Umbilical cord mesenchymal stem cell preparation preserving fluid and preparation method thereof

Abstract

The invention discloses a preserving fluid for an umbilical cord mesenchymal stem cell preparation. The preserving fluid comprises 0.9% sodium chloride injection, human serum albumin and thymopentin for injection. The preserving fluid can stably preserve stem cells for 72 hours at the room temperature of 15-30 DEG C, and the cell viability is maintained to be 80% or above; meanwhile, the preserving fluid is simple in component, does not contain complex additives such as a pH buffering agent and an antioxidant, and is simple and convenient in preparation process and easy in cost control.

Description

Technical Field

[0001] This invention relates to the field of mesenchymal stem cell technology, and in particular to a preservation solution for umbilical cord mesenchymal stem cell preparation and its preparation method. Background Technology

[0002] Human Umbilical Cord Mesenchymal Stem Cells (hUC-MSCs) are a type of pluripotent stem cells found in umbilical cord tissue. They possess multipotent differentiation potential and can regulate immune responses by secreting various bioactive factors, exhibiting anti-inflammatory and immunosuppressive properties. In recent years, stem cell therapy has rapidly developed and gained widespread attention. HUC-MSCs have stronger proliferative and differentiation capabilities, low immunogenicity, convenient sourcing, and no ethical restrictions, making them the most commonly used seed cells in clinical cell therapy. Current technologies utilizing HUC-MSCs for treatment aim to preserve their viability as much as possible, typically by storing cell preparations at 2-8°C to improve cell survival. However, in practice, cell preparations may need to be transported at room temperature. How to improve the viability of HUC-MSCs during long-term preservation at room temperature is a pressing issue. Furthermore, existing preservation solutions are often complex, using multiple reagents such as pH buffers, antioxidants, and osmotic stabilizers, lacking safety and convenience, and increasing cost and complexity.

[0003] This invention optimizes the composition of mesenchymal stem cell preservation solution, enabling long-term preservation of mesenchymal stem cells at room temperature, improving cell viability, extending the shelf life of cell preparations, facilitating long-distance transportation and direct intravenous reinfusion without special processing, and making it convenient for clinical applications. Summary of the Invention

[0004] The purpose of this invention is to provide a preservation solution for umbilical cord mesenchymal stem cells and its preparation method, so as to solve the technical problems existing in the prior art.

[0005] To achieve the above objectives, the specific technical solution of the present invention is as follows:

[0006] A preservation solution for umbilical cord mesenchymal stem cells, comprising the following components: 0.9% sodium chloride injection, human serum albumin, and thymopentin for injection.

[0007] Preferably, the concentration of human serum albumin in the preservation solution is 20 g / L, the concentration of thymopentin for injection is 5-25 mg / L, and the remainder is 0.9% sodium chloride injection.

[0008] A method for preparing a preservation solution for umbilical cord mesenchymal stem cells includes the following steps:

[0009] Step 1: Under sterile conditions, take 10mg of thymopentin for injection, add 10ml of 0.9% sodium chloride injection, and dissolve it thoroughly to prepare a 1mg / ml thymopentin stock solution.

[0010] Step 2: Add 10ml of human serum albumin and 0.5-2.5ml of the thymopentin stock solution prepared in Step 1 to 70-85ml of 0.9% sodium chloride injection, and mix well.

[0011] Step 3: Add 0.9% sodium chloride injection to the mixture from Step 2 to a final volume of 100ml, filter, seal in a sterile container, and store at 2-8℃.

[0012] A method for preserving umbilical cord mesenchymal stem cells includes the following steps:

[0013] Step a: Prepare the preservation solution for umbilical cord mesenchymal stem cells according to the method described above;

[0014] Step b: Mix the P3-6 generation umbilical cord mesenchymal stem cells with the cell preparation preservation solution prepared in step a to prepare a cell preparation. The cell density in the cell preparation is 0.8-1.2 × 10⁻⁶. 6 Cells / ml; cell preparations should be stored at 15-30℃.

[0015] A preparation of umbilical cord mesenchymal stem cells is prepared by mixing the above-mentioned preservation solution with P3-6 generation umbilical cord mesenchymal stem cells, wherein the cell density in the preparation is 0.8-1.2 × 10⁻⁶. 6 per ml.

[0016] The present invention has the following advantages:

[0017] 1. This invention can achieve stable preservation of stem cells for 72 hours under room temperature conditions of 15-30℃, with cell viability maintained at over 80%.

[0018] 2. This invention contains only three components: sodium chloride injection, human serum albumin, and thymopentin, making the composition simple. Detailed Implementation

[0019] To better understand the purpose and function of this invention, the following provides a more detailed description of the preservation solution for umbilical cord mesenchymal stem cells and its preparation method.

[0020] Example 1

[0021] This invention provides a preservation solution for umbilical cord mesenchymal stem cell preparations, comprising: human serum albumin, thymopentin for injection, and 0.9% sodium chloride injection. The human serum albumin is 10g / 50ml and was purchased from Grifols Pharmaceuticals (Shanghai) Co., Ltd.; the thymopentin for injection is 10mg / vial and was purchased from Beijing Shuanglu Pharmaceutical Co., Ltd.; and the 0.9% sodium chloride injection is 0.9g / 100ml and was purchased from Zhejiang Dubang Pharmaceutical Co., Ltd.

[0022] The steps for preserving umbilical cord mesenchymal stem cells using the above-mentioned umbilical cord mesenchymal stem cell preparation preservation solution are as follows:

[0023] Step a: Preparation of preservation solution for umbilical cord mesenchymal stem cells:

[0024] First, in a sterile environment, dissolve 10 mg of thymopentin for injection in 10 ml of 0.9% sodium chloride injection to prepare a 1 mg / ml thymopentin stock solution. Then, add 10 ml of human serum albumin and 0.5 ml of thymopentin stock solution to 70-85 ml of 0.9% sodium chloride injection and mix thoroughly. Finally, add 0.9% sodium chloride injection to bring the mixture to 100 ml, filter, seal in a sterile container, and store at 2-8°C.

[0025] Step b: Mix and prepare cell preparation:

[0026] P3-6 generation umbilical cord mesenchymal stem cells were mixed with the above-mentioned cell preparation preservation solution to prepare a cell preparation with a cell density of 0.8-1.2 × 10⁻⁶. 6 Cells / ml; cell preparations should be stored at 15-30℃.

[0027] The 0.9% sodium chloride injection in the preservation solution is a commonly used drug solvent in clinical practice. It can protect cells by maintaining osmotic pressure and ensuring cell viability. Human serum albumin, whose main component is serum albumin, provides essential nutrients to cells, maintains their physiological state, and functions to maintain cellular microenvironment stability, promote substance exchange, and support cell metabolism. Human serum albumin is widely used clinically, and the concentration used in this project is within the safe range for clinical use and will not produce toxic effects on the human body.

[0028] Thymopentin is a polypeptide composed of five amino acids: arginine, lysine, aspartic acid, valine, and tyrosine. It regulates and enhances the body's cellular immune function, promotes cell proliferation, scavenge free radicals to prevent oxidative damage to biomolecules, protects against cell damage, and maintains cell viability. Related patents (application number: 202410793618.1, invention title: Application of Active Peptide Composition in the Preparation of Drugs Promoting Umbilical Cord Hematopoietic Stem Cell Proliferation) demonstrate that thymopentin can promote the growth of umbilical cord hematopoietic stem cells and enhance their proliferative activity. Thymopentin can protect against cell damage by scavenging free radicals and thus maintaining cell viability (Reference: "Study on the Antitumor and Antioxidant Effects of Thymopentin").

[0029] Example 2: Compared with Example 1, the amount of thymopentin stock solution added in step a is 1 ml, and the other steps and raw materials are the same as in Example 1.

[0030] Example 3: Compared with Example 1, the amount of thymopentin stock solution added in step a is 1.5 ml, and the other steps and raw materials are the same as in Example 1.

[0031] Example 4: Compared with Example 1, the amount of thymopentin stock solution added in step a is 2 ml, and the other steps and raw materials are the same as in Example 1.

[0032] Example 5: Compared with Example 1, the amount of thymopentin stock solution added in step a is 2.5 ml, and the other steps and raw materials are the same as in Example 1.

[0033] Comparative Example: Compared with Example 1, no thymopentin for injection was added in the comparative example, and the other steps and raw materials were the same as in Example 1.

[0034] Cell performance testing: Umbilical cord mesenchymal stem cells from each embodiment were sampled every 24 hours. Cells were stained with trypan blue solution to detect the total number of cells and the number of viable cells, and the cell viability was calculated. The results are shown in Tables 1-3. The data in the tables show that, using this cell preservation solution, the mesenchymal stem cells preserved for 72 hours showed higher activity, increased viable cell count, and a cell viability maintained above 80% compared to traditional preservation solutions. Using the above cell preservation solution is simple to operate, produces high cell viability, and allows for longer cell preservation at room temperature.

[0035]

[0036] The beneficial effects of this invention are:

[0037] 1. This invention can achieve stable preservation of stem cells for 72 hours at room temperature of 15-30℃, with cell viability maintained at over 80%. It solves the core pain point of traditional preservation solutions that rely on refrigeration at 2-8℃ and cannot be transported over long distances, thus meeting the needs of cross-regional clinical transportation.

[0038] 2. This invention contains only three components: sodium chloride injection, human serum albumin, and thymopentin. The composition is simple, and these are all clinically approved pharmaceutical excipients or drugs. It does not contain complex additives such as pH buffers and antioxidants. The preparation process is simple and the cost is easy to control.

[0039] It is understood that the present invention has been described through some embodiments, and those skilled in the art will recognize that various changes or equivalent substitutions can be made to these features and embodiments without departing from the spirit and scope of the invention. Furthermore, under the teachings of the present invention, these features and embodiments can be modified to adapt to specific situations and materials without departing from the spirit and scope of the invention. Therefore, the present invention is not limited to the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of this application are within the protection scope of the present invention.

Claims

1. A preservation solution for umbilical cord mesenchymal stem cell preparation, characterized in that, The preservation solution for umbilical cord mesenchymal stem cells consists of the following components: 0.9% sodium chloride injection, human serum albumin, and thymopentin for injection.

2. The preservation solution for umbilical cord mesenchymal stem cell preparation according to claim 1, characterized in that, The preservation solution contains 20 g / L human serum albumin, 5-25 mg / L thymopentin for injection, and the remainder is 0.9% sodium chloride injection.

3. A method for preparing a preservation solution for umbilical cord mesenchymal stem cells, characterized in that, Includes the following steps: Step 1: Under sterile conditions, take 10mg of thymopentin for injection, add 10ml of 0.9% sodium chloride injection, and dissolve it thoroughly to prepare a 1mg / ml thymopentin stock solution. Step 2: Add 10ml of human serum albumin and 0.5-2.5ml of the thymopentin stock solution prepared in Step 1 to 70-85ml of 0.9% sodium chloride injection, and mix well. Step 3: Add 0.9% sodium chloride injection to the mixture from Step 2 to a final volume of 100ml, filter, seal in a sterile container, and store at 2-8℃.

4. A method for preserving umbilical cord mesenchymal stem cells, characterized in that, Includes the following steps: Step a: Prepare a preservation solution for umbilical cord mesenchymal stem cell preparation according to the method described in claim 3; Step b: Mix the P3-6 generation umbilical cord mesenchymal stem cells with the cell preparation preservation solution prepared in step a to prepare a cell preparation. The cell density in the cell preparation is 0.8-1.2 × 10⁻⁶. 6 Cells / ml; cell preparations should be stored at 15-30℃.

5. A preparation of umbilical cord mesenchymal stem cells, characterized in that, The preparation is made by mixing the preservation solution described in claim 2 with P3-6 generation umbilical cord mesenchymal stem cells, wherein the cell density in the preparation is 0.8-1.2 × 10⁻⁶. 6 per ml.

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