A cell protection solution, protection method and application

Abstract

The application relates to the technical field of cell protection, and provides a cell protection solution, a protection method and application. The cell protection solution comprises leonurine, human serum albumin and physiological saline. The cell protection method 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; and S3, resuspending and preserving collected mesenchymal stem cells or immune cells by using the cell protection solution. The cell protection solution is applied to mesenchymal stem cells and immune cells. The cell protection solution, the protection method and the application can maintain cell activity after mesenchymal stem cells and immune cells are prepared into cell preparations and have no toxic side effects on the cells.

Description

Technical Field

[0001] This invention relates to the field of cell protection technology, specifically to a cell protection solution, protection method, and application. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cell, possessing all the common characteristics of stem cells, namely self-renewal and multipotent differentiation capabilities. They are also the most widely used in clinical practice. Combined use with hematopoietic stem cells can improve transplant success rates and accelerate hematopoietic reconstitution. Immune cells refer to cells that participate in or are related to the immune response, including lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, mast cells, etc. Immune cells can be divided into various types, each playing an important role in the human body.

[0003] Currently, mesenchymal stem cells and immune cells often face problems such as reduced cell activity and impaired function during the preservation and application of cell preparations. Existing cell protectants or methods have limitations, including limited effectiveness, high cost, and potential toxic side effects. For example, some commonly used chemical protectants may be toxic to cells or affect the biological characteristics of cells after long-term storage.

[0004] Therefore, finding a way to maintain cell viability and reduce cytotoxic side effects after mesenchymal stem cells and immune cells are prepared into cell preparations is an urgent problem to be solved. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a cell protection solution, protection method, and application that can maintain cell viability and prevent toxic side effects after mesenchymal stem cells and immune cells are prepared into cell preparations.

[0006] This invention provides a cell protection solution comprising leonurine, human serum albumin, and physiological saline.

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

[0008] Furthermore, the concentration of the leonurine is n μM, where 0 < n ≤ 100.

[0009] This invention provides a cell protection method, comprising the following steps:

[0010] S1. Add the human serum albumin to physiological saline to prepare a cell preservation solution;

[0011] S2. Add the leonurine to the cell preservation solution to prepare a cell protection solution;

[0012] S3. The collected mesenchymal stem cells or immune cells are resuspended and preserved using the cell protection solution.

[0013] Further, before step S2, the leonurine is dissolved in DMSO to prepare a leonurine solution.

[0014] Furthermore, in step S2, the temperature of the cell preservation solution is 4°C, and the pH value of the cell preservation solution is in the range of 7.2-7.4.

[0015] Furthermore, the leonurine is synthesized artificially.

[0016] This invention provides the application of cell protection solutions in cell preparations.

[0017] Furthermore, the cell protection solution extends the activity maintenance time of the cell preparation to more than 24 hours.

[0018] Furthermore, the cell preparation is at least one of stem cell preparations, immune cell preparations, and somatic cell preparations.

[0019] Furthermore, the stem cell preparation is a UCMSC cell preparation, and the immune cell preparation is an NK cell preparation.

[0020] The beneficial effects of this invention are as follows: The cell protection solution, protection method, and application provided by this invention can significantly improve the cell viability of mesenchymal stem cells and immune cells after they have been prepared into cell preparations, resulting in a significantly higher survival rate of mesenchymal stem cells and immune cells compared to the control group without added leonurine. Furthermore, leonurine, as a natural product or its synthetic form, has better biocompatibility and a lower risk of toxic side effects compared to some chemically synthesized cell protectants, which is beneficial for its widespread application in clinical and other fields. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0022] Figure 1 A schematic diagram illustrating the effect of leonurine on the viability of mesenchymal stem cells;

[0023] Figure 2 A schematic diagram illustrating the effect of leonurine on the activity of immune cells;

[0024] Figure 3This is a schematic diagram illustrating the effects of multiple cell protection solutions on the viability of mesenchymal stem cells.

[0025] Figure 4 A schematic diagram illustrating the effects of multiple cell protection solutions on the viability of immune cells;

[0026] Figure 5 This diagram illustrates the effect of multiple cell protection solutions without human serum albumin on the viability of immune cells. Detailed Implementation

[0027] It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] The cell protection solution consists of leonurine, human serum albumin, and physiological saline. The concentration of leonurine 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%.

[0030] Example 1, Composition of cell protection solution:

[0031] The concentration of leonurine was 0 μM (i.e., leonurine-free), the volume percentage of human serum albumin was 3%, and the volume percentage of physiological saline was 97%.

[0032] Example 2, Composition of cell protection solution:

[0033] The concentration of leonurine was 5 μM, the volume percentage of human serum albumin was 3%, and the volume percentage of physiological saline was 97%.

[0034] Example 3, Composition of cell protection solution:

[0035] The concentration of leonurine was 10 μM, the volume percentage of human serum albumin was 3%, and the volume percentage of physiological saline was 97%.

[0036] Example 4, Composition of cell protection solution:

[0037] The concentration of leonurine was 25 μM, the volume percentage of human serum albumin was 3%, and the volume percentage of physiological saline was 97%.

[0038] Example 5, Composition of cell protection solution:

[0039] The concentration of leonurine was 50 μM, the volume percentage of human serum albumin was 3%, and the volume percentage of physiological saline was 97%.

[0040] Example 6, Composition of cell protection solution:

[0041] The concentration of leonurine was 100 μM, the volume percentage of human serum albumin was 3%, and the volume percentage of physiological saline was 97%.

[0042] Collected mesenchymal stem cells (MSCs) were added to a cell preservation solution to detect the effect of leonurine on MSC viability. This experiment used the CCK8 assay to measure the OD value of the CCK8 reagent, which reacts with metabolites within the MSCs to form a soluble yellow product. The viability of the MSCs was determined by measuring the absorbance of this yellow product.

[0043] like Figure 1 As shown, six groups of mesenchymal stem cell viability assays were performed simultaneously. Using 96-well plates, the same number of mesenchymal stem cells were seeded in each group. After adhesion, leonurine concentrations of 0 μM, 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM were added, respectively. After culturing for 24 h and 48 h, CCK8 assays were performed. The results showed that different concentrations of leonurine had no effect on the viability of mesenchymal stem cells, indicating that leonurine has no toxic effect on the growth of mesenchymal stem cells.

[0044] like Figure 2 As shown, six groups of immune cell viability assays were performed simultaneously. Using 96-well plates, the same number of mesenchymal stem cells were seeded in each group. After adhesion, leonurine concentrations of 0 μM, 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM were added, respectively. After culturing for 24 h and 48 h, CCK8 assays were performed. The results showed that most concentrations of leonurine had no effect on immune cell viability. Only 100 μM leonurine at 24 h and 10 μM at 48 h enhanced immune cell viability. This indicates that leonurine not only has no toxic effect on immune cell growth but may also enhance cell viability.

[0045] Collected mesenchymal stem cells were added to a cell preservation solution to detect the effect of leonurine on mesenchymal stem cell viability. This experiment used various methods, including cell viability assays (such as the MTT assay and trypan blue rejection assay), to verify the protective effect of leonurine on mesenchymal stem cells or immune cells.

[0046] like Figure 3As shown, six groups of mesenchymal stem cell viability assays were performed simultaneously. First, leonurine was added to the same volume of cell preservation solution in each of the six groups to prepare six cell preservation solutions with concentrations of leonurine of 0 μM, 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM, respectively. Then, the same number of mesenchymal stem cells from each of the six groups were added to the aforementioned six cell preservation solutions. The effect of different concentrations of leonurine on the viability of mesenchymal stem cells over 24 hours was observed using cell viability assays (such as the MTT assay and trypan blue rejection assay). In addition, a control group was added, with a leonurine concentration of 0 μM, a human serum albumin volume percentage of 0%, and a physiological saline volume percentage of 100%.

[0047] from Figure 3 It can be seen that the viability of mesenchymal stem cells in the control group was always at its lowest point within 0-24 hours. Therefore, the cell protection solution of the present invention can significantly improve the viability of mesenchymal stem cells and play a role in protecting immune cells. At different time points, different concentrations of leonurine also have different effects on the viability of mesenchymal stem cells.

[0048] like Figure 4 As shown, six groups of immune cell viability assays were performed simultaneously. First, leonurine was added to six equal volumes of cell preservation solution to prepare six cell preservation solutions with concentrations of leonurine: 0 μM, 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM, respectively. The volume percentage of human serum albumin in the cell preservation solution was 3%. Then, the same number of immune cells were added to each of the six cell preservation solutions. The effect of different concentrations of leonurine on the viability of immune cells over 24 hours was observed using cell viability assays (such as the MTT assay and trypan blue rejection assay).

[0049] from Figure 4 It can be seen that within 0-24 hours, the viability of immune cells in the cell protection solution with a concentration of 0 μM of leonurine is always at its lowest point. Therefore, the cell protection solution of the present invention can significantly improve the viability of immune cells and play a role in protecting immune cells. At different time points, the effect of different concentrations of leonurine on the viability of immune cells is also different.

[0050] At the same time, such as Figure 5 As shown, in the six groups of cell preservation solutions, the volume percentage of human serum albumin was 0% (i.e., no human serum albumin), the volume percentage of physiological saline was 100%, and the concentrations of leonurine were 0 μM, 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM, respectively.

[0051] from Figure 5 As can be seen, in the absence of human serum albumin, the viability of immune cells remained at its lowest point within 0-24 hours in a cell preservation solution containing 0 μM leonurine. This indicates that using a cell preservation solution containing only leonurine can also improve the viability of immune cells and exert a protective effect on them. At different time points, different concentrations of leonurine also had varying effects on the viability of immune cells.

[0052] This invention provides a cell protection method, comprising the following steps:

[0053] S1. Add human serum albumin to physiological saline to prepare a cell preservation solution.

[0054] S2. Add leonurine to the cell preservation solution to prepare a cell protection solution.

[0055] S3. Resuspend and preserve the collected mesenchymal stem cells or immune cells using cell preservation solution.

[0056] In one embodiment, prior to step S2, leonurine is dissolved in DMSO to prepare a leonurine solution, which is then stored at -20°C. DMSO is a good solvent that effectively dissolves leonurine, thus preparing the leonurine solution. In step S2, the leonurine solution is added to the cell preservation solution to prepare the cell protection solution.

[0057] In one embodiment, in step S2, the temperature of the cell preservation solution is 4°C, and the pH value of the cell preservation solution is in the range of 7.2-7.4.

[0058] In one embodiment, leonurine is synthesized artificially.

[0059] This invention provides the application of cell protection solutions in cell preparations.

[0060] In one embodiment, the cell preservation solution extends the activity of the cell preparation to more than 24 hours. This application achieves the following technical effects:

[0061] I. Expanding the clinical application scope of cell preparations

[0062] The duration of activity of cell preparations is a key bottleneck restricting their transition from preparation to clinical application. Traditional cell preparations have only an 8-hour activity window, which greatly limits their practical value: medical institutions need to complete the entire process of preparation, quality testing, and patient infusion in a very short time. If there are delays in testing, temporary changes in the patient's condition, or logistical disruptions, the cell preparation will be rendered unusable due to loss of activity, resulting in a serious waste of medical resources and potentially delaying the patient's optimal treatment time.

[0063] Cell protection solutions containing leonurine extend the activity maintenance time of cell preparations to 24 hours, providing a three-fold safety buffer for clinical applications. This breakthrough completely breaks the time constraint on the application scenarios of cell preparations, enabling them to handle complex clinical procedures with ease, significantly reducing medical risks and resource waste caused by insufficient time, and laying a core foundation for the standardized and large-scale clinical application of cell preparations.

[0064] II. Expanding application boundaries and empowering cross-regional healthcare

[0065] The 8-hour activity window limitation strictly restricts the application of cell preparations to the vicinity of the preparation facility, making cross-city and cross-regional distribution of cell preparations virtually impossible. This results in an uneven distribution of high-quality cell preparation therapeutic resources, often leaving patients in remote areas unable to access advanced treatments in a timely manner. The 24-hour activity maintenance capability makes cross-regional distribution of cell preparations feasible: cell preparations can be transported to medical institutions hundreds of kilometers away via standardized cold chain logistics, enabling the cross-regional allocation of high-quality therapeutic resources.

[0066] III. Optimize production processes and improve industrial transformation efficiency

[0067] At the industrialization level, the short activity duration necessitates a highly efficient production, quality control, packaging, and distribution process for cell therapy products. Production planning has extremely low tolerance for error; a delay in any stage can render the entire batch ineffective, significantly increasing production costs and quality control difficulties. The application of cell protectants containing leonurine can improve this situation: on the industrial side, it can optimize production processes, extend production cycle flexibility, reduce quality control costs, improve product qualification rates and production capacity stability, and accelerate the transition of cell therapy technology from the laboratory to industrialization.

[0068] IV. Reduce medical costs and promote the widespread adoption of cell therapy technology

[0069] With an 8-hour activity limit, the time loss of cell preparations has become an important part of medical costs: on the one hand, ineffective cell preparations directly lead to the waste of resources such as raw materials, manpower, and equipment; on the other hand, in order to shorten the circulation time, medical institutions need to invest additional costs to build dedicated logistics channels or set up preparation centers nearby, which significantly increases the construction and operation costs of medical facilities.

[0070] The ability to maintain activity for 24 hours effectively reduces the aforementioned costs: by reducing formulation failure and waste, it directly saves on medical resource investment; by expanding the transportation radius, it eliminates the need to establish a preparation center at each medical site, reducing infrastructure construction costs; at the same time, the standardized long-term storage and transportation model can form a large-scale supply chain, further reducing the unit product cost. This cost reduction enables cell therapy technology to benefit more patients, promoting this high-end medical technology to become a widely accessible routine treatment method, with significant social and economic benefits.

[0071] In one embodiment, the cell preparation is at least one of stem cell preparations, immune cell preparations, and somatic cell preparations.

[0072] In one embodiment, the stem cell preparation is a UCMSC cell preparation, and the immune cell preparation is an NK cell preparation.

[0073] UCMSCs (umbilical cord mesenchymal stem cells) have multi-lineage differentiation potential and immunomodulatory functions, and have important application value in tissue engineering, organ regeneration and treatment of autoimmune diseases; NK cells (natural killer cells) are an important component of the immune system, and can kill tumor cells and virus-infected cells without prior sensitization, playing a key role in anti-tumor and immune surveillance.

[0074] Leonuridine is one of the active ingredients of the traditional Chinese medicine Leonurus japonicus. It has been proven to have various pharmacological effects such as anti-inflammation, but there are no reports on its application in the protection of UCMSC cells or NK cells.

[0075] This embodiment innovatively applies leonurine to the protection of UCMSC cells or NK cells. Experiments have shown that leonurine can significantly improve the activity of UCMSC cells and NK cells after they are prepared into cell preparations, resulting in a significantly higher survival rate of UCMSC cells and NK cells than the control group without leonurine.

[0076] Furthermore, leonine, as a natural product or its synthetic form, has better biocompatibility and lower risk of toxic side effects compared to some chemically synthesized cell protectants, which is conducive to its widespread application in clinical and other fields.

[0077] In one embodiment, at 2-8°C, the cell protection solution extends the activity of the cell preparation to more than 24 hours.

[0078] In one embodiment, the activity of the cell preparation is primarily evaluated based on cell viability.

[0079] The above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technologies or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. A cell protection solution suitable for mesenchymal stem cells or immune cells, characterized in that: It includes leonurine, human serum albumin and physiological saline, wherein the volume percentage of human serum albumin is 2-5% and the concentration of leonurine is 0 < n ≤ 100 μM.

2. The cell protection solution according to claim 1, characterized in that: The mesenchymal stem cells are umbilical cord mesenchymal stem cells, and the immune cells are natural killer cells.

3. The cell protection solution according to claim 2, characterized in that: The leonurine mentioned is either artificially synthesized or naturally extracted.

4. A cell protection method, applied to the cell protection solution according to any one of claims 1-3, characterized in that, Includes the following steps: S1. Add the human serum albumin to physiological saline to prepare a cell preservation solution; S2. After dissolving leonurine in DMSO to prepare a solution, add it to the cell preservation solution to prepare a cell protection solution. The temperature of the cell protection solution is 4℃ and the pH range is 7.2-7.

4. S3. The collected mesenchymal stem cells or immune cells are resuspended and preserved using the cell protection solution.

5. The application of the cell protection solution according to any one of claims 1-3 in cell preparations, characterized in that, The cell protection solution extends the activity of the cell preparation to more than 24 hours.

6. The application according to claim 5, characterized in that, The cell preparation is stored at a temperature of 2-8℃, and cell activity is evaluated by cell viability.

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