阅读说明：本技术 用于干细胞、脐带血的低温保存液及其制备方法 (Low-temperature preservation solution for stem cells and umbilical cord blood and preparation method thereof ) 是由 魏东兵 陈哲 罗嘉 王建飞 于 2020-12-29 设计创作，主要内容包括：本发明公开了一种用于干细胞、脐带血的低温保存液及其制备方法,按重量比包括以下物料：NaCl 0.2-0.6％,Dextran40 3-6％,DMSO 45-51％,纯化水43-48％。并经称量混合后,制得保存液。本品制备流程简单,成分少,对保存细胞的安全性具有更大的保证,安全性高,且细胞复苏活率高,使用效果好。(The invention discloses a low-temperature preservation solution for stem cells and umbilical cord blood and a preparation method thereof, wherein the low-temperature preservation solution comprises the following materials in parts by weight: NaCl 0.2-0.6%, Dextran 403-6%, DMSO 45-51%, and purified water 43-48%. And weighing and mixing to obtain the preservation solution. The product has simple preparation process, few components, high safety, high cell recovery and activity rate, and good use effect, and can ensure cell preservation safety.)

1. A low-temperature preservation solution for stem cells and umbilical cord blood is characterized by comprising the following materials in parts by weight: NaCl 0.2-0.6%, Dextran 403-6%, DMSO 45-51%, and purified water 43-48%.

2. The cryopreservation liquid for stem cells and umbilical cord blood as claimed in claim 1, which comprises the following materials by weight: NaCl 0.3-0.5%, Dextran 404-5%, DMSO 48-50%, and purified water 44-46%.

3. The cryopreservation liquid for stem cells and umbilical cord blood as claimed in claim 1, which comprises the following materials by weight: NaCl 0.398%, Dextran 404.502%, DMSO 49.499%, and purified water 45.601%.

4. The cryopreservation liquid for stem cells and umbilical cord blood as claimed in claim 1, which comprises the following materials by weight: NaCl 0.408%, dextran 404.527%, DMSO 49.796%, purified water 45.269%.

5. A method for preparing a cryopreservation solution for stem cells and umbilical cord blood as claimed in any one of claims 1 to 4, comprising the steps of: (1) weighing the materials according to the weight ratio; (2) mixing the above materials, and making into preservative solution.

6. The method for preparing a cryopreservation solution for stem cells and umbilical cord blood according to claim 5, wherein the mixing method comprises: adding purified water → adding NaCl for dissolution → adding Dextran40 for dissolution → slowly adding DMSO; mixing operation standard: no precipitate is generated during mixing, and no large amount of heat is released.

Technical Field

The invention relates to the technical field of cell preservation solution, in particular to low-temperature preservation solution for stem cells and umbilical cord blood and a preparation method thereof.

Background

Cord blood refers to the blood remaining in the placenta and umbilical cord after the fetus is delivered, the umbilical cord is ligated and disconnected. Cord blood contains a large number of stem cells, including hematopoietic stem cells and a variety of other stem cells, collectively known as cord blood stem cells. Cord blood hematopoietic stem cells have had a history of saving lives for over 30 years. There are also many clinical studies on cord blood in regenerative medicine, aiming to promote the repair of damage to tissues and organs of the body by using cord blood stem cells.

For convenient storage and clinical application, the cord blood is subjected to a separation preparation process to remove red blood cells and blood plasma as much as possible, and nucleated cell components of the cord blood are enriched. The enriched cells contain various stem cells, and have self-renewal capacity and the potential of differentiating into various mature cells; meanwhile, the traditional Chinese medicine composition also contains a large number of immune cells, and has good immune regulation and anti-infection effects. The enriched cord blood cells will be finally stored in a liquid nitrogen tank at a deep low temperature in a dormant state.

In the preservation process of stem cells and umbilical cord blood, low-temperature preservation liquid is needed to protect the stem cells and the umbilical cord blood so as to ensure the recovery survival rate of the cells. However, the prior art has the following defects: 1) the use effect of similar products is unstable, and the batch difference is large; 2) the survival rate of the cells after recovery is reduced; 3) high requirements for storage conditions lead to a series of problems in use. Therefore, a low-temperature preservation solution with high comprehensive performance for stem cells and umbilical cord blood is needed.

Disclosure of Invention

The invention aims to provide a low-temperature preservation solution for stem cells and umbilical cord blood, which has the advantages of higher guarantee on the safety of preserved cells, high safety and high cell recovery and survival rate.

In order to solve the technical problems, the technical scheme of the invention is as follows: a low-temperature preservation solution for stem cells and umbilical cord blood comprises the following materials in percentage by weight: NaCl 0.2-0.6%, Dextran 403-6%, DMSO 45-51%, and purified water 43-48%.

As a preferred embodiment, the low-temperature preservation solution for stem cells and umbilical cord blood comprises the following materials in percentage by weight: NaCl 0.3-0.5%, Dextran 404-5%, DMSO 48-50%, and purified water 44-46%.

As a preferred embodiment, the low-temperature preservation solution for stem cells and umbilical cord blood comprises the following materials in percentage by weight: NaCl 0.398%, Dextran 404.502%, DMSO 49.499%, and purified water 45.601%.

As a preferred embodiment, the low-temperature preservation solution for stem cells and umbilical cord blood comprises the following materials in percentage by weight: NaCl 0.408%, dextran 404.527%, DMSO 49.796%, purified water 45.269%.

The invention also provides a preparation method of the low-temperature preservation solution for stem cells and umbilical cord blood, and the production flow is simplified.

A preparation method of a low-temperature preservation solution for stem cells and umbilical cord blood comprises the following steps: (1) weighing the materials according to the weight ratio; (2) mixing the above materials, and making into preservative solution.

As a preferred embodiment, the mixing method is: adding purified water → adding NaCl for dissolution → adding Dextran40 for dissolution → slowly adding DMSO; mixing operation standard: no precipitate is generated during mixing, and no large amount of heat is released.

After the preservation solution is prepared, the effectiveness and the safety of the product on the cryopreservation of stem cells and umbilical cord blood are ensured through all-round quality inspection.

By adopting the technical scheme, all raw materials in the product formula accord with pharmacopoeia grades, and the formula is clear; the formula flow is simple, the cost is low, and the production flow is simplified; by adopting the formula, the product has few components and little influence on the activity of the cord blood stem cells, so that the safety of the preserved cells is ensured; quality inspection is carried out from appearance of the product to detection of mycoplasma, viruses and the like, so that the effectiveness and safety of the product on stem cell cryopreservation can be ensured; through the test experiment of freezing and storing the cord blood stem cells by the cell preservation solution, the cell recovery survival rate is over 90 percent. And the product has less same product and large market potential.

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

Example 1

The low-temperature preservation solution for stem cells and umbilical cord blood comprises the following materials in percentage by weight: NaCl 0.2%, Dextran40 (low molecular weight Dextran) 3%, DMSO 51%, purified water 45.8%.

Example 2

The low-temperature preservation solution for stem cells and umbilical cord blood comprises the following materials in percentage by weight: NaCl 0.6%, Dextran 406%, DMSO 46%, purified water 47.4%.

Example 3

The low-temperature preservation solution for stem cells and umbilical cord blood comprises the following materials in percentage by weight: NaCl 0.4%, Dextran 405%, DMSO 48%, purified water 46.6%.

Example 4

The low-temperature preservation solution for stem cells and umbilical cord blood comprises the following materials in percentage by weight: NaCl 0.55%, dextran 405.56%, DMSO 50.58%, and purified water 43.31%.

Example 5

The low-temperature preservation solution for stem cells and umbilical cord blood comprises the following materials in percentage by weight: NaCl 0.3%, Dextran 405%, DMSO 50%, purified water 44.7%.

Example 6

The low-temperature preservation solution for stem cells and umbilical cord blood comprises the following materials in percentage by weight: NaCl 0.5%, dextran 404.5%, DMSO 49.5%, purified water 45.5%.

Example 7

The low-temperature preservation solution for stem cells and umbilical cord blood comprises the following materials in percentage by weight: NaCl 0.45%, dextran 404.55%, DMSO 48.75%, and purified water 46.25%.

Example 8

The low-temperature preservation solution for stem cells and umbilical cord blood comprises the following materials in percentage by weight: NaCl 0.446%, dextran 404.727%, DMSO 48.889%, and purified water 45.938%.

Example 9

The low-temperature preservation solution for stem cells and umbilical cord blood comprises the following materials in percentage by weight: NaCl 0.408%, dextran 404.527%, DMSO 49.796%, purified water 45.269%.

Example 10

The low-temperature preservation solution for stem cells and umbilical cord blood comprises the following materials in percentage by weight: NaCl 0.398%, Dextran 404.502%, DMSO 49.499%, and purified water 45.601%.

The above examples all employ the following preparation methods: the method comprises the following steps: (1) weighing the materials according to the weight ratio; (2) mixing the above materials, and making into preservative solution.

The mixing sequence and method are as follows: adding purified water → adding NaCl for dissolution → adding Dextran40 for dissolution → slowly adding DMSO;

the operating criteria during the mixing process were: no precipitate is generated during mixing, and no large amount of heat is released.

The development process is as follows:

mixing DMSO, low molecular dextran and normal saline according to different proportions, wherein the content of the DMSO is 45% -55%, and the content of the low molecular dextran is 3% -15%; mixing the solutions according to different mixing sequences, and observing whether the physical change of the product has a large amount of heat release and precipitates;

and the following modes are found to be optimal through experiments: weighing → adding purified water → adding NaCl for dissolving → adding low molecular dextran for dissolving → slowly adding DMSO, the operation flow ensures that no precipitate is generated and no large amount of heat is released when the raw materials are mixed;

strictly controlling the quality of the cell freezing solution, and detecting after filtering the product: bacterial endotoxin is less than 0.06EU/mL, and the bacterial endotoxin is sterile and mycoplasma negative, so that the quality of the frozen stock solution product is guaranteed to be qualified;

further researching the cryopreservation effect of the cell cryopreservation liquid with different proportions on the cells; explore the corresponding cryopreservation program: 1) cooling by a program; 2) direct freezing effect on cells at-80 ℃ and-196 ℃ and the like;

after the research experiment, the result shows that the product can ensure that the cell recovery activity rate is more than 90% in the formula range. Further, the optimal formula and preparation method of the cell frozen stock solution are obtained.

The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.