CN108990964B

CN108990964B - Cell cryopreservation liquid

Info

Publication number: CN108990964B
Application number: CN201810902354.3A
Authority: CN
Inventors: 蔡海波; 陈焕芸; 周乐豪; 谭文松
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2018-08-09
Filing date: 2018-08-09
Publication date: 2022-01-28
Anticipated expiration: 2038-08-09
Also published as: CN108990964A

Abstract

The cell cryopreservation liquid contains a basic culture medium, glycerol, fetal calf serum and an impermeable protective agent, and the contents of the components are as follows: 80-94 v/v% of a basic culture medium, 5-10 v/v% of glycerol, 1-10 v/v% of fetal bovine serum and 0.01-0.10 w/v% of an impermeable protective agent; the basal medium is RPMI-1640, MEM, high-sugar DMEM, low-sugar DMEM or DMEM-F12; the non-permeable protective agent is sodium alginate, sodium hyaluronate or gamma-polyglutamic acid; the invention does not contain dimethyl sulfoxide, obtains good freezing effect by different collocation of the non-permeable protective agent, glycerol and fetal calf serum, has strong water molecule complexing ability, is similar to the dimethyl sulfoxide in the freezing function, but can reduce the formation of ice crystals in cells in the freezing process; as a cell cryopreservation solution, the cell cryopreservation solution has the advantages of definite added components, high safety, small damage to cells, consistent shape before and after cell cryopreservation, high cell survival rate after cell recovery and good proliferation capacity.

Description

Cell cryopreservation liquid

Technical Field

The invention relates to the technical field of cell and bioengineering, in particular to a cell cryopreservation solution.

Background

Cell cryopreservation is one of important links in biological research, and plays an important role in cryopreservation of artificially cultured cells, prevention of cell degeneration caused by cell subculture, prevention of bacterial pollution in subculture, and solution of a series of problems in the process of maintaining subculture. However, freezing of cells can also damage cells to some extent.

In the field of cell cryopreservation, a refrigerant (namely Cryo-protective Agent, CPA for short) is widely adopted at present. The refrigerant comprises an osmotic refrigerant and a non-osmotic refrigerant, wherein the osmotic refrigerant mainly comprises dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, acetamide and methanol; the protection mechanism is as follows: before the cell freezing suspension is completely solidified, the refrigerant can enter the cells to form a certain molar concentration inside and outside the cells, so that the electrolyte concentration in the unfrozen solution inside and outside the cells is reduced, the cells are protected from being damaged by high-concentration electrolyte, meanwhile, the water in the cells cannot be excessively infiltrated, and the cells can be prevented from being excessively dehydrated and shrunk. The refrigerant can effectively reduce the damage of the solute and the damage of the ice crystal of the cells in the process of freezing and storing the cells. Among the currently used refrigerants, dimethyl sulfoxide (DMSO) is the most commonly used refrigerant in cell preservation, but it also has a certain toxicity to cells and can denature intracellular proteins at normal temperature, and therefore, these disadvantages limit its further application.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a cell cryopreservation solution on the basis of the existing research results, which does not contain dimethyl sulfoxide, obtains a better freezing effect by different combinations of non-permeable protective agents such as sodium alginate, hyaluronic acid and gamma-polyglutamic acid, glycerol and fetal calf serum, can be used for low-temperature cryopreservation of cells, and has the positive effects of consistent shape before and after cell cryopreservation, high survival rate after cell recovery and good proliferation capacity.

In order to achieve the purpose, the invention adopts the following technical scheme.

The cell cryopreservation liquid is characterized by comprising a basic culture medium, glycerol, fetal calf serum and an impermeable protective agent, wherein the contents of the components are as follows:

further, the basic culture medium is RPMI-1640, MEM, high-sugar DMEM, low-sugar DMEM or DMEM-F12.

Further, the non-permeable protective agent is Sodium alginate (Sodium alginate), Sodium hyaluronate (Hyaluronic acid Sodium salt) or gamma-polyglutamic acid (gamma-polyglutamic acid).

The cell freezing solution has the positive effects that:

(1) does not contain dimethyl sulfoxide, obtains better freezing effect by different collocation of non-permeable protective agents, namely sodium alginate, sodium hyaluronate, gamma-polyglutamic acid, glycerol and fetal calf serum, has strong water molecule complexing ability, is similar to the dimethyl sulfoxide in the freezing function, can reduce the formation of ice crystals in cells in the freezing process, and can be used for low-temperature freezing storage of the cells.

(2) As a cell cryopreservation solution, the cell cryopreservation solution has the positive effects of consistent morphology before and after cell cryopreservation, high survival rate after cell recovery and good proliferation capacity of cells after cell cryopreservation recovery.

(3) The additive components are definite, the safety is high, and the damage to cells is small; can be used for replacing the most commonly used dimethyl sulfoxide refrigerating fluid at present.

Drawings

FIG. 1 is a photograph under a microscope of HeLa cells before cryopreservation.

FIG. 2 is a photograph under a microscope of the first day of the cryopreservation of HeLa cells recovered by the cell cryopreservation solution of the present invention.

FIG. 3 is a photograph under a microscope of HeLa cells on the third day after cryopreservation and recovery using the cell cryopreservation solution of the present invention.

Detailed Description

The following describes embodiments of the cell culture medium of the present invention with reference to the drawings, and provides 7 examples and 2 comparative examples. However, it should be noted that the present invention is not limited to the following embodiments.

Example 1

A cell cryopreservation liquid comprises RPMI-1640 culture medium as basic component, and the following additive components:

glycerol (glycerinum) 10 v/v%;

fetal bovine serum (Fetal calf serum) 10 v/v%;

sodium alginate (Sodium alginate) 0.10 w/v%.

Example 2

A cell freezing medium comprises DMEM-F12 as basic component, and comprises the following components:

10 v/v% of glycerol;

10 v/v% of fetal bovine serum;

sodium alginate 0.01 w/v%.

Example 3

10 v/v% of glycerol;

10 v/v% of fetal bovine serum;

sodium hyaluronate (Hyaluronic acid sodium salt) 0.10 w/v%.

Example 4

A cell cryopreservation solution comprises MEM as basic component, and the following components:

10 v/v% of glycerol;

10 v/v% of fetal bovine serum;

0.01 w/v% of sodium hyaluronate.

Example 5

A cell freezing medium comprises low-sugar DMEM medium as basic components and is added with the following components:

10 v/v% of glycerol;

10 v/v% of fetal bovine serum;

0.10 w/v% of gamma-Polyglutamic acid (gamma-Polyglutamic acid).

Example 6

A cell freezing medium comprises high-sugar DMEM medium as basic component, and comprises the following components:

10 v/v% of glycerol;

10 v/v% of fetal bovine serum;

0.01 w/v% of gamma-polyglutamic acid.

Example 7

5 v/v% of glycerol;

1 v/v% of fetal bovine serum;

0.01 w/v% of gamma-polyglutamic acid.

Comparative example 1

A cell cryopreservation solution comprises the following components: RPMI-1640 medium 80%; 10% of fetal bovine serum; 10% of dimethyl sulfoxide; as a control frozen stock solution 1 to the cell frozen stock solution of the present invention.

Comparative example 2

A cell cryopreservation solution comprises the following components: RPMI-1640 medium 90%; 10% of glycerol; as a control frozen stock solution 2 to the cell frozen stock solution of the present invention.

Effect verification of cell cryopreservation solution

Cell freezing and recovery experiments were performed using the cell freezing solutions prepared in examples 1 to 7 of the present invention and comparative freezing solutions 1 and 2 prepared in comparative examples 1 to 2, respectively, in the following manner.

Freezing and storing HeLa cells: HeLa cells grown as a monolayer were cultured, and in the logarithmic growth phase, the cells were digested with 0.25% trypsin solution for 4 minutes, 15ml of a culture medium (RPMI-1640 medium 90% and FBS 10%) was added, the cells were homogenized by gently pipetting, centrifuged at 1000rpm/min for 5 minutes, the supernatant was discarded, and the cell lysates prepared in examples 1 to 7 and comparative example were added and mixed. At 1 × 10⁶The concentration of cells/ml, 1ml system is put into a 1.5ml freezing tube; storing at 4 deg.C for 30min, storing at-20 deg.C for 2 hr, transferring to refrigerator at-80 deg.C for long-term storage: respectively freezing and storing for 1 month; and 3, three months.

Cell recovery: the frozen cells are taken out from a refrigerator at the temperature of minus 80 ℃, quickly transferred to a water bath kettle at the temperature of 37 ℃ for unfreezing, then transferred to a 1.5ml EP tube, centrifuged for 5min at 1000r/min, resuspended by a culture medium and inoculated to a T75 culture flask for culture. Cell viability after 1 and 3 months of cryopreservation is shown in table 1.

TABLE 1 survival of cells after 1 and 3 months of cryopreservation

。

The anchorage rate of the cells after 1 month and 3 months of cryopreservation on the first day after cell recovery is shown in table 2.

TABLE 2 anchorage rate of the first day after cell recovery after 1 and 3 months of cryopreservation

。

The survival rate of the cells after 3 months of cryopreservation and the anchorage rate of the cells on the first day after recovery are shown in table 3.

TABLE 3 cell survival rate by cryopreservation for 3 months using the cell cryopreservation solution of example 7 and cell adherence rate on the first day after recovery

。

From the above results of cell survival and anchorage rate at the first day after cell recovery, it can be seen that:

(1) the cell cryopreservation solution disclosed by the invention can achieve the effect of freezing and preserving cells by DMSO (dimethyl sulfoxide), can reduce the toxic effect on the cells, and has the advantages of unchanged cell form and high survival rate after the freezing and restoring.

(2) The cell freezing solution does not contain DMSO, is safe and non-toxic, does not bring harm to the health and the environment of experimenters, adopts three non-permeable protective agents which are widely used cosmetic raw materials, and has convenient and stable sources.

(3) The cell cryopreservation solution disclosed by the invention is simple and clear in components, convenient to configure and use and capable of being widely applied to the field of cell cryopreservation.

The foregoing is only a preferred embodiment of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the scope of the invention as defined by the appended claims.

Claims

1. The application of a cell cryopreservation liquid in the cryopreservation and resuscitation of Hela cells, wherein the cell cryopreservation liquid contains a basal culture medium, glycerol, fetal calf serum and an impermeable protective agent, and the contents of the components are as follows:

wherein the basic culture medium is RPMI-1640, MEM, high-sugar DMEM, low-sugar DMEM or DMEM-F12;

the non-permeable protective agent is sodium alginate, sodium hyaluronate or gamma-polyglutamic acid;

the cell freezing medium does not contain dimethyl sulfoxide;

the cryopreservation conditions of the HeLa cells comprise: storing at 4 deg.C for 30min, storing at-20 deg.C for 2 hr, and transferring to refrigerator at-80 deg.C for long-term storage;

the recovery conditions of the HeLa cells comprise: taking out the frozen HeLa cells from a refrigerator at the temperature of-80 ℃, and quickly transferring the HeLa cells into a water bath kettle at the temperature of 37 ℃ for unfreezing.