CN110862962A - Method for culturing and amplifying NK cells in vitro by using gallic acid - Google Patents

The invention discloses a method for in vitro culture and amplification of NK cells by gallic acid, which can establish stable and large-scale high-purity and high-immunocompetence NK cells without serum, feeder cell stimulation and pre-coating of a culture bottle, and is simple and feasible; gallic acid and IL-2 are used together for stimulating NK cell expansion culture, and can reduce T cell number, improve NK cell purity and enhance NK cell activity, and can make CD56 with immunoregulation function in the culture method^highThe CD16-NK cell subset proportion is increased, so the culture method is expandedThe NK cell product of (1) can be emphatically applied to the medicines of immunomodulators, and the culture method does not reduce the activity and the quantity of NK cells.

Method for culturing and amplifying NK cells in vitro by using gallic acid

The technical field is as follows:

the invention relates to the field of immune cell therapy, in particular to a method for in vitro stable, large-scale expansion of high-purity and high-cytotoxic-activity NK cells of autologous or allogeneic peripheral blood mononuclear cells, and more particularly relates to application and a method for in vitro stable, large-scale expansion of high-purity and high-cytotoxic-activity NK cells of gallic acid in autologous or allogeneic peripheral blood mononuclear cells.

Background art:

cellular immunotherapy is one of the most promising tumor therapy methods at present, and achieves the purpose of killing tumor cells by in vitro amplification or reinjection into a patient after modification or enhances the autoimmune function of the tumor patient by activating the immune system of the body so as to resist tumors. At present, NK cell immunotherapy is receiving more and more attention. NK cells account for 5-15% of human peripheral blood lymphocytes, and their phenotype is generally defined as CD3^-CD56⁺NK cells can be further subdivided into two major sub-groups: CD56 with immunoregulation function^highCD16^-Cell and CD56 with cytotoxic activity^dimCD16⁺A cell. NK cells play an important immune monitoring function in early immune response of resisting virus infection and tumors, and can directly and quickly play cytotoxic activity without identifying tumor specific antigens. Particularly important is that NK cells can effectively eliminate tumor stem cell-like cells in organisms and inhibit the growth and metastasis of tumors. Recent research shows that blocking of an immunodetection point receptor TIGIT on the surface of the NK cell can effectively prevent the exhaustion of the NK cell, promote the tumor immunity depending on the NK cell and inhibit the growth of various malignant tumors of mice. Blocking the PD-1/PD-L1 signal channel can activate NK cells and T cells, reverse the tumor immune microenvironment and enhance the endogenous anti-tumor immune effect of the organism. Therefore, NK cells play an essential role in the immune system of the body in response to tumor resistance, and the dysfunction of NK cells in the body is one of the possible causes of tumor occurrence and development. Autologous or allogeneic NK cells have been used to treat malignant lymphoma, refractory non-hodgkin lymphoma, relapsed/refractory acute leukemia, acute myeloid leukemia, childhood relapsed/refractory neuroblastoma, advanced gastric cancer, colorectal cancer/pancreatic cancer liver metastasis, and a variety of relapsed, metastatic solid tumors. Clinically, the medicine shows good tolerance and certain degreeThe therapeutic effect of (1). The therapeutic effect of the NK cells is positively correlated with the purity, the quantity and the activity of the returned NK cells.

Cellular immunotherapy is one of the most promising tumor therapy methods at present, and achieves the purpose of killing tumor cells by in vitro amplification or reinjection into a patient after modification or enhances the autoimmune function of the tumor patient by activating the immune system of the body so as to resist tumors.

There are some currently available NK cell expansion techniques for clinical use, but most of them use feeder cells (e.g., IL-15 and 41BB ligand-expressing K562 cells, EBV transformed lymphoblastoid cell lines, Wilm's tumor cells, radiation-irradiated PBMCs, etc.) to stimulate Peripheral Blood Mononuclear Cells (PBMCs) or purify enriched NK cells to obtain a large number of fully functional NK cells. The existing NK cell amplification method has the defects that the amplification quantity of NK cells is low, and the clinical application requirements are difficult to meet; or a feeder cell culture method is adopted, certain risk exists in clinical application, the steps of NK cell amplification culture are complicated, the cost is high, the proportion of NK cells is relatively low, residual T cells and B cells exist, and the risk of causing GVHD exists. At present, some feeder cell-free NK cell amplification methods reported in patent documents mostly adopt anti-CD3 antibodies and anti-CD16 antibodies to coat cell culture flasks to stimulate and activate NK cells, the cell culture methods are complicated and unstable, and the purity of the amplified NK cells is low. Therefore, there is an urgent need to develop a stable, serum-free, feeder cells-free NK cell expansion culture system.

The invention content is as follows:

in order to solve the defects of the prior art and the defects of complicated NK cell culture technology, low NK cell purity, weak killing function and unstable culture system in the prior art, the invention provides a serum-free and feeder cell-free NK cell in-vitro amplification method, which can stably amplify a large number of NK cells with high purity and high cytotoxic activity under in-vitro culture conditions, thereby effectively providing the NK cells required by clinical application.

In order to achieve the aim, the invention designs a serum-free and feeder cell-free NK cell in-vitro amplification method, which comprises the following steps:

(1) isolating the mononuclear cells;

(2) depletion of CD3 from monocytes by immunoselection⁺T lymphocytes;

(3) adding a serum-free cell culture medium for NK cell culture, and inoculating IL-2 with the final concentration of 500-;

(4) replacing NK cells;

(5) and (5) harvesting the NK cells.

The mononuclear cells are obtained by collecting peripheral blood through intravenous puncture of a heparin anticoagulant sterile disposable blood collection tube and then through Ficoll density gradient centrifugation or single-core cells collected through a single-core collection machine; or from mononuclear cells obtained by inducing differentiation of cord blood, bone marrow and iPSCs.

The immunoselection removes CD3 from mononuclear cells⁺The T lymphocyte can adopt methods such as immunomagnetic beads, membrane vesicle immunoselection, flow cytometry immunoselection and the like.

The concentration of interleukin-2 used in the cytokine combination is preferably 1000-1800U/ml.

The concentration of gallic acid is preferably 0.5-50 μ g/ml.

The NK cell exchange solution is prepared by supplementing NK cells containing cytokines required by NK cell amplification to adjust the concentration of the NK cells to 1 x 10 through serum-free culture⁶cells/ml。

The prepared NK cell is applied to the preparation of immunomodulators and/or antitumor drugs.

The NK cell prepared by the method is applied to immune diseases.

The invention has the beneficial effects that:

(1) compared with the existing NK cell amplification method, the NK cell amplification method can establish stable, large-amount and high-purity and high-cytotoxic activity NK cells through amplification without serum, feeder cell stimulation or pre-coating of a culture bottle, and is simple and feasible.

(2) Gallic acid and IL-2 are used together for stimulating NK cell expansion culture, and can reduce T cell number, improve NK cell purity and enhance NK cell activity, and can make CD56 with immunoregulation function in the culture method^highCD16^-An increased proportion of NK cell subsets, CD56^highCD16^-The NK cell subgroup accounts for the main group, so the NK cell product cultured by the culture method can be mainly applied to immunoregulation medicine application, and the activity and the number of the NK cells cannot be reduced by the culture method, so that the defects in the traditional technical method are overcome.

Description of the drawings:

FIG. 1 is a growth curve of amplified cultured NK cells.

The specific implementation mode is as follows:

the technical solutions in the embodiments of the present invention are clearly and completely described below with reference to specific embodiments. It should be understood that the described embodiments are part of the present invention, and are intended to be illustrative only and not limiting in scope. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.

EXAMPLE 1NK cell in vitro expansion-isolation of monocytes

1. Isolation of monocytes

1.2 uniformly diluting the peripheral whole blood of a patient and DPBS in proportion, and slowly injecting the diluted peripheral whole blood and DPBS into the upper layer of the liquid surface of the lymphocyte separation solution in a centrifugal tube;

centrifuging at 400 Xg at 1.320 deg.C for 30 min;

1.4 using a sterile suction tube to suck the plasma layer in the separation tube, putting the plasma into a 50ml sterile centrifuge tube, carrying out water bath at 56 ℃ for 30 minutes to inactivate, carrying out centrifugation at 800g multiplied by 10 minutes, and transferring the upper plasma layer into a new 50ml sterile centrifuge tube;

1.5 sucking and separating the obtained mononuclear cell layers (PBMCs) and putting the PBMCs into a 50ml sterile centrifuge tube;

1.6 adding DPBS, mixing uniformly, centrifuging for 10 minutes at 20 ℃ at 400 Xg, and washing for 1 time;

Example 2NK cell in vitro expansion-CD 3⁺T cell depletion

2.2 Easysep^TMBlending RapidSphereTM50100 in Human CD3 Positive Selection Kit II, adding 90 μ l/ml sample into the flow tube in step 2.1, blending, standing at room temperature for 3 min;

2.3 the volume of the cell suspension in the 5ml sterile flow tube in the step 2.2 is filled to 2.5ml by using the solution containing 0.5 percent HSA, 1mM EDTA and DPBS, and the mixture is gently mixed;

2.4 Place the 5ml sterile flow tube in a magnet at room temperature for 3min, pick up the magnet, pour the cells into a 15ml sterile centrifuge tube, and count 20. mu.l of the cell suspension in a 1.5ml EP tube.

EXAMPLE 3 NK cell in vitro expansion-NK cell culture

3.2 discarding the supernatant, centrifuging at 20 ℃ for 5min at 400 Xg;

3.4 cells were incubated at 37 ℃ with 5.0% CO at saturated humidity₂Culturing in an incubator.

3.7 Place the cells at 37 deg.C, saturated humidity, 5.0% CO₂And continuing culturing in the incubator.

3.8 days 5-21, observing cell growth state every day, supplementing L500 culture medium containing cytokine (if enough plasma is available, 10% autologous plasma content can be maintained), and maintaining cell concentration 1 × 10⁶cells/ml, 5.0% CO at 37 ℃ under saturated humidity₂The incubator continues to culture and cells are harvested until the desired number of cells is reached. Meanwhile, if the cell volume exceeds 240ml, the cells are transferred to a flask for culture or transferred to a cell culture bag for subculture.

EXAMPLE 4 NK cell in vitro expansion-NK cell culture

4.2 discarding the supernatant, centrifuging at 20 ℃ for 5min at 400 Xg;

4.4 cells were incubated at 37 ℃ with 5.0% CO at saturated humidity₂Culturing in an incubator.

4.7 the cells were incubated at 37 ℃ with 5.0% CO at saturation humidity₂And continuing culturing in the incubator.

4.8 days 5-21 of culture, observing cell growth state every day, supplementing L500 culture medium containing cytokine (if enough plasma is available, 10% autologous plasma content can be maintained), and maintaining cell concentration 1 × 10⁶cells/ml, 5.0% CO at 37 ℃ under saturated humidity₂The incubator continues to culture until the required number of cells is reached and the cells are harvestedAnd (4) cells. Meanwhile, if the cell volume exceeds 240ml, the cells are transferred to a flask for culture or transferred to a cell culture bag for subculture.

The group is a control experiment group, 700 ten thousand cells are inoculated, the total number of the cells can reach about 7.5 hundred million after being amplified for 21 days, and the requirement of clinical application cannot be met.

EXAMPLE 5 NK cell in vitro expansion-NK cell culture

5.2 discarding the supernatant, centrifuging at 20 ℃ for 5min at 400 Xg;

5.4 cells were incubated at 37 ℃ with 5.0% CO at saturated humidity₂Culturing in an incubator.

5.7 the cells were incubated at 37 ℃ with 5.0% CO at saturation humidity₂And continuing culturing in the incubator.

5.8 days 5-21, observing cell growth state every day, supplementing L500 culture medium containing cytokine (if enough plasma is available, 10% autologous plasma content can be maintained), and maintaining cell concentration 1 × 10⁶cells/ml, 5.0% CO at 37 ℃ under saturated humidity₂The incubator continues to culture until the required number of cells is reached and the cells are harvestedAnd (4) cells. Meanwhile, if the cell volume exceeds 240ml, the cells are transferred to a flask for culture or transferred to a cell culture bag for subculture.

The group is a conventional NK cell culture experimental group, the number of inoculated cells and the number of cells subjected to amplification culture are counted on 0 th, 4 th, 6 th, 8 th, 10 th, 12 th, 14 th, 16 th, 18 th and 21 th days of culture, the cells grow normally, 700 ten thousand cells are inoculated and 21 days of amplification are carried out, the total number of the cells can reach about 45 hundred million, and the requirement of clinical application is met.

Example 6 NK cell phenotype detection

6. NK cell phenotype detection by amplification culture according to the invention

6.2 adding 1mLDPBS to wash once, centrifuging at 400 Xg for 5 minutes, removing the supernatant;

6.4 DPBS washes twice, discards the supernatant, and finally resuspends the cells using 150. mu.L PBS.

Culturing 21-day cells, detecting cultured NK cells by using an AECANovocyte flow cytometer, analyzing data, taking the average value of 5 experimental results, wherein the purities of the NK cells of an experimental group, a control group and a conventional group respectively reach 99.73%, 97.28% and 98.56%, and the purity difference of the NK cells cultured in each group is not large in purity, so that the purity requirements of clinical application can be met. Wherein the phenotype test results of each group of cells are shown in Table 1:

table 1NK cell immunophenotype:

the cell phenotype was examined by the above experiment, and it can be seen that the group of experimentsThe culture method can improve CD56 in NK cells^highCD16^-NK cell subset number ratio, CD56 in control group^highCD16^-The ratio of the number of NK cell subsets is reduced, and the gallic acid and IL-2 can effectively improve CD56^highCD16^-NK cell subpopulation number ratio.

The NK cell subtype cultured by the culture method can be suitable for regulating the body of an immune system or regulating or treating diseases of the immune system.

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.