CN112359015B - Induced amplification method of NK-T cells - Google Patents
Induced amplification method of NK-T cells Download PDFInfo
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Abstract
The invention discloses an induction amplification method of CIK2 (NK NK NK-T) cells, which comprises the steps of induction and activation of immune cells CIK2 (NK NK-T) and large-capacity culture amplification of the immune cells CIK2 (NK NK-T); also comprises a step of detecting immune cell CIK2 (NK NK NK-T)And (5) carrying out the steps. The induced amplification method of CIK2 (NK NK NK-T) cells provided by the invention can stably improve CD16 + /56 + CD3 ‑ Cells (NK cells) and CD16 + /56 + CD3 + Percentage of cells (NK-like T cells) and number of cells expanded by the total cell culture. The immune cell provided by the invention maintains the characteristic that NK cells have stronger effect of killing tumor cells and virus-infected cells than CIK1 cells; and the low expansion rate of NK cells is compensated, and the new immune cell CIK2 is also named as NK NK-T cells.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an induced amplification method of CIK2 (NK NK NK-T) cells.
Background
The 'cytokine induced killer cell' (CIK) is an immune cell which is induced and activated by a CD3 monoclonal antibody, gamma-interferon, interleukin-1, interleukin-2 and other cytokines from lymphocytes separated from human peripheral blood, has the functions of identifying and killing tumor cells, inhibiting viruses and regulating the immunity of the organism and can be cultured and amplified in a large quantity, has strong proliferation capacity and mainly comprises NK-like T cells (CD 56) + CD3 + )、T(CD3 + CD8 + ) A predominant cell population. Has the advantages of both the antitumor activity of T fine lymphocyte and the non-MHC restriction killing of NK cell. The cell has recognition and killing effects on various tumor cells without damaging normal cells in vivo, has remarkable infusion effect after operation or radiotherapy and chemotherapy of patients, can eliminate residual tiny metastasis focus, is commonly used for patients with ineffective radiotherapy and chemotherapy and targeted therapy, or is combined with conventional therapy to improve the life quality and prolong the life cycle of tumor patients. The "cytokine induced killer cells" (CIK) national drug administration issued clinical research lots, lot No.: 2004L02376.
The 'cytokine induced killer cells' (CIK) have significant 'immunoregulation' and somatic cell repair effects. While treating tumors, most patients, especially patients after radiotherapy and chemotherapy, have the appearance of 'youthful appearance' such as reduction or disappearance of digestive tract symptoms, glossy skin, fading of black spots, disappearance of varicosity, stop of hair loss, even hair growth or blackening of local white hair and the like. Theoretically, NK (CD 56) + CD3 - ) The cell has stronger and more effective effect of killing tumor and virus infected cells, butThe number of NK cells in blood is far lower than that of T cells, and the content of NK cells is still very low after the cytokine induced killer Cells (CIK) are cultured and activated.
The "cytokine-induced killer cell" (CIK) is an individualized immune cell activation technology, and due to individual differences, lymphocytes of many individuals are activated and amplified to obtain the "cytokine-induced killer cell" (CIK) and CD16 thereof + 56 + /CD3 + The population of cells (NK-like T cells) does not meet the theoretical or expected requirement, i.e., CD16 cannot be stably induced + 56 + /CD3 + The large cell population has individual curative effect difference clinically.
NK cells are a subgroup of 'cytokine induced killer cells' (CIK), and the content of the NK cells is low; for example, NK cells are simply induced and amplified, the amplification rate is low, the number of the cells is difficult to meet the clinical requirement, and in order to meet the clinical requirement, NK (CD 56) needs to be developed at present + CD3 - ) Cells and NK-T (CD 16) + 56 + /CD3 + ) The cell content and the amplification rate are high, and the novel induction method is used for solving the clinical requirement.
Disclosure of Invention
To overcome the defects and shortcomings of the prior art, the invention aims to provide a method for inducing and amplifying CIK2 (NK NK-T) cells so as to stably improve CD16 + 56 + /CD3 - Cells (NK cells) and CD16 + 56 + /CD3 + Percentage of cells (NK-like T cells) and number of cells expanded by the total cell culture.
In order to achieve the above objects, according to a first aspect of the present invention, there is provided a method for induced expansion of CIK2 (NK NK NK-T) cells, the method comprising the steps of induction and activation of the immune cells CIK2 (NK NK-T) and large-volume culture expansion of the immune cells CIK2 (NK NK-T);
wherein, the induction and activation of the immune cell CIK2 (NK NK NK-T) comprise the following steps:
s01: drawing 50ml of anticoagulated peripheral blood: anticoagulation is carried out by adopting 1ml of heparin sodium with 12,500 units, and sodium citrate anticoagulation is eliminated;
s02, coating a T75 culture bottle with 7ml of a CD16 monoclonal antibody (25-35 mu g) and herceptin (1-2 mg) inducer for 24 hours, and sucking out a coating solution for later use;
s03, separating the 50ml of peripheral blood to obtain 30ml of plasma (without inactivation);
s04 separating the peripheral blood to obtain 3-5 × 10 lymphocytes 7 Put into a coated T75 culture flask, and add 30ml of complete culture solution: the AIM-V serum-free culture medium contains recombinant human gamma-interferon (IFN-r) for injection, recombinant human interleukin-2 (IL-2) for injection, recombinant human interleukin-15 (IL-15), recombinant human interleukin-21 (IL-21), recombinant human stem cell growth factor (SCF), adenosine disodium triphosphate injection (ATP) and coenzyme A for injection, wherein the cytokines of IFN-r, IL-2, IL-15, IL-21 and SCF are all 1000U/ml;
and 3ml autologous plasma was added; s05, after 24 hours, adding 25-35 mu g of CD3 monoclonal antibody;
the large-capacity culture amplification of immune cells CIK2 (NK NK NK-T) comprises the following steps:
s201: cell replacement induced after 72 hours 30ml (containing 3ml autologous plasma) complete medium;
s202: transferring to a T175 culture flask X1 on the 5 th day;
s203: transferring to a T175 culture flask multiplied by 2 on the 7 th day;
s204: transferring to a T175 culture flask multiplied by 4 on the 9 th day, wherein the total amount of the fluid infusion reaches 1000ml;
s205: transferring into culture bags with volume of 2000ml multiplied by 2 on day 11, wherein the total amount of the supplementary liquid in each bag is 1000ml;
s206: the total amount of the liquid supplement of each culture bag on the 13 th day is 4000ml;
s207: cells can be harvested at day 15 with a total quantity of immune cells CIK2 (NK NK-T) of > 1X 10 10 。
Further, the method also comprises an assay step of immune cell CIK2 (NK NK NK-T):
s301: the asepsis, endotoxin and mycoplasma of the harvested cells are qualified according to the verification of China pharmacopoeia III;
s302: flow cytophenotypic assay of immune cells CIK2 (NK NK NK-T):
Through the technical scheme, compared with the original CIK1 cell, the immune cell CIK2 or NK NK NK-T cell provided by the invention has high and stable NK phenotype expression, certain adhesiveness and CD16 + 56 + CD 3-or NK cell + CD16 + 56 + /CD3 + Or NK-T cell phenotype stably greater than 35%; CIK1 cells: CD16 + 56 + /CD3 + Due to individual difference of cells, a plurality of individuals are difficult to stably reach, so that the immune cell provided by the invention keeps the characteristic that NK cells have stronger tumor killing and virus infection effects than CIK1 cells; but also makes up the low expansion rate of NK cells. Can be applied to the national drug administration for the clinical research of new drugs, and brings new hope for tumor patients.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is a schematic diagram illustrating the process of killing tumor cells by CIK2 cells in an exemplary embodiment of the invention;
figure 2 shows donor 1 lymphocyte-induced CIK1: CD16 + 56 + /CD3 - (NK)+CD16 + 56 + /CD3 + (NK-T) 7.2%;
figure 3 shows donor 2 lymphocyte-induced CIK1: CD16 + 56 + /CD3 - (NK)+CD16 + 56 + /CD3 + (NK-T) 26.9%;
FIG. 4 shows CD16 expression by CIK2 cells induced by donor 1 lymphocytes + 56 + /CD3 - (NK)+CD16 + 56 + /CD3 + (NK-T) 46.3% flow phenotype higher than CIK1;
FIG. 5 shows donor 3 lymphocyte-induced expression of CD16 by CIK2 cells + 56 + /CD3 - (NK)+CD16 + 56 + /CD3 + (NK-T) 38.6% flow phenotype diagram;
FIG. 6 illustrates a first exemplary embodiment of the present invention; induced by donor 1CIK2：CD16 + 56 + /CD3 - (NK)+CD16 + 56 + /CD3 + (NK-T) 46.3% flow phenotype diagram;
FIG. 7 illustrates a second exemplary embodiment of the present invention; CIK2 induced by donor 2: CD16 + 56 + /CD3 - (NK)+CD16 + 56 + /CD3 + (NK-T) 35.0% flow phenotype diagram;
FIG. 8 illustrates a third exemplary embodiment of the present invention; CIK2 induced by donor 3: CD16 + 56 + /CD3 - (NK)+CD16 + 56 + /CD3 + (NK-T) 38.6% flow phenotype diagram;
FIG. 9 shows a fourth exemplary embodiment of the present invention; donor 4 induced CIK2: CD16 + 56 + /CD3 - (NK)+CD16 + 56 + /CD3 + (NK-T) is 47.8% flow phenotype diagram.
Detailed Description
The present invention will be described in further detail below with reference to specific embodiments and examples and the accompanying drawings. It should be noted that, in the present application, the embodiments and features of the embodiments may be combined with each other without conflict. The present invention will be described in detail below with reference to the accompanying drawings in conjunction with embodiments.
According to an exemplary embodiment of the present invention, there is provided a method for induced expansion of CIK2 (NK NK-T) cells, the method comprising steps of induction and activation of the immune cells CIK2 (NK NK-T) and large-capacity culture expansion of the immune cells CIK2 (NK-T);
wherein, the induction and activation of the immune cell CIK2 (NK NK NK-T) comprise the following steps:
s01: drawing 50ml of anticoagulated peripheral blood: adopting 1ml of heparin sodium with 12,500 units for anticoagulation, and removing sodium citrate for anticoagulation;
s02, coating a T75 culture bottle with 7ml of a CD16 monoclonal antibody (25-35 mu g) and herceptin (1-2 mg) inducer for 24 hours, and sucking out a coating solution for later use;
s03, separating 50ml of peripheral blood to obtain 30ml of plasma (without inactivation);
s04, separating the peripheral blood to obtain 3-5 × 10 lymphocytes 7 Put into a coated T75 culture flask, and add 30ml of complete culture solution: the AIM-V serum-free culture medium contains recombinant human gamma-interferon (IFN-r) for injection, recombinant human interleukin-2 (IL-2) for injection, recombinant human interleukin-15 (IL-15), recombinant human interleukin-21 (IL-21), recombinant human stem cell growth factor (SCF), adenosine disodium triphosphate injection (ATP) and coenzyme A for injection, wherein the cytokines IFN-r, IL-2, IL-15, IL-21 and SCF are all 1000U/ml;
and 3ml autologous plasma was added; s05, after 24 hours, adding 25-35 mu g of CD3 monoclonal antibody;
the large-capacity culture amplification of immune cells CIK2 (NK NK NK-T) comprises the following steps:
s201: cell replacement induced after 72 hours 30ml (containing 3ml autologous plasma) complete medium;
s202: transferring to a T175 culture flask multiplied by 1 on the 5 th day;
s203: transferring to a T175 culture flask multiplied by 2 on the 7 th day;
s204: transferring to a T175 culture flask multiplied by 4 on day 9, wherein the total amount of the supplementary solution reaches 1000ml;
s205: transferring into culture bags with the volume of 2000ml multiplied by 2 on day 11, wherein the total amount of the supplementary liquid in each bag is 1000ml;
s206: the total amount of the liquid supplement of each culture bag on the 13 th day is 4000ml;
s207: cells can be harvested at 15 days, and the total amount of immune cells CIK2 (NK NK NK-T) is more than or equal to 1 × 10 10 。
Preferably, the method further comprises an assay step of immune cells CIK2 (NK NK NK-T):
s301: the asepsis, endotoxin and mycoplasma of the harvested cells are qualified according to the verification of China pharmacopoeia III;
s302: flow cytophenotypic assay of immune cells CIK2 (NK NK NK-T):
FIG. 1 is a schematic diagram showing the process of killing tumor cells by CIK2 cells in an exemplary embodiment of the invention. The new immune cell CIK2 is also named NK NK-T cell.
The following examples are provided to further illustrate the advantageous effects of the present invention.
Example (b):
example 1
Induction and activation of immune cells (CIK 2):
the induction and activation of immune cell CIK2 (NK NK NK-T) comprises the following steps:
s01: and (3) extracting 50ml of anticoagulated peripheral blood: adopting 1ml of heparin sodium with 12,500 units for anticoagulation, and removing sodium citrate for anticoagulation;
s02, coating a T75 culture bottle with 7ml of a inducer, namely 'CD 16 monoclonal antibody (25-35 mu g) and herceptin (trastuzumab 1-2 mg)', for 24 hours, and sucking coating liquid for later use;
s03, separating 50ml of peripheral blood to obtain 30ml of plasma (without inactivation);
s04 lymphocyte 3-5 x 10 separated from peripheral blood 7 Put into a coated T75 flask, and add 30ml of complete culture solution: the AIM-V serum-free culture medium contains recombinant human gamma-interferon (IFN-r) for injection, recombinant human interleukin-2 (IL-2) for injection, recombinant human interleukin-15 (IL-15), recombinant human interleukin-21 (IL-21), recombinant human stem cell growth factor (SCF), adenosine disodium triphosphate injection (ATP) and coenzyme A for injection, wherein the cytokines of IFN-r, IL-2, IL-15, IL-21 and SCF are all 1000U/ml;
and 3ml of autologous plasma was added;
(II) 24 hours later, adding 25-35 mu g of CD3 monoclonal antibody;
large-volume culture expansion of immune cells CIK2 (NK NK NK-T) comprises the following steps:
s201: cell replacement induced after 72 hours 30ml (containing 3ml autologous plasma) complete medium;
s202: transferring to a T175 culture flask multiplied by 1 on the 5 th day;
s203: transferring to a T175 culture flask multiplied by 2 on the 7 th day;
s204: transferring to a T175 culture flask multiplied by 4 on day 9, wherein the total amount of the supplementary solution reaches 1000ml;
s205: transferring into culture bags with volume of 2000ml multiplied by 2 on day 11, wherein the total amount of the supplementary liquid in each bag is 1000ml;
s206: the total amount of the liquid supplement of each culture bag on the 13 th day is 4000ml;
s207: cells can be harvested at day 15, and the total amount of immune cells CIK2 (NK NK NK-T) is > 1 × 10 10 。
(III) detection of immune cells (CIK 2):
s301: the asepsis, endotoxin and mycoplasma of the harvested cells are qualified according to the verification of China pharmacopoeia III,
s302: flow cytophenotypic assay of immune cells CIK2 (NK NK NK-T):
CD16 + /56 + CD3 - (NK)31.9％CD16 + /56 + CD3 + (NK-T)14.4％。
as shown in FIG. 6, the flow cytophenotype of CIK2 (NK NK NK-T) as donor 1 immune cell, CD16 + 56 + /CD3 - (NK)+CD16 + 56 + /CD3 + (NK-T) was 46.3%, that is, NK + NK-T was 46.3%.
Example 2
Induction and activation of immune cells (CIK 2):
the induction and activation of immune cell CIK2 (NK NK NK-T) comprises the following steps:
s01: and (3) extracting 50ml of peripheral blood anticoagulation step: adopting 1ml of heparin sodium with 12,500 units for anticoagulation, and removing sodium citrate for anticoagulation;
s02, coating a T75 culture bottle with 7ml of an inducer, namely 25-35 mu g of CD16 monoclonal antibody and 1-2mg of herceptin (trastuzumab), for 24 hours, and sucking coating liquid for later use;
s03, separating 50ml of peripheral blood to obtain plasma (without inactivation);
s04 lymphocytes (3-5X 10) 7 Put into a coated T75 flask, and add 30ml of complete culture solution: the AIM-V serum-free culture medium contains recombinant human gamma-interferon (IFN-r) for injection, recombinant human interleukin-2 (IL-2) for injection, recombinant human interleukin-15 (IL-15), recombinant human interleukin-21 (IL-21), recombinant human stem cell growth factor (SCF), adenosine disodium triphosphate injection (ATP) and coenzyme A for injection,wherein the cytokines IFN-r, IL-2, IL-15, IL-21 and SCF are all 1000U/ml;
and 3ml of autologous plasma was added;
s05, after 24 hours, adding 25-35 mu g of CD3 monoclonal antibody;
(II) large-capacity culture amplification of immune cells (CIK 2):
s201: cell replacement induced after 72 hours 30ml (containing 3ml autologous plasma) complete medium;
s202: transferring to a T175 culture flask X1 on the 5 th day;
s203: transferring to a T175 culture flask multiplied by 2 on the 7 th day;
s204: transferring to a T175 culture flask multiplied by 4 on the 9 th day, wherein the total amount of the fluid infusion reaches 1000ml;
s205: transferring into culture bags with the volume of 2000ml multiplied by 2 on day 11, wherein the total amount of the supplementary liquid in each bag is 1000ml;
s206: the total amount of the liquid supplement of each culture bag on the 13 th day is 4000ml;
s207: cells can be harvested at 15 days, and the total quantity of immune cells CIK2 (NK NK-T) is more than or equal to 1 × 10 10 。
(III) detecting immune cells (CIK 2):
s301: the asepsis, endotoxin and mycoplasma of the harvested cells are qualified according to the verification of China pharmacopoeia III;
s302: flow cytophenotypic assay of immune cells CIK2 (NK NK NK-T):
FIG. 7 shows the flow cytotypic phenotype, CD16, of CIK2 (NK NK NK-T) which is also a donor 2 immune cell + 56 + /CD3 - (NK)+CD16 + 56 + /CD3 + (NK-T) was 35.0%, that is, NK + NK-T was 35.0%.
Example 3
Induction and activation of immune cells (CIK 2):
the induction and activation of immune cell CIK2 (NK NK NK-T) comprises the following steps:
s01: and (3) extracting 50ml of peripheral blood anticoagulation step: adopting 12500 units of heparin sodium for anticoagulation, and excluding sodium citrate for anticoagulation;
s02, coating a T75 culture bottle with 7ml of an inducer, namely 25-35 mu g of CD16 monoclonal antibody and 1-2mg of herceptin (trastuzumab), for 24 hours, and sucking coating liquid for later use;
s03, separating 50ml of peripheral blood to obtain plasma (without inactivation);
s04 lymphocytes (3-5X 10) 7 Put into a coated T75 culture flask, and add 30ml of complete culture solution: the AIM-V serum-free culture medium contains recombinant human gamma-interferon IFN-r for injection, recombinant human interleukin-2 (IL-2) for injection, recombinant human interleukin-15 (IL-15), recombinant human interleukin-21 (IL-21), recombinant human stem cell growth factor (SCF), adenosine disodium triphosphate injection (ATP) and coenzyme A for injection, wherein the cytokines IFN-r, IL-2, IL-15, IL-21 and SCF are all 1000U/ml;
and 3ml autologous plasma was added;
s05, after 24 hours, adding 25-35 mu g of CD3 monoclonal antibody;
(II) large-capacity culture amplification of immune cells (CIK 2):
s201: cell replacement induced after 72 hours 30ml (containing 3ml autologous plasma) complete medium;
s202: transferring to a T175 culture flask X1 on the 5 th day;
s203: transferring to a T175 culture flask multiplied by 2 on the 7 th day;
s204: transferring to a T175 culture flask multiplied by 4 on the 9 th day, wherein the total amount of the fluid infusion reaches 1000ml;
s205: transferring into culture bags with volume of 2000ml multiplied by 2 on day 11, wherein the total amount of the supplementary liquid in each bag is 1000ml;
s206: the total amount of the liquid supplement of each culture bag on the 13 th day is 4000ml;
s207: cells can be harvested at 15 days, and the total quantity of immune cells CIK2 (NK NK-T) is more than or equal to 1 × 10 10 。
(III) detection of immune cells (CIK 2):
s301: the asepsis, endotoxin and mycoplasma of the harvested cells are qualified according to the verification of China pharmacopoeia III;
s302: flow cytometric phenotypic detection of immune cells CIK2 (NK NK NK-T):
FIG. 8, showing the flow cytometric phenotype of immune cells CIK2 (NK NK NK-T) of the same donor 3, CD16 + 56 + /CD3 - (NK)+CD16 + 56 + /CD3 + (NK-T) was 38.6%, that is, NK + NK-T was 38.6%.
Example 4
Induction and activation of immune cells (CIK 2):
the induction and activation of immune cell CIK2 (NK NK NK-T) comprises the following steps:
s01: and (3) extracting 50ml of peripheral blood anticoagulation step: adopting heparin sodium with 12,500 units for anticoagulation, and removing sodium citrate anticoagulation;
s02, coating a T75 culture bottle with 7ml of an inducer, namely 25-35 mu g of CD16 monoclonal antibody and 1-2mg of herceptin (trastuzumab), for 24 hours, and sucking coating liquid for later use;
s03, separating 50ml of peripheral blood to obtain plasma (without inactivation);
s04 lymphocytes (3-5X 10) 7 Placing into a coated T75 culture flask, adding
Complete culture solution 30ml: the AIM-V serum-free culture medium contains recombinant human gamma-interferon (IFN-r) for injection, recombinant human interleukin-2 (IL-2) for injection, recombinant human interleukin-15 (IL-15), recombinant human interleukin-21 (IL-21), recombinant human stem cell growth factor (SCF), adenosine disodium triphosphate injection (ATP) and coenzyme A for injection, wherein the cytokines IFN-r, IL-2, IL-15, IL-21 and SCF are all 1000U/ml;
and 3ml of autologous plasma was added; (ii) a
S05, after 24 hours, adding 25-35 mu g of CD3 monoclonal antibody;
(II) large-capacity culture amplification of immune cells (CIK 2):
s201: cell replacement induced after 72 hours 30ml (containing 3ml autologous plasma) of complete medium and;
s202: transferring to a T175 culture flask multiplied by 1 on the 5 th day;
s203: transferring to a T175 culture flask multiplied by 2 on the 7 th day;
s204: transferring to a T175 culture flask multiplied by 4 on the 9 th day, wherein the total amount of the fluid infusion reaches 1000ml;
s205: transferring into culture bags with volume of 2000ml multiplied by 2 on day 11, wherein the total amount of the supplementary liquid in each bag is 1000ml;
s206: the total amount of the liquid supplement of each culture bag on the 13 th day is 4000ml;
s207: cells can be harvested at 15 days, and the total quantity of immune cells CIK2 (NK NK-T) is more than or equal to 1 × 10 10 。
(III) detection of immune cells (CIK 2):
s301: the asepsis, endotoxin and mycoplasma of the harvested cells are qualified according to the verification of China pharmacopoeia III;
s302: flow cytometric phenotypic detection of immune cells CIK2 (NK NK NK-T):
as figure 9 shows the flow cytometric phenotype of immune cell CIK2 (NK-T), donor 4 induced CIK2: CD16 + 56 + /CD3 - (NK)+CD16 + 56 + /CD3 + (NK-T) was 47.8%, that is, NK + NK-T was 47.8%.
CIK1 induced by donor 1 lymphocytes shown in figure 2: CD16 + /56 + CD3 - (NK)+CD16 + 56 + /CD3 + (NK-T) 7.2%; lower expression was seen by flow phenotyping. Figure 3 shows donor 2 lymphocyte-induced CIK1: CD16 + 56 + /CD3 - (NK)+CD16 + 56 + /CD3 + (NK-T) 26.9%; the flow type phenotype schematic diagram shows that the expression is high, and shows that although the same induction method is adopted, different individuals have large expression difference amplitude and are unstable. FIG. 4 shows CD16 expression by CIK2 cells induced by donor 1 lymphocytes + 56 + /CD3 - (NK)+CD16 + 56 + /CD3 + (NK-T) is a 46.3% flow phenotype diagram, and the flow phenotype diagram analysis shows that the expression of CIK2 or NK-T is higher than that of CIK1. FIG. 5 shows donor 3 lymphocyte-induced expression of CD16 by CIK2 cells + 56 + /CD3 - (NK)+CD16 + 56 + /CD3 + (NK-T) 38.6% flow phenotype diagram; CD16 shown by FIGS. 6, 7, 8, 9 + 56 + /CD3 - (NK)+CD16 + 56 + /CD3 + (NK-T) flow phenotype diagram shows that the flow phenotype diagram analysis shows that the expression of CIK2 or NK-T is higher than that of CIK1.
In a word, compared with the original CIK1 cell, the CIK2 or NK NK NK-T cell provided by the invention has high and stable expression of NK phenotype and CD16 + 56 + CD 3-or NK cell + CD16 + 56 + /CD3 + Or NK-T cell phenotype stably greater than 35%; CIK1 cells: CD16 + 56 + /CD3 + Due to individual difference of cells, a plurality of individuals are difficult to stably reach, so that the immune cell provided by the invention keeps the characteristic that NK cells have stronger tumor killing and virus infection effects than CIK1 cells; but also compensates for the low expansion rate of NK cells. Can report the clinical research of new drugs to the national drug administration, and brings new hope for tumor patients.
The present invention is capable of other embodiments, and various changes and modifications can be made by one skilled in the art without departing from the spirit and scope of the invention.
Claims (1)
1. An induced amplification method of NK-T cells is characterized by comprising the steps of induction and activation of immune cell NK-T and large-capacity culture amplification of the immune cell NK-T;
wherein, the induction and activation of the immune cells NK NK-T comprise the following steps:
s01: drawing 50ml of anticoagulated peripheral blood: anticoagulation is carried out by adopting 1ml of heparin sodium with 12,500 units, and sodium citrate anticoagulation is eliminated;
s02: coating a T75 culture bottle with 25-35 mug of ' CD16 monoclonal antibody and 7ml of trastuzumab ' 1-2mg ' of an inducer for 24 hours, and sucking out a coating solution for later use;
s03: 30ml of plasma is obtained by separating the 50ml of peripheral blood without inactivation;
s04: the above-mentionedSeparating peripheral blood to obtain lymphocyte 3-5 × 10 7 Put into a coated T75 culture flask, and add 30ml of complete culture solution: the AIM-V serum-free culture medium contains recombinant human gamma-interferon (IFN-r) for injection, recombinant human interleukin-2 (IL-2) for injection, recombinant human interleukin-15 (IL-15), recombinant human interleukin-21 (IL-21), recombinant human stem cell growth factor (SCF), adenosine disodium triphosphate injection (ATP) and coenzyme A for injection, wherein the cytokines of IFN-r, IL-2, IL-15, IL-21 and SCF are all 1000U/ml;
and 3ml autologous plasma was added;
s05: after 24 hours, adding 25-35 microgram of the CD3 monoclonal antibody;
the large-capacity culture and expansion of the immune cells NK NK-T comprises the following steps:
s201: cell replenisher induced after 72 hours contained 30ml complete medium of 3ml autologous plasma;
s202: transferring to a T175 culture flask X1 on the 5 th day;
s203: transferring to a T175 culture flask multiplied by 2 on the 7 th day;
s204: transferring to a T175 culture flask multiplied by 4 on day 9, wherein the total amount of the supplementary solution reaches 1000ml;
s205: transferring into culture bags with volume of 2000ml multiplied by 2 on day 11, wherein the total amount of the supplementary liquid in each bag is 1000ml;
s206: the total amount of the liquid supplement of each culture bag on the 13 th day is 4000ml;
s207: cells can be harvested at day 15, and the total amount of NK-T of immune cells is > 1X 10 10 ；
Also comprises a step of detecting NK-T of the immune cells:
s301: the asepsis, endotoxin and mycoplasma of the harvested cells are qualified according to the verification of China pharmacopoeia III;
s302: flow cytometric phenotypic detection of immune cells NK NK-T:
CD16 + 56 + /CD3 - NK cell + CD16 + 56 + /CD3 + NK-T cells are more than or equal to 35 percent.
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