阅读说明：本技术 一种免疫细胞的冻存液及冻存方法 (Frozen stock solution and frozen stock method for immune cells ) 是由 赵辉 高萧芳 孙宪凯 段书娇 王艳琦 于 2021-08-27 设计创作，主要内容包括：本发明涉及免疫细胞的冻存液及冻存方法,尤其涉及本发明的一种免疫细胞的冻存液及冻存方法,包括FBS、DMSO、1640培养液、低分子肝素钙和抗氧化；一种免疫细胞的冻存液的冻存方法,包括以下步骤：步骤一、存放；把免疫细胞放入冷冻管中,然后加入冻存液,缓慢吹打混匀,把冷冻管放入降温室；步骤二、第一次降温；先降至3°且温度保持4min；然后以-2℃/min的速度降温至-3℃；最后以-11℃/min的速度降温至-40℃；步骤三、第一次升温先以-16℃/min的速度升温至-30℃；第四步、第二次降温；先以-30℃保持4min；然后以-2℃/min的速度降温至-50℃；最后先以-10℃/min的速度降温至-80℃；-80℃保持5d；第五步、存储；最后存入液氮罐中冻存。(The invention relates to a frozen stock solution and a frozen stock method of immune cells, in particular to a frozen stock solution and a frozen stock method of immune cells, which comprise FBS, DMSO, 1640 culture solution, low molecular heparin calcium and antioxidation; a cryopreservation method of immune cell cryopreservation liquid comprises the following steps: step one, storing; placing the immune cells into a freezing tube, then adding the freezing solution, slowly blowing and uniformly mixing, and placing the freezing tube into a cooling chamber; step two, cooling for the first time; firstly reducing the temperature to 3 ℃ and keeping the temperature for 4 min; then cooling to-3 ℃ at the speed of-2 ℃/min; finally, cooling to-40 ℃ at the speed of-11 ℃/min; step three, firstly heating up to-30 ℃ at the speed of-16 ℃/min for the first time; fourthly, cooling for the second time; keeping at-30 deg.C for 4 min; then cooling to-50 ℃ at the speed of-2 ℃/min; finally, the temperature is reduced to minus 80 ℃ at the speed of minus 10 ℃/min; -80 ℃ for 5 days; fifthly, storing; and finally storing the mixture in a liquid nitrogen tank for freezing.)

1. A frozen stock solution and a frozen stock method of immune cells are characterized in that: comprises FBS, DMSO, 1640 culture solution, low molecular heparin calcium and antioxidant.

2. The cryopreservation solution and method for immune cells according to claim 1, wherein: the volume parts of the FBS are 10-20.

3. The cryopreservation solution and method for immune cells according to claim 1, wherein: the volume part of the DMSO is 10-20.

4. The cryopreservation solution and method for immune cells according to claim 1, wherein: the 1640 culture solution is 40-60 parts by volume.

5. The cryopreservation solution and method for immune cells according to claim 1, wherein: the volume part of the low molecular heparin calcium is 8-5.

6. The cryopreservation solution and method for immune cells according to claim 1, wherein: the volume part of the antioxidant is 2-5.

7. The cryopreservation solution and method for immune cells according to claim 1, wherein: the antioxidant is vitamin C injection.

8. The cryopreservation solution for immune cells and the cryopreservation method thereof according to any one of claims 1 to 7, wherein: preparing the frozen stock solution according to the following proportion: according to the volume parts, 10-20 parts of FBS, 10-20 parts of DMSO, 40-60 parts of 1640 culture solution, 8-5 parts of low molecular heparin calcium with the concentration of 1500-.

9. A cryopreservation method of immune cell cryopreservation liquid is characterized by comprising the following steps:

step one, storage

Placing the immune cells into a freezing tube, then adding the freezing solution, slowly blowing and uniformly mixing, and placing the freezing tube into a cooling chamber;

step two, cooling for the first time

Firstly reducing the temperature to 3 ℃ and keeping the temperature for 4 min;

then cooling to-3 ℃ at the speed of-2 ℃/min;

finally, cooling to-40 ℃ at the speed of-11 ℃/min;

step three, first temperature rise

Firstly, heating to-30 ℃ at the speed of-16 ℃/min;

the fourth step, the second cooling

Keeping at-30 deg.C for 4 min;

then cooling to-50 ℃ at the speed of-2 ℃/min;

finally, the temperature is reduced to minus 80 ℃ at the speed of minus 10 ℃/min;

-80 ℃ for 5 days;

the fifth step, storage

And finally storing the mixture in a liquid nitrogen tank for freezing.

Technical Field

The invention relates to a preparation method of a Miao medicine, namely a Chinese mugwort moxa stick, and particularly relates to frozen stock solution and a frozen stock method of immune cells.

Background

Cell freezing is a technique of placing cells in a low-temperature environment to slow down cell metabolism so as to achieve long-term storage. The principle is that the cells are placed in liquid nitrogen at the temperature of 196 ℃ below zero for ultralow temperature preservation, so that the cells can be temporarily separated from the growth state to preserve the characteristics of the cells, and the cells can be used by reviving the cells when needed. When the cells are frozen, the protective agent is added into the cell suspension, so that the freezing point of the solution can be lowered, water in the cells permeates out of the cells under the condition of slow freezing, and the formation of ice crystals in the cells is reduced, thereby avoiding the damage of the cells in the freezing process. At present, the cryopreservation of cells is mainly performed under the ultralow temperature condition of liquid nitrogen at the temperature of-196 ℃, the metabolic function of the cells is arrested under the ultralow temperature condition, the cells can be preserved for a long time, and when the cells are needed, the cryopreserved cells are taken out and quickly thawed in water bath at the temperature of 37 ℃, so that the activity and the function of the cells can be recovered. However, the existing immune cell cryopreservation method only solves the cryopreservation of stem cells and tumor cells, but the immune cell cryopreservation method cannot be well solved. Due to the cell structure and the cell characteristics of immune cells, when the conventional cell cryopreservation solution and the conventional cell cryopreservation method are used for cryopreserving the immune cells, the immune cells are easily damaged by ice crystals, the survival rate of the recovered cells is low, the cell proliferation quantity is insufficient, the cells are easy to age, the efficiency is low, the cryopreservation quality is low, the activity and the survival rate of the recovered cells are poor, and the like.

Disclosure of Invention

The invention provides a frozen stock solution and a frozen stock method for immune cells.

In order to solve the problems, the invention provides a frozen stock solution of immune cells and a freezing method thereof, wherein the frozen stock solution comprises FBS, DMSO, 1640 culture solution, low-molecular heparin calcium and antioxidation.

The cryopreservation solution and the cryopreservation method for the immune cells, provided by the invention, further have the following technical characteristics:

further, the volume part of the FBS is 10-20. More preferably the parts by volume of FBS are 11, 12, 13, 14, 15, 16, 17, 18, 19.

Further, the volume fraction of the DMSO is 10-20. More preferably the volume fraction of DMSO is 11, 12, 13, 14, 15, 16, 17, 18, 19.

Furthermore, the 1640 culture solution is 40-60 parts by volume. More preferably 1640 parts of culture medium are 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59.

Further, the volume fraction of the low molecular heparin calcium is 8-5. More preferably 1640 culture medium parts by volume 7, 6.

Further, the volume part of the antioxidant is 2-5. More preferably the antioxidant is present in 3, 4 parts by volume.

Further, the antioxidant is vitamin C injection.

Further, the frozen stock solution is prepared according to the following proportion: according to the volume parts, 10-20 parts of FBS, 10-20 parts of DMSO, 40-60 parts of 1640 culture solution, 8-5 parts of low molecular heparin calcium with the concentration of 1500-.

Further, the cryopreservation method of the immune cell cryopreservation solution comprises the following steps:

step one, storage

Placing the immune cells into a freezing tube, then adding the freezing solution, slowly blowing and uniformly mixing, and placing the freezing tube into a cooling chamber;

step two, cooling for the first time

Firstly reducing the temperature to 3 ℃ and keeping the temperature for 4 min;

then cooling to-3 ℃ at the speed of-2 ℃/min;

finally, cooling to-40 ℃ at the speed of-11 ℃/min;

step three, first temperature rise

Firstly, heating to-30 ℃ at the speed of-16 ℃/min;

the fourth step, the second cooling

Keeping at-30 deg.C for 4 min;

then cooling to-50 ℃ at the speed of-2 ℃/min;

finally, the temperature is reduced to minus 80 ℃ at the speed of minus 10 ℃/min;

-80 ℃ for 5 days;

the fifth step, storage

And finally storing the mixture in a liquid nitrogen tank for freezing.

The invention has the beneficial effects that: the cryopreservation solution and the cryopreservation method for the immune cells are simple in formula, each component has respective functions, and the components are mutually matched and cooperated, so that the cryopreservation effect of the immune cells can be obviously improved, the intracellular water can not be crystallized when the cells are close to a freezing point, and the activity and the cell proliferation capacity of the recovered cells are not influenced.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a graph showing the ratio of components of a frozen stock solution of an immune cell according to the present invention;

FIG. 2 is a flow chart of a method for freezing immune cell freezing medium according to 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.

The invention relates to a frozen stock solution and a frozen stock method of immune cells, in particular to a frozen stock solution and a frozen stock method of immune cells, which comprise FBS, DMSO, 1640 culture solution, low molecular heparin calcium and antioxidant.

The volume parts of the FBS are 10-20.

The volume part of the DMSO is 10-20.

The 1640 culture solution is 40-60 parts by volume.

The volume part of the low molecular heparin calcium is 8-5.

The volume part of the antioxidant is 2-5.

The antioxidant is vitamin C injection.

Preparing the frozen stock solution according to the following proportion: according to the volume parts, 10-20 parts of FBS, 10-20 parts of DMSO, 40-60 parts of 1640 culture solution, 8-5 parts of low molecular heparin calcium with the concentration of 1500-.

A cryopreservation method of immune cell cryopreservation liquid comprises the following steps:

step one, storage

Placing the immune cells into a freezing tube, then adding the freezing solution, slowly blowing and uniformly mixing, and placing the freezing tube into a cooling chamber;

step two, cooling for the first time

Firstly reducing the temperature to 3 ℃ and keeping the temperature for 4 min;

then cooling to-3 ℃ at the speed of-2 ℃/min;

finally, cooling to-40 ℃ at the speed of-11 ℃/min;

step three, first temperature rise

Firstly, heating to-30 ℃ at the speed of-16 ℃/min;

the fourth step, the second cooling

Keeping at-30 deg.C for 4 min;

then cooling to-50 ℃ at the speed of-2 ℃/min;

finally, the temperature is reduced to minus 80 ℃ at the speed of minus 10 ℃/min;

-80 ℃ for 5 days;

the fifth step, storage

And finally storing the mixture in a liquid nitrogen tank for freezing.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.