had a certain effect on enhancing cell activity.

[0032] The collected mesenchymal stem cells are added to the cell protection solution to detect the effect of Leonurine on the activity of mesenchymal stem cells. The effect of Leonurine on the activity of mesenchymal stem cells is detected by detecting the OD value of CCK8. In the CCK8 experiment, the CCK8 reagent will react with the metabolic products in the mesenchymal stem cells to form a soluble yellow product. The activity of mesenchymal stem cells is determined by measuring the absorbance of the yellow product.

[0033] As shown in Figure 3The six groups of mesenchymal stem cell viability detection experiments are carried out at the same time. First, leonuride is added in the same volume of cell preservation solution in six groups to prepare six groups of cell protection solutions, and the concentration of leonuride in the six groups of cell protection solutions is 0 μM, 5 μM, 10 μM, 20 μM, 50 μM and 100 μM respectively. Then, the same number of mesenchymal stem cells in six groups are added into the above six groups of cell protection solutions. Through cell viability detection (such as MTT method, trypan blue staining method, etc.), the influence of the above different concentrations of leonuride on the viability of mesenchymal stem cells within 24 hours is observed. In addition, an additional control group is added, and the concentration of leonuride in the control group is 0 μM, the volume percentage of human serum albumin is 0%, and the volume percentage of physiological saline is 100%.

[0034] From Figure 3 It can be seen that the viability of mesenchymal stem cells in the control group is always at the lowest point within 0-24 hours, so the cell protection solution of the present application can significantly improve the viability of mesenchymal stem cells and play a role in protecting immune cells. The influence of different concentrations of leonuride on the viability of mesenchymal stem cells is also different at different time nodes.

[0035] As Figure 4 shown, six groups of immune cell viability detection experiments are carried out at the same time. First, leonuride is added in the same volume of cell preservation solution in six groups to prepare six groups of cell protection solutions, and the concentration of leonuride in the six groups of cell protection solutions is 0 μM, 5 μM, 10 μM, 20 μM, 50 μM and 100 μM respectively. Then, the same number of immune cells in six groups are added into the above six groups of cell protection solutions. Through cell viability detection (such as MTT method, trypan blue staining method, etc.), the influence of the above different concentrations of leonuride on the viability of immune cells within 24 hours is observed.

[0036] From Figure 4 It can be seen that the viability of immune cells in the cell protection solution with a leonuride concentration of 0 μM is always at the lowest point within 0-24 hours, so the cell protection solution of the present application can significantly improve the viability of immune cells and play a role in protecting immune cells. The influence of different concentrations of leonuride on the viability of immune cells is also different at different time nodes.

[0037] At the same time, as Figure 5 shown, the volume percentage of human serum albumin in the six groups of cell protection solutions is 0% (i.e. no human serum albumin is contained), the volume percentage of physiological saline is 100%, and the concentration of leonuride is 0 μM, 5 μM, 10 μM, 20 μM, 50 μM and 100 μM respectively.

[0038] FromFigure 5 It can be seen that in the absence of human serum albumin, the viability of immune cells in the cell protection solution with a leonuride concentration of 0 μM is always at the lowest point within 0-24 hours, indicating that the use of a cell protection solution containing only leonuride can also improve the viability of immune cells and play a protective role on immune cells. Different concentrations of leonuride have different effects on the viability of immune cells at different time points.

[0039] The present application provides a cell protection method, comprising the following steps: S1, adding human serum albumin to physiological saline to prepare a cell preservation solution.

[0040] S2, adding leonuride to the cell preservation solution to prepare a cell protection solution.

[0041] S3, resuspending and preserving the collected mesenchymal stem cells or immune cells with the cell protection solution.

[0042] In one embodiment, before step S2, leonuride is dissolved in DMSO to prepare a leonuride solution, and the leonuride solution is stored at-20℃. DMSO is a good solvent that can better dissolve leonuride to prepare a leonuride solution. In step S2, the staff can add the leonuride solution to the cell preservation solution to prepare the cell protection solution.

[0043] In one embodiment, in step S2, the temperature of the cell protection solution is 4℃, and the pH value of the cell protection solution ranges from 7.2 to 7.4.

[0044] In one embodiment, leonuride is artificially synthesized.

[0045] The present application provides the application of the cell protection solution in cell preparations.

[0046] In one embodiment, the cell protection solution prolongs the activity maintenance time of the cell preparation to more than 24 hours. This application can achieve the following technical effects: I. Expand the clinical application radiation range of cell preparations The activity maintenance time of cell preparations is a key bottleneck that restricts their preparation to clinical application. Traditional cell preparations have only an 8-hour activity window, which greatly limits the actual use value of the preparations: medical institutions need to complete the whole process of preparation, quality testing and patient infusion within a very short time. Once there is a delay in testing, temporary changes in patient conditions or poor logistics connection, the cell preparations will be scrapped due to loss of activity, not only causing serious waste of medical resources, but also possibly delaying the best treatment opportunity for patients.

[0047] The cell protection solution containing leonuride can maintain the activity of the cell preparation for 24 hours, which is equivalent to providing a 3 times longer safety buffer period for clinical application. This breakthrough completely breaks the time constraints on the application scenarios of cell preparations, enabling cell preparations to handle complex clinical processes with ease, significantly reducing medical risks and resource waste due to insufficient time, and laying a core foundation for the standardization and large-scale clinical application of cell preparations.

[0048] II. Expanding application boundaries and empowering cross-regional medical care The 8-hour activity window period limits the application of cell preparations to the vicinity of the preparation institution, making it almost impossible to achieve cross-city and cross-regional cell preparation deployment, resulting in the difficulty of balanced coverage of high-quality cell preparation treatment resources, and patients in remote areas often cannot obtain advanced treatment in time. The 24-hour activity maintenance capability enables the feasibility of cross-regional circulation of cell preparations: cell preparations can be transported to medical institutions hundreds of kilometers away through standardized cold chain logistics, realizing the cross-regional deployment of high-quality treatment resources.

[0049] III. Optimizing production processes and improving industrial transformation efficiency In terms of industrial transformation, the short activity duration requires that the production, quality control, packaging, and delivery of cell preparations must be highly compact, with very low tolerance for production plans. Any delay in one link will cause the entire batch of products to fail, greatly increasing production costs and quality control difficulties. The application of cell protection solution containing leonuride can improve this situation: the industry can optimize the production process, extend the production cycle flexibility, reduce the quality control cost, improve the product qualification rate and production capacity stability, and accelerate the process of cell therapy technology from the laboratory to industrialization.

[0050] IV. Reducing medical costs and promoting the popularization of cell therapy technology Under the 8-hour activity restriction, the time loss of cell preparations becomes an important part of medical costs: on the one hand, the failure of cell preparations directly leads to the waste of resources such as raw materials, manpower, and equipment; on the other hand, to shorten the circulation time, medical institutions need to invest additional costs to build special logistics channels or set up preparation centers nearby, greatly increasing the construction and operation costs of medical facilities.

[0051] The 24-hour activity maintenance capability effectively reduces the above costs: by reducing the loss of ineffective preparations, it directly saves medical resource investment; by expanding the transportation radius, it reduces the infrastructure construction cost without the need to layout preparation centers at each medical point; at the same time, the standardized long-term storage and transportation mode can form a large-scale supply chain, further reducing the unit product cost. The reduction of cost enables cell therapy technology to benefit more patients, and promotes this high-end medical technology to become a common treatment method that can be popularized, with significant social and economic benefits.

[0052] In one embodiment, the cell preparation is at least one of a stem cell preparation, an immune cell preparation, a somatic cell preparation.

[0053] In one embodiment, the stem cell preparation is a UCMSC cell preparation, and the immune cell preparation is an NK cell preparation.

[0054] UCMSC cells (umbilical cord mesenchymal stem cells) have multi-directional differentiation potential and immunomodulatory function, and have important application value in tissue engineering, organ regeneration, and treatment of autoimmune diseases; NK cells (natural killer cells) are an important part of the immune system, and can kill tumor cells and virus-infected cells without prior sensitization, and play a key role in anti-tumor and immune surveillance.

[0055] Leonurine is one of the active components of Chinese medicine Leonurus japonicus Houtt, which has been proven to have various pharmacological effects such as anti-inflammatory, but there is no report on its application in UCMSC cell or NK cell protection.

[0056] The present embodiment innovatively applies Leonurine in the protection of UCMSC cells or NK cells, and through experiments, it is found that Leonurine can significantly improve the activity of UCMSC cells and NK cells after being prepared into a cell preparation, and the survival rate of UCMSC cells and NK cells is significantly higher than that of the control group without adding Leonurine.

[0057] Moreover, as a natural product or its artificial synthetic product, Leonurine has better biocompatibility and lower risk of toxic side effects compared to some chemically synthesized cell protective agents, which is conducive to the promotion and application of Leonurine in the field of clinical treatment, etc.

[0058] In one embodiment, under the condition of 2-8℃, the cell protective solution can maintain the activity of the cell preparation for more than 24 hours.

[0059] In one embodiment, the activity of the cell preparation is mainly evaluated by cell viability.

[0060] The above description is only the preferred embodiments of the present application, and it should be understood that the present application is not limited to the forms disclosed herein, and should not be considered as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concepts described herein by the above teachings or related technical or knowledge. Any modification and change made by those skilled in the art without departing from the spirit and scope of the present application shall be within the protection scope of the appended claims of the present application.

Claims

1. A cell protective solution, characterized by: leucine, human serum albumin and physiological saline.

2. The cell protective solution of claim 1, wherein: The volume percentage of the human serum albumin is 2-5%.

3. The cell protective solution of claim 2, wherein: The concentration of the leucine is n μM, wherein 0 4. A method for protecting cells, using the cell-protecting solution according to claim 3, characterized by: The method comprises the following steps: S1, adding the human serum albumin into physiological saline to prepare a cell preservation solution; S2, adding the leucine into the cell preservation solution to prepare a cell protection solution; S3, resuspending and preserving the collected mesenchymal stem cells or immune cells with the cell protection solution.

5. A method of cytoprotection according to claim 4, wherein: Before step S2, the leucine is dissolved in DMSO to prepare the leucine solution.

6. The method of claim 4, wherein: In step S2, the temperature of the cell protection solution is 4°C, and the pH value of the cell protection solution ranges from 7.2 to 7.

4.

7. The cell protection solution of any one of claims 1-3 is used in a cell preparation.

8. Use of a cell protective fluid according to claim 7 in a cell preparation, characterized in that: The cell protection solution prolongs the activity maintenance time of the cell preparation to more than 24 hours.

9. Use of a cell protective fluid according to claim 8 in a cell preparation, characterized in that: The cell preparation is at least one of a stem cell preparation, an immune cell preparation and a somatic cell preparation.

10. Use of a cell protective fluid according to claim 9 in a cell preparation, characterized in that: The stem cell preparation is a UCMSC cell preparation, and the immune cell preparation is an NK cell preparation.

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