CN114149969A - NK cell in-vitro culture method for selectively amplifying same-species reactive NK cells - Google Patents

Abstract

The invention discloses an NK cell in-vitro culture method for selectively amplifying homologus reactive NK cells, which comprises the step of culturing the NK cells in a culture solution containing feeder cells; the feeder cells are PMBC which are not matched with the HLA-Cw locus of the NK cells. According to the invention, PMBC which is not matched with NK cell Cw sites is used as a feeder cell, so that NK cell subsets which express KIR phenotypes and can not be combined with the NK cell Cw sites on the surface of the feeder cell in NK cells are activated, and advantage amplification is generated; therefore, in the mating stage before organ transplantation, a donor which is not matched with the recipient Cw site is selected, recipient PMBC is used as a feeder cell to feed donor NK cells, so that the same kind of reactive NK cells can be preferentially expanded, the number of the NK cells can be expanded to 40-70 times of the initial number after 2-3 weeks of culture, and the purity of the same kind of reactive NK cells can reach more than 90%.

Description

NK cell in-vitro culture method for selectively amplifying same-species reactive NK cells

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an NK cell in-vitro culture method for selectively amplifying same-species reactive NK cells.

Background

Natural killer cells (NK cells) are a class of large granular lymphocytes different from T, B lymphocytes, which are the main effector cells of the natural immune system, do not require antigen pre-sensitization, exhibit an immediate effect, and are therefore located at the first line of defense of the body against tumor and viral infection; meanwhile, the immune system can regulate the acquired immunity by secreting various cytokines and chemokines, so the immune system is also a bridge for connecting the natural immunity and the acquired immunity. The activity of NK cells is exerted by virtue of the interaction of various receptor molecules expressed on the surface of the NK cells and corresponding ligands of target cells, wherein the most important is killer cell immunoglobulin-like receptor (KIR).

KIR belongs to immunoglobulin superfamily receptors and is expressed on the surface of NK cells; KIRs are divided into two groups, inhibitory and active, each with their specific extracellular domains, and each binding to a specific ligand (HLA class i antigen), mediating inhibitory or activating responses, respectively. Wherein KIR2DL2/2DL3 and 2DS2 recognize HLA-Cw1, -Cw3, -Cw7, -Cw8, -Cw12, -Cw13, -Cw14 and-Cw 16, and the common characteristic of the group of HLA-class I antigens is that the 80 th amino acid residue is aspartic acid (group C1); and KIR2DL1/2DS1 recognizes HLA-Cw2, -Cw4, -Cw5, -Cw6, -Cw15, Cw-1602, -Cw17, -Cw18, and the common feature of the group of HLA-class I antigens is that the 80 th amino acid residue is lysine (group C2). NK cells recognize autologous HLA-I antigens through KIR to achieve self immune tolerance, when heterogenous cells do not have HLA-I antigens recognized by NK cell surface inhibitory KIR receptors, homoreactivity NK cells are generated, and the homoreactivity NK cells are activated and amplified to kill the heterogenous cells.

In recent years, due to the intensive recognition of NK cell receptor recognition patterns, the role of homoreactive NK cells in allogeneic hematopoietic stem cell transplantation (Allo-HSCT), particularly in mismatched Allo-HSCT, has received much attention.

Ruggeri, 2002, reported in the science journal that when donor KIR was not matched with recipient MHC-I (MHC, major histocompatibility complex, human MHC is referred to as HLA) receptor, the incidence of GVHD (graft versus host disease) was reduced and survival was significantly prolonged without recurrence 5 years after transplantation in the bone marrow transplantation study of acute myeloid leukemia. This significant finding suggests that homoreactive NK resulting from KIR-ligand incompatibility in the donor would bring new hopes for fundamentally solving the problems of GVHD and GVL (graft versus leukemia effect) in bone marrow transplantation. The activity of homotypic reactive NK cells triggered by KIR-ligand incompatibility between a donor and a recipient theoretically has a plurality of beneficial effects on HSCT, and NK cells can effectively reduce the occurrence of GVHD by killing antigen presenting cells such as dendritic cells of the recipient; preventing graft rejection by killing host T cells, facilitating graft implantation; meanwhile, because the activated NK cells have strong anti-tumor effect, the GVL effect can be enhanced, and relapse after transplantation is avoided.

For example, chinese patent application publication No. CN109593711A discloses a method for preparing a transplantable Natural Killer (NK) cell fraction, the method comprising: (a) obtaining a CD3 depleted NK cell fraction that is HLA haploid-matched or HLA mismatched to a subject; (b) culturing said CD3 depleted NK cell fraction in vitro under a plurality of conditions that allow cell proliferation, wherein said plurality of conditions comprise providing a plurality of nutrients, serum, IL-15, and nicotinamide in an amount of 1.0 to 10 millimoles; (c) supplementing said CD3 depleted NK cell fraction with fresh nutrients, serum, IL-15, and nicotinamide 8 to 10 days after step (b) to produce an expanded CD3 depleted NK cell fraction; (d) collecting the expanded CD3 depleted NK cell fraction 14 to 16 days after step (b); and (e) washing and concentrating the expanded CD3 depleted NK cell fraction of step (d); thereby producing a transplantable fraction of NK cells.

However, in this method, the acquisition of "a CD 3-depleted NK cell fraction that is HLA haploid matched or HLA mismatched with a subject" depends on the donation of the donor, which is inconvenient to find the donation source, and on the one hand, the proportion of NK cells required in the donation source is generally low, and the general purification or enrichment method has poor effect.

Disclosure of Invention

The invention aims to provide an NK cell in-vitro culture method for selectively amplifying same-species reactive NK cells, and the method can be used for quickly obtaining the same-species reactive NK cells with the purity of more than 90%.

In order to achieve the purpose, the technical scheme of the invention is as follows:

an NK cell in vitro culture method for selectively amplifying homotypic reactive NK cells comprises the step of culturing NK cells in a culture solution containing feeder cells;

the feeder cells are peripheral blood mononuclear cells which are not matched with the HLA-Cw site of the NK cells.

According to the invention, peripheral blood mononuclear cells which are not matched with the HLA-Cw sites of the NK cells are used as feeder cells, so that NK cell subsets which express KIR phenotypes and can not be combined with the HLA-Cw sites on the surfaces of the feeder cells in the NK cells are activated, and advantage amplification is generated; the part of preferentially amplified NK cell sub-population is the same reactive NK cell, the same reactive NK cell can preferentially kill target cells of the same HLA-Cw sub-population expressed by feeder cells, including tumor cells and activated and proliferated T cells, and can exert graft-versus-leukemia effect and inhibit graft-versus-host reaction.

Therefore, in the matching stage before organ transplantation, a donor which is not matched with HLA-Cw sites of a recipient is selected, and donor NK cells can be directly fed by using peripheral blood mononuclear cells of the recipient as feeder cells, so that the homoreactivity NK cells for transplantation can be preferentially expanded, the time consumption is short, the number of the NK cells can be expanded to 40-70 times of the initial number after 2-3 weeks of culture, the purity of the homoreactivity NK cells can reach more than 90%, and the homoreactivity NK cells have stronger cytotoxicity on activated T cells and tumor cells which are not matched with HLA-Cw sites.

In the above-mentioned NK cell in vitro culture method for selectively amplifying homoreactive NK cells, the feeder cells are previously subjected to a deproliferation treatment before the addition of a culture solution. The optimal state of the feeder cells is a state without proliferation capacity but with the survival of the cells, the survival of the feeder cells can stimulate the preferential amplification of the homoreactivity NK cells which are not matched with the HLA-Cw sites, and the feeder cells without proliferation capacity can not rob the culture resources of the NK cells and can not influence the proliferation of the NK cells.

Preferably, in the above method for in vitro culture of NK cells for selectively amplifying homoreactive NK cells, the feeder cells are treated with mitomycin to disable the proliferation ability of the cultured cells;

alternatively, gamma rays or Co are used₆₀Feeder cells were radio-inactivated to render the cultured cells proliferation incompetent.

Wherein, mitomycin is adopted to treat the feeder cells without a radioactive source, so the implementation is more convenient and safer.

Specifically, the method for treating the feeder cells by using mitomycin comprises the following steps: adjusting the concentration of the feeder cells to 1X 10 with a culture medium⁶～2×10⁶cells/mL, and mitomycin at a final concentration of 15-40. mu.g/mL for 15-45min, followed by washing with PBS to remove mitomycin.

Preferably, the concentration of said feeder cells is adjusted to 2X 10 with a culture medium⁶cells/mL, and mitomycin was added to a final concentration of 25. mu.g/mL for 30min, followed by at least five washes with PBS to remove mitomycin.

Preferably, in the above method for culturing NK cells selectively amplifying homoreactive NK cells in vitro, the feeder cells are stimulated to proliferate to a predetermined amount by phytohemagglutinin, and then the feeder cells are processed for their proliferative capacity. Namely, after the feeder cells are activated and proliferated to a certain number, the feeder cells are processed with the capacity of proliferation removal so as to meet the number requirement and ligand requirement for stimulating the preferential amplification of the same reactive NK cells.

The research of the invention finds that the phytohemagglutinin can stimulate lymphocytes in peripheral blood mononuclear cells to be activated and proliferated, and the expression of ligands MICA/MICB, ULBP-1 and ULBP-4 of NKG2D on the surface of the activated lymphocytes is up-regulated; the expression of the ligand PVR of DNAM-1 is up-regulated, and the expression of ligands ICAM-1 and ICAM-2 of LFA-1 are up-regulated to different degrees; and the up-regulation of the expression of the feeder cell surface ligand can provide certain activation signals for cultured NK cells, and is helpful for promoting the in-vitro amplification of the same reactive NK cells.

In the above method for culturing NK cells selectively amplifying homoreactive NK cells in vitro, the method for stimulating the proliferation of feeder cells by phytohemagglutinin comprises the following steps: the concentration of the feeder cells is adjusted by using a culture solutionAdjusted to 1 × 10⁶～2×10⁶cells/mL, and adding phytohemagglutinin with final concentration of 3-8 μ g/mL and interleukin 2 with final concentration of 100-300U/mL, culturing for 72-96 h.

Preferably, the concentration of said feeder cells is adjusted to 2X 10 with a culture medium⁶cells/mL, and phytohemagglutinin at a final concentration of 5. mu.g/mL and interleukin 2 at a final concentration of 200U/mL were added and cultured for 96 h.

In the method for in vitro culturing of NK cells for selectively amplifying homoreactive NK cells, the concentration ratio of NK cells to feeder cells in the culture solution is 1 (3-7). The appropriate feeder cell concentration can provide a more appropriate microenvironment for the expansion of the NK cells, including the strength of an activating signal, the opportunity of the activating receptor of the NK cells to be combined with a corresponding ligand and the like.

Preferably, the concentration ratio of NK cells to feeder cells in the culture solution is 1: 5.

Preferably, the method for culturing NK cells selectively amplifying homoreactive NK cells in vitro of the present invention comprises the steps of:

(1) enriching NK cells from the donor's peripheral blood;

(2) respectively inoculating NK cells and feeder cells into the culture solution, adding interleukin 2 with the final concentration of 300-800U/mL and interleukin 15 with the final concentration of 50-200ng/mL into the culture solution, and then placing the mixture at 37 ℃ and 5% CO₂And culturing for 2-3 weeks under the condition of saturated humidity.

Preferably, in the above-mentioned NK cell in vitro culture method for selectively amplifying homoreactive NK cells, in step (2), the medium is changed every 3 to 4 days to renew the feeder cells, interleukin 2 and interleukin 15.

Compared with the prior art, the invention has the beneficial effects that:

(1) according to the invention, peripheral blood mononuclear cells which are not matched with the HLA-Cw sites of the NK cells are used as feeder cells, NK cell subsets which express KIR phenotypes and cannot be combined with the HLA-Cw sites on the surfaces of the feeder cells in the NK cells are activated to generate advantage amplification, and the preferentially amplified NK cell subsets are the same reactive NK cells; therefore, in the matching stage before organ transplantation, a donor which is not matched with HLA-Cw sites of a recipient is selected, and donor NK cells can be directly fed by using peripheral blood mononuclear cells of the recipient as feeder cells, so that the homoreactive NK cells for transplantation can be preferentially expanded, the time consumption is short, the number of the NK cells can be expanded to 40-70 times of the initial number after 2-3 weeks of culture, the purity of the obtained homoreactive NK cells can reach more than 90%, and the homoreactive NK cells have stronger cytotoxicity on activated T cells and tumor cells which are not matched with HLA-Cw sites.

(2) In the invention, the feeder cells are subjected to the treatment of the proliferation capacity in advance before being added into the culture solution, the optimal state of the feeder cells is the state without proliferation capacity but the survival state of the cells is maintained, the feeder cells can stimulate the preferential amplification of the same kind of reactive NK cells which are not matched with the HLA-Cw sites of the feeder cells only when the feeder cells survive, and the feeder cells without proliferation capacity can not rob the culture resources of the NK cells and can not influence the proliferation of the NK cells.

(3) In the invention, phytohemagglutinin is adopted to stimulate feeder cells to proliferate to a preset number, and then proliferation removing capacity treatment is carried out; the research of the invention finds that the phytohemagglutinin can stimulate lymphocytes in peripheral blood mononuclear cells to be activated and proliferated, and the expression of ligands MICA/MICB, ULBP-1 and ULBP-4 of NKG2D on the surface of the activated lymphocytes is up-regulated; the expression of the ligand PVR of DNAM-1 is up-regulated, and the expression of ligands ICAM-1 and ICAM-2 of LFA-1 are up-regulated to different degrees; and the up-regulation of the expression of the feeder cell surface ligand can provide certain activation signals for cultured NK cells, and is helpful for promoting the in-vitro amplification of the same reactive NK cells.

Drawings

FIG. 1 is a diagram of gating lymphocytes for flow cytometry analysis using flow cytometer forward scatter and side scatter parameters;

wherein, FS: : FS Lin represents forward scattered light; and SS: : SS Lin represents side scattered light; FS Lin, SS Lin subset represent lymphocyte subpopulations;

FIG. 2 is a graph of the proportion of NK cells in peripheral blood before enrichment by RosetteSep immunodensity gradient centrifugation;

wherein, FITC-CD56 denotes FITC-stained CD56⁺The fluorescence intensity of the cells; PE-CD3 denotes PE-stained CD3^-The fluorescence intensity of the cells; CD56 at Q3⁺CD3^-The cells are NK cells, and the percentage shown is the percentage of the NK cells in all lymphocyte populations; the same applies below;

FIG. 3 is a graph of the proportion of NK cells in the pool after enrichment by RosetteSep immunodensity gradient centrifugation;

FIG. 4 is a graph of the efficiency of NK cell expansion by different feeder cells;

wherein 0W, 1W, 2W and 3W represent, in order, 0 week, 1 week, 2 weeks and 3 weeks; the ordinate represents the fold increase in NK cell number; AUTO denotes the use of autologous PMBC as feeder cells; ALLO indicates the use of allogeneic PMBC as feeder cells; k562 indicates the use of K562 cells as feeder cells; the same applies below;

FIG. 5 is CD3 after NK cell expansion by different feeder cells^-CD56⁺The proportion of NK cells;

wherein the ordinate represents CD3 in the culture system^-CD56⁺Percentage of NK cells;

FIG. 6 is a graph of the effect of expanding NK cells with autologous PMBC as feeder cells on KIR distribution on NK cells;

wherein FITC-KIR2DL2/3 represents FITC-stained KIR2DL 2/3-expressing NK cell subsets, PE-KIR2DL1 represents PE-stained KIR2DL 1-expressing NK cell subsets, the same applies below;

FIG. 7 is a graph of the effect of amplifying NK cells with allogeneic PMBC as feeder cells on KIR distribution on NK cells;

FIG. 8 is a graph of the effect of expanding NK cells with K562 cells as feeder cells on KIR distribution on NK cells;

FIG. 9 is a graph of the gating of all cells for flow cytometry analysis using forward and side scatter parameters for degranulation activity detection;

wherein FSC-H represents forward scattered light; SSC-H denotes side scattered light; FSC-H, SSC-H subset represents the cell population for degranulation analysis;

FIG. 10 is a graph showing the degranulation effect of homoreactive NK cells when leukemia primary cells of patients with Cw site genotype C1/C1 subgroup are used as target cells;

wherein PE-CD107a represents PE-stained CD107a in NK cells⁺The fluorescence intensity of the cells; APC-CD56 represents APC-stained CD56⁺The fluorescence intensity of the cells; at Q2 is CD56⁺CD107a⁺The percentage of cells, which is the percentage of NK cells with degranulation activity in total NK cells, is the same as below;

FIG. 11 is a graph showing the degranulation effect of homoreactive NK cells when PHA-stimulated PBMCs of patients with a Cw locus genotype of C1/C1 subgroup were used as target cells.

Detailed Description

The technical solution of the present invention will be further described in detail with reference to the accompanying drawings and the detailed description.

Example 1

This embodiment is a method for culturing NK cells in vitro, which selectively amplifies homoreactive NK cells, comprising the following steps:

(1) enriching NK cells from the donor's peripheral blood;

enriching NK cells from peripheral blood of a donor by adopting a RosetteSep immune density gradient centrifugation method, wherein the sorting result is shown in a figure 1-3;

as can be seen from FIGS. 1-3, the proportion of NK cells in the donor's peripheral blood prior to sorting was only 10.5%; after enrichment by a RosetteSep immune density gradient centrifugation method, the proportion of NK cells reaches 88.3 percent;

(2) respectively inoculating NK cells and feeder cells into the culture solution, adding interleukin 2 with the final concentration of 300-800U/mL and interleukin 15 with the final concentration of 50-200ng/mL into the culture solution, and then placing the mixture at 37 ℃ and 5% CO₂Culturing under the condition of saturated humidity to obtain the same reactive NK cells;

specifically, the method comprises the following steps:

selecting donor autologous peripheral blood mononuclear cells (autologous PMBC), allogeneic peripheral blood mononuclear cells (allogeneic PMBC) which are not matched with HLA-Cw sites of the donor and K562 cells as feeder cells respectively, and adjusting the concentration of the feeder cells to 2 x 10 by using the culture solution⁶cell/mL, and adding plant with final concentration of 5 μ g/mLHemagglutinin and interleukin 2 of 200U/mL, and culturing for 96h for standby (K562 cells are not treated);

in this example, NK cells were from a 29 year old healthy male donor with HLA-Cw at the C1/C2 subgroup, autologous PMBC were from the same donor as NK cells, and allogeneic PMBC were from peripheral blood in remission from leukemia patients with HLA-Cw at the C1/C1 subgroup.

HLA-C site matching and KIR genotype information are shown in Table 1.

TABLE 1

② continuously adjusting the concentration of the feeder cells after the phytohemagglutinin treatment to 2 x 10 by using the culture solution⁶cell/mL, adding mitomycin C with the final concentration of 25 mug/mL for treatment for 30min, and washing for at least five times by PBS to remove the mitomycin C for later use;

③ adopting SCGM culture solution containing 10% AB serum to adjust the concentration of the enriched NK cells to 5 × 10⁵cell/mL, then inoculating in 6-hole cell culture plate, inoculating 2mL per hole; the feeder cells treated by mitomycin C are added into each cell hole according to the number ratio of NK cells to feeder cells of 1:5, interleukin 2 with the final concentration of 500U/mL and interleukin 15 with the final concentration of 100ng/mL are added into each cell hole, and then the mixture is placed at 37 ℃ and 5% CO₂Culturing under the condition of saturated humidity;

changing the liquid once every 3.5 days, and updating feeder cells, interleukin 2 and interleukin 15;

expansion of dynamic detection cell number in culture process and CD3 in NK cells^-CD56⁺The purity of the cells and the results of the assay are shown in FIGS. 4 and 5.

As shown in fig. 4, the NK cells were grown less at the first week and significantly increased at the second week in the in vitro culture of NK cells; in the third week of culture, the increase in the number of NK cells was even higher, wherein when autologous PMBC was used as feeder cells, the increase in the number of NK cells reached 70 times the initial one, when K562 cells were used as feeder cells, the increase in the number of NK cells reached about 65 times the initial one, and when allogeneic PMBC was used as feeder cells, the increase in the number of NK cells reached about 55 times the initial one.

As shown in FIG. 5, as the culture time was prolonged, CD3 was found in NK cells^-CD56⁺The proportion of cells was gradually increased, wherein the proportion of CD3-CD56+ cells among NK cells expanded when allogeneic PMBC was used as feeder cells was higher, reaching about 95%, compared to autologous PMBC as feeder cells after 2-3 weeks of culture.

As is clear from the above test results, using allogeneic PBMC as a feeder cell has no advantage in promoting the increase of the total number of NK cells, but can preferentially expand CD3-CD56+ cells (i.e., a subgroup of NK cells having KIR subtype mismatched with HLA-CW sites of the feeder cell) in NK cells, so that CD3 in NK cells^-CD56⁺The proportion of cells gradually increases; and the NK cell subset can preferentially kill target cells expressing the same Cw subset as the feeder cells, including tumor cells and activated proliferating T cells, exerting graft-versus-leukemia effect and inhibiting graft-versus-host reaction.

After the culture is completed, KIR2DL1, KIR2DL2/3, KIR3L1 subgroup distribution and NKG2A expression are detected by a flow cytometer, and the detection results are shown in FIG. 6, FIG. 7 and FIG. 8; and the CD107a degranulation test is adopted to detect the functions of the same reactive NK cells, and the detection results are shown in figure 9, figure 10 and figure 11.

As can be seen from FIGS. 6, 7 and 8, since NK cells in this example were derived from healthy donors having HLA-Cw sites of C1/C2 subgroup, autologous PBMC were derived from the same donor as NK cells, while allogeneic PMBC were derived from peripheral blood in remission stage of leukemia patients having HLA-Cw sites of C1/C1 subgroup. When allogeneic PMBC is used as feeder cells, C1 ligand from the surface of the feeder cells binds to a subpopulation of NK cells expressing KIR2DL2/2DL3 recipients, transmitting inhibitory signals, thereby inhibiting proliferation of the subpopulation of NK cells; while the NK cell subset expressing KIR2DL1/2DS1 is preferentially amplified because it lacks the corresponding ligand to bind to it and therefore does not produce inhibitory signals.

For leukemia patients with Cw genotype of C1/C1 subgroup, the amplified NK cell subgroup expressing KIR2DL1/2DS1 is the NK cell subgroup with the same reactivity, and the NK cell subgroup can kill and express tumor cells to play a graft-versus-tumor effect and prevent disease recurrence; but also can kill and activate the proliferated T cells and inhibit graft-versus-host reaction.

As can be seen from FIGS. 9, 10 and 11, when leukemia primary cells of patients with HLA-Cw locus genotype C1/C1 subgroup or PBMCs after PHA stimulation were used as target cells, the degranulation activity of NK cells expanded by C1/C1 allogeneic feeder cells on leukemia primary cells of patients with C1/C1 subgroup or PBMCs after PHA stimulation was 34.1% and 26.5%, respectively.

Claims (10)

1. An NK cell in vitro culture method for selectively amplifying homotypic reactive NK cells is characterized by comprising the step of culturing NK cells in a culture solution containing feeder cells;

the feeder cells are peripheral blood mononuclear cells which are not matched with the HLA-Cw site of the NK cells.

2. The method according to claim 1, wherein said feeder cells are subjected to a degroliferation treatment prior to the addition of said culture medium.

3. The method for in vitro culturing of NK cells for selectively amplifying homoreactive NK cells according to claim 2, wherein the feeder cells are treated with mitomycin to disable proliferation of the cultured cells;

alternatively, gamma rays or Co are used₆₀Feeder cells were radio-inactivated to render the cultured cells proliferation incompetent.

4. The method according to claim 3, wherein the concentration of said feeder cells is adjusted to 1X 10 by using a culture medium⁶～2×10⁶cells/mL, and mitomycin at a final concentration of 15-40. mu.g/mL for 15-45min, followed by washing with PBS to remove mitomycin.

5. The method according to claim 2, wherein the feeder cells are stimulated to proliferate to a predetermined amount by phytohemagglutinin, and then treated for de-proliferative capacity.

6. The method according to claim 5, wherein the concentration of said feeder cells is adjusted to 1X 10 by using a culture medium⁶～2×10⁶cells/mL, and adding phytohemagglutinin with final concentration of 3-8 μ g/mL and interleukin 2 with final concentration of 100-300U/mL, culturing for 72-96 h.

7. The method according to claim 1, wherein the concentration ratio of NK cells to feeder cells in the culture medium is 1 (3-7).

8. The method according to claim 7, wherein the ratio of the concentration of NK cells to feeder cells in the culture medium is 1: 5.

9. The method for culturing NK cells for selectively amplifying homoreactive NK cells according to any one of claims 1 to 8, comprising the steps of:

(1) enriching NK cells from the donor's peripheral blood;

(2) respectively inoculating NK cells and feeder cells into the culture solution, adding interleukin 2 with the final concentration of 300-800U/mL and interleukin 15 with the final concentration of 50-200ng/mL into the culture solution, and then placing the mixture at 37 ℃ and 5% CO₂And culturing for 2-3 weeks under the condition of saturated humidity.

10. The method for in vitro culturing NK cells that selectively amplifies homoreactive NK cells according to claim 9, wherein in the step (2), the medium is changed every 3 to 4 days and the feeder cells, interleukin 2 and interleukin 15 are renewed.

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