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Refrigeration protection liquid and preservation method for mesenchymal stem cells

A technology of mesenchymal stem cells and preservation method, which is applied in the field of mesenchymal stem cell cryopreservation protection solution and preservation, can solve the problems of limited clinical application and short preservation time, and achieves the effects of good cell protection, high safety, and reduced dependence.

Active Publication Date: 2022-04-29
北京中卫医正科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the storage time of cold-preserved cells is very short and usually needs to be used within 12 hours, which is also very limited in clinical application.

Method used

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  • Refrigeration protection liquid and preservation method for mesenchymal stem cells
  • Refrigeration protection liquid and preservation method for mesenchymal stem cells
  • Refrigeration protection liquid and preservation method for mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1-3

[0035] Prepare cryopreservation solutions containing different concentrations (w / v) of trehalose, and the dosage of each group of reagents is shown in the table below. Filter and sterilize each of the prepared cryopreservation solutions with a 0.22 μm filter membrane, and pre-cool the filtrate at 4°C (the temperature of the filtrate is 4°C). Will 1×10 7 After the umbilical cord mesenchymal stem cells were mixed with 1ml of cryoprotectant solution, they were transferred to a 4°C refrigerator for refrigerated storage.

[0036]

[0037] Take out the cell suspension of each group from the refrigerator on day 0, day 1, day 2, day 3, day 4, day 5, and day 6, mix well, draw 50 μL, dilute to an appropriate multiple, and stain with 0.4% trypan blue , added to a cell counting board for cell counting, and calculated the cell viability (see the table below), and analyzed the relationship between the viable cell rate, storage time and trehalose, and each group of experiments was repeat...

Embodiment 4-6

[0041] Prepare cryopreservation solutions containing different concentrations (v / v) of human serum albumin injection, and the dosage of each group of reagents is shown in the table below. Filter and sterilize each of the prepared cryopreservation solutions with a 0.22 μm filter membrane, and pre-cool the filtrate at 4°C (the temperature of the filtrate is 4°C). Will 1×10 7 After the umbilical cord mesenchymal stem cells were mixed with 1ml of cryoprotectant solution, they were transferred to a 4°C refrigerator for refrigerated storage.

[0042]

[0043] Take out the cell suspension of each group from the refrigerator on day 0, day 1, day 2, day 3, day 4, day 5, and day 6, mix well, draw 50 μL, dilute to an appropriate multiple, and stain with 0.4% trypan blue , add a cell counting board to count the cells, and calculate the cell viability (see the table below), analyze the relationship between the viable cell rate, storage time and human albumin injection, and repeat 3 tim...

Embodiment 7

[0047] Prepare the refrigerated protective solution according to the formula in the table below (Example 2), filter and sterilize the prepared protective solution with a 0.22 μm filter membrane, and pre-cool the filtrate at 4°C (the temperature of the filtrate is 4°C). Will 1×10 7 Each umbilical cord mesenchymal stem cell was mixed with 1ml of cryoprotectant solution, pre-cooled at -20°C for 0-9min, and then transferred to a 4°C refrigerator for storage.

[0048]

[0049] After the cells were refrigerated for 5 days, 100 μL was drawn, diluted to an appropriate multiple, stained with 0.4% trypan blue, added to a cell counting plate for cell counting, and the cell viability was calculated (see the table below). Each experiment was repeated 3 times.

[0050] -20℃ pre-cooling time 0min 1min 3min 5min 7min 9min Cell Viability% 76.6±1.8 78.4±1.4 84.3±1.4 86.2±1.6 67.3±1.2 50.1±1.0

[0051] From the comparison of cell viability, it can be seen...

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Abstract

The invention provides a mesenchymal stem cell refrigeration protection liquid and a preservation method. The refrigeration protection liquid comprises the following components: trehalose, a low-molecular heparin calcium injection, a dextran 40 sodium chloride injection, human serum albumin and a compound electrolyte injection. The cold storage protection liquid does not contain toxicological components such as DMSO (dimethylsulfoxide) and the like, and the components are all injection-grade medicine auxiliary materials approved by the country and can be directly transfused back into veins. By adopting a pre-cooling type refrigeration preservation method, the cells and the protection liquid can quickly enter a low-temperature equilibrium state so as to reduce cell damage, the method can be used for long-time preservation of the mesenchymal stem cells under the refrigeration condition of 4 DEG C, is convenient to transport, and reduces the dependence of stem cell products on regions.

Description

technical field [0001] The invention belongs to the technical field of stem cell biology, and in particular relates to a mesenchymal stem cell cryopreservation protection solution and a preservation method. Background technique [0002] Mesenchymal stem cells (MSCs) are pluripotent stem cells with high self-renewal ability and multi-directional differentiation potential derived from the mesoderm in the early stage of development. They are widely distributed in various tissues throughout the body and can be cultured and expanded in vitro. And it can differentiate into nerve cells, osteoblasts, chondrocytes, muscle cells and adipocytes under specific conditions. These characteristics make it have important application value in the treatment of tissue damage diseases and immune diseases. [0003] Stem cell preparation techniques and treatment programs are diverse, complex and specific. Stem cells are different from other medicines. It is difficult to maintain long-term surviva...

Claims

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Application Information

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IPC IPC(8): A01N1/02
CPCA01N1/021A01N1/0221A01N1/0284
Inventor 修冰水王宏李帅民刘世红
Owner 北京中卫医正科技有限公司
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