阅读说明：本技术 一种造血干细胞冻存液及其制备方法和使用方法 (Hematopoietic stem cell cryopreservation liquid and preparation method and application method thereof ) 是由 黄亚非 代建波 余昆洪 李捡平 于 2018-08-09 设计创作，主要内容包括：本发明涉及一种造血干细胞冻存液及其制备方法和使用方法,包括卵磷脂、卵清蛋白、肌球蛋白、血小板裂解物和缓冲液；其中,每100ml所述冻存液中,所述卵磷脂的质量范围为1g至4g,所述卵清蛋白的质量范围为0.8g至1.5g,所述肌球蛋白的质量范围为0.8g至1.5g,所述血小板裂解物的质量至少为所述肌球蛋白质量的7.5倍,余量为所述缓冲液。本发明的有益效果是：本发明解决了传统细胞冻存液成分的毒副作用、细胞冻存存活率低的问题,既通过改变细胞内外电解质平衡,又通过增强细胞膜张力、武装细胞膜结构,提高细胞在冻存时的存活率。(The invention relates to a hematopoietic stem cell frozen stock solution and a preparation method and a using method thereof, wherein the hematopoietic stem cell frozen stock solution comprises lecithin, ovalbumin, myosin, platelet lysate and buffer solution; wherein, in every 100ml of the frozen stock solution, the mass range of the lecithin is 1g to 4g, the mass range of the ovalbumin is 0.8g to 1.5g, the mass range of the myosin is 0.8g to 1.5g, the mass of the platelet lysate is at least 7.5 times of the mass of the myosin, and the balance is the buffer solution. The invention has the beneficial effects that: the invention solves the problems of toxic and side effect of the components of the traditional cell freezing solution and low cell freezing survival rate, and improves the survival rate of cells during freezing by changing the balance of electrolytes inside and outside the cells, enhancing the tension of cell membranes and armed cell membrane structure.)

1. A hematopoietic stem cell cryopreservation liquid is characterized in that: including lecithin, ovalbumin, myosin, platelet lysate and buffers;

wherein, in every 100ml of the frozen stock solution, the mass range of the lecithin is 1g to 4g, the mass range of the ovalbumin is 0.8g to 1.5g, the mass range of the myosin is 0.8g to 1.5g, the mass of the platelet lysate is at least 7.5 times of the mass of the myosin, and the balance is the buffer solution.

2. The hematopoietic stem cell cryopreserved fluid according to claim 1, wherein: the mass ratio of the lecithin to the myosin ranges from 4:1 to 1:1, and the mass ratio of the ovalbumin to the myosin ranges from 8:10 to 11:10, per 100ml of the frozen stock solution.

3. The hematopoietic stem cell cryopreserved fluid according to claim 2, wherein: the mass ratio of the lecithin to the myosin was 3:1 per 100ml of the frozen stock solution.

4. The hematopoietic stem cell cryopreserved fluid according to claim 2, wherein: the mass ratio of the ovalbumin to the myosin is 1:1 per 100ml of the frozen stock solution.

5. The hematopoietic stem cell cryopreserved fluid according to claim 1, wherein: the buffer solution is Bomaili A injection or PBS buffer solution.

6. A method for preparing a hematopoietic stem cell frozen stock solution, wherein each 100ml of the frozen stock solution is prepared, comprising the steps of:

s01: uniformly mixing 1g to 4g of lecithin, 0.8g to 1.5g of ovalbumin and 0.8g to 1.5g of myosin in 2ml of Bomai A injection buffer solution to obtain a mixed solution;

s02: standing the mixed solution obtained in the step S01 for 15min at normal temperature in the dark;

s03: adding platelet lysate with the mass at least 7.5 times that of the myosin into the mixed solution, and adding a certain amount of Bomai force A injection buffer solution for constant volume to ensure that the volume of the frozen stock solution reaches 100 ml.

7. The method according to claim 6, wherein the method comprises the steps of: in the step S01, the mass ratio of the lecithin to the myosin is 3: 1.

8. The method according to claim 6, wherein the method comprises the steps of: in the step S01, the mass ratio of the ovalbumin to the myosin is 1: 1.

9. A method for using a hematopoietic stem cell frozen stock solution is characterized by comprising the following steps: the method comprises the following steps of adding the prepared hematopoietic stem cell suspension into the frozen stock solution, and directly storing in a refrigerator at-80 ℃.

10. The method of using a hematopoietic stem cell cryopreserved fluid according to claim 9, wherein the method comprises the steps of: the volume ratio of the freezing medium to the hematopoietic stem cell suspension is 1:1 or 2: 1.

Technical Field

The invention relates to the technical field of hematopoietic stem cell cryopreservation, in particular to a hematopoietic stem cell cryopreservation liquid and a preparation method and a using method thereof.

Background

The freezing solution is a substance capable of protecting cells from freezing damage, the cells can be protected from solution damage and ice crystal damage by adding the freezing solution into the cell suspension, and the concentration of electrolyte in unfrozen solution inside and outside the cells can be reduced by maintaining a certain molar concentration inside and outside the cells, so that the cells are protected from damage of solute.

When the traditional cell freezing solution is stored in a refrigerator at the temperature of minus 80 ℃, low molecular or high molecular chemicals are added, and the cell survival rate of the cells during low-temperature storage is stabilized by changing the balance of electrolytes inside and outside the cells and osmotic pressure; for example, the conventional cell freezing solution is added with 10% of dimethyl sulfoxide, albumin and hydroxyethyl starch composition, and some low molecular substances such as dimethyl sulfoxide have cytotoxicity and other toxic and side effects, the survival rate of frozen cells is not very high, and the conventional cell freezing protective solution has many defects in cell freezing on the whole.

Disclosure of Invention

The invention aims to solve the technical problem of the prior art and provides a hematopoietic stem cell frozen stock solution, a preparation method and a use method thereof.

The technical scheme for solving the technical problems is as follows:

according to one aspect of the present invention, there is provided a hematopoietic stem cell cryopreserved comprising lecithin, ovalbumin, myosin, platelet lysate and buffer;

wherein, in every 100ml of the frozen stock solution, the mass range of the lecithin is 1g to 4g, the mass range of the ovalbumin is 0.8g to 1.5g, the mass range of the myosin is 0.8g to 1.5g, the mass of the platelet lysate is at least 7.5 times of the mass of the myosin, and the balance is the buffer solution.

The invention has the beneficial effects that: the heavy chain of myosin can transport the ovalbumin into the cell, so that the ovalbumin is combined with water molecules in advance to change the viscosity in the cell, thereby preventing the formation of ice crystals when cooling, in addition, the combined action of myosin and lecithin can enhance the tension and the compressive resistance of cell membranes, thereby ensuring that the cell is not easy to be punctured by the ice crystals, thereby ensuring that the cell can be directly frozen and stored in a refrigerator at the temperature of-80 ℃ without program control cooling when being frozen and being convenient to operate; the platelet lysate can provide required ATP and nutrient substances for the transportation of myosin, and also can be used for regulating the electrolyte balance together with a buffer solution; the invention solves the problems of toxic and side effect of the components of the traditional cell freezing solution and low cell freezing survival rate, and improves the survival rate of cells during freezing by changing the balance of electrolytes inside and outside the cells, enhancing the tension of cell membranes and armed cell membrane structure.

On the basis of the technical scheme, the invention can be further improved as follows.

Further: the mass ratio of the lecithin to the myosin ranges from 4:1 to 1:1, and the mass ratio of the ovalbumin to the myosin ranges from 8:10 to 11:10, per 100ml of the frozen stock solution.

Further: the mass ratio of the lecithin to the myosin was 3:1 per 100ml of the frozen stock solution.

The beneficial effects of the further scheme are as follows: the efficiency of the cooperation of lecithin and myosin is improved, the formation of ice crystals is reduced as far as possible, and the cell cryopreservation does not need program control cooling and is convenient to operate.

Further: the mass ratio of the ovalbumin to the myosin is 1:1 per 100ml of the frozen stock solution.

The beneficial effects of the further scheme are as follows: the combined action efficiency of the ovalbumin and the myosin is improved, the tension and the compressive resistance of the cell membrane are enhanced as much as possible, the cells are prevented from being punctured by ice crystals, and the survival rate of the cells is improved.

Further: the buffer solution is Bomaili A injection or PBS buffer solution.

The beneficial effects of the further scheme are as follows: the functions of constant volume and electrolyte balance adjustment are achieved.

According to another aspect of the present invention, there is provided a method for preparing a frozen stock solution of hematopoietic stem cells, each 100ml of the frozen stock solution being prepared, comprising the steps of:

s01: uniformly mixing 1g to 4g of lecithin, 0.8g to 1.5g of ovalbumin and 0.8g to 1.5g of myosin in 2ml of Bomai A injection buffer solution to obtain a mixed solution;

s02: standing the mixed solution obtained in the step S01 for 15min at normal temperature in the dark;

s03: adding platelet lysate with the mass at least 7.5 times that of the myosin into the mixed solution, and adding a certain amount of Bomai force A injection buffer solution for constant volume to ensure that the volume of the frozen stock solution reaches 100 ml.

The invention has the beneficial effects that: lecithin, ovalbumin and myosin are evenly mixed in 2ml of Bomaili A injection buffer solution and are kept stand in the dark at normal temperature, so that biological action time is provided for the mutual combination of the lecithin, the myosin and the ovalbumin, and the cooperation efficiency of the lecithin, the myosin and the ovalbumin is improved.

On the basis of the technical scheme, the invention can be further improved as follows.

Further: in the step S01, the mass ratio of the lecithin to the myosin is 3: 1.

The beneficial effects of the further scheme are as follows: the efficiency of the cooperation of lecithin and myosin is improved, the formation of ice crystals is reduced as far as possible, and the cell cryopreservation does not need program control cooling and is convenient to operate.

Further: in the step S01, the mass ratio of the ovalbumin to the myosin is 1: 1.

The beneficial effects of the further scheme are as follows: the combined action efficiency of the ovalbumin and the myosin is improved, the tension and the compressive resistance of the cell membrane are enhanced as much as possible, the cells are prevented from being punctured by ice crystals, and the survival rate of the cells is improved.

According to another aspect of the present invention, there is provided a method for using a hematopoietic stem cell frozen stock solution, comprising the steps of adding the prepared hematopoietic stem cell suspension to the frozen stock solution, and directly storing in a refrigerator at-80 ℃.

The invention has the beneficial effects that: when the cells are frozen, the cells do not need to be cooled by program control, can be directly frozen in a refrigerator with the temperature of minus 80 ℃, and are convenient to operate.

On the basis of the technical scheme, the invention can be further improved as follows.

Further: the volume ratio of the freezing medium to the hematopoietic stem cell suspension is 1:1 or 2: 1.

The beneficial effects of the further scheme are as follows: the cells can fully contact and interact with the active ingredients of the cryopreservation liquid, so that the optimal cryopreservation effect is achieved, and the cell survival rate is improved.

Detailed Description

The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

A hematopoietic stem cell cryopreserved comprising lecithin, ovalbumin, myosin, platelet lysate, and a buffer;

wherein, in every 100ml of the frozen stock solution, the mass range of the lecithin is 1g to 4g, the mass range of the ovalbumin is 0.8g to 1.5g, the mass range of the myosin is 0.8g to 1.5g, the mass of the platelet lysate is at least 7.5 times of the mass of the myosin, and the balance is the buffer solution.

The mass ratio of the lecithin to the myosin ranges from 4:1 to 1:1, and the mass ratio of the ovalbumin to the myosin ranges from 8:10 to 11:10, per 100ml of the frozen stock solution.

Preferably, the mass ratio of lecithin to myosin is 3:1 per 100ml of the frozen stock solution.

Preferably, the mass ratio of the ovalbumin to the myosin is 1:1 per 100ml of the frozen stock solution.

The buffer solution is Bo Mai Li A injection solution or PBS buffer solution, and in the embodiment, Bo Mai Li A injection buffer solution is preferred.

The Bomaili A injection buffer was purchased from Shanghai Baite medical supplies, Inc.

Preferably, the frozen stock solution contains 3g of lecithin, 1g of ovalbumin, 1g of myosin and 7.5g of platelet lysate per 100ml, and the balance is Bo Mai injection buffer.

A method for preparing a hematopoietic stem cell frozen stock solution, wherein each 100ml of the frozen stock solution is prepared, and the method comprises the following steps:

s01: uniformly mixing 1g to 4g of lecithin, 0.8g to 1.5g of ovalbumin and 0.8g to 1.5g of myosin in 2ml of Bomai A injection buffer solution to obtain a mixed solution;

s02: standing the mixed solution obtained in the step S01 for 15min at normal temperature in the dark;

s03: adding platelet lysate with the mass at least 7.5 times that of the myosin into the mixed solution, and adding a certain amount of Bomai force A injection buffer solution for constant volume to ensure that the volume of the frozen stock solution reaches 100 ml.

In step S01, the mass ratio of lecithin to myosin is 3:1, and the mass ratio of ovalbumin to myosin is 1: 1.

A method for using the frozen stock solution of hematopoietic stem cells includes such steps as adding the prepared hematopoietic stem cell suspension to said frozen stock solution, and directly storing in-80 deg.C refrigerator.

Further, the volume ratio of the freezing medium to the hematopoietic stem cell suspension is 1:1 or 2: 1. In this embodiment, the volume ratio of the cryopreservation solution to the hematopoietic stem cell suspension is preferably 2: 1.