CN118575808A - Immune cell freezing solution and freezing method - Google Patents

Abstract

The invention provides immune cell cryopreservation liquid and a cryopreservation method. An immune cell cryopreservation solution comprises a cell basal medium and a cell cryopreservation protective agent, wherein the cell cryopreservation protective agent comprises the following raw materials: polyethylene glycol, phosphatidylglycerol, linolenic acid, beta-ecdysterone, punicalagin, and dihydromyricetin. The invention also provides application of the immune cell cryopreservation liquid and an immune cell cryopreservation method. The invention can effectively protect immune cells, effectively improve the recovery survival rate of immune cells when freezing the immune cells, and the recovered cells still have better proliferation capacity and better biological activity after freezing.

Description

Immune cell freezing solution and freezing method

Technical Field

The present invention relates to the technical field of immune cell cryopreservation, and in particular to an immune cell cryopreservation solution and a cryopreservation method.

Background Art

Cell cryopreservation is a method for preserving cells, which is usually used in cell culture, medical research and biotechnology applications. This technology can preserve cells for a long time at extremely low temperatures (usually liquid nitrogen temperature) without inactivation. Cell cryopreservation mainly adopts the preservation scheme under ultra-low temperature conditions of -196℃. Under ultra-low temperature conditions, the metabolic function of cells stagnates, but they are not dead. Cell recovery is the process of rapid thawing of frozen cells at 36-40℃ to restore the activity and function of cells. It is of great significance both in clinical and basic research, especially the cryopreservation technology of immune cells. Immune cells, such as lymphocytes and dendritic cells, play an important role in the prevention and treatment of diseases. By freezing and preserving immune cells, the activity of cells can be preserved for a long time, so that they can be used at any time when needed, which provides convenience for immunotherapy and vaccine development. Immune cell therapy, such as CAR-T cell therapy, has become an important treatment for diseases such as cancer. Cryopreservation of immune cells can solve the problems of preparation and storage, making this treatment technology more flexible and feasible.

Early cell freezing methods were relatively simple, and basic cryogens such as glycerol were usually used for cell freezing. The effect of this method was unstable and the cell survival rate was low, which limited its wide application in scientific research and applications. With the in-depth study of cell biology, scientists began to develop more complex freezing fluids, such as freezing fluids containing cryoprotectants such as dimethyl sulfoxide (DMSO) and mannitol. The emergence of these freezing fluids significantly improved the efficiency and survival rate of cell freezing. Traditional cell freezing fluids usually contain ingredients such as fetal bovine serum, but the use of serum may have certain problems, such as batch differences, infectious disease risks, etc. Therefore, some studies have begun to try to develop serum-free cell freezing fluids to improve the safety and stability of cells.

Although cell freezing technology has been widely used and continuously improved, there are still some technical problems. During the freezing and thawing process, cells may be damaged, resulting in increased cell mortality or impaired function. This is due to factors such as the destruction of cell structure by ice crystals produced during the freezing process and the toxicity of cryopreservatives in the cryopreservative solution to cells. Different types of cells have different tolerances to cryopreservatives in the cryopreservative solution, so an optimized cryopreservative solution formula is required for different types of cells. At the same time, the concentration and ratio of cryopreservatives in the cryopreservative solution may also affect the freezing and thawing effects of cells. Therefore, it is of great significance to develop a cell cryopreservative solution that can improve the survival rate of cell cryopreservation and recovery.

Summary of the invention

In view of this, the present invention provides an immune cell freezing solution and a freezing method to solve the above problems.

The technical solution of the present invention is achieved in this way:

An immune cell freezing solution comprises a cell basal culture medium and a cell freezing protective agent, wherein the cell freezing protective agent comprises the following raw materials: polyethylene glycol, phosphatidylglycerol, linolenic acid, β-ecdysterone, punicalagin, and dihydromyricetin.

Preferably, based on the final volume of the freezing solution, the content of each component in the cell cryoprotectant is: polyethylene glycol 4-6 mg/mL, phosphatidylglycerol 2.5-5 mg/mL, linolenic acid 1-1.5 mg/mL, β-ecdysterone 25-40 μg/mL, punicalagin 16-25 μg/mL, and dihydromyricetin 8-12 μg/mL.

Preferably, based on the final volume of the freezing solution, the content of each component in the cell cryoprotectant is: polyethylene glycol 5 mg/mL, phosphatidylglycerol 4 mg/mL, linolenic acid 1.2 mg/mL, β-ecdysterone 35 μg/mL, punicalagin 20 μg/mL, and dihydromyricetin 10 μg/mL.

Preferably, the basal culture medium is serum-free GT-T551 culture medium.

Preferably, the present invention also provides a use of an immune cell freezing solution in freezing immune cells.

Preferably, the present invention also provides a method for freezing immune cells, comprising the following steps:

S1: Immune cells were extracted from whole blood;

S2: preparing the above immune cell freezing solution;

S3: Cryopreservation of immune cells.

Preferably, the S3 immune cell cryopreservation is specifically as follows: the extracted immune cells are added to the prepared immune cell cryopreservation solution, mixed to obtain a cell suspension, transferred into a sterile cryopreservation tube, first stored at 0-5°C for 2-4 hours, then stored at -80°C for 12-24 hours, and finally transferred and stored in liquid nitrogen for cryopreservation.

Preferably, the concentration of cells in the cell suspension is 1×10 ⁷ -3×10 ⁷ cells/mL.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The immune cell freezing solution provided by the present invention uses a compound of polyethylene glycol, phosphatidylglycerol, and linolenic acid to replace DMSO, which changes the permeability of the cell membrane and protects the cell membrane, reduces the damage to the cells caused by ice crystals formed in the cells during the freezing process, maintains a stable osmotic pressure in the extracellular environment, and avoids the increase of local electrolyte concentration after cell dehydration, which causes the disorder of the internal space structure of the cell.

(2) The serum-free cryopreservation solution of the present invention is also added with β-ecdysterone, punicalagin, and dihydromyricetin, which helps to quickly restore the activity of cells after recovery. The immune cell cryopreservation solution of the present invention improves the survival rate of cells after freezing through the synergistic effect of polyethylene glycol, phosphatidylglycerol, linolenic acid, β-ecdysterone, punicalagin, and dihydromyricetin. The cells recovered after freezing still have good activity and maintain good proliferation ability.

(3) The immune cell cryopreservation solution of the present invention uses safer components and does not add exogenous serum and DMSO, thereby reducing the potential risk of destroying cell activity; and the immune cell cryopreservation solution of the present invention does not contain components such as antibodies and complement, and has few impurities, thereby reducing the impact on the activity of immune cells.

DETAILED DESCRIPTION

In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

The β-ecdysterone (CAS No. 5289-74-7) described in the present invention was purchased from Shanghai Bolson Biotechnology Co., Ltd., BES2027ST.

The punicalagin (CAS No. 65995-63-3) described in the present invention was purchased from Shanghai Bolson Biotechnology Co., Ltd., BES2007SA.

The dihydromyricetin (CAS No. 27200-12-0) described in the present invention was purchased from Shanghai Bolson Biotechnology Co., Ltd., BES2011SE.

Example 1

An immune cell freezing solution comprises a cell basal culture medium and a cell cryoprotectant, wherein the basal culture medium is a serum-free GT-T551 culture medium, and the contents of the components in the cell cryoprotectant are as follows, based on the total volume of the freezing solution: 4 mg/mL polyethylene glycol, 2.5 mg/mL phosphatidylglycerol, 1 mg/mL linolenic acid, 25 μg/mL β-ecdysterone, 16 μg/mL punicalagin, and 8 μg/mL dihydromyricetin.

A method for freezing immune cells, comprising the following steps:

S1. Extract immune cells from whole blood: (1) Take peripheral blood and place it in a centrifuge tube, centrifuge it at 25°C for 20 minutes, and separate the upper plasma layer; (2) Take the lower layer of concentrated blood and add PBS buffer at a volume ratio of 1:1 and mix well; (3) Centrifuge for 20 minutes, aspirate the peripheral blood mononuclear cell layer, add PBS buffer and mix well; (4) Centrifuge for 10 minutes and remove the supernatant; (6) Collect the cell precipitate, which is the immune cell;

S2. Mix the components to prepare immune cell freezing solution under a sterile environment;

S3. Cryopreservation of immune cells: Add the extracted immune cells to the prepared immune cell freezing solution, mix well to obtain a cell suspension, wherein the cell concentration in the cell suspension is 1×10 ⁷ cells/mL, transfer to a sterile cryopreservation tube, first store at 0°C for 2 hours, then store at -80°C for 12 hours, and finally transfer to liquid nitrogen for cryopreservation.

Example 2

An immune cell freezing solution comprises a cell basal culture medium and a cell cryoprotectant, wherein the basal culture medium is a serum-free GT-T551 culture medium, and the contents of the components in the cell cryoprotectant are as follows, based on the total volume of the freezing solution: 5 mg/mL polyethylene glycol, 4 mg/mL phosphatidylglycerol, 1.2 mg/mL linolenic acid, 35 μg/mL β-ecdysterone, 20 μg/mL punicalagin, and 10 μg/mL dihydromyricetin.

A method for freezing immune cells, comprising the following steps:

S1. Extract immune cells from whole blood: (1) Take peripheral blood and place it in a centrifuge tube, centrifuge it at 25°C for 20 minutes, and separate the upper plasma layer; (2) Take the lower layer of concentrated blood and add PBS buffer at a volume ratio of 1:1 and mix well; (3) Centrifuge for 20 minutes, aspirate the peripheral blood mononuclear cell layer, add PBS buffer and mix well; (4) Centrifuge for 10 minutes and remove the supernatant; (6) Collect the cell precipitate, which is the immune cell;

S2. Mix the components to prepare immune cell freezing solution under a sterile environment;

S3. Cryopreservation of immune cells: Add the extracted immune cells to the prepared immune cell freezing solution, mix well to obtain a cell suspension, the cell concentration in the cell suspension is 2×10 ⁷ cells/mL, transfer to a sterile cryopreservation tube, first store at 5°C for 2 hours, then store at -80°C for 24 hours, and finally transfer to liquid nitrogen for cryopreservation.

Example 3

An immune cell freezing solution comprises a cell basal culture medium and a cell cryoprotectant, wherein the basal culture medium is a serum-free GT-T551 culture medium, and the contents of the components in the cell cryoprotectant are as follows, based on the total volume of the freezing solution: 6 mg/mL polyethylene glycol, 5 mg/mL phosphatidylglycerol, 1.5 mg/mL linolenic acid, 40 μg/mL β-ecdysterone, 25 μg/mL punicalagin, and 12 μg/mL dihydromyricetin.

A method for freezing immune cells, comprising the following steps:

S1. Extract immune cells from whole blood: (1) Take peripheral blood and place it in a centrifuge tube, centrifuge it at 25°C for 20 minutes, and separate the upper plasma layer; (2) Take the lower layer of concentrated blood and add PBS buffer at a volume ratio of 1:1 and mix well; (3) Centrifuge for 20 minutes, aspirate the peripheral blood mononuclear cell layer, add PBS buffer and mix well; (4) Centrifuge for 10 minutes and remove the supernatant; (6) Collect the cell precipitate, which is the immune cell;

S2. Mix the components to prepare immune cell freezing solution under a sterile environment;

S3. Cryopreservation of immune cells: Add the extracted immune cells to the prepared immune cell freezing solution, mix well to obtain a cell suspension, the cell concentration in the cell suspension is 3×10 ⁷ cells/mL, transfer to a sterile cryopreservation tube, first store at 5°C for 4 hours, then store at -80°C for 24 hours, and finally transfer to liquid nitrogen for cryopreservation.

Comparative Example 1

This example provides an immune cell freezing solution, which is different from Example 2 in that the cell freezing protective agent in this example does not contain polyethylene glycol, the amount of phosphatidylglycerol is adjusted to 6.5 mg/mL, and the amount of linolenic acid is adjusted to 3.7 mg/mL. The rest is the same as Example 2. The immune cell freezing method of this comparative example is the same as the method in Example 2.

Comparative Example 2

This example provides an immune cell freezing solution, which is different from Example 2 in that the cell freezing protective agent in this example does not contain phosphatidylglycerol, the amount of polyethylene glycol is adjusted to 7 mg/mL, and the amount of linolenic acid is adjusted to 3.2 mg/mL. The rest is the same as Example 2. The immune cell freezing method of this comparative example is the same as the method in Example 2.

Comparative Example 3

This example provides an immune cell freezing solution, which is different from Example 2 in that the cell freezing protective agent in this example does not contain linolenic acid, the amount of polyethylene glycol is adjusted to 5.6 mg/mL, and the amount of phosphatidylglycerol is adjusted to 4.6 mg/mL, and the rest is the same as Example 2. The immune cell freezing method of this comparative example is the same as the method in Example 2.

Comparative Example 4

This example provides an immune cell freezing solution, which is different from Example 2 in that the cell freezing protective agent in this example does not contain β-ecdysterone, and the rest is the same as Example 2. The immune cell freezing method of this comparative example is the same as the method in Example 2.

Comparative Example 5

This example provides an immune cell freezing solution, which is different from Example 2 in that the cell freezing protective agent in this example does not contain punicalagin, and the rest is the same as Example 2. The immune cell freezing method of this comparative example is the same as the method in Example 2.

Comparative Example 6

This example provides an immune cell freezing solution, which is different from Example 2 in that the cell freezing protective agent in this example does not contain dihydromyricetin, and the rest is the same as Example 2. The immune cell freezing method of this comparative example is the same as the method in Example 2.

Comparative Example 7

This example provides an immune cell freezing solution, including 10% DMSO, 10% fetal bovine serum, and 80% serum-free GT-T551 culture medium in volume fraction; the immune cell freezing method of this comparative example is the same as the method in Example 2.

Experimental Example 1: Test of the recovery performance of immune cells

The frozen immune cells frozen in liquid nitrogen for 3 months, 9 months and 24 months in Examples 1-3 and Comparative Examples 1-7 were revived, and the immune cell resuscitation included the following steps: taking out the cryopreservation tube and quickly placing it in a 36°C constant temperature water bath for 4 minutes, shaking until the cell suspension was completely melted, centrifuging, and washing 3 times with physiological saline to obtain revived immune cells. The revived CIK cells were stained with trypan blue to count the cell survival rate (three cryopreservation tubes were counted each time and the average value was taken), and the cell survival rate = (total number of cells-number of stained cells)/total number of cells × 100%, and the results are shown in Table 1.

Table 1 Cell recovery survival rate of different immune cell cryopreservation solutions

The results of Table 1 show that for the same freezing time, the CIK cell survival rate in Examples 1 to 3 is higher, and as the freezing time is extended, the survival rate of CIK cell recovery in Examples 1 to 3 does not decrease significantly. After omitting one of polyethylene glycol, phosphatidylglycerol, and linolenic acid in Comparative Examples 1-3, the survival rate of frozen cells is not as good as that in Examples 1 to 3, indicating that the freezing solution of the present invention is compounded by adding polyethylene glycol, phosphatidylglycerol, and linolenic acid to change the permeability of the cell membrane while protecting the cell membrane, reducing the formation of ice crystals in the cell during the freezing process. Damage to the cells. In Comparative Examples 3 to 6, one of β-ecdysterone, punicalagin, and dihydromyricetin is omitted, and the cell survival rate after freezing is lower than that in Examples 1 to 3, indicating that the present invention uses three components of saponin polysaccharide, sodium carboxymethyl cellulose, and polyglycerol fatty acid esters to work synergistically, which helps to maintain the integrity and stability of the cell membrane, has a regulatory effect on the metabolic process of the cell, helps to maintain the osmotic pressure of the extracellular environment stable, avoids cell dehydration and local electrolyte concentration increases, and reduces the occurrence of cell death. The cryopreservation solution of Comparative Example 7 is added with DMSO and fetal bovine serum, while the cryopreservation solution of the present invention is not added with exogenous serum and DMSO, thereby reducing the possibility of animal pathogen contamination, avoiding the possible side effects of DMSO, and providing higher safety for immune cells and a higher survival rate of immune cell recovery.

Experimental Example 2: Post-resuscitation proliferation performance test of immune cells

CIK cells revived after being frozen for 24 months in Example 2 and freshly prepared CIK cells that were not frozen were taken as control groups and expanded under the same culture conditions. The process was as follows: CIK cells were added to CIK cell culture medium to prepare a cell suspension with a cell concentration of 1×10 ⁵ cells/mL. The above culture medium was transferred to a cell culture bottle and placed in an incubator at 37°C and 5% CO ₂ for culture. During the culture process, the CIK cells were counted every day, and the culture medium was supplemented in time to maintain the cell concentration at 1.0×10 ⁵ cells/mL. Trypan blue staining was used to count the number of cells and calculate the cell proliferation multiple. The results are shown in Table 2.

Table 2 Expansion times of immune cell cryopreservation cells after thawing

It can be seen from Table 2 that the expansion multiples of CIK cells after cryopreservation in the immune cell cryopreservation solution of Example 2 are similar to those of CIK cells that have not been cryopreserved and are directly cultured, indicating that the cryopreservation solution of the present invention can maintain the activity of CIK cells, and the proliferation ability of the cells after recovery is basically unaffected.

Experimental Example 3: Post-resuscitation expression performance test of immune cells

The CIK cells frozen and thawed in the immune cell cryopreservation solution of Example 1-3 were detected by flow cytometry for CIK surface markers CD3 and CD56; the expression rate of cell surface markers after thawing was compared with the value of cells before freezing. The results of cell surface marker expression rate detection are shown in Table 3 below.

Table 3 CIK cell surface marker expression rate detection results

From the results in Table 3, it can be seen that the expression rate of surface markers of the immune cells frozen in the immune cell freezing solution of Examples 1-3 of the present invention after thawing and recovery did not change significantly compared with before freezing, indicating that the cells were not seriously damaged during the freezing and recovery process and maintained their active state.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (8)

1. The immune cell cryopreservation liquid is characterized by comprising a cell basal medium and a cell cryopreservation protective agent, wherein the cell cryopreservation protective agent comprises the following raw materials: polyethylene glycol, phosphatidylglycerol, linolenic acid, beta-ecdysterone, punicalagin, and dihydromyricetin.

2. The immune cell cryopreservation solution according to claim 1, wherein the content of each component in the cell cryopreservation protective agent is as follows, based on the total volume of the cryopreservation solution: 4-6mg/mL of polyethylene glycol, 2.5-5mg/mL of phosphatidylglycerol, 1-1.5mg/mL of linolenic acid, 25-40 mug/mL of beta-ecdysterone, 16-25 mug/mL of punicalagin and 8-12 mug/mL of dihydromyricetin.

3. The immune cell cryopreservation solution according to claim 2, wherein the content of each component in the cell cryopreservation protective agent is as follows, based on the total volume of the cryopreservation solution: polyethylene glycol 5mg/mL, phosphatidylglycerol 4mg/mL, linolenic acid 1.2mg/mL, beta-ecdysterone 35 μg/mL, punicalagin 20 μg/mL, dihydromyricetin 10 μg/mL.

4. The frozen stock solution of claim 1, wherein the basal medium is a serum-free GT-T551 medium.

5. Use of an immune cell cryopreservation solution according to any one of claims 1 to 4 for cryopreserving immune cells.

6. A method for cryopreserving immune cells, comprising the steps of:

S1: extracting immune cells by using whole blood;

S2: preparing the immune cell cryopreservation solution according to any one of claims 1 to 4;

S3: the immune cells were frozen.

7. The method for cryopreserving immune cells according to claim 6, wherein the step of cryopreserving the S3 immune cells comprises the steps of: adding the extracted immune cells into the prepared immune cell cryopreservation solution, mixing to obtain cell suspension, transferring into a sterile cryopreservation tube, preserving for 2-4 hours at 0-5 ℃, preserving for 12-24 hours at-80 ℃, and transferring and preserving in liquid nitrogen for cryopreservation.

8. The method of claim 7, wherein the concentration of cells in the cell suspension is 1 x 10 ⁷-3×10⁷ cells/mL.