CN115053891A

CN115053891A - Cell freezing medium and implementation method

Info

Publication number: CN115053891A
Application number: CN202210810693.5A
Authority: CN
Inventors: 余鸿楷; 刘鸿基; 曾筱恬; 黄淑宇; 符维萂; 游慧; 何香荔
Original assignee: Guangzhou Xingwang Medical Technology Co ltd
Current assignee: Guangzhou Xingwang Medical Technology Co ltd
Priority date: 2022-07-11
Filing date: 2022-07-11
Publication date: 2022-09-16

Abstract

The invention discloses a cell cryopreservation solution and an implementation method thereof, wherein the formula comprises the following components: normal saline, glycerol, glucose and trehalose, and the implementation method comprises the following steps: step one, weighing raw materials; step two, preparing a frozen stock solution; step three, sterilizing; step four, freezing and storing the cells; in the third step, ultraviolet rays are adopted for sterilization treatment, and the irradiation time of an ultraviolet lamp is 10-20 min; in the fourth step, the cell pretreatment specifically comprises: firstly, removing old culture solution, and washing cells by using PBS (phosphate buffer solution); then removing PBS, adding a proper amount of trypsin to digest the cells growing in the monolayer, and removing the trypsin for later use after centrifugal treatment; the freezing point of the frozen stock solution prepared by the invention is reduced by adopting glycerol and trehalose to protect cells, the hypertonic action of the glycerol is reduced by physiological saline, and the cells are provided with nutrition by glucose.

Description

Cell freezing medium and implementation method

Technical Field

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

Background

The cell freezing storage means that cells are placed in a low-temperature environment to achieve the effect of reducing cell metabolism, so that the purpose of storing the cells for a long time is achieved, and the cells are used for subsequent experiments or clinics. The common cryoprotectant such as DMSO can rapidly penetrate into cells, reduce the freezing point of the cells, improve the permeability of cell membranes to water, promote the water in the cells to seep out of the cells, and reduce the formation of ice crystals in the cells, thereby reducing the damage of the ice crystals to the cells; when unfreezing, the extracellular water is promoted to enter the cells, the osmotic swelling is relieved, the electrolyte in the unfrozen solution inside and outside the cells is diluted, and the damage to the solute is reduced.

However, the existing cryoprotectants have the following defects: DMSO has serious toxic effect, reacts with a protein hydrophobic group to cause protein denaturation, has vascular toxicity and liver and kidney toxicity, and cannot be used for clinically freezing protective bacteria liquid to be used for patients; fetal calf serum is also one of the most common extracellular protective agents for cell cryopreservation at present, but animal serum has the problems of pathogen infection and immunological rejection reaction, difference, instability and the like.

Disclosure of Invention

The present invention aims to provide a cell cryopreservation solution and an implementation method thereof, so as to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme: a cell cryopreservation liquid comprises the following components: the saline solution, the glycerol, the glucose and the trehalose comprise the following components in percentage by mass: 85% of normal saline, 10% of glycerol, 3% of glucose and 2% of trehalose.

An implementation method of a cell cryopreservation solution comprises the following steps of weighing raw materials; step two, preparing a frozen stock solution; step three, sterilizing; step four, freezing and storing the cells;

in the first step, 85% of normal saline, 10% of glycerol, 3% of glucose and 2% of trehalose are weighed for later use, wherein the sum of the mass percentages of all the components is 1;

adding the physiological saline weighed in the step one into a beaker, sequentially adding glycerol, glucose and trehalose, and stirring and mixing to obtain a frozen stock solution;

in the third step, the frozen stock solution prepared in the second step is taken and sterilized for later use;

and in the fourth step, pretreating the cells to be cryopreserved, adding a proper amount of cryopreservation liquid into the cells, adjusting the cell density, subpackaging the cells into cryopreservation tubes, cooling the cells to the temperature of-70 ℃ in a gradient manner for 1-1.5ml per tube, preserving the temperature for 12-24 hours, and finally transferring the cells into a liquid nitrogen container for preservation.

Preferably, in the second step, a magnetic stirrer is used for stirring, and the stirring time is 30 min.

Preferably, in the third step, ultraviolet rays are adopted for sterilization treatment, and the irradiation time of an ultraviolet lamp is 10-20 min.

Preferably, in the fourth step, the cell pretreatment specifically comprises: firstly, removing old culture solution, and washing cells by using PBS (phosphate buffer solution); then removing PBS, adding a proper amount of trypsin to digest the cells growing in the monolayer, centrifuging, and removing the trypsin for later use.

Preferably, in the fourth step, the cell density is adjusted to 5 × 10 ⁶ -5×10 ⁷ One per ml.

Preferably, in the fourth step, the cooling rate of the gradient cooling is 5-10 ℃/min.

Compared with the prior art, the invention has the beneficial effects that: the freezing point of the frozen stock solution prepared by the invention is reduced by adopting glycerol and trehalose to protect cells, the hypertonic action of the glycerol is reduced by physiological saline, and the cells are provided with nutrition by glucose.

Drawings

FIG. 1 is a flow chart of the method of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1, a technical solution provided by the present invention:

example (b):

a cell cryopreservation liquid comprises the following components: the saline solution, the glycerol, the glucose and the trehalose comprise the following components in percentage by mass: 85% of normal saline, 10% of glycerol, 3% of glucose and 2% of trehalose.

adding the physiological saline weighed in the step one into a beaker, sequentially adding glycerol, glucose and trehalose, and stirring for 30min by using a magnetic stirrer to obtain a frozen stock solution;

in the third step, the frozen stock solution prepared in the second step is irradiated by an ultraviolet lamp for 20min for later use;

taking cells to be frozen, removing old culture solution, and washing the cells by using PBS (phosphate buffer solution); then go toRemoving PBS, adding appropriate amount of trypsin to digest monolayer-grown cells, centrifuging, removing trypsin, adding appropriate amount of frozen stock solution, and adjusting cell density to 5 × 10 ⁶ -5×10 ⁷ And (2) packaging each tube per ml, then subpackaging into freezing tubes, wherein each tube is 1-1.5ml, performing gradient cooling to-70 ℃ at a cooling rate of 5-10 ℃/min, preserving heat, standing for 12-24h, and finally transferring into a liquid nitrogen container for preservation. Comparative example 1:

a cell cryopreservation liquid comprises the following components: the saline solution, the glycerol and the glucose comprise the following components in percentage by mass: 87% of normal saline, 10% of glycerol and 3% of glucose; the method of implementation is the same as in the examples.

Comparative example 2:

a cell cryopreservation liquid comprises the following components: the physiological saline, the glycerol and the trehalose comprise the following components in percentage by mass: 88% of normal saline, 10% of glycerol and 2% of trehalose; the method of implementation is the same as in the examples.

Comparative example 3:

a cell cryopreservation liquid comprises the following components: the saline solution, the glucose and the trehalose comprise the following components in percentage by mass: 95% of normal saline, 3% of glucose and 2% of trehalose; the method of implementation is the same as in the examples.

The cryopreservation solution prepared in the above examples and comparative examples was used to cryopreserve oral cells, and the cells were taken out and revived after being cryopreserved for 14 days, and the cell viability rate was calculated, and the results are as follows:

	examples	Comparative example 1	Comparative example 2	Comparative example 3
					Normal saline/%)	85	87	88	95
Glycerol/%)	10	10	10	0
					Glucose/%)	3	3	0	3
Trehalose/%)	2	0	2	2
					Cell viability/%)	94.8	57.3	71.6	2.6

Based on the above, the trehalose adopted by the invention can interact with cell membranes, the membrane protein structure is stabilized in the processes of cryopreservation and recovery, a glassy matrix can be formed to reduce the damage of ice crystals formed outside cells to the cells, the physiological saline can properly reduce the hypertonicity of glycerol, the glucose can properly increase the nutrient content of the cryopreservation solution, the glycerol can well play a role in reducing the freezing point, DMSO and animal serum are not added into the cryopreservation solution, most risks are avoided, the advantages of the four components are integrated, the effects of protecting the activity and the number of the cells can be effectively achieved, and the survival rate of cell recovery can be improved.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Claims

1. A cell cryopreservation liquid comprises the following components: normal saline, glycerol, glucose and trehalose, its characterized in that: the weight percentage of each component is as follows: 85% of normal saline, 10% of glycerol, 3% of glucose and 2% of trehalose.

2. An implementation method of a cell cryopreservation solution comprises the following steps of weighing raw materials; step two, preparing a frozen stock solution; step three, sterilizing; step four, freezing and storing the cells; the method is characterized in that:

in the first step, 85% of normal saline, 10% of glycerol, 3% of glucose and 2% of trehalose are weighed for later use, wherein the sum of the mass percentages of the components is 1;

3. The method according to claim 2, wherein the cell cryopreservation solution comprises: and in the second step, stirring for 30min by adopting a magnetic stirrer.

4. The method according to claim 2, wherein the cell cryopreservation solution comprises: in the third step, ultraviolet rays are adopted for sterilization treatment, and the irradiation time of an ultraviolet lamp is 10-20 min.

5. The method according to claim 2, wherein the cell cryopreservation solution comprises: in the fourth step, the cell pretreatment specifically comprises: firstly, removing old culture solution, and washing cells by using PBS; then PBS is removed, a proper amount of trypsin is added to digest the cells growing in the monolayer, and after centrifugal treatment, the trypsin is removed for standby.

6. The method according to claim 2, wherein the cell cryopreservation solution comprises: in the fourth step, the cell density is adjusted to 5 × 10 ⁶ -5×10 ⁷ One per ml.

7. The method according to claim 2, wherein the cell cryopreservation solution comprises: in the fourth step, the cooling rate of gradient cooling is 5-10 ℃/min.