CN114521549A - Immune cell cryopreservation liquid and application thereof, cell cryopreservation method and cell recovery method - Google Patents
Immune cell cryopreservation liquid and application thereof, cell cryopreservation method and cell recovery method Download PDFInfo
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Abstract
The invention provides an immune cell cryopreservation solution and application thereof, a cell cryopreservation method and a recovery method based on the problem of low survival rate of immune cells during cryopreservation, and relates to the technical field of biology. An immune cell cryopreservation liquid comprises the following components in percentage by volume: 10-15% of glycerol, 20-25% of dextran, 5-10% of cell culture medium, 5-10% of radix scutellariae, 3-6% of ginseng and wheat, 23-6% of IL-23, 2-8% of gamma-interferon, 5-10% of human albumin and 10-20% of human autologous plasma. The invention also provides application of the immune cell cryopreservation liquid, a cryopreservation method and a recovery method of immune cells. The invention can effectively protect immune cells and effectively improve the survival rate of the immune cells when the immune cells are frozen.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an immune cell cryopreservation solution and application thereof, a cell cryopreservation method and a cell recovery method.
Background
Cellular immunotherapy refers to a method of effectively treating a patient with a tumor by directly or indirectly using immune cells; cellular immunotherapy of great interest has progressed from the early days of traditional or non-specific cell therapies such as lymphokine-activated killer cells, multiple cytokine-induced killer cells, cytotoxic t lymphocytes, natural killer cells, etc., to specific cell therapies such as car-nk (chimeric antigen receptor-modified nk cells) and tcr-t (t-cell receptor-chimeric t cells); with the widespread clinical application of cell therapy, cell preparation preservation and cryo-transport present significant challenges.
In a room temperature environment, cell metabolism is vigorous, and the physiological activities generate free radicals and lipid peroxides to cause the change of the intracellular environment, so that cells swell and die; during cell transportation, the problem is more prominent; in the liquid nitrogen freezing state, the cells almost stop life activities and metabolism, so the cells are usually transported by liquid nitrogen or dry ice after being frozen; however, when the cells are frozen and thawed, the cells are easily damaged by solution and ice crystals, and cell death is caused. Therefore, there is a need to protect cells with cell freezing solutions when freezing cells to reduce solution damage and ice crystal damage, thereby reducing cell death.
At present, most of immune cell frozen stock solution on the market is prepared from fetal calf serum, high-proportion DMSO, patient autologous plasma and non-clinical application-level reagents, animal serum has potential risk of introducing animal pathogenic bacteria pollution, DMSO has certain toxicity, and the survival rate of immune cells is low when the immune cells are frozen.
Disclosure of Invention
The first object of the present invention is to provide an immune cell cryopreservation solution which can effectively protect immune cells and effectively increase the survival rate of immune cells when freezing the immune cells.
The second purpose of the invention is to provide an application of immune cell frozen stock solution in freezing immune cells.
The third object of the present invention is to provide a method for cryopreserving immune cells, which comprises cryopreserving immune cells using an immune cell cryopreserving solution.
The fourth object of the present invention is to provide a method for recovering immune cells, which recovers cryopreserved immune cells.
The embodiment of the invention is realized by the following technical scheme:
an immune cell cryopreservation liquid comprises the following components in percentage by volume: 10-15% of glycerol, 20-25% of dextran, 5-10% of cell culture medium, 5-10% of radix scutellariae, 3-6% of ginseng and wheat, 23-6% of IL-23, 2-8% of gamma-interferon, 5-10% of human albumin and 10-20% of human autologous plasma.
The invention combines the radix scutellariae, the ginseng, the IL-2 and the gamma-interferon together, can promote the proliferation and the differentiation of lymphocytes, and enhance the activity of immunocytes and the resistance to viruses and germs. And effectively ensures the stability of the immune cell frozen stock solution.
Furthermore, the addition amount of the IL-2 is 2-5 ten thousand IU, and the addition amount of the gamma-interferon is 2-5 ten thousand IU.
Further, the cell culture medium is an IMDM medium, an RPMI-1640 medium or an McCoy5A medium.
An application of frozen immune cell liquid in freezing immune cells.
Further, the immune cell is a T lymphocyte, a B lymphocyte, a K lymphocyte, or an NK cell.
An immune cell cryopreservation method comprises the following steps:
s1: extracting immune cells by using whole blood;
s2: preparing immune cell cryopreservation liquid;
s3: adding the extracted immune cells into the prepared immune cell freezing solution in proportion, preserving at 4-8 ℃ for 2-4 h, preserving at-80 ℃ for 10-12 h, and transferring and preserving in liquid nitrogen, namely freezing the immune cells.
Further, the specific method for extracting immune cells from whole blood in step S1 is as follows: (1) taking 0.5ml of whole blood for inspection and counting, taking 50-100 ml of whole blood to centrifuge for 10-15 min at 900-1200 g, and separating out upper plasma; (2) taking the concentrated blood in the lower layer, subpackaging with 20 ml/tube, adding normal saline according to the volume ratio of 1:1, and mixing uniformly; (3) centrifuging at 400-600 g for 20-25 min, and sucking leucocyte layer mononuclear cells; (4) washing the leucocyte with 1 time of physiological saline, centrifuging for 5-10 min at 900-1200 g, and removing supernatant; (5) washing the cell precipitate with 40ml of normal saline, centrifuging for 5-10 min at 900-1200 g, and removing the supernatant; (6) collecting the cell precipitate to obtain the immune cell.
Further, in the step S3, 1.5-2 ml of immune cell freezing medium is added to every 1000-1500 ten thousand IU of immune cells.
An immunocyte recovery method comprises thawing frozen immunocyte in 38-40 deg.C water bath for 30-50 s, mixing with culture medium, and recovering 5% CO at 37 deg.C2And culturing for 20-24 h in a constant-temperature incubator to obtain the revived immune cells.
Further, the culture medium is T551 culture medium supplemented with 15% volume ratio human autologous plasma.
The technical scheme of the embodiment of the invention at least has the following advantages and beneficial effects:
1. the main component of the immune cell frozen stock solution adopts glycerol instead of dimethyl sulfoxide, the glycerol is colorless, odorless, sweet and clear viscous liquid, the glycerol is a skeleton component of triglyceride molecules, and the final products of triglyceride metabolism are glycerol and fatty acid, so that the immune cell frozen stock solution is non-toxic and harmless to cells.
2. The immune cell freezing solution disclosed by the invention adopts safer components, so that the potential risk of destroying the cell activity is reduced; in addition, the immune cell freezing medium does not contain components such as antibodies, complements and the like, has less foreign proteins and reduces the influence on the activity of immune cells.
3. The immune cell frozen stock solution disclosed by the invention is simple in adopted components, simple in preparation method and low in cost, and is only 1/5-1/3 of the cost of the cell frozen stock solution on the market.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The anti-tumor combination composition provided by the embodiment of the invention, and the preparation method and application thereof are specifically described below.
Example 1
The embodiment provides an immune cell cryopreservation solution, which comprises the following components in percentage by volume: 10% of glycerol, 25% of dextran, 10% of IMDM culture medium, 5% of radix scutellariae, 6% of ginseng and wheat, 26% of IL-26%, 8% of gamma-interferon, 10% of human serum albumin and 20% of human autologous plasma. Wherein the addition amount of the IL-2 is 2 ten thousand IU, and the addition amount of the gamma-interferon is 2 ten thousand IU.
An immune cell cryopreservation method comprises the following steps:
s1: the method for extracting the immune cells by using the whole blood comprises the following steps: (1) collecting 0.5ml whole blood, inspecting, counting, collecting 50ml whole blood, centrifuging at 900g for 10min, and separating upper layer plasma; (2) taking the concentrated blood in the lower layer, subpackaging with 20 ml/tube, adding normal saline according to the volume ratio of 1:1, and mixing uniformly; (3) centrifuging at 400g for 20min, and sucking the leucocyte layer mononuclear cells; (4) washing leucocyte with 1 time physiological saline, centrifuging at 900g for 5min, and removing supernatant; (5) washing the cell precipitate with 40ml of normal saline, centrifuging at 900g for 5min, and removing the supernatant; (6) collecting cell sediment to obtain immune cells;
s2: mixing the components in an aseptic environment to prepare immune cell frozen stock solution;
s3: adding 1.5ml immune cell freezing medium into every 1000 ten thousand IU immune cells, preserving at 4 ℃ for 2h, preserving at-80 ℃ for 10h, and transferring and preserving in liquid nitrogen, namely freezing and preserving the immune cells.
A method for recovering immunocytes comprises thawing frozen immunocytes in water bath at 38 deg.C for 50s, mixing with T551 culture medium containing 15 vol% human autoplasma, and recovering immunocytes at 37 deg.C under 5% CO2Culturing in a constant temperature incubator for 20h to obtain the revived immune cells.
Example 2
The embodiment provides an immune cell cryopreservation solution, which comprises the following components in percentage by volume: 15% of glycerol, 20% of dextran, 10% of RPMI-1640 culture medium, 10% of radix scutellariae, 6% of ginseng and wheat, 26% of IL-26%, 8% of gamma-interferon, 10% of human albumin and 15% of human autologous plasma. Wherein the addition amount of the IL-2 is 5 ten thousand IU, and the addition amount of the gamma-interferon is 3 ten thousand IU.
An immune cell cryopreservation method comprises the following steps:
s1: the method for extracting the immune cells by using the whole blood comprises the following steps: (1) collecting 0.5ml whole blood, inspecting, counting, centrifuging 100ml whole blood at 1200g for 15min, and separating upper layer plasma; (2) taking the concentrated blood in the lower layer, subpackaging with 20 ml/tube, adding normal saline according to the volume ratio of 1:1, and mixing uniformly; (3) centrifuging at 600g for 25min, and sucking the leucocyte layer mononuclear cells; (4) washing leucocyte with 1 time of physiological saline, centrifuging at 1200g for 10min, and removing supernatant; (5) washing the cell precipitate with 40ml of normal saline, centrifuging at 1200g for 10min, and removing the supernatant; (6) collecting cell sediment to obtain immune cells;
s2: mixing the components in an aseptic environment to prepare immune cell frozen stock solution;
s3: adding 2ml of immune cell freezing medium into every 1500 ten thousand IU of immune cells, preserving at 8 ℃ for 4h, preserving at-80 ℃ for 12h, and finally transferring and preserving in liquid nitrogen, namely freezing and preserving the immune cells.
A method for recovering immunocytes comprises thawing frozen immunocytes in 40 deg.C water bath for 30s, mixing with 15 vol% of T551 culture medium containing human autologous plasma, and recovering 5% CO at 37 deg.C2Culturing in a constant temperature incubator for 20h to obtain the revived immune cells.
Example 3
The embodiment provides an immune cell cryopreservation solution, which comprises the following components in percentage by volume: 12% of glycerol, 24% of dextran, 8% of McCoy5A culture medium, 8% of radix scutellariae, 5% of ginseng and wheat, 25% of IL-25%, 8% of gamma-interferon, 10% of human serum albumin and 20% of human autologous plasma. Wherein the addition amount of the IL-2 is 4 ten thousand IU, and the addition amount of the gamma-interferon is 3 ten thousand IU.
An immune cell cryopreservation method comprises the following steps:
s1: the method for extracting the immune cells by using the whole blood comprises the following steps: (1) collecting 0.5ml whole blood, inspecting, counting, collecting 80ml whole blood, centrifuging at 1000g for 12min, and separating upper layer plasma; (2) taking the concentrated blood in the lower layer, subpackaging with 20 ml/tube, adding normal saline according to the volume ratio of 1:1, and mixing uniformly; (3) centrifuging at 500g for 22min, and sucking tunica albuginea mononuclear cells; (4) washing leucocyte with 1 time of physiological saline, centrifuging at 1000g for 8min, and removing supernatant; (5) washing the cell precipitate with 40ml of normal saline, centrifuging at 1000g for 8min, and removing the supernatant; (6) collecting cell precipitate to obtain immune cell;
s2: mixing the components in an aseptic environment to prepare immune cell frozen stock solution;
s3: adding 2ml of immune cell freezing medium into each 1000 ten thousand IU of immune cells, preserving for 3h at 5 ℃, preserving for 12h at-80 ℃, and finally transferring and preserving in liquid nitrogen, namely freezing and preserving the immune cells.
A method for recovering immunocytes comprises thawing frozen immunocytes in 40 deg.C water bath for 40s, mixing with 15 vol% of T551 culture medium containing human autologous plasma, and recovering 5% CO at 37 deg.C2Culturing in a constant temperature incubator for 22h to obtain the recovered immune cells.
Example 4
The embodiment provides an immune cell cryopreservation solution, which comprises the following components in percentage by volume: 12% of glycerol, 23% of dextran, 10% of RPMI-1640 culture medium, 7% of radix scutellariae, 6% of ginseng and wheat, 26% of IL-gamma-interferon, 10% of human albumin and 20% of human autologous plasma. Wherein the addition amount of the IL-2 is 4 ten thousand IU, and the addition amount of the gamma-interferon is 4 ten thousand IU.
An immune cell cryopreservation method comprises the following steps:
s1: the method for extracting the immune cells by using the whole blood comprises the following steps: (1) collecting 0.5ml whole blood, inspecting, counting, collecting 75ml whole blood, centrifuging at 1100g for 10min, and separating upper layer plasma; (2) taking the concentrated blood in the lower layer, subpackaging with 20 ml/tube, adding normal saline according to the volume ratio of 1:1, and mixing uniformly; (3) centrifuging at 400g for 20min, and sucking the leucocyte layer mononuclear cells; (4) washing leucocyte with 1 time of physiological saline, centrifuging at 1100g for 10min, and removing supernatant; (5) washing the cell precipitate with 40ml of normal saline, centrifuging at 1100g for 10min, and removing the supernatant; (6) collecting cell sediment to obtain immune cells;
s2: mixing the components in an aseptic environment to prepare immune cell frozen stock solution;
s3: adding 1.5ml immune cell freezing medium into every 1500 ten thousand IU immune cells, preserving at 6 ℃ for 4h, preserving at-80 ℃ for 12h, and transferring and preserving in liquid nitrogen, namely freezing and preserving the immune cells.
A method for recovering immunocytes comprises thawing frozen immunocytes in water bath at 38 deg.C for 45s, mixing with T551 culture medium containing 15% human autoblood plasma, and recovering immunocytes at 37 deg.C and 5% CO2Culturing in a constant temperature incubator for 24h to obtain the resuscitated immune cells.
Comparative example 1
The comparative example provides an immune cell cryopreservation solution, which comprises the following components in percentage by volume: 15% of dimethyl sulfoxide, 25% of dextran, 24% of RPMI-1640 culture medium, 26% of IL-albumin, 10% of human serum albumin and 20% of human autologous plasma.
The freezing and thawing method of the immune cells of this comparative example was the same as that of example 1.
Comparative example 2
The embodiment provides an immune cell cryopreservation solution, which comprises the following components in percentage by volume: 15% of glycerol, 25% of dextran and 60% of RPMI-1640 culture medium.
The freezing and thawing method of the immune cells of this comparative example was the same as that of example 1.
Comparative example 3
The embodiment provides an immune cell cryopreservation solution, which comprises the following components in percentage by volume: 45% of dimethyl sulfoxide, 35% of fetal bovine serum culture medium and 20% of human autologous plasma.
The freezing and thawing method of the immune cells of this comparative example was the same as that of example 1.
Examples of the experiments
The cell concentrations and cell numbers of the cryopreserved immune cells and the resuscitated immune cells obtained in examples 1 to 4 and comparative examples 1 to 3 were measured by the MTT method. The results are shown in table 1:
TABLE 1 cell concentration and cell number Table
As can be seen from Table 1, the cell survival rates of the cryopreserved immune cells and the resuscitated immune cells in the embodiments 1 to 4 of the invention are higher than the cell survival rates of the comparative examples 1 to 3; the immune cell frozen stock solution of the comparative example 1 only adopts dimethyl sulfoxide, dextran, RPMI-1640 culture medium, IL-2, human serum albumin and human autologous plasma; the immune cell frozen stock solution of the comparative example 2 only adopts glycerol, dextran and RPMI-1640 culture medium; the immune cell freezing medium of comparative example 3 only used dimethyl sulfoxide, fetal bovine serum and human autologous plasma; proved by adopting the immune cell cryopreservation liquid provided by the invention, the immune cells can be effectively protected when being frozen and preserved, and the survival rate of the immune cells is effectively improved.
In conclusion, the immune cell cryopreservation solution provided by the invention can effectively protect immune cells and effectively improve the survival rate of the immune cells when the immune cells are frozen by combining glycerol, dextran, a cell culture medium, radix scutellariae, ginseng, IL-2, gamma-interferon, human serum albumin and human autologous plasma in a certain proportion.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. The immune cell cryopreservation liquid is characterized by comprising the following components in percentage by volume: 10-15% of glycerol, 20-25% of dextran, 5-10% of cell culture medium, 5-10% of radix scutellariae, 3-6% of ginseng and wheat, 23-6% of IL-23, 2-8% of gamma-interferon, 5-10% of human albumin and 10-20% of human autologous plasma.
2. The immune cell cryopreservation solution of claim 1, wherein the IL-2 is added in an amount of 2 to 5 ten thousand IU, and the interferon-gamma is added in an amount of 2 to 5 ten thousand IU.
3. The immune cell cryopreservation liquid of claim 1, wherein the cell culture medium is an IMDM medium, an RPMI-1640 medium or an McCoy5A medium.
4. Use of the immune cell cryopreservation solution of any one of claims 1 to 3 in cryopreservation of immune cells.
5. The use of the immune cell cryopreservation solution of claim 4, wherein the immune cells are T lymphocytes, B lymphocytes, K lymphocytes or NK cells.
6. An immune cell cryopreservation method is characterized by comprising the following steps:
s1: extracting immune cells by using whole blood;
s2: preparing an immune cell cryopreservation solution according to any one of claims 1 to 3;
s3: adding the extracted immune cells into the prepared immune cell freezing solution in proportion, preserving at 4-8 ℃ for 2-4 h, preserving at-80 ℃ for 10-12 h, and transferring and preserving in liquid nitrogen, namely freezing the immune cells.
7. The method for cryopreserving an immune cell according to claim 6, wherein 1.5 to 2ml of the immune cell cryopreserving solution is added to every 1000 to 1500 ten thousand IU of the immune cell in the step S3.
8. A method for recovering immune cells, comprising the step of obtaining the antibody of claim 6The frozen immune cells are quickly thawed in water bath at 38-40 ℃ for 30-50 s, mixed with a culture medium, and subjected to 5% CO treatment at 37 DEG C2And culturing for 20-24 h in a constant-temperature incubator to obtain the revived immune cells.
9. The method for recovering immune cells according to claim 8, wherein the culture medium is T551 culture medium supplemented with 15% by volume of human autologous plasma.
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| CN107251894A (en) * | 2017-06-30 | 2017-10-17 | 太仓东浔生物科技有限公司 | A kind of cell freezing method of high viability |
| CN109526943A (en) * | 2018-12-30 | 2019-03-29 | 深圳光彩生命工程技术有限公司 | A kind of immunocyte cryopreservation methods |
| TW202121984A (en) * | 2019-08-29 | 2021-06-16 | 美國德州系統大學評議委員會 | Cell cryopreservation medium |
| CN111670897A (en) * | 2020-05-28 | 2020-09-18 | 太东(镇江)生物科技有限公司 | Bovine sperm cryopreservation liquid and preparation method thereof |
| CN112753695A (en) * | 2021-01-04 | 2021-05-07 | 浙江卫未生物医药科技有限公司 | Immune cell cryopreservation method |
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