CN106701681B - A kind of external evoked amplification of immunocyte, the method for freezing and recovering - Google Patents

Abstract

The invention discloses the external evoked amplification of immunocyte, the method for freezing and recovering.This method includes：Culture vessel, the culture vessel after being coated with are coated with using CD16 antibody；The first induced amplification culture is carried out in the culture vessel that mononuclearcell is placed in after being coated with using immune cell activation culture medium, obtains the immunocyte of preliminary induced amplification；The immunocyte of preliminary induced amplification is subjected to the second induced amplification culture, the immunocyte broken up using immune cell expansion culture medium；The immunocyte of differentiation is subjected to the 3rd induced amplification culture using immunocyte scale amplification culture medium, obtains the immunocyte of large-scale function activation；Immunocyte is frozen using immunocyte frozen stock solution, the immunocyte frozen；The immunocyte water-bath frozen is melted, and mixed with the Cryopreservation liquid of immunocyte, obtains recovery cell mixture；Recovery cell mixture is subjected to centrifugal treating, the immunocyte recovered.

Description

A method for inducing expansion, cryopreservation and recovery of immune cells in vitro

technical field

The invention relates to the field of biotechnology, in particular to methods for inducing expansion, cryopreservation and recovery of immune cells in vitro.

Background technique

In recent years, biological therapy has become the fifth largest treatment mode after surgery, radiotherapy and chemotherapy, and endocrine therapy, and has gradually received attention. Adoptive cellular immunotherapy (ACI) is one of the methods of cell biological therapy. It refers to the infusion of immune cells with anti-tumor activity to tumor patients, directly killing tumor cells or stimulating the body's immune response to kill tumor cells, so as to achieve the goal of treating tumors. Purpose. Currently, adoptive immune cells in clinical use include DC-CIK cells, TIL cells, LAK cells and NK cells, among which CIK cells, LAK cells and A-NK cells are all anti-cancer systems with natural killer cells as the main body.

Natural killer cells (natural killer cells), also called NK cells, are mainly derived from bone marrow CD34+ lymphocytes, distributed in bone marrow, peripheral blood and spleen, accounting for 10%-20% of peripheral blood lymphocytes. NK cells play an important role in tumor immunity and elimination of non-self cells: it is the main component of natural immune defense and is located in the first line of defense of the body's defense system. The killing activity of NK cells is not limited by MHC and does not depend on antibodies. Antigen pre-sensitization can recognize and kill tumor and virus-infected cells, and directly exert cytotoxic effect on tumor cells and kill tumor cells through perforin-granzyme pathway and Fas-FasL pathway; at the same time, it can secrete multiple Various cytokines and chemokines such as TNF-α, IFN-γ and IL-1, etc. These cytokines are involved in anti-cancer and regulation of acquired immune response, so NK cells are also a bridge connecting innate immunity and adaptive immunity. Although the safety and curative effect of NK cell's anti-cancer effect have been affirmed, because it only accounts for 10%-20% of peripheral blood lymphocytes, how to obtain high-purity and high-quality NK cell products is the key to NK therapy. In recent years, it has been found that relatively large-scale NK cells can be prepared through in vitro stimulation culture. It is known that cytokines such as IL-2, IL-15, IL-18 and IL-7 play an important role in the in vitro expansion of NK cells. The multiples of their in vitro expansion of NK cells range from several times to tens of times. IL-2 is an important cytokine that induces NK cell proliferation, it can activate NK cells, promote NK cell proliferation and cytokine production. The role of IL-15 and IL-7 is similar to that of IL-2. At the same time, they can also promote the directional differentiation of hematopoietic stem cells into NK cells by binding to the complex receptor γ chain expressed on the surface of NK cells, and have a positive effect on the development and differentiation of NK cells. It plays an important role in maintaining long-term in vitro survival. The synergistic effect of IL-15 and IL-2 also makes the two combined for the in vitro expansion of NK cells, which is currently the most traditional combination of cytokines for the in vitro expansion of NK cells. It has been reported that IL-18 can not only induce activated THi cells to secrete a large amount of IFN-γ, but also enhance the cytotoxicity of NK cells by promoting the opening of the Fas-FasL pathway in a dose-dependent manner. However, there are already NK cell therapy products on the market, but the recovery effect after cryopreservation is poor, the culture system used is complicated, the amplification multiple and tumor killing effect are not good, and some culture systems have safety risks and other problems.

Therefore, the methods of culture, cryopreservation and recovery of natural killer cells need to be improved.

Contents of the invention

The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, an object of the present invention is to propose a method for inducing expansion, freezing and recovery of immune cells in vitro, which induces and expands immune cells, has high induction efficiency, fast expansion speed, high safety and low cost , and the large-scale functionally activated immune cells obtained by induction and expansion still maintain good cell characteristics after long-term storage and recovery.

According to one aspect of the present invention, the present invention provides a method for inducing expansion, cryopreservation and recovery of immune cells in vitro. According to an embodiment of the invention, the method includes:

Coating the culture container with CD16 antibody to obtain the coated culture container;

placing the mononuclear cells in the coated culture container by using the immune cell activation medium for the first induction and expansion culture, so as to obtain the initially induced and expanded immune cells;

performing a second induction and expansion culture on the initially induced and expanded immune cells by using an immune cell expansion medium, so as to obtain differentiated immune cells;

performing a third induction and expansion culture of the differentiated immune cells by using the medium for large-scale expansion of immune cells, so as to obtain large-scale functionally activated immune cells;

Freezing the large-scale functionally activated immune cells using an immune cell freezing solution, so as to obtain frozen immune cells;

thawing the frozen immune cells in a water bath at 37-42°C, and mixing them with the immune cell cryopreservation solution to obtain a recovery cell mixture; and

centrifuging the recovered cell mixture to obtain recovered immune cells,

in,

The immune cell activation medium is a serum-free lymphocyte medium added with the first plasma, interleukin-2 and sapelin;

The immune cell expansion medium is a serum-free lymphocyte medium added with interleukin-2 and sapelin;

The immune cell scale expansion medium is a serum-free lymphocyte medium supplemented with interleukin-2;

The immune cell cryopreservation solution comprises 2-10% by volume of DMSO, 2-15% by volume of HSA, 20-88% by volume of the second plasma and the rest of the freezing medium;

Liquorgrass is the whole herb of Conyza japonica (Thunb.) Less in the Compositae family. Yan Shilun et al. have reported that the compound Liquor saponin R was isolated from Liquor grass, and its structure was identified as 3-O-β-D-glucopyranosylmedicagenic acid 28 -O-β-D-apiofuranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-[β-D-apiofuranosyl-(1→3)]-α-L-rhamnopyranosyl-(1→ 2)-α-L-arabinopyranosyl ester, its structural formula is:

The inventors have also surprisingly found that a small amount of liquor grass saponin R, blood plasma and HSA (human serum albumin, HumanSerum Albumin) combined with DMSO to prepare the cryopreservation solution of immune cells can reduce the amount of DMSO used, and this cryopreservation solution is beneficial to Cryopreserved cells have a better protective effect, and the frozen cells still maintain good cell viability after recovery, and the cell recovery rate is high.

According to one aspect of the present invention, the present invention provides an immune cell cryopreservation solution, which comprises 0.2-0.8 volume % of DMSO; 2-15 volume % of HSA; 0.001 g/ml of Liquor saponin R, 20-88 volume % plasma; and balance media. The inventors surprisingly found that the cryopreservation solution can be effectively used for cryopreservation of immune cells, and the cells still maintain good cell viability after recovery, and the cell recovery rate is high.

The frozen recovery fluid contains albumin and dextran.

The inventors have surprisingly found that large-scale immune cells can be obtained by inducing expansion culture of mononuclear cells by using this method, which has the advantages of high induction efficiency, fast expansion speed, high safety and low cost. Moreover, the large-scale immune cells cryopreserved by this method have a long effective storage time, the cells still maintain good cell viability after recovery, and the cell recovery rate is high, thus meeting the needs of clinical treatment of a large number of immune cells.

In addition, the method of in vitro induced expansion, cryopreservation and recovery of immune cells according to the above-mentioned embodiments of the present invention may also have the following additional technical features:

According to an embodiment of the present invention, the concentration of sapelin in both the immune cell activation medium and the immune cell expansion medium is 0.007-0.013KE/ml, preferably 0.01KE/ml.

According to an embodiment of the present invention, in the immune cell activation medium, the immune cell expansion medium and the immune cell scale expansion medium, the concentration of the interleukin-2 is 700-1300IU/ml , preferably 1000 IU/ml.

According to an embodiment of the present invention, in the immune cell activation medium, the immune cell expansion medium and the immune cell scale expansion medium, the serum-free lymphocyte medium is OpTmizer ^TM CTS ^TM Serum Free Medium or SuperCulture TM L500 Human Lymphocyte Serum Free Medium.

According to an embodiment of the present invention, in the immune cell activation medium, the concentration of the first plasma is 7-13% by volume, preferably 10% by volume.

According to an embodiment of the present invention, both the first plasma and the second plasma are autologous plasma.

According to an embodiment of the present invention, in the immune cell cryopreservation solution, the concentration of DMSO is 10% by volume; the concentration of HSA is 5% by volume; the concentration of the second plasma is 40% by volume; The freezing medium is 1640 medium or Stemspan medium.

According to an embodiment of the present invention, in the frozen resuscitation fluid, the albumin is human albumin, and optionally, the human albumin is derived from commercially available human serum albumin or autologous plasma.

According to an embodiment of the present invention, the content of the albumin is 1-5%, preferably 2.5%.

According to an embodiment of the present invention, the dextran is dextran 40.

According to an embodiment of the present invention, the content of the dextran is 2-8% by weight, preferably 5% by weight.

According to an embodiment of the present invention, the immune cells are natural killer cells and dendritic cells.

According to an embodiment of the present invention, when the CD56 expression of the initially induced and expanded immune cells exceeds 70%, the second induction and expansion culture is performed.

According to an embodiment of the present invention, when the expression of CD56 of the differentiated immune cells exceeds 90%, the third induction expansion culture is performed.

According to an embodiment of the present invention, during the first induction and expansion culture process, the cells are subcultured every 1 day.

According to an embodiment of the present invention, during the second induction expansion culture process, passage is performed every 2 days.

According to an embodiment of the present invention, during the third induction expansion culture process, passage is performed every 2 days.

According to an embodiment of the present invention, 1 ml of the immune cell cryopreservation solution is used for every 10 ⁶ -10 ⁹ large-scale functionally activated immune cells.

According to an embodiment of the present invention, the volume ratio of the cryopreserved immune cells mixed with the frozen resuscitation solution after thawing is 1:0.5-5, preferably 1:1.

Additional aspects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

The benefits of the present invention are:

The present invention proposes a method for inducing expansion, cryopreservation and recovery of immune cells in vitro. The method induces and expands immune cells, has high induction efficiency, fast expansion speed, high safety and low cost, and the obtained Large-scale functionally activated immune cells still maintain good cell properties after long-term preservation and recovery.

Description of drawings

The above and/or additional aspects and advantages of the present invention will become apparent and comprehensible from the description of the embodiments in conjunction with the following drawings, wherein:

Figure 1 shows a schematic diagram of the results of the number of peripheral blood mononuclear cells and cell activity cultured for different times according to an embodiment of the present invention;

Figure 2 shows a schematic diagram of the results of the proportion of lymphocytes in peripheral blood mononuclear cells before and after culture for 14 days according to an embodiment of the present invention;

Figure 3 shows a schematic diagram of the results of the number of peripheral blood mononuclear cells and cell activity cultured for different times according to yet another embodiment of the present invention;

Figure 4 shows a schematic diagram of the results of the proportion of lymphocytes in peripheral blood mononuclear cells before and after culture for 12 days according to an embodiment of the present invention;

Fig. 5 shows a schematic diagram of the results of a tumorigenic experiment in nude mice according to an embodiment of the present invention.

detailed description

Embodiments of the present invention are described in detail below, examples of which are shown in the drawings, wherein the same or similar reference numerals designate the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the figures are exemplary only for explaining the present invention and should not be construed as limiting the present invention.

It should be noted that the terms "first" and "second" are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of indicated technical features. Thus, a feature defined as "first" and "second" may explicitly or implicitly include one or more of these features. Further, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

According to one aspect of the present invention, the present invention provides a method for inducing expansion, cryopreservation and recovery of immune cells in vitro. According to an embodiment of the present invention, the method includes: using CD16 antibody to coat the culture container to obtain the coated culture container; using an immune cell activation medium to place mononuclear cells in the coated culture container for the first induction Expansion culture to obtain primary induced and expanded immune cells; use the immune cell expansion medium to conduct the second induction and expansion culture of the initially induced and expanded immune cells to obtain differentiated immune cells; use immune cell scale expansion culture The differentiated immune cells are subjected to the third induction and expansion culture to obtain large-scale functionally activated immune cells; the large-scale functionally activated immune cells are cryopreserved by using the immune cell cryopreservation medium to obtain cryopreserved immune cells; The stored immune cells are thawed in a water bath at 37-42°C, and mixed with the frozen recovery solution of immune cells to obtain a recovery cell mixture; and the recovery cell mixture is centrifuged to obtain recovery immune cells.

The inventors have surprisingly found that large-scale immune cells can be obtained by inducing expansion culture of mononuclear cells by using this method, which has the advantages of high induction efficiency, fast expansion speed, high safety and low cost. Moreover, the large-scale immune cells cryopreserved by this method have a long effective storage time, the cells still maintain good cell viability after recovery, and the cell recovery rate is high, thus meeting the needs of clinical treatment of a large number of immune cells.

According to an embodiment of the present invention, the immune cell activation medium is a serum-free lymphocyte medium supplemented with first plasma, interleukin-2 and sapelin; the immune cell expansion medium is a serum-free lymphocyte medium supplemented with interleukin-2 and sapelin. Serum lymphocyte medium; medium for large-scale expansion of immune cells is a serum-free lymphocyte medium supplemented with interleukin-2. The medium group is used to induce expansion and culture of mononuclear cells to obtain large-scale immune cells, which has the advantages of high induction efficiency, fast expansion speed, high safety and low cost, so as to meet the needs of clinical treatment of a large number of immune cells.

According to an embodiment of the present invention, the immune cell cryopreservation solution comprises: 2-10 vol% DMSO, 2-15 vol% HSA, 20-88 vol% second plasma and the rest cryopreservation medium. Therefore, the second plasma is added to the immune cell cryopreservation solution, and the ratio of each component is appropriate, the effective storage time of the immune cells is long, the cells still maintain good cell viability after recovery, and the cell recovery rate is high.

Wherein, it should be noted that when the sum of the volume percentages of DMSO, HSA and plasma is less than 1, the immune cell cryopreservation solution further includes the remaining volume of culture medium. For example, in the immune cell cryopreservation liquid, when the concentration of DMSO is 10% by volume, the concentration of HSA is 2% by volume, and the concentration of plasma is 88% by volume, the immune cell cryopreservation liquid does not contain medium; when the concentration of DMSO When the concentration is 5% by volume, the concentration of HSA is 2% by volume, and the concentration of plasma is 88% by volume, the immune cell cryopreservation solution contains 5% by volume of the culture medium; when the concentration of DMSO is 10% by volume, the concentration of HSA is 5% by volume, when the plasma concentration is 40% by volume, the immune cell cryopreservation solution contains 45% by volume of medium.

According to an embodiment of the present invention, the frozen resuscitation fluid contains albumin and dextran. Thus, using the frozen recovery solution including albumin and dextran to dilute the thawed immune cells, at normal temperature, not only reduces the damage of DMSO in the cell freezing solution to the immune cells, but also the albumin and dextran in the frozen recovery solution have the effect on immune cells. Thawed immune cells regulate osmotic pressure and nutrition.

According to an embodiment of the present invention, the concentration of sapelin in both the immune cell activation medium and the immune cell expansion medium is 0.007-0.013KE/ml. Therefore, the induction and proliferation of immune cells are fast, the expansion efficiency is high, the obtained immune cells are of high purity, and have better immune functions, which can be used for clinical treatments such as anti-infection, anti-tumor, and immunity enhancement.

According to a preferred embodiment of the present invention, the concentration of sapelin in both the immune cell activation medium and the immune cell expansion medium is 0.01KE/ml. Therefore, the induction and proliferation of immune cells are faster, the expansion efficiency is high, the obtained immune cells are of high purity, and have better immune functions, which can be used for clinical treatments such as anti-infection, anti-tumor, and immunity enhancement.

According to an embodiment of the present invention, the concentration of interleukin-2 in the immune cell activation medium, immune cell expansion medium and immune cell scale expansion medium is 700-1300 IU/ml. Therefore, the induction and proliferation of immune cells are fast, the expansion efficiency is high, the obtained immune cells are of high purity, and have better immune functions, which can be used for clinical treatments such as anti-infection, anti-tumor, and immunity enhancement.

According to a preferred embodiment of the present invention, the concentration of interleukin-2 in the immune cell activation medium, immune cell expansion medium and immune cell scale expansion medium is 1000 IU/ml. Therefore, the induction and proliferation of immune cells are faster, the expansion efficiency is high, the obtained immune cells are of high purity, and have better immune functions, which can be used for clinical treatments such as anti-infection, anti-tumor, and immunity enhancement.

According to an embodiment of the present invention, among the immune cell activation medium, immune cell expansion medium and immune cell scale expansion medium, the serum-free lymphocyte medium is: OpTmizer ^TM CTS ^TM serum-free medium or SuperCulture ^TM L500 serum-free medium for human lymphocytes. Thus, it is beneficial to the high-efficiency expansion and culture of immune cells in vitro, and maintains high functional activity.

According to an embodiment of the present invention, in the immune cell activation medium, the concentration of the first plasma is 7-13% by volume. Thus, it is beneficial to the high-efficiency expansion and culture of immune cells in vitro, and maintains high functional activity.

According to a preferred embodiment of the present invention, the concentration of the first plasma in the immune cell activation medium is 10% by volume. As a result, the induction and proliferation speed and efficiency of immune cells are significantly improved, and the obtained immune cells have high purity and better immune function.

According to an embodiment of the present invention, both the first plasma and the second plasma are autologous plasma. As a result, immune cells have little rejection of plasma, the induction and proliferation speed and efficiency of immune cells are significantly improved, and the obtained immune cells have high purity and better immune function.

Wherein, it should be noted that the term "autologous plasma" as used herein refers to the plasma from the same source as the cells to be induced to expand and cryopreserved. In other words, the plasma and the mononuclear cells to be induced to expand are obtained from the same individual. Therefore, the rejection effect on autologous immune cells is small, which is beneficial to the protection of cryopreserved cells.

According to an embodiment of the present invention, in the immune cell cryopreservation solution, the concentration of DMSO is 10% by volume; the concentration of HSA is 5% by volume; the concentration of the second plasma is 40% by volume; the freezing medium is 1640 medium or Stemspan Medium. Therefore, the osmotic pressure adjustment effect is good during the freezing and thawing process, and the protective effect on the cells is outstanding. After the cells are thawed, they still maintain good cell viability, which can effectively protect the cells, improve the survival rate of the cells, and reasonably control the cost of the cryopreservation solution. . According to an embodiment of the present invention, the albumin in the frozen resuscitation solution is human albumin. Albumin in the blood has functions of transportation, maintenance of osmotic pressure and nutrition. Albumin has the effect of protecting cells in vitro. Both human albumin and mononuclear cells are blood components, which are more conducive to protecting cells, making cells more active, promoting the induction of mononuclear cells into CIK cells, and obtaining CIK cells for clinical use, with little rejection by patients.

According to the embodiments of the present invention, the source of human albumin is not particularly limited, as long as human albumin can be provided, it can be derived from commercially available human serum albumin or human plasma. Human mononuclear cells have little rejection of human serum albumin or plasma, which is conducive to the recovery of frozen cells.

According to the embodiment of the present invention, the content of albumin is not particularly limited, as long as it can improve the induction efficiency, amplification factor or activity of mononuclear cells induced into CIK cells. According to some embodiments of the present invention, the content of albumin is 1-5% or 1-5%. Thus, the proliferation rate of the recovered cells is fast, the induction differentiation efficiency is high, and the cell activity is good. According to a preferred embodiment of the present invention, the content of albumin is 2.5. Thus, the proliferation rate of the revived cells is faster, the induction of differentiation efficiency is high, and the cell activity is better.

It should be noted that the content of albumin can be either volume percentage or weight percentage, which is adjusted according to the state of albumin. If albumin is solid, the content of albumin is 1-5% by weight. The albumin is in a liquid state, and the albumin content is 1-5% by volume.

According to an embodiment of the present invention, the dextran is dextran 40. Thereby, it is beneficial to improve the recovery effect of cells.

According to an embodiment of the present invention, the content of dextran is 2-8% by weight. Thus, the proliferation rate of the recovered cells is fast, the induction differentiation efficiency is high, and the cell activity is good. According to a preferred embodiment of the present invention, the content of the dextran is 5% by weight. Thus, the proliferation rate of the revived cells is faster, the induction of differentiation efficiency is high, and the cell activity is better. According to another aspect of the present invention, the present invention provides a freezing recovery kit for killing cells induced by various cytokines. According to an embodiment of the present invention, the kit includes the aforementioned frozen resuscitation solution for killing cells induced by various cytokines. Thus, in the subsequent induction process, the mononuclear cells recovered by using the frozen recovery kit can induce the formation of various cytokine-induced killer cells with fast proliferation rate, high induction differentiation efficiency, and good cell activity.

According to an embodiment of the present invention, the immune cells are natural killer cells and dendritic cells. Thus, the induction amplification efficiency is high.

According to an embodiment of the present invention, when the expression of CD56 of the activated immune cells exceeds 70%, the second induction expansion culture is performed. Thus, when most of the mononuclear cells are induced to differentiate to form immune cells, the second induction expansion culture can be carried out, so that the purity of immune cells is further improved, the number of cells is significantly increased, and the function of immune cells is further enhanced during the expansion process.

According to an embodiment of the present invention, when the expression of CD56 of the differentiated immune cells exceeds 90%, the third large-scale induction expansion culture is performed. Therefore, when most of the mononuclear cells are induced to differentiate into immune cells, the third induction and expansion culture can be carried out, so that the immune cells can be further expanded on a large scale while maintaining immune function, providing sufficient resources for possible clinical immunotherapy. cell.

According to an embodiment of the present invention, during the process of activating the culture medium for immune cells, passage is performed every 1 day. Thus, during the process of activating the immune cell culture medium, the immune cells are significantly activated and effectively expanded, and the proportion of other cells is significantly reduced.

According to an embodiment of the present invention, during the second induction and expansion culture process, the cells are subcultured every 2 days. Thus, during the second induction expansion culture process, the purity of immune cells is further improved, while the ratio of other cells is further reduced, and the function of immune cells is enhanced.

According to an embodiment of the present invention, during the third induction and expansion culture process, the cells are subcultured every 2 days. Thus, during the third induction and expansion culture process, the immune cells can achieve large-scale expansion with high purity, and sufficient functionally activated immune cells can be obtained for possible clinical immunotherapy.

According to an embodiment of the present invention, 1 ml of immune cell cryopreservation solution is used for every 10 ⁶ -10 ⁹ large-scale functionally activated immune cells. Thus, the cryopreservation of immune cells can be effectively realized, and the concentration of the cryopreserved cells is high, which is suitable for the cryopreservation of large-scale immune cells, and the cryopreservation cost is low and the effect is good. According to a specific example of the present invention, 1 ml of the immune cell cryopreservation solution is used for every 10 ⁷ -10 ⁸ immune cells. Therefore, the concentration of cryopreserved cells is high, suitable for large-scale cryopreservation of immune cells, the cost of cryopreservation is low, and the effect of cell cryopreservation is good.

According to an embodiment of the present invention, the cryopreserved immune cells are thawed and mixed with the frozen resuscitation solution at a volume ratio of 1:0.5-5. Thus, it is ensured that the DMSO concentration after dilution of the frozen resuscitation fluid is low, and the damage to immune cells is small. According to a preferred embodiment of the present invention, after thawing, the cryopreserved immune cells are mixed with the frozen resuscitation solution at a volume ratio of 1:1. Therefore, under the premise of ensuring that the concentration of DMSO after dilution is low and the damage to immune cells is small, the amount of freezing and resuscitation fluid is small, and the cost of freezing and resuscitation is low.

The present invention will be described below with reference to specific embodiments. It should be noted that these embodiments are only illustrative and should not be construed as limiting the present invention.

The solutions of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used, whose manufacturers are not indicated, are conventional products that can be purchased from the market, for example, they can be purchased from Sigma Company.

Example 1:

Using the method for inducing expansion of immune cells in the embodiment of the present invention, the isolated mononuclear cells are induced and expanded into immune cells, and the activity of the immune cells is detected.

1. Experimental method

1. Prepare anti-human CD16 coated T75 vials

1.1 Add 5 mL of 2.5 μg/mL anti-human CD16 monoclonal antibody dissolved in medical normal saline into a sterile culture bottle, shake the culture bottle gently to make the antibody cover the culture surface, and overnight at 4°C in the dark.

1.2 Recover the antibody coating solution before use, wash the culture flask once with 5 mL of normal saline, and then wash once with 5 mL of T cell expansion medium (OpTmizer ^TM CTS ^TM T-cell expansion SFM).

2. Collect peripheral blood, separate peripheral blood plasma and mononuclear cells

2.1 Collect about 100ml of human peripheral blood with a sterile blood collection bag added with anticoagulant, and reserve 1ml of peripheral blood for quick test and blood type identification. After passing the quick test, the peripheral blood will be sent to the GMP laboratory. If the quick test fails, the blood sample will be discarded.

2.2 Take out the blood bag in the GMP laboratory, disinfect the blood collection bag with alcohol, observe the absence of coagulation and hemolysis, open the blood bag in the ultra-clean bench, transfer the blood to a 50ml sterile centrifuge tube (≤40ml/tube), and centrifuge at 2500rpm for 15min .

2.3 Transfer the upper layer plasma to another sterile centrifuge tube, centrifuge at 3500rpm for 15min, collect the supernatant plasma into a new sterile centrifuge tube, seal the centrifuge tube mouth with the mouth film, and use the blood cells to separate mononuclear cells.

2.4 Put the plasma in a water bath at 56°C for 30-50 minutes to inactivate the complement, centrifuge at 3500rpm for 15 minutes to remove the complement, put 10ml each into 15ml sterile centrifuge tubes, freeze at -20°C for later use; and set aside 7.5ml of plasma Carry out slow inspection: five items of virus, mycoplasma, endotoxin and microorganism, among which the virus shall be tested by a third party.

2.5 Resuspend the blood cell pellet in step 2 with an equal volume of plasma saline and transfer it to a 250ml sterile glass bottle, add 1/3 of the total blood volume of hydroxyethyl starch, shake the saline bottle gently to mix evenly, Let the red blood cells settle.

2.6 After the erythrocyte layer has settled and stratified, gently suck the milky white suspension in the upper layer into a sterile centrifuge tube, centrifuge at 1800rpm for 5min, discard the supernatant, and resuspend the precipitate with 10ml of normal saline.

2.7 Take 2 sterile 15ml centrifuge tubes, add 5ml normal temperature human peripheral blood lymphocyte separation medium to each, and gently add 5ml cell suspension to the upper layer respectively. Centrifuge slowly at 2000 rpm at room temperature for 25 minutes.

2.8 Gently take out the centrifuge tube, carefully absorb the white blood cells in the cloud layer in the middle of the interface into a new 15ml centrifuge tube, add physiological saline to wash twice.

2.9 Resuspend the cells in 1ml of normal saline, take 5μl of cells and add them to 245μl of normal saline to dilute 50 times, count, and use trypan blue staining to count the cell viability. The bloody dirt after the experiment was soaked overnight with bromogeramine before being discarded.

2.10 Reserve 4.5x106 cells for flow cytometric antibody labeling to detect the proportion of NK cells; the remaining mononuclear cells are inoculated into a culture flask with 20ml of medium, and the medium ratio is: 18ml OpTmizer ^TM CTS ^TM T-cellexpansion SFM medium + 2ml of autologous plasma (Autologous plasma) + final concentration of 1000IU/ml Pepro TechIL-2 + final concentration of 0.01KE/ml Sapelin (OK432), 37 ° C, 5% CO2 sterile culture, recorded as day 0 .

2.11 Observe the cells every day, and carry out appropriate passage and expansion. The medium ratio is: OpTmizer ^TM CTS ^TM T-cell expansion SFM medium + 10% autologous plasma (Autologous plasma) + final concentration 1000IU/ml Pepro Tech IL-2. When the autologous plasma has been used up, no more plasma is added, that is, OpTmizer ^TM CTS ^TM T-cell expansion SFM medium + final concentration of 1000IU/ml PeproTech IL-2.

2.12 When the culture system has reached 2L, replace the culture system, that is, SuperCulture TM L500 human lymphocyte serum-free medium + domestic IL-2 with a final concentration of 1000IU/ml, observe the cells every day, and carry out appropriate passage. After 14 days, the cells were recovered, and 10 ml of the medium supernatant was collected for detection of secreted factors by Elisa.

2.13 Resuspend the recovered cells with 200ml of normal saline, add 10ml of human serum albumin, mix well; take 10ml of the cell suspension for testing, and inject the remaining cells into the reinfusion bag for reinfusion. Take 10ml of cell suspension from the reinfusion bag for detection: five items of mycoplasma, endotoxin, microorganism and virus.

3. Detection of Lymphocyte Lineage in Cells by Flow Cytometry

The 10ml cell suspension that was taken out was centrifuged at 1800rpm to recover the cells for flow cytometry antibody labeling. Set isotype control, single standard sample and staining tubes, the number of cells in each tube is about 5×105, and then add the corresponding antibody for staining. Place it at 4°C for 30 minutes, wash with physiological saline, and then analyze the proportion of NK cells in the lymphocyte population on a computer.

4. Aliquot the remaining 10ml cell suspension supernatant to detect five items of virus, endotoxin, mycoplasma and microorganism.

5. Nude mouse tumorigenicity test SPF-grade female BALB/c nude mice were purchased from the Institute of Experimental Animals, Chinese Academy of Medical Sciences. They were 4-6 weeks old and weighed 16-20 g. Drinking water, standard feed and other articles in contact with animals are all sterilized. Take the positive control Ragi cells and K562 cells and the NK cells to be tested on the 21st day of in vitro induction and differentiation at 3×107 cells/0.2ml and inoculate the ribs of nude mice subcutaneously, mark with picric acid, and observe the tumor formation for 2 months .

6. The medium supernatant of the harvested cells was tested for secreted factors by Elisa, that is, IFN-γ, TNF-α and Perforin.

2. Experimental results

1 The experimental results of culturing for 14 days

1.1 After 14 days of culture, the cells can be expanded by 150 times

The 6×107 peripheral blood mononuclear cells separated by the peripheral blood lymphocyte separation medium were inoculated into a 20ml culture system, and the cells quickly entered the logarithmic growth phase. After 14 days of culture, the culture system was expanded to 4L, the number of cells was expanded to 9×109, the amplification factor was up to 150 times, and the number of living cells was above 90%. The results are detailed in Figure 1.

1.2 The proportion of NK cells in the expanded peripheral blood mononuclear cells was significantly increased

After peripheral blood mononuclear cells were expanded for 14 days, the proportion of NK cells (CD3-CD56+) in lymphocytes increased from 18.15% to 95.27%, while the proportion of T lymphocytes (CD3+) decreased from 72.15% to 4.61%. Both helper T cells (Th, CD3+CD4+) and cytotoxic T cells (Tc, CD3+CD8a+) were reduced, B lymphocytes (CD3-CD19+) basically disappeared, and the cell uniformity was significantly improved; combined with the cells in Figure 1 Counting calculation shows that NK cells expanded 780 times after 14 days of culture, and the results of the lymphocyte ratio in peripheral blood mononuclear cells before and after 14 days of culture are shown in Figure 2, where NK cells: CD3-CD56+; T cells: CD3+; helper Sexual T cells (Th): CD3+CD4+; Cytotoxic T cells (Tc cells): CD3+CD8a+; B cells: CD3-CD9+.

1.3 No pathogen infection was found in the detection of cell products obtained from culture expansion

We entrusted the detection platform of cultured cells and cell suspensions to detect hepatitis B surface antigen, biscuit antigen, human immunodeficiency virus antibody, Treponema pallidum specific antibody, macrophage virus, mycoplasma, bacteria and endotoxin, and the test results were all positive. Negative, indicating that this batch of products is safe, and no pollution was caused during the cultivation process. See Table 1 for the results.

Table 1 NK cell clinical safety test after culturing for 14 days

2. Experimental results of 12 days of cultivation

2.1 After 12 days of culture, the cells can expand 75 times

Take 9×107 peripheral blood mononuclear cells separated by the peripheral blood lymphocyte separation medium and inoculate them into a 20ml culture system, and the cells quickly enter the logarithmic growth phase. After 12 days of culture, the culture system expanded to 4L, the number of cells expanded to 6.76×109, the amplification factor reached 75 times, and the number of living cells was above 90%. The experimental results are shown in Figure 3.

2.2 The proportion of NK cells in the expanded peripheral blood mononuclear cells was significantly increased

After 12 days of expansion of peripheral blood mononuclear cells, the proportion of NK cells (CD3-CD56+) in lymphocytes increased from 32.87% on day 7 to 92.19% on day 12, while the proportion of T lymphocytes (CD3+) decreased to 3.24%, helper T cells (Th, CD3+CD4+) and cytotoxic T cells (Tc, CD3+CD8a+) decreased, B lymphocytes (CD3-CD19+) basically disappeared, and the cell uniformity was significantly improved. The proportion of lymphocytes in the peripheral blood mononuclear cells before and after 12 days is shown in Figure 4, wherein, NK cells: CD3-CD56+; T cells: CD3+; helper T cells (Th): CD3+CD4+; cytotoxic T cells ( Tc cells): CD3+CD8a+; B cells: CD3-CD9+.

2.3 No pathogen infection was found in the detection of cell products obtained from culture expansion

We entrusted the detection platform of cultured cells and cell suspensions to detect hepatitis B surface antigen, biscuit antigen, human immunodeficiency virus antibody, Treponema pallidum specific antibody, macrophage virus, mycoplasma, bacteria and endotoxin, and the test results were all positive. Negative, indicating that this batch of products is safe, and no pollution was caused during the cultivation process. The results are shown in Table 2.

Table 2 NK cell clinical safety test after culturing for 12 days

Table 3. Elisa detection of culture supernatant

Experiment 1 Experiment 2 IFN-γ 1.48x10 ⁵ pg/ml ^1.435x105 pg/ml TNF-α 61.1pg/ml 48.75pg/ml Perforin 19ng/ml 17.48ng/ml

All the cultured cells secrete IFN-γ, TNF-α, Perforin.

3. The cultured and expanded NK cells will not form tumors in vivo

The results of the nude mouse tumorigenic experiment are shown in Figure 5, wherein, A normal saline control group (number of tumor-forming mice/number of mice in the group, 0/5), B Raji cell control group (3/5), C K562 Cell control group (4/5), D NK cell group (0/5), that is, subcutaneous injection of 0.2ml normal saline group and 3×107/0.2ml NK cell group cultured for 21 days within 2 months of observation There was no tumor formation in any of the mice, and visible tumors were formed in 3/5 and 4/5 mice injected with the same volume of Raji cells and K562 cells, respectively. This result shows that even after 21 days of culture, NK cells are still safe and effective, and will not cause tumor formation.

Example 2

In this example, 6 kinds of cryopreservation solutions without plasma and 6 kinds of cryopreservation solutions containing plasma were used respectively to freeze the NK cells induced and expanded in vitro in Example 1, and compare the cryopreservation effects so as to observe the effect of plasma on Effects of cryopreservation of NK cells, and the possibility of reducing DMSO concentrations to reduce cytotoxicity. details as follows:

1. Use plasma-free cryopreservation medium to freeze NK cells

1.1. NK cell cryopreservation and recovery methods:

The NK cells obtained on the 10th day of in vitro induction and expansion in Example 1 were cryopreserved in four freezing solutions containing HSA, and the components of the four freezing solutions were as follows:

Freezing solution 1: 5 vol% DMSO+10 vol% HSA;

Freezing solution 2: 5 vol% DMSO+15 vol% HSA;

Freezing solution 3: 10% by volume DMSO + 10% by volume HSA;

Freezing solution 4: 10 vol% DMSO + 15 vol% HS

Freezing solution 5: 0.2 vol% DMSO + 15 vol% HSA, after the preparation is completed, add liquoriasaponin R to a final concentration of 0.001 g/ml;

Freezing solution 6: 0.4 volume % DMSO + 10 volume % HSA, after the preparation is completed, add liquoriasaponin R to a final concentration of 0.001 g/ml; A,

Among them, the remaining volume in the cryopreservation solution 1-6 is 1640 or Stemspan medium.

Cryopreserve NK cells according to the following steps: After mixing the cryopreservation solution with the cells, quickly transfer it into a cryopreservation tube and put it into a cryopreservation box, program cooling at -70°C overnight, and transfer to liquid nitrogen the next day. Among them, 1ml of cryopreservation solution was used for every 107 NK cells.

NK cells were cryopreserved for 30 days and then revived.

Detect the survival rate of cells before and after cryopreservation, and the recovery rate of cells after thawing. Specifically, the calculation method of the cell survival rate before cryopreservation and after cryopreservation and recovery is: [number of living cells/(number of living cells+number of dead cells)]×100%. The calculation method of the cell recovery rate after recovery is: (the number of viable cells after recovery/the number of viable cells during cryopreservation) × 100%.

1.2. Results of NK cell resuscitation:

The results showed that the survival rate of cells after recovery was lower than that before cryopreservation. Specifically, the survival rate of cells preserved in cryopreservation solutions 3 and 4 was significantly better than that of cryopreservation solutions 1 and 2, and there was no difference between cryopreservation solutions 3 and 4, indicating that 10% by volume of DMSO had a better effect on NK cells. There is no significant difference in the protective effect of different concentrations of HSA on NK cells; the cell recovery rate of cryopreservation solution 3 and 4 is also better than that of cryopreservation solution 1 and 2. The cell recovery rate of cryopreservation solution 5 and 6 is also better than that of cryopreservation solution 1 and 2, especially cryopreservation solution 5, the cell recovery rate is as high as 99.3%.

The cell recovery rate of each group of cryopreserved solution is:

Freezing solution 1: 62.3%;

Freezing solution 2: 61.2%;

Freezing solution 3: 73.5%;

Freezing solution 4: 75.3%,

Freezing solution 5: 99.3%;

Freezing solution 6: 77.2%;

2. Use plasma-containing cryopreservation medium to freeze NK cells

2.1. Separate and obtain umbilical cord blood mononuclear cells, inoculate the mononuclear cells into a culture bottle with 20ml of culture medium, the ratio of the culture medium is: 18ml ^{OpTmizerTM CTSTM} T-cell expansion SFM medium+2ml autologous plasma (Autologous plasma) + final concentration 1000IU/ml Pepro Tech IL-2 + final concentration 0.01KE/ml Sapelin (OK432), 37°C, 5% CO2 sterile culture, recorded as day 0. Observe the cells every day, and carry out appropriate passage and expansion. The medium ratio is: OpTmizer ^TM CTS ^TM T-cell expansion SFM medium + 10% autologous plasma (Autologous plasma) + final concentration 1000IU/ml Pepro Tech IL-2, induced and expanded to obtain NK cells.

2.2. NK cell cryopreservation and recovery methods:

The above-mentioned NK cells obtained by induction and expansion in vitro for 10 days were cryopreserved by using four kinds of cryopreservation solutions containing plasma respectively, and the components of the four kinds of cryopreservation solutions containing plasma were as follows:

Freezing solution 9: 5 vol% DMSO+40 vol% plasma;

Freezing solution 10: 10 vol% DMSO+40 vol% plasma;

Freezing solution 11: 5 vol% DMSO+5 vol% HSA+40 vol% plasma;

Freezing solution 12: 10 vol% DMSO + 5 vol% HSA + 40 vol% plasma,

Freezing solution 13: 0.2 vol% DMSO + 15 vol% HSA + 40 vol% plasma, after the preparation is completed, add liquoriasaponin R to a final concentration of 0.001 g/ml;

Freezing solution 14: 0.4 vol% DMSO + 10 vol% HSA + 40 vol% plasma, after the preparation is complete, add liquoriasaponin R to a final concentration of 0.001 g/ml;

Among them, in cryopreservation solutions 9-14, the plasma is autologous plasma, and the remaining volume is 1640 or Stemspan medium.

Among them, the NK cells were cryopreserved according to the following steps: after mixing the cryopreservation solution and the cells, they were quickly transferred into cryopreservation tubes and placed in a cryopreservation box, cooled overnight at -70°C, and transferred to liquid nitrogen the next day. Among them, 1ml of cryopreservation solution was used for every 107 NK cells.

NK cells were cryopreserved for 30 days, and then resuscitated, wherein the resuscitation solution contained 2.5% plasma and 5% Dextran40.

The survival rate of cells before and after cryopreservation, the expression of cell surface markers, and the recovery rate of cells after thawing were detected. Specifically, the calculation method of the cell survival rate before cryopreservation and after cryopreservation and recovery is: [number of living cells/(number of living cells+number of dead cells)]×100%. The calculation method of the cell recovery rate after recovery is: (the number of viable cells after recovery/the number of viable cells during cryopreservation) × 100%. The expression of NK cell surface marker CD3-CD56+ was detected by flow cytometry.

2.3. Results of NK cell resuscitation:

The results showed that the survival rate and expression of cell surface markers of immune cells preserved in the six preservation solutions were not significantly different from those before cryopreservation, and the cell recovery rate was higher than that of the cryopreservation solution without plasma in "step 1". Moreover, cells frozen in cryopreservation solutions 5 and 6, 9 and 10 all had a certain proliferation ability after thawing, and cells frozen in solutions 13 and 14 had strong reproduction ability. Thus, it was shown that blood plasma and liquoriasaponin R had a good protective effect on cells.

The cell recovery rate of each group of cryopreserved solution is:

Freezing solution 7: 71.5%;

Freezing solution 8: 72.3%;

Freezing solution 9: 83.3%;

Freezing solution 10: 83.2%,

Freezing solution 11: 99.7%;

Freezing solution 12: 92.3%;

In summary, the inventors used DMSO, HAS, white wine grass saponin R and different concentration ratios in plasma to preserve the immune cells induced by cord blood mononuclear cells, and observed the effect of cryopreservation solution. The results showed that 10 volume % DMSO had better cell Protective effect; while different concentrations of HSA had no significant difference in protective effect on immune cells. However, the combination of autologous plasma and HSA and DMSO in the embodiment of the present invention, or further combined with the cryopreservation solution of liquoria saponin R to preserve the immune cells induced in vitro, has a high recovery rate of cells after resuscitation, good cell activity, and has a certain proliferation ability , while the recovery rate of cells preserved in frozen liquid in other groups was low.

Example 3

The NK cells frozen in the freezing solution 9 of Example 2 were resuscitated, and the specific method was as follows

1. Recovery process

(1) The NK cells cryopreserved in the cryopreservation solution 7 of Example 2 were taken out from the liquid nitrogen, and rapidly thawed in a water bath at 37-42°C.

(2) The cells were treated with an equal volume of X-VIVO15 medium (referred to as group P) and an equal volume of recovery solution containing 2.5% HSA and 5% Dextran40 (referred to as group F1).

(3) Centrifuge, take the cells separately, count with a cell viability analyzer, and measure the cell viability.

2. Culture after recovery

Cells in group P and group F1 were cultured in vitro using immune cell scale expansion medium, SuperCulture TML500 human lymphocyte serum-free medium + domestic IL-2 with a final concentration of 1000IU/ml, and the cell technology and Vitality analysis.

3. Calculate

Since the number of initial cells is inconsistent when resuscitating cells, it is necessary to quote the correction factor to calculate the number of CD3/CD56 positive cells. The calculation method is as follows:

(1) Calculate the average value of the starting cell numbers of the two groups;

(2) Divide the average number by the starting cell number of each group to obtain the correction factor K of each group;

(3) Multiply the final cell number of each group by the corresponding correction factor K and the CD3/CD56 positive percentage, which is the number of CD3/CD56 positive cells.

4. Results

(1) Results of recovery experiment

Table 4 shows the results of cell number and cell viability of NK cells recovered in "step 1".

Table 4 Effect of resuscitation solution on cell number and cell viability

group cell number cell viability P (0.90±0.08)×10 ⁷ (80.30±0.01)% F1 (0.95±0.04)×10 ⁷ (90.25±0.02)%

It can be seen from Table 4 that there is no significant difference in cell number (p>0.05) between the revived cells treated with medium P or resuscitation solution F1, but there is a significant difference in cell viability (p<0.05). The results showed that, compared with the control group P resuscitation fluid, the F1 resuscitation fluid could well enhance the viability of the resuscitated NK cells.

(2) Recovery culture results

In "step 2", the results of NK cell culture in group P and group F1 on the next day are shown in Table 5.

The impact of table 5 resuscitation solution on the cell number and cell viability of cultivating 24h

group cell number cell viability P (0.89±0.08)×10 ⁷ (90.20±0.01)% F1 (0.96±0.04)×10 ⁷ (93.10±0.02)%

It can be seen from Table 5 that there was no significant difference in the number of cells and cell viability between the two groups after being treated with culture medium P or resuscitation solution F1 for 24 hours in vitro (p>0.05).

5 Conclusion

The above results show that after large-scale in vitro culture and expansion of NK cells and cryopreservation, the cryopreserved cells were treated with resuscitation fluid in group P and group F1. Although there was no significant difference in the number of the two, the viability of the cells treated in group F1 was higher than that in group P group, and further proved that compared with the frozen recovery solution of the ordinary medium of the P group, the frozen recovery solution of the F1 group (containing 2.5% HSA, 5% Dextran40) can better promote the recovery of the cells during the cell recovery process.

In the description of this specification, descriptions referring to the terms "one embodiment", "some embodiments", "example", "specific examples", or "some examples" mean that specific features described in connection with the embodiment or example , structure, material or characteristic is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Although the embodiments of the present invention have been shown and described, those skilled in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principle and spirit of the present invention. The scope of the invention is defined by the claims and their equivalents.

Claims (9)

1. A method for in vitro induced expansion, cryopreservation and recovery of immune cells, characterized in that it comprises: Coating the culture container with CD16 antibody to obtain the coated culture container; placing the mononuclear cells in the coated culture container by using the immune cell activation medium for the first induction and expansion culture, so as to obtain the initially induced and expanded immune cells; performing a second induction and expansion culture on the initially induced and expanded immune cells by using an immune cell expansion medium, so as to obtain differentiated immune cells; performing a third induction and expansion culture of the differentiated immune cells by using the medium for large-scale expansion of immune cells, so as to obtain large-scale functionally activated immune cells; Freezing the large-scale functionally activated immune cells using an immune cell freezing solution, so as to obtain frozen immune cells; thawing the frozen immune cells in a water bath at 37-42°C, and mixing them with the immune cell cryopreservation solution to obtain a recovery cell mixture; and centrifuging the recovered cell mixture to obtain recovered immune cells, in, The immune cell activation medium is a serum-free lymphocyte medium added with the first plasma, interleukin-2 and sapelin; The immune cell expansion medium is a serum-free lymphocyte medium added with interleukin-2 and sapelin; The immune cell scale expansion medium is a serum-free lymphocyte medium supplemented with interleukin-2; The immune cell cryopreservation solution comprises 2-10% by volume of DMSO, 2-15% by volume of HSA, 20-88% by volume of the second plasma and the rest of the freezing medium; The frozen resuscitation fluid contains albumin and dextran; The concentration of said sapelin is 0.007-0.013KE/ml, In the immune cell activation medium, the immune cell expansion medium and the immune cell scale expansion medium, the concentration of the interleukin-2 is 700-1300IU/ml, In the immune cell activation medium, the immune cell expansion medium and the immune cell scale expansion medium, the serum-free lymphocyte medium is OpTmizer ^™ CTS ^™ serum-free medium or SuperCulture ^™ L500 human lymphocyte serum-free medium, In the immune cell activation medium, the concentration of the first plasma is 7-13% by volume; The freezing medium is 1640 medium or Stemspan medium. 2. The method according to claim 1, wherein both the first plasma and the second plasma are autologous plasma. 3. The method according to claim 1, characterized in that, in the immune cell cryopreservation solution, The concentration of the DMSO is 10% by volume; The concentration of the HSA is 5% by volume; The concentration of the second plasma is 40% by volume. 4. The method according to claim 1, characterized in that, in the frozen resuscitation fluid, the albumin is human albumin, optionally, the human albumin is derived from commercially available human serum albumin or autologous plasma , Optionally, the content of the albumin is 1-5%, Optionally, the dextran is dextran 40, Optionally, the content of the dextran is 2-8% by weight. 5. The method according to claim 1, wherein the immune cells are natural killer cells and dendritic cells. 6. The method according to claim 1, characterized in that, when the expression of CD56 of the initially induced and expanded immune cells exceeds 70%, the second induction and expansion culture is carried out, Optionally, when the expression of CD56 of the differentiated immune cells exceeds 90%, the third induction expansion culture is performed. 7. The method according to claim 1, characterized in that, in the first induction expansion culture process, passage once every 1 day, Optionally, during the second induction expansion culture process, passaging once every 2 days, Optionally, during the third induction and expansion culture process, passage is performed every 2 days. 8 . The method according to claim 1 , wherein 1 ml of the immune cell cryopreservation solution is used for every 10 ⁶ -10 ⁹ large-scale functionally activated immune cells. 9 . The method according to claim 1 , wherein the volume ratio of the cryopreserved immune cells mixed with the frozen resuscitation fluid after thawing is 1:0.5-5.