CN113122498A - Method for in vitro induced amplification, cryopreservation and recovery of immune cells - Google Patents
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Abstract
The invention provides a method for in vitro induction amplification, cryopreservation and resuscitation of immune cells, and relates to the technical field of in vitro amplification of immune cells. The method for in vitro induction amplification, cryopreservation and recovery of the immune cells mainly comprises the steps of performing activation culture on mononuclear cells by using an immune cell activation culture medium, performing primary induction amplification culture on a primary amplification culture medium, performing secondary amplification culture on a secondary amplification culture medium, performing cryopreservation treatment on a cryopreservation solution, and mixing and unfreezing the cryopreservation solution. The invention overcomes the defects of the prior art, effectively improves the induction efficiency of immune cells, has high in-vitro amplification speed and high safety, and ensures that the cells still have good cell characteristics after long-term storage.
Description
Technical Field
The invention relates to the technical field of immune cell in-vitro amplification, in particular to a method for in-vitro induced amplification, cryopreservation and resuscitation of immune cells.
Background
Cancer is one of the most threatening diseases to human beings in modern society, and the treatment of cancer has been the focus of modern medicine. The tumor immunotherapy is recognized as the fourth major tumor therapy by medical circles at home and abroad, wherein the autoimmune cell (T cell, NK cell) therapy technology belongs to the third medical technology which is allowed to be clinically applied by national Ministry of health. In recent years, natural killer cell (NK) immunotherapy is becoming a reliable anticancer therapy worldwide, suitable for clinical treatment of various malignant tumors with few side effects and without damaging normal tissues. NK cells have entered the human sight in the 70 th 20 th century, and have been developed for nearly 40 years. NK cells are defined as Large Granular Lymphocytes (LGL), which are differentiated from lymphoid progenitors and constitute three broad classes of immune cells with B and T lymphocytes. NK cells kill target cells (such as virus-infected host cells and tumor cells) without prior exposure to antigen, and since the killing activity of NK cells is MHC-free, it is called natural killing activity, which plays an important role in immune surveillance and early anti-infectious immune processes.
NK cells are mainly derived from lymphocytes of bone marrow CD34+, distributed in bone marrow, peripheral blood and spleen, and account for 10% -20% of peripheral blood lymphocytes. NK cells play an important role in tumor immunity, clearing of non-self-growing cells and the like: it is the main component of natural immune defense, is positioned on the first defense line of a body defense system, the killing activity of NK cells has no MHC restriction, does not depend on antibodies, can identify and kill tumor and virus infected cells without antigen pre-sensitization, and directly exerts cytotoxic effect on the tumor cells through a perforin-granzyme pathway and an Fas-FasL pathway to kill the tumor cells; meanwhile, the recombinant human immunodeficiency virus also can secrete various cytokines and chemokines such as TNF-alpha, IFN-gamma, IL-1 and the like in the early stage of pathogenesis, and the cytokines participate in anticancer and adjustment of acquired immune response, so that NK cells are also a bridge for connecting natural immunity and acquired immunity. Although the safety and efficacy of NK cell anticancer effect are confirmed, since it only accounts for 10% -20% of peripheral blood lymphocytes, how to obtain high purity, high quality NK cell product is the key of NK treatment.
However, the existing NK cell in vitro culture problems mainly lie in that the NK cell in vitro amplification speed is low, a large number of T cells are mixed, the cultured NK cell performance is poor, the bioactivity is insufficient, and the application No. CN201611233042.5 discloses a method for inducing and amplifying immune cells in vitro, wherein the induction efficiency and the amplification efficiency of the immune cells are improved by adding substances such as interleukin-2 and the like into a culture medium, but the cell growth efficiency in the actual production process is still low, and the production cost is also high.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for in vitro induced amplification, cryopreservation and recovery of immune cells, which effectively improves the induction efficiency of the immune cells, has high in vitro amplification speed and high safety, and ensures that the cells still have good cell characteristics after long-term storage.
In order to achieve the above purpose, the technical scheme of the invention is realized by the following technical scheme:
a method for in vitro induced amplification, cryopreservation and resuscitation of immune cells comprises the following steps:
(1) coating the culture container: coating a culture container with the CD16 antibody, and obtaining the coated culture container for later use;
(2) monocyte activation culture: placing the single nuclear cells into the culture container coated in the step (1) by adopting an immune cell activation culture medium, and performing activation culture for 24-36h to obtain activated cells for later use, wherein the immune cell activation culture medium comprises a serum-free lymphocyte culture medium, autologous plasma, IL-2, gamma interferon and saperin;
(3) primary induction and amplification: performing primary amplification culture on the activated cells by using a primary amplification culture medium, and adding EPO (erythropoietin) into the culture medium in the culture process, wherein the primary amplification culture medium comprises a serum-free lymphocyte culture medium, autologous plasma and IL-10;
(4) and (3) secondary amplification: performing secondary amplification on the immune cells subjected to the primary amplification by adopting a secondary amplification culture medium, and adding IL-2 into the culture medium in the amplification process, wherein the secondary amplification culture medium comprises a serum-free lymphocyte culture medium, autologous plasma and parathyroid hormone, and a large amount of activated immune cells are obtained after the secondary amplification is finished;
(5) freezing treatment: freezing the activated immune cells by adopting a freezing medium to obtain frozen immune cells;
(6) cell recovery: and adding the frozen immune cells into frozen liquid at the temperature of 40-45 ℃, mixing, stirring and dissolving, and centrifuging the solution after dissolving to obtain the recovered immune cells.
Preferably, the content of autologous plasma in the immune cell activation medium in the step (2) is 7-13 vol%, the concentration of IL-2 is 700-1300IU/mL, the concentration of gamma interferon is 300-400U/mL, and the concentration of sapelin is 0.007-0.013 KE/mL.
Preferably, the content of autologous plasma in the primary amplification medium in step (3) is 15 vol% -22 vol%, and the content of IL-10 is 500-1000U/ml.
Preferably, the addition amount of EPO in the step (3) is 1.2-2.0ng/ml, and EPO is added after 2h of primary induced amplification.
Preferably, in the step (4), the content of autologous plasma in the secondary amplification medium is 20-24 vol%, and the content of parathyroid hormone is 0.52-0.78 ug/ml.
Preferably, the amount of IL-2 added in step (4) is 500-600IU/ml, and IL-2 is added after 2h of secondary amplification.
Preferably, the frozen stock solution in the step (5) comprises 5-8% of DMSO, 1-2% of HSA, 10-15% of autologous plasma, 1-2% of aminoethanol and the balance of serum-free lymphocyte culture medium.
Preferably, the secondary expansion in step (4) is performed when the expression of CD56 of the differentiated immune cells exceeds 90%.
Preferably, the immune cell is a natural killer cell.
The invention provides a method for in vitro induction amplification, cryopreservation and resuscitation of immune cells, which has the advantages compared with the prior art that:
(1) according to the invention, autologous plasma, IL-2, gamma interferon and sabelin are added in the activation process of immune cells, so that the activation efficiency of the immune cells is effectively improved, and proliferation and amplification are rapidly completed, thereby achieving the effect of promoting the amplification of the immune cells;
(2) in the invention, autologous plasma, IL-10, autologous plasma and parathyroid hormone adding culture media are respectively adopted for culture in the primary amplification and the secondary amplification, so that the growth and the amplification of immune cells are further stimulated, EPO is added in the primary amplification process, IL-2 is added in the secondary amplification process, the amplification efficiency is accelerated, the cells are stimulated again, the growth is accelerated, and the cell growth efficiency is ensured.
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 are clearly and completely described below in conjunction with the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all 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.
Experimental materials:
medium No. 1: prepared by mixing 7 volume percent of autologous plasma with 93 volume percent of SuperCultureTML500 human lymphocyte serum-free culture medium, and IL-2 with the concentration of 700IU/mL, gamma interferon with the concentration of 300 and 400U/mL and sapelin with the concentration of 0.007 to 0.013KE/mL are added in the culture medium;
medium No. 2: the preparation method comprises the following steps of mixing 13 volume percent of autologous plasma with 87 volume percent of SuperCultureTML500 human lymphocyte serum-free culture medium, and adding IL-2 with the concentration of 1300IU/mL, gamma interferon with the concentration of 400U/mL and sapelin with the concentration of 0.013KE/mL into the culture medium;
medium No. 3: the preparation method comprises the following steps of mixing 10 volume percent of autologous plasma with 90 volume percent of SuperCultureTML500 human lymphocyte serum-free culture medium, and adding IL-2 with the concentration of 1000IU/mL, gamma interferon with the concentration of 350U/mL and sapelin with the concentration of 0.010KE/mL into the culture medium;
medium No. 4: mixing 15 vol% of autologous plasma with 85 vol% of SuperCultureTML500 human lymphocyte serum-free medium, wherein the medium is added with IL-10 with the content of 500U/ml;
medium No. 5: mixing 22 vol% of autologous plasma with 78 vol% of SuperCultureTML500 human lymphocyte serum-free medium, wherein the medium is added with IL-10 with the content of 1000U/ml;
medium No. 6: the preparation method comprises the following steps of mixing 19 volume percent of autologous plasma with 81 volume percent of SuperCultureTML500 human lymphocyte serum-free culture medium, wherein IL-10 with the content of 800U/ml is added into the culture medium;
medium No. 7: the preparation method comprises the following steps of mixing 20 volume percent of autologous plasma with 80 volume percent of SuperCultureTML500 human lymphocyte serum-free culture medium, wherein parathyroid hormone with the content of 500IU/ml is added into the culture medium;
medium No. 8: mixing 24 volume percent of autologous plasma with 76 volume percent of SuperCultureTML500 human lymphocyte serum-free culture medium, wherein parathyroid hormone with the content of 600IU/ml is added into the culture medium;
medium No. 9: prepared by mixing 22 volume percent of autologous plasma with 78 volume percent of SuperCultureTML500 human lymphocyte serum-free culture medium, and parathyroid hormone with the content of 550IU/ml is added into the culture medium;
no. 1 frozen stock solution: 5% of DMSO, 1% of HSA, 10% of autologous plasma, 1% of aminoethanol and the balance of SuperCultureTML500 human lymphocyte serum-free culture medium;
no. 2 frozen stock solution: 8% of DMSO, 2% of HSA, 15% of autologous plasma, 2% of aminoethanol and the balance of SuperCultureTML500 human lymphocyte serum-free culture medium;
no. 3 frozen stock solution: 7% of DMSO, 1.5% of HSA, 13% of autologous plasma, 1.5% of aminoethanol and the balance of SuperCultureTML500 human lymphocyte serum-free culture medium;
example 1:
placing the mononuclear cells in a No. 1 culture medium in a coated culture container for activated culture for 24h, then adopting a No. 4 culture medium, adding EPO according to 1.2ng/ml after culturing for 2h, then adopting a No. 7 culture medium for continuous culture until the expression of CD56 of differentiated immune cells exceeds 90%, adding IL-2 according to 500IU/ml after secondary amplification culture exceeds 2h, and then continuing culture; cells were observed daily, on day 0, and passaged every 2-3 days, depending on the color of the medium.
Example 2:
placing the mononuclear cells in a No. 2 culture medium in a coated culture container for activated culture for 24h, then adopting a No. 5 culture medium, adding EPO according to 2.0ng/ml after culturing for 2h, then adopting a No. 8 culture medium for continuous culture until the expression of CD56 of differentiated immune cells exceeds 90%, adding IL-2 according to 600IU/ml after secondary amplification culture exceeds 2h, and then continuing culture; cells were observed daily, on day 0, and passaged every 2-3 days, depending on the color of the medium.
Example 3:
placing the mononuclear cells in a No. 3 culture medium in a coated culture container for activated culture for 24h, then adopting a No. 6 culture medium, adding EPO according to 1.6ng/ml after culturing for 2h, then adopting a No. 9 culture medium for continuous culture until the expression of CD56 of differentiated immune cells exceeds 90%, adding IL-2 according to 550IU/ml after secondary amplification culture exceeds 2h, and then continuing culture; cells were observed daily, on day 0, and passaged every 2-3 days, depending on the color of the medium.
Detection 1:
the cells cultured in the above examples 1 to 3 were examined for cell activity for 0 to 14 days (shown in Table 1 below) and the NK cell proportion and the cell expansion fold (shown in Table 2 below) in the lymphocyte population after 14 days:
TABLE 1
TABLE 2
| Group of | Fold expansion of cells | NK cell proportion |
| Example 1 | 232 | 98.36 |
| Example 2 | 226 | 98.27 |
| Example 3 | 228 | 97.98 |
And (3) detection 2:
the NK cells cultured for 14 days in the example 3 are frozen by using the number 1 frozen stock solution, the number 2 frozen stock solution and the number 3 frozen stock solution respectively, the frozen stock solution and the cells are uniformly mixed, then the mixture is quickly transferred into a frozen tube and placed into a frozen box, the temperature is reduced by a program at-70 ℃ overnight, and the mixture is transferred into liquid nitrogen the next day, wherein 1ml of the frozen stock solution is used for every 107 NK cells. And (3) freezing and storing the NK cells for 60 days, and then recovering, wherein the recovery mode is that similar freezing solutions are heated to 40-45 ℃, and then slowly dropwise added into the freezing solutions after freezing until the cells are completely thawed. And detecting the survival rate of the cells before and after the freezing and the recovery rate of the cells after the recovery. Specifically, the method for calculating the cell survival rate before cryopreservation and after cryopreservation and recovery comprises the following steps: [ viable cell count/(viable cell count + dead cell count) ] X100%. The calculation method of the recovery rate of the cells after recovery comprises the following steps: (number of viable cells after recovery/number of viable cells at the time of cryopreservation). times.100%, the results are shown in Table 3 below:
table 3:
| frozen stock solution | Cell viability (%) | Cell yield (%) |
| No. 1 frozen stock solution | 98.77 | 96.37 |
| No. 2 frozen stock solution | 99.19 | 97.32 |
| No. 3 frozen stock solution | 98.87 | 96.72 |
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (9)
1. The method for in vitro induced amplification, cryopreservation and recovery of immune cells is characterized by comprising the following steps of:
(1) coating the culture container: coating a culture container with the CD16 antibody, and obtaining the coated culture container for later use;
(2) monocyte activation culture: placing the single nuclear cells into the culture container coated in the step (1) by adopting an immune cell activation culture medium, and performing activation culture for 24-36h to obtain activated cells for later use, wherein the immune cell activation culture medium comprises a serum-free lymphocyte culture medium, autologous plasma, IL-2, gamma interferon and saperin;
(3) primary induction and amplification: performing primary amplification culture on the activated cells by using a primary amplification culture medium, and adding EPO (erythropoietin) into the culture medium in the culture process, wherein the primary amplification culture medium comprises a serum-free lymphocyte culture medium, autologous plasma and IL-10;
(4) and (3) secondary amplification: performing secondary amplification on the immune cells subjected to the primary amplification by adopting a secondary amplification culture medium, and adding IL-2 into the culture medium in the amplification process, wherein the secondary amplification culture medium comprises a serum-free lymphocyte culture medium, autologous plasma and parathyroid hormone, and a large amount of activated immune cells are obtained after the secondary amplification is finished;
(5) freezing treatment: freezing the activated immune cells by adopting a freezing medium to obtain frozen immune cells;
(6) cell recovery: and adding the frozen immune cells into frozen liquid at the temperature of 40-45 ℃, mixing, stirring and dissolving, and centrifuging the solution after dissolving to obtain the recovered immune cells.
2. The method for inducing expansion, cryopreservation and resuscitation of immune cells in vitro as claimed in claim 1, wherein: in the step (2), the content of autologous plasma in the immune cell activation medium is 7-13 vol%, the concentration of IL-2 is 700-1300IU/mL, the concentration of gamma interferon is 300-400U/mL, and the concentration of sapelin is 0.007-0.013 KE/mL.
3. The method for inducing expansion, cryopreservation and resuscitation of immune cells in vitro as claimed in claim 1, wherein: the content of autologous plasma in the primary amplification medium in the step (3) is 15-22 vol%, and the content of IL-10 is 500-1000U/ml.
4. The method for inducing expansion, cryopreservation and resuscitation of immune cells in vitro as claimed in claim 1, wherein: the addition amount of the EPO in the step (3) is 1.2-2.0ng/ml, and the EPO is added after 2 hours of primary induced amplification.
5. The method for inducing expansion, cryopreservation and resuscitation of immune cells in vitro as claimed in claim 1, wherein: in the step (4), the content of autologous plasma in the secondary amplification culture medium is 20-24 vol%, and the content of parathyroid hormone is 0.52-0.78 ug/ml.
6. The method for inducing expansion, cryopreservation and resuscitation of immune cells in vitro as claimed in claim 1, wherein: the addition amount of IL-2 in the step (4) is 500-600IU/ml, and IL-2 is added after the secondary amplification is carried out for 2 h.
7. The method for inducing expansion, cryopreservation and resuscitation of immune cells in vitro as claimed in claim 1, wherein: the frozen stock solution in the step (5) comprises 5-8% of DMSO, 1-2% of HSA, 10-15% of autologous plasma, 1-2% of aminoethanol and the balance of serum-free lymphocyte culture medium.
8. The method for inducing expansion, cryopreservation and resuscitation of immune cells in vitro as claimed in claim 1, wherein: performing the secondary expansion in step (4) when the expression of CD56 of the differentiated immune cells exceeds 90%.
9. The method for inducing expansion, cryopreservation and resuscitation of immune cells in vitro as claimed in claim 1, wherein: the immune cell is a natural killer cell.
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| CN116751745A (en) * | 2023-08-09 | 2023-09-15 | 北京圣美细胞生命科学工程研究院有限公司 | Combined immune cell exosome polypeptide regeneration factor and application thereof |
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| CN116751745A (en) * | 2023-08-09 | 2023-09-15 | 北京圣美细胞生命科学工程研究院有限公司 | Combined immune cell exosome polypeptide regeneration factor and application thereof |
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Application publication date: 20210716 |