Special cryopreservation solution for hepatocytes and preparation method therefor

Abstract

The present invention relates to a special cryopreservation solution for hepatocytes and a preparation method therefor, and belongs to the technical field of cell cryopreservation and thawing. The special cryopreservation solution for hepatocytes comprises the following components: a cryoprotectant, a biofilm protectant and an antioxidant. The biofilm protectant comprises an inhibitor Y27632, glycerol, human serum albumin, collagen and a dextran 40 sodium chloride injection. The special cryopreservation solution for hepatocytes of the present invention can effectively improve the hydrophobicity of the biofilm and reduce the fluidity of the biofilm, so that the damage to the biofilm of hepatocytes caused by ice crystals during the freezing and thawing processes is minimized, and the cell viability of hepatocytes after thawing is significantly improved.

Description

A special freezing solution for hepatocytes and preparation method thereof Technical Field

[0001] The invention belongs to the technical field of stem cell cryopreservation and resuscitation, and relates to a special freezing solution for liver cells and a preparation method thereof. Background Art

[0002] Traditional cryopreservation fluids typically contain DMSO (dimethyl sulfoxide) and mannitol and are used to preserve various cell types. However, as a specialized cell type, hepatocytes have more stringent requirements for freezing conditions and preservation fluids. Traditional cryopreservation fluids may not provide adequate protection, causing damage to hepatocytes during the freezing and thawing process, reducing their viability and function. Therefore, to meet the special needs of hepatocytes, it is necessary to develop a cryopreservation fluid specifically for hepatocytes to improve their freezing effect and maintain their integrity and function. A dedicated hepatocyte cryopreservation fluid and preparation method that can meet the critical quality attribute requirements for clinical applications and industrial production is urgently needed. At the same time, the preparation method must also take into account the special characteristics of hepatocytes to ensure that no damage is caused to hepatocytes during the preparation process. Technical issues

[0003] Therefore, in response to the special needs of hepatocytes, the present invention proposes a hepatocyte-specific freezing solution and a preparation method thereof to solve the problems existing in the prior art and improve the freezing effect and preservation quality of hepatocytes. Technical Solutions

[0004] In order to solve the above problems, the present invention proposes the following technical solutions:

[0005] A cryopreservation solution specifically for hepatocytes, comprising a cryoprotectant, a biofilm protectant, and an oxidant. The biofilm protectant comprises inhibitor Y-27632, human serum albumin, collagen, glycerol, and dextran 40 sodium chloride injection.

[0006] The concentration of the inhibitor Y-27632 in the freezing solution is 1.6-6.4 mg / L, the concentration of the human serum albumin in the freezing solution is 5%-40% by mass, the concentration of the collagen in the freezing solution is 150-250 mg / L, and the concentration of the glycerol in the freezing solution is 0.5%-5% by mass.

[0007] Furthermore, the antioxidant is composed of mercaptoethanol, vitamin C, and dextran 40 sodium chloride injection. Furthermore, the volume percentage of mercaptoethanol in the freezing solution is 0.05-0.1%, and the concentration of vitamin C in the freezing solution is 9-35 mg / L.

[0008] Furthermore, the cryoprotectant is composed of dimethyl sulfoxide and dextran 40 sodium chloride injection. The volume percentage of dimethyl sulfoxide in the cryopreservation solution is 2-10%, and the mass fraction of dextran 40 sodium chloride injection is 30-80%.

[0009] Furthermore, the concentration of the inhibitor Y-27632 in the freezing solution is 3.2~4.8 mg / L, the concentration of the human serum albumin in the freezing solution is 10%~30% by mass, and the concentration of the collagen in the freezing solution is 175~225 mg / L.

[0010] Furthermore, the volume percentage of the mercaptoethanol in the freezing solution is 0.075-0.1%, the concentration of the vitamin C in the freezing solution is 18-27 mg / L; and the volume percentage of the dimethyl sulfoxide in the freezing solution is 3-8%.

[0011] The present invention provides a method for preparing a special freezing solution for hepatocytes, comprising the following steps:

[0012] (1) Inhibitor Y-27632, human serum albumin, collagen, glycerol, and dextran 40 sodium chloride injection were mixed, stirred, and filtered for sterilization to obtain a biofilm protective agent;

[0013] (2) Mixing mercaptoethanol, vitamin C, and dextran 40 sodium chloride injection, stirring, filtering and sterilizing, to obtain an antioxidant;

[0014] (3) Mix dimethyl sulfoxide and dextran 40 sodium chloride injection, stir, filter and sterilize to obtain a cryoprotectant;

[0015] (4) Separately package the biofilm protectant, antioxidant, and cryoprotectant and store them in a 4°C refrigerator. Beneficial effects

[0016] Beneficial effects of the present invention: (1) The biofilm protectant of the present invention is composed of inhibitor Y-27632, human serum albumin, collagen and dextran 40 sodium chloride injection. It is a groundbreaking discovery that the use of a biofilm protectant containing inhibitor Y-27632, human serum albumin, collagen and glycerol can effectively reduce the damage of ice crystals to the biofilm of liver cells during cryopreservation and greatly improve the cell viability after recovery. (2) The present invention is a cryopreservation fluid specifically for liver cells, which solves some of the disadvantages of existing cryopreservation fluids when used for cryopreservation of liver cells and fills the gap in the current field of stem cell cryopreservation and recovery technology specifically for liver cell cryopreservation. (3) The present invention has sufficient quality research, good cryopreservation effect and good stability. The cryopreservation fluid specifically for liver cells of the present invention can be used to stably freeze liver cells under liquid nitrogen conditions for up to 48 months. Best Mode for Carrying Out the Invention

[0017] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, the specific implementation methods, structures, features and effects of the present invention are described in detail below in combination with the embodiments.

[0018] Example 1

[0019] The contents of the components of the hepatocyte cryopreservation solution are as follows: inhibitor Y-27632 3.2 mg / L, 10% human serum albumin solution, collagen 175 mg / L, 2% glycerol, 0.08% mercaptoethanol, 13 mg / L vitamin C, and 5% dimethyl sulfoxide.

[0020] The preparation method of the hepatocyte cryopreservation solution is as follows: Taking a total volume of 1000 ml as an example:

[0021] (1) 3.2 mg of inhibitor Y-27632, 5% human albumin, 175 mg of collagen, 2% glycerol, and 350 ml of dextran 40% sodium chloride injection were mixed, placed on a magnetic stirrer with a speed of 35 r / min and a stirring time of 8 min, and filtered and sterilized using a filter with a pore size of 0.2 μm to obtain a biofilm protectant;

[0022] (2) 0.8 ml of mercaptoethanol, 13 mg of vitamin C, and 250 ml of dextran 40% sodium chloride injection were mixed, placed on a magnetic stirrer with a speed of 35 r / min and a stirring time of 8 min, and sterilized by filtration using a filter with a pore size of 0.2 μm to obtain an antioxidant;

[0023] (3) Mix 50 ml of dimethyl sulfoxide and 250 ml of dextran 40% sodium chloride injection, place on a magnetic stirrer with a speed of 35 r / min and a stirring time of 8 min, and filter and sterilize using a filter with a pore size of 0.2 μm to obtain a cryoprotectant;

[0024] (4) Separately package the biofilm protectant, antioxidant, and cryoprotectant and store them in a 4°C refrigerator;

[0025] (5) Use the above-mentioned hepatocyte-specific freezing solution to perform cryopreservation and resuscitation operations on the hepatocytes, and record the cell viability after resuscitation.

[0026] Example 2

[0027] The preparation method of the hepatocyte cryopreservation solution is as follows: Taking a total volume of 1000 ml as an example:

[0028] (1) 64.8 mg of inhibitor Y-27632, 20% human serum albumin solution, 200 mg of collagen, 2% glycerol, and 330 ml of dextran 40 sodium chloride injection were mixed, placed on a magnetic stirrer with a speed of 35 r / min and a stirring time of 8 min, and filtered and sterilized using a filter with a pore size of 0.2 μm to obtain a biofilm protectant;

[0029] (2) 0.9 ml of mercaptoethanol, 22 mg of vitamin C, and 250 ml of dextran 40% sodium chloride injection were mixed, placed on a magnetic stirrer with a speed of 35 r / min and a stirring time of 8 min, and filtered and sterilized using a filter with a pore size of 0.2 μm to obtain an antioxidant;

[0030] (3) Mix 75 ml of dimethyl sulfoxide and 250 ml of dextran 40% sodium chloride injection, place on a magnetic stirrer with a speed of 35 r / min and a stirring time of 8 min, and filter and sterilize using a filter with a pore size of 0.2 μm to obtain a cryoprotectant;

[0031] (4) Separately package the biofilm protectant, antioxidant, and cryoprotectant and store them in a 4°C refrigerator;

[0032] (5) Use the above-mentioned hepatocyte-specific freezing solution to perform cryopreservation and resuscitation operations on the hepatocytes, and record the cell viability after resuscitation.

[0033] Comparative Example 1: No human serum albumin was added, and the amount of inhibitor Y-27632 added was adjusted to 70.8 mg. The rest of the configuration and operation methods were the same as those in Example 2.

[0034] Comparative Example 2: No inhibitor Y-27632 was added, and the amount of human albumin added was adjusted to 10% by mass. The rest of the configuration and operation methods were the same as those in Example 2.

[0035] Comparative Example 3: No inhibitor Y-27632 and human serum albumin were added, and the remaining configurations and operating methods were the same as those in Example 2.

[0036] Comparative Example 4 Unfrozen hepatocytes of the same passage were used as control cells, and frozen and thawed hepatocytes were used as test cells. After thawed for 12 months, viable cells were counted and cell viability was detected according to the experimental method of Examples 1 and 2 of the present invention.

[0037] The results showed that the unique formula design combination of the hepatocyte-specific freezing solution provided by the present invention achieved a better freezing and thawing effect.

[0038] The cell viability of the hepatocytes of Examples 1 and 2 after being frozen for 48 months and then revived was maintained at above 80%, among which the cell viability in Example 2 was the highest, and the cell viability after being frozen for 48 months and revived was still maintained at 88%, which was the best cryopreservation and resuscitation effect, achieving the intended purpose of the present invention; the cell viability of the hepatocytes of Comparative Examples 1 to 3 after being frozen for only 12 months and revived was less than 80%, and the cryopreservation and resuscitation effect was significantly worse than that of Examples 1 and 2.

[0039] The above experiments fully demonstrate that the inhibitor Y-27632, human serum albumin and collagen can significantly improve the cell viability of hepatocytes after cryopreservation and recovery through synergistic effects.

[0040] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the scope of the technical solution of the present invention, make some changes or modifications to equivalent embodiments using the technical contents disclosed above. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention. Industrial Applicability

[0041] The hepatocyte-specific freezing solution of the present invention can effectively improve the hydrophobicity of the biofilm and reduce the fluidity of the biofilm, thereby minimizing the damage of the hepatocyte biofilm caused by ice crystals during the freezing and thawing process, significantly improving the cell viability of the hepatocytes after thawing, and can be widely used in the cell freezing process.

Claims

1. A cryopreservation solution specifically for hepatocytes, characterized in that, the cryopreservation solution specifically for hepatocytes comprises: a cryoprotectant, a biomembrane protectant, and an antioxidant.

2. The cryopreservation solution specifically for hepatocytes according to claim 1, characterized in that, the biomembrane protectant consists of inhibitor Y-27632, human serum albumin, collagen, glycerol, and dextran 40 sodium chloride injection.

3. The cryopreservation solution specifically for hepatocytes according to claim 2, characterized in that, the concentration of inhibitor Y-27632 in the cryopreservation solution is 1.6 - 6.4 mg / L, the concentration of human serum albumin in the cryopreservation solution is 5% - 40% by mass fraction, the concentration of collagen in the cryopreservation solution is 150 - 250 mg / L, and the concentration of glycerol in the cryopreservation solution is 0.5% - 5% by mass fraction.

4. The cryopreservation solution specifically for hepatocytes according to claim 1, characterized in that, the antioxidant consists of mercaptoethanol, vitamin C, and dextran 40 sodium chloride injection.

5. The cryopreservation solution specifically for hepatocytes according to claim 4, characterized in that, the volume percentage of mercaptoethanol in the cryopreservation solution is 0.05 - 0.1%, and the concentration of vitamin C in the cryopreservation solution is 9 - 35 mg / L.

6. The cryopreservation solution specifically for hepatocytes according to claim 1, characterized in that, the cryoprotectant consists of dimethyl sulfoxide and dextran 40 sodium chloride injection.

7. The cryopreservation solution specifically for hepatocytes according to claim 6, characterized in that, the volume percentage of dimethyl sulfoxide in the cryopreservation solution is 2 - 10%, and the dextran 40 sodium chloride injection is 30% - 80% by mass fraction dextran 40 sodium chloride injection.

8. The cryopreservation solution specifically for hepatocytes according to claim 1, characterized in that, the concentration of inhibitor Y-27632 in the cryopreservation solution is 3.2 - 4.8 mg / L, the concentration of human serum albumin in the cryopreservation solution is 10% - 30% by mass fraction, and the concentration of collagen in the cryopreservation solution is 175 - 225 mg / L.

9. The cryopreservation solution specifically for hepatocytes according to claim 1, characterized in that, the volume percentage of mercaptoethanol in the cryopreservation solution is 0.075 - 0.1%, the concentration of vitamin C in the cryopreservation solution is 18 - 27 mg / L; the volume percentage of dimethyl sulfoxide in the cryopreservation solution is 3 - 8%.

10. A preparation method of a cryopreservation solution specifically for hepatocytes as described in any one of claims 1 - 9, characterized in that, it comprises the following steps: (1) Mix inhibitor Y-27632, human serum albumin, collagen, glycerol, and dextran 40 sodium chloride injection, stir, and filter to sterilize to obtain a biomembrane protectant; (2) Mix mercaptoethanol, vitamin C, and dextran 40 sodium chloride injection, stir, and filter to sterilize to obtain an antioxidant; (3) Mix dimethyl sulfoxide and dextran 40 sodium chloride injection, stir, and filter to sterilize to obtain a cryoprotectant; (4) Encapsulate the biofilm protectant, antioxidant, and cryoprotectant separately and store them in a refrigerator at 4°C.