CN114958740A - Method for in vitro culture and enrichment of human NK cells - Google Patents

Method for in vitro culture and enrichment of human NK cells Download PDF

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CN114958740A
CN114958740A CN202210630711.1A CN202210630711A CN114958740A CN 114958740 A CN114958740 A CN 114958740A CN 202210630711 A CN202210630711 A CN 202210630711A CN 114958740 A CN114958740 A CN 114958740A
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CN114958740B (en
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张卫红
靳文静
陈瑜
牟颖慧
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Beijing Zhongke Weiyuan Biotechnology Co ltd
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    • C12N5/0646—Natural killers cells [NK], NKT cells
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Abstract

The invention belongs to the field of cell culture, particularly relates to a method for culturing NK cells in vitro, and more particularly relates to a method for directionally amplifying a large number of NK cells from human peripheral blood mononuclear cells by using a combination of stimulators. The technical scheme of the invention has simple components and simple and convenient operation, and the obtained NK cells have high proportion, more proliferation and good state.

Description

Method for in vitro culture and enrichment of human NK cells
Technical Field
The invention belongs to the field of cell culture, particularly relates to a method for in vitro culture and enrichment of human NK cells, and more particularly relates to a method for directionally amplifying a large number of NK cells from human peripheral blood mononuclear cells by using a stimulus.
Background
In 2011, cancer surpasses heart disease, and becomes the first leading cause of death worldwide. WHO published 12 months in 2013, the number of newly added cancer patients worldwide has exceeded 1400 million every year, which is greatly increased compared to 1270 million patients as a statistical result in 2008. At the same time, the number of deaths among cancer patients has increased, from 760 to 820 million in the past. According to the statistics data of the Cancer in 2015 published by the Cancer hospital of Chinese medical academy of sciences, Hejiemitsu of national Cancer center, professor Chenwangqing of national Cancer registration center and the like in the Journal of CA: A Cancer Journal for Clinicians: in 2015, 429.2 ten thousand new cases of tumors are found in our country. It was reported that by 2030, new cases of Cancer increased by 50% to 2160 million people per year (Chen W, Zheng R, Baade P D, et al. Cancer statistics in China,2015[ J ]. CA: A Cancer Journal for Clinicians,2016,66: 118.). The 2016 chinese cancer morbidity and mortality reported by heijic in 2022 showed 406.4 million new cases and 241.4 million total deaths in 2016. (Zheng R, Zhang S, Zeng H, et al Cancer introduction and movement in China,2016[ J ]. Journal of the National Cancer Center,2022,5: 3-4.). The American Cancer society and the International Cancer research institute, United in 2021, proposed in 2020 Global statistical analysis of Cancer published in Journal for Clinicians of CA: A Cancer Journal for providing 1929 million new Cancer cases in 2020, wherein 456 million new cancers in China account for 23.7% of the world; 996 million cases of global death, with 290 million Chinese Cancer deaths accounting for 29% of the total Cancer deaths, with Cancer deaths located first worldwide (Bray F, Sun H, Ferlay J, et al. Global Cancer statistics 2020: GLOBOCAN observations of infections and Cancer works for 36cancers in 185 countries [ J ]. CA: A Cancer Journal for clinics, 2021,71: 212.).
Aiming at tumors, the traditional surgical excision, chemotherapy and radiotherapy have damage to normal tissues, and have limitations and limited effect. The targeted therapy appeared in recent years designs corresponding therapeutic drugs aiming at the well-defined carcinogenic sites on the cellular molecular level, and the drugs enter the body to specifically select the carcinogenic sites to combine with the carcinogenic sites and take effect, so that tumor cells are specifically killed, and normal tissue cells around the tumor are not injured. However, the molecular targeted drugs have the following problems: the effectiveness is low, and certain medicine can only act on specific mutant genotype tumors; the long-term therapeutic effect of targeted drugs is reduced in cases where tumor gene mutations create drug tolerance; may cause serious adverse reactions; some tumors cannot be effectively treated by targeted drugs.
Immunotherapy is just able to solve the above problems. When the immune system is weakened by itself or by tumor cells, it provides an advantage for the development of tumors; the tumor immunotherapy enhances the anti-tumor capability of the tumor microenvironment by mobilizing the immune system of the organism, thereby controlling and killing tumor cells. Tumor immunotherapy is a therapeutic method for controlling and eliminating tumors by restarting and maintaining tumor-immune circulation and restoring normal anti-tumor immune response of the body.
The immune anticancer therapy in 2013 is judged as the first breakthrough of 10 years of Science journal. Since 2013, the immunotherapy has continuously obtained breakthrough progress, and clinical research has also succeeded to a certain extent, so that the immunotherapy is the most promising treatment means in the current tumor treatment field and is expected to become a new conventional treatment method following surgery and chemoradiotherapy methods.
With the rapid development of stem cell biology, immunology, molecular technology, tissue engineering technology and the like, cellular immunotherapy has more and more prominent effects in the treatment of tumors and the like as a safe and effective treatment means. Currently, research and development of novel cell therapy techniques have become an important research field for solving related diseases such as tumor. The cellular immunity treatment is to collect the autoimmune cells of human body, increase the number of the autoimmune cells by thousands of times through in vitro culture, enhance the target killing function, then transfuse the autoimmune cells to human body to kill pathogens, cancer cells and mutated cells in blood and tissues, break immune tolerance, activate and enhance the immunocompetence of the organism, and take the double effects of treatment and health care into consideration.
Tumor immune Cell therapy has experienced a history of nearly 30 years, starting with the 20 th century 80 s treatment of malignant melanoma with IL-2 stimulation of first generation lymphokine activated LAK cells (lymphokine activated Killer cells) found by the Rosenberg team of the national NIH tumor institute, to second generation Cytokine Induced CIK cells (Cytokine activated Killer cells) and natural Killer cells NK, and third generation optimized DC-CIK cells (dendritic Cell-Cytokine activated Killer cells) and TIL cells (tumor infiltrating lymphocytes), until currently heat-contained targeted and well-defined chimeric antigen receptor T cells (CAR-T) and TCR-engineered T cells (TCR-T).
NK cells, natural killer cells, are the core cells of the natural immune system, are the first line of immunological defense of the body against tumor and viral infection, and participate in the immunoregulation function of the body. Unlike specific killing by the adaptive immune system, NK cells are not directed against any specific tumor marker, but are recognized by some features common to tumors, such as MHC-I receptor down-regulation and expression of injury-associated proteins, so that cell therapy drugs developed based on NK cells have broad-spectrum adaptability. After identifying the tumor cells, the NK cells can be independently and rapidly killed directly at the first time without the instruction of an immune system or the cooperation of other immune cells.
Based on the above advantages, NK cells from various sources have been applied to immunotherapy of tumors, including autologous sources, allogeneic sources, cell line sources, umbilical cord blood sources, and iPSC sources, among others.
In the developed clinical researches, the clinical application of NK cells in hemangioma is the most, a plurality of clinical researches aiming at acute myelogenous leukemia all observe clear clinical effects, the total response rate can reach more than 50%, and partial patients can achieve Complete Remission (CR). Good clinical results were also observed for other hematological tumors, such as multiple myeloma, non-hodgkin lymphoma, and some solid tumors, and more importantly, NK cell therapy is highly safe with little observation of any graft versus host reaction (GVHD) and associated toxicity.
The efficacy of NK cells is definite, the safety is high, and the NK cells are favored by clinical researchers for a long time, but the problem that the researchers are troubled is how to obtain enough number of NK cells meeting the clinical requirement. The early mainstream method is to use mitomycin to treat or irradiate inactivated feeder layer cells for co-culture, and has the advantages of good amplification effect and capability of meeting the clinical application requirements, however, the minor residue of mitomycin can cause adverse effects on human bodies in the subsequent clinical application, the irradiation mode has poor use convenience and is not easy to expand, and the feeder cells which are not completely treated are always hidden troubles in the subsequent application. In the later period, with the progress of research, the culture method using pure cytokines is more and more inclined. IL-2 (interleukin-2) is the most used cytokine, and the IL-2 used in large amount in the current culture process is important for the proliferation of NK cells, however, the proportion of regulatory T cells (Treg cells) generated by IL-2 induction is higher relative to the in vivo environment, and the large amount of T cells generated after adoptive immune cell therapy is also one of the important factors for limiting the treatment effect. Therefore, the proportion of NK cells is increased and the proportion of T cells is reduced in the culture process. Researches such as Choi YH and Pencheva-Demireva M prove that the addition of IL-15/IL-18 in a culture system can effectively increase the number of obtained NK cells and remarkably improve the capacity of killing tumor cells in vitro; the studies of M.S.Gokhale et al show that the addition of low doses of IL-12 contributes to the increase of NK cell number; other cytokines, including IL-21, IL-27, etc. are also used, but currently, the cytokine method for culturing NK has not formed a classical method in general, and the finally obtained number is limited, so that the clinical use requirement is difficult to achieve.
Therefore, the development of a high-quality NK cell culture technology can simultaneously meet clinical requirements and ensure safety, has a wide market space, and is very important for improving the cell treatment effect of tumors.
Disclosure of Invention
In order to solve the above problems, it is an object of the present invention, in one aspect, to provide a method for culturing enriched human NK cells, comprising the steps of:
1) PBMCs (peripheral blood mononuclear cells) were isolated from human peripheral blood;
2) magnetically sorting said PBMCs to obtain a suspension of CD3 negative cells depleted of T lymphocytes;
3) culturing the cell suspension in a cell culture flask pre-coated with CD16 antibody by using lymphocyte serum-free medium containing 0-15%, preferably 1-10% human self plasma to obtain the target NK cell suspension,
the concentration of the CD16 antibody used in pre-coating is 5-20 μ g/mL, and IL-2 with a concentration of 100-2000IU/mL, IL-15 with a concentration of 10-100ng/mL, IL-18 with a concentration of 10-100ng/mL, and IL-21 with a concentration of 10-100ng/mL are added to the culture medium.
Preferably, in the method, the culture is carried out by supplementing an activated medium of the same volume as the culture system from day 0 to day 5, particularly from day 3 to day 5 of the culture, and the culture is carried out by supplementing an amplification medium to the culture from day 5 to day 3 of the culture in accordance with the total number of cells, each time the amplification medium is supplemented, so that the cell density is maintained at 0.5X 10 6 /mL-1×10 6 /mL，
The activation culture medium is a lymphocyte serum-free culture medium which is added with IL-2 with the concentration of 100-2000IU/mL, IL-15 with the concentration of 10-100ng/mL, IL-18 with the concentration of 10-100ng/mL and IL-21 with the concentration of 10-100ng/mL and contains 5-10% of human autologous plasma,
the amplification culture medium is a lymphocyte serum-free culture medium containing human autologous plasma, wherein only IL-2 with the concentration of 100-2000IU/mL is added as a cytokine, the human autologous plasma in the amplification culture medium is added by 5-10% at the early stage, and 0-2%, preferably 0-1%, particularly 1% of the human autologous plasma is added from the 7 th day till the human autologous plasma is used.
Preferably, in the method, the culture time is 10-20 days, preferably 12-16 days.
Preferably, in the method, the activation medium is added with IL-2 with the concentration of 500-1500IU/mL, IL-15 with the concentration of 10-30ng/mL, IL-18 with the concentration of 30-70ng/mL and IL-21 with the concentration of 30-70ng/mL, the IL-2 in the amplification medium is 500-1500IU/mL, and the CD16 antibody used in pre-coating is 5-10 mu g/mL.
Preferably, in the method, the activation medium is supplemented with IL-2 at a concentration of 1000IU/mL, IL-15 at a concentration of 15ng/mL, IL-18 at a concentration of 50ng/mL, and IL-21 at a concentration of 50ng/mL, the amplification medium has IL-2 at a concentration of 1000IU/mL, and the CD16 antibody used in the pre-coating has a concentration of 10 μ g/mL.
Preferably, in this method, the activation medium is free of IL-12.
Preferably, in the method, wherein the seeding density of the cell suspension is 0.5X 10 6 /mL-2×10 6 /mL。
Preferably, in the method, the processing of step 1) is: freshly collected peripheral blood was plasma-separated, diluted four-fold with 0.9% sodium chloride injection at ambient temperature, and the diluted blood samples were diluted at a rate of 5:3, centrifuging the upper layer of the human peripheral blood lymphocyte separation solution at 400g for 20 minutes at 20 ℃, sucking out the middle mononuclear cell layer to a new sterile centrifuge tube, diluting the middle mononuclear cell layer with equal volume of 0.9% sodium chloride injection, and centrifuging the middle mononuclear cell layer for 5 minutes at 800g at 25 ℃; the supernatant was discarded, diluted and centrifuged repeatedly once, and after resuspension with an appropriate amount of sodium chloride injection, isolated PBMC were obtained.
Preferably, in the method, the cell culture flask pre-coated with the CD16 antibody is realized by the following method: 0.9% sodium chloride injection containing CD16 antibody at a concentration of 5-10. mu.g/mL was added to the cell culture flasks and incubated at 37 ℃ for 2-6 hours or at 4 ℃ overnight.
Preferably, in the method, the sorting in step 2) is performed by a sorting column.
Preferably, in the method, the sorting is performed by placing the sorting column in a strong magnetic field.
Preferably, in this method, the proportion of NK cells in the obtained objective NK cell suspension to all cells is 80% or more, preferably 90% or more, more preferably 92% or more, 95% or more, 97% or more.
Preferably, in the method, the proliferation multiple of the NK cells in the obtained target NK cell suspension is 1000-3500 times, preferably 2000-3000 times.
In another aspect, the present invention aims to provide a system for culturing an enriched human NK cell suspension comprising: IL-2 at a concentration of 500-1500 IU/mL; IL-15 at a concentration of 10-30 ng/mL; IL-18 at a concentration of 30-70 ng/mL; and IL-21 at a concentration of 30-70 ng/mL.
According to the technical scheme, the obtained NK cells in the cell suspension are large in proliferation quantity and good in activity, and meanwhile, the content proportion of the NK cells is high and the proportion of T cells is small in the target cell suspension based on the total cell number.
Drawings
FIG. 1 is a flow chart showing the results of analysis of NK cell proportion by in vitro stimulation culture using treatment methods A and B.
FIG. 2 is a graph showing the number of amplified NK cells obtained by in vitro stimulation using the treatment methods A and B.
FIG. 3(a) is a flow chart showing the results of NK cell ratio analysis obtained by in vitro stimulation of samples from volunteers V199 cultured with culture medium comprising different combinations of stimuli A1, B1, C1.
FIG. 3(B) is a flow chart showing the results of NK cell ratio analysis obtained by in vitro stimulation of samples from volunteers V200 cultured with culture medium comprising different combinations of stimuli A1, B1, C1.
FIG. 4 is a graph showing the number of expanded NK cells cultured by in vitro stimulation using a culture medium comprising different combinations of stimulators A1, B1, and C1.
FIG. 5(a) is a flow chart showing the results of NK cell proportion analysis obtained by in vitro stimulation of samples from volunteers V201 cultured with culture medium comprising the optimized different stimulus combinations A2, B2, C2.
FIG. 5(B) is a flow chart showing the results of NK cell proportion analysis obtained by in vitro stimulation of samples from volunteers V202 cultured with culture medium comprising the optimized different stimulus combinations A2, B2, C2.
FIG. 6 is a graph showing the expanded number of NK cells cultured by in vitro stimulation with a culture medium comprising the optimized different combinations of stimulators A2, B2, C2.
Detailed Description
The technical scheme of the invention is further illustrated by specific examples, but a person skilled in the art can understand that: the following detailed description and examples are intended to illustrate the invention and should not be construed as limiting the invention in any way. It will be apparent to those skilled in the art that many modifications can be made to the present invention without departing from the spirit thereof, and such modifications are intended to be within the scope of the invention.
The following experimental methods are all the ones which are conventional in the art unless otherwise specified, and are all room temperature (20 ℃ C. to 37 ℃ C.) unless otherwise specified. The experimental materials used are those which can be easily obtained from commercial companies, unless otherwise specified, and the antibodies used in the present invention are easily available from commercial companies.
Example 1 volunteer autologous plasma isolation and peripheral blood lymphocyte (PBMC) isolation
The lymphocytes used in the present invention are derived from the venous peripheral blood of an individual. After the screened individuals are qualified by the physical examination of a clinician, the testers inform the specific project flow and the required blood volume, the volunteers agree and sign an informed consent, and the clinical medical staff take blood from the volunteers. In the experiment, 2 healthy volunteers were selected in each batch, about 50mL of heparin sodium anticoagulation-containing 10mL disposable vacuum blood collection tubes (purchased from BD company, product number: 367874) were used for blood collection, and the blood samples were immediately turned upside down and mixed to prevent blood coagulation.
1) Transferring freshly collected peripheral blood into a 50mL centrifuge tube, centrifuging for 15 minutes at 800g, heating upper plasma at 56 ℃ for 30 minutes for inactivation, centrifuging for 10 minutes at 800g, standing for 15 minutes at low temperature, centrifuging for 20 minutes at 4000g, taking supernatant, and storing at 4 ℃ for later use;
2) the lower layer cell fraction was diluted four-fold with 0.9% sodium chloride injection, and the diluted blood sample was added to a sample previously filled with 15mL of a human peripheral blood lymphocyte separation medium (purchased from tianjin third-year biotechnology limited, product number: HY2015) to avoid interface confusion;
3) the above-mentioned centrifuge tube containing the human peripheral blood lymphocyte separation liquid and the blood sample was centrifuged at 20 ℃ by a high-speed refrigerated centrifuge (purchased from thermo fisher, trade name: 75004524), setting the speed to be increased to 1 gear and decreased to 4 gears, and centrifuging for 20 minutes at 400 g;
dividing the centrifuged sample into four layers, namely a red blood cell layer, a lymphocyte separation liquid layer, a white cloudy mononuclear cell (including lymphocytes and monocytes) layer and a plasma layer from the bottom of the tube to the top in sequence, sucking and discarding the uppermost layer of plasma by using a serum pipette, carefully sucking out the mononuclear cell layer and transferring the mononuclear cell layer to a new sterile centrifuge tube, thereby obtaining crude pure PBMC cells;
4) diluting crude pure PBMC cells with an equal volume of 0.9% sodium chloride injection, followed by centrifugation at 800g for 5 minutes at 25 deg.C; discarding the supernatant, and repeating the above steps once; adding a proper amount of sodium chloride injection (containing human serum albumin with the final concentration of 0.2%) to resuspend the cells, and then counting; isolated PBMC cells were obtained for use.
Example 2T cell depletion
The T lymphocytes in the PBMC cells separated by the method are removed by an immunomagnetic bead method for subsequent culture.
1) The isolated PBMC cells obtained in example 1 were first treated at 1X 10 intervals 7 The cells were mixed with 20. mu.L of CD3 antibody-coupled magnetic beads (purchased from Meitian whirlwind Biotechnology Co., Ltd., Germany, trade name: 130-. Then for each 10 7 1-2mL of buffer was added to each cell and centrifuged at 800g for 5 minutes at room temperature.
2) Carefully aspirate the cell supernatant with a pipette and resuspend the cell pellet by adding 500. mu.L buffer (typically less than 1.25X 10) 8 Resuspend individual cells in 500. mu.L buffer); a 30 mu m disposable sterile filter mesh (purchased from German America whirlwind biotechnology limited, commodity number: 130-; the cell suspension was then added and allowed to flow down naturally, thereby obtaining CD 3-negative PBMC cells, as follows.
3) The LD separation column was placed under a strong magnetic field (OctMACS Separator, purchased from 130-090-976, America, whirlwind, Inc., Germany), and a 30 μm filter was placed thereon, which was rinsed 1 time with 2mL of buffer solution until the buffer solution completely flowed out of the separation column, to obtain a treated LD separation column for use.
4) Slowly adding the PBMC cells obtained in the step 2) and combined with the CD3 antibody coupled magnetic beads into the LD sorting column processed in the step 3), wherein the T cells combined with the CD3 antibody coupled magnetic beads are adsorbed in the LD sorting column under the action of a magnetic field, and other cells not combined with the magnetic beads flow out of the sorting column, namely flow-through cell suspension. And after the cell suspension completely naturally flows down, washing the sorting column by using 1mL of buffer solution, and after the cell suspension completely flows down, repeatedly washing once to ensure that the cells which are not combined with the magnetic beads in the sorting column are completely eluted out of the sorting column, thereby obtaining the cell suspension with negative CD3 (CD 3-).
5) The resulting cell suspension was centrifuged at 800g for 5 minutes at room temperature and resuspended in lymphocyte serum-free medium (purchased from Corning, Inc., 88-581-CM) containing 10% human autologous plasma as described in example 1 and IL-2 (purchased from Beijing Tetracycline biopharmaceutical) at a final concentration of 1000IU/mL, counted to a concentration of 0.5-2X 10 6 cells/mL of cell suspension, i.e. CD3 negative cell suspension, in a total volume of no less than 20mL and no more than 30mL for subsequent stimulation treatment.
Example 3 in vitro stimulation of NK cell expansion
The solution of the CD16 antibody for coating was prepared simultaneously with the preparation of the cells obtained in step 5) of example 2, specifically, CD16 antibody (concentration of 1mg/mL, purchased from the same marine company) was diluted to 8mL of 0.9% sodium chloride injection at a final concentration of 10 μ g/mL, and the prepared 0.9% sodium chloride injection containing CD16 antibody at a final concentration of 10 μ g/mL was added to a cell culture flask, followed by incubation at 37 ℃ for 2-6 hours or overnight incubation at 4 ℃ for coating. The coating solution was removed by aspiration and rinsed 1 time with 10ml of 0.9% sodium chloride injection to obtain a T75 cell culture flask coated with CD16 antibody.
The cells obtained in step 5) of example 2 were placed in a T75 cell culture flask pre-coated with CD16 antibody, wherein the culture medium used was a lymphocyte serum-free medium (purchased from Corning, 88-581-CM) containing IL-12, IL-15, IL-18, and IL-21 at final concentrations of 10ng/mL, 15ng/mL, 50ng/mL, respectively; at 37 deg.C, 5% CO 2 And 100% humidityCulturing under the condition; the two treatment methods of A, B are respectively adopted for fluid replacement culture:
A. culturing from day 0 to day 16, and maintaining at cell density of 0.5-1 × 10 every 2-3 days 6 cell/mL (wherein the specific cell density is determined by the proliferation rate of the sample cells, and the cell density is maintained at 0.5X 10 when the proliferation rate of the sample cells is high 6 Cell density was maintained at 1X 10 cells/mL when the proliferation rate of the sample cells was high 6 cells/mL) was replenished, and each replenishment was performed using an activation medium containing IL-2 at a final concentration of 1000IU/mL, IL-12 at 10ng/mL, IL-15 at 15ng/mL, IL-18 at 50ng/mL, and IL-21 at 50ng/mL in a lymphocyte serum-free medium (purchased from Corning, 88-581-CM), 10% human autologous plasma was added on day 3, 5% human autologous plasma was added on day 5, and 1% human autologous plasma was added on day 7 until completion;
B. supplementing the culture medium by using the same volume of the activated culture medium as that of the culture system on day 0 (initial culture volume) from day 3, and continuing the culture until day 5; the day 5 fluid replacement was changed to an amplification medium containing IL-2 at a final concentration of 1000IU/mL in lymphocyte serum-free medium (purchased from Corning, 88-581-CM), and the culture was continued until day 16, and the cell density was maintained at 0.5-1X 10 per 2-3 days 6 Supplementing the solution in a cell/mL mode, adding 10% of human autologous plasma on day 3, adding 5% of human autologous plasma on day 5, and adding 1% of human autologous plasma on day 7 till the human autologous plasma is used up;
after the above culture expansion to 14-16 days, respectively counting the cells cultured by A, B treatment modes, each group taking not less than 5 × 10 5 The individual cells are ready for subsequent flow cytometry analysis and detection, and the quantity and the cell proportion of NK cells generated by stimulation are qualitatively and quantitatively detected.
Example 4 Effect of mode of addition of stimulators on the proportion of NK cells after expansion by in vitro stimulation
According to the culture method provided in example 3, samples were taken at day 14 after the culture of NK cells for flow cytometry, respectively, to evaluate the cultured NK cell subsets.
Taking cultured cells 5X 10 5 After centrifugation at 600g for 5 minutes at room temperature and discarding the supernatant, the cells were washed with 1mL of 0.9% sodium chloride injection, centrifuged and the supernatant discarded, and then resuspended with 100. mu.L of 0.9% sodium chloride injection, 1. mu.L each of FITC-anti-CD3 antibody (available from Biolegend, trade name: 300306), PE-anti-CD56 antibody (available from Biolegend, trade name: 362508) and APC-anti-CD16 antibody (available from Biolegend, trade name: 302012) was added, incubated at room temperature for 15 minutes, washed with 1mL of 0.9% sodium chloride injection, and the supernatant discarded after centrifugation at 600g for 5 minutes. Resuspend with 500. mu.L of 0.9% sodium chloride injection, add to flow tube (from Haiman) and detect in a dual laser flow cytometer (from Beckman Coulter, Inc.: CytoFLEX, the same below), and analyze the data with CytoExpert. The results are shown in figure 1 and table 1 below.
TABLE 1 analysis of NK cell proportion after in vitro stimulation culture
(the unit is the percentage of the cells in all cells)
Figure BDA0003679560760000091
Wherein V193, V194 represent samples from different volunteers, the same applies below.
The result shows that the NK cell proportion of B treatment is higher and the purity is better. Compared with the treatment mode A in which the activation medium is added all the time to the 16 th day during fluid replacement, the treatment mode B in which the activation medium is added to the 5 th day and the amplification medium is changed to the 16 th day during fluid replacement on the 5 th day has better effect.
Example 5 Effect of mode of addition of stimuli on the number of cells expanded by in vitro stimulation
The cell suspensions at 14 days after the culture in example 3 were counted, respectively, and analyzed and detected using a cell counter. The results are shown in figure 2 and table 2 below.
TABLE 2 results of cell number analysis after in vitro stimulation
(the unit is the multiplication factor of the cells, i.e., the ratio of the number of cells after culture to the number of cells before culture.)
V193 V194
A treatment 1330.61 2469.98
B treatment 2245.28 3757.65
The results show that proliferation is better with B treatment. That is, it is demonstrated that NK cell proliferation is better when the culture is continued to the 14 th to 16 th days by using the activation medium and then the amplification medium.
Example 6 irritant combination comparative 1
The cells obtained in step 5) of example 2 were treated with the following combinations of stimuli a1, B1, and C1, respectively, in order to compare NK cell proliferation levels and cell ratios under different combinations of stimuli. The specific addition or culture conditions are as follows:
A1. culturing in a cell culture flask pre-coated with CD16 antibody: while preparing the cells obtained in step 5) of example 2, CD16 antibody (concentration 1mg/mL, purchased from Hokkaido) was diluted to a final concentration of 10. mu.g/mL in 8mL of 0.9% sodium chloride injection and incubated at 37 ℃ for 1-3 hours; the coating solution was removed by aspiration, 10mL of 0.9% sodium chloride injection was added, and the mixture was rinsed 1 time. Examples of the invention2, step 5) according to 0.5X 10 6 /mL-2×10 6 Adding the concentration of/mL into a T75 culture flask pre-coated with a CD16 antibody for culture, wherein the culture medium uses the activation medium in example 3;
B1. removing IL-15 on the basis of A1;
C1. removing IL-18 on the basis of A1;
the cells treated with the three activation media A1, B1 and C1 respectively at 37 deg.C and 5% CO 2 And cultured under 100% humidity conditions for 5 days. The amplification medium in example 3 was changed to continue amplification for 14-16 days. Counting the cultured cells, each at least 5 × 10 5 The individual cells are ready for subsequent flow cytometry analysis and detection, and the quantity and the cell proportion of NK cells generated by stimulation are qualitatively and quantitatively detected. The results of measuring the NK cell ratio are shown in fig. 3(a), fig. 3(b), and table 3 below.
TABLE 3 analysis of NK cell proportion after in vitro stimulation culture
(the unit is the percentage of the cells in all cells)
Figure BDA0003679560760000101
FIG. 3(a) is a flow chart showing the results of analysis of the proportion of NK cells by in vitro stimulation culture using a culture medium containing different combinations of stimulators A1, B1 and C1. In particular to the results of the CD3-CD56+ and CD16+ CD56+ ratio of plasma from volunteer V199 separated, magnetically sorted and cultured by adding different cytokines to day 14. FIG. 3(B) is a flow chart showing the results of analysis of the proportion of NK cells by in vitro stimulation culture using a culture medium containing different combinations of stimulators A1, B1 and C1. In particular to the results of the ratio of CD3-CD56+ and CD16+ CD56+ when plasma from volunteer V200 was separated, magnetically sorted and cultured by adding different cytokines to day 14.
The results show that the proportion of each group of NK cells is not greatly different on the 14 th day after treatment and culture, the proportion of CD3-CD56+ NK cells is more than 95%, and the purity is high. Of these, the proportion of CD16+ CD56+ NK cells was slightly lower upon a1 treatment.
In addition, NK cell number results are shown in FIG. 4 and Table 4 below.
TABLE 4 results of cell number analysis after in vitro stimulation
(the unit is the multiplication factor of the cells, i.e., the ratio of the number of cells after culture to the number of cells before culture.)
V199 V200
A1 645.95 2601.63
B1 2091.50 3542.93
C1 304.30 1225.13
The results show that, at 14 days after the treatment and culture, the B1 group, namely the culture medium (without IL-15) containing IL-2 with the final concentration of 1000IU/mL, IL-12 with the final concentration of 10ng/mL, IL-18 with the final concentration of 50ng/mL and IL-21 with the final concentration of 50ng/mL, has better cell proliferation and remarkable advantages, and compared with the cell number before the culture, the cell number after the culture can reach more than 2000 times.
From example 6 it can be seen that the CD16 antibody pre-coated and using only IL-2 and IL-12, IL-18, IL-21 added medium can produce a higher number of NK cells, further following the other stimulus combinations compared to the currently optimal combination.
Example 7 combination of stimulants comparative 2
The cells obtained in step 5) of example 2 were treated with the following combinations of stimuli a2, B2, and C2, respectively, in order to compare NK cell proliferation levels and cell ratios under different combinations of stimuli. The specific addition or culture conditions are as follows:
A2. culturing in a cell culture flask pre-coated with CD16 antibody: while preparing the cells obtained in step 5) of example 2, the CD16 antibody (concentration 1mg/mL, purchased from Hokkaido) was diluted to a final concentration of 10. mu.g/mL in 8mL of 0.9% sodium chloride injection and incubated at 37 ℃ for 2 hours; before use, 10mL of 0.9% sodium chloride injection was added and rinsed 1 time. The cells obtained in step 5) of example 2 were cultured at 0.5X 10 6 /mL-2×10 6 The culture was carried out by adding to a T75 flask pre-coated with a CD16 antibody at a concentration of/mL, using the B1 stimulus-combined activation medium preferred in example 6, i.e., a medium containing IL-2 (final concentration: 1000IU/mL), IL-12 (final concentration: 10ng/mL), IL-18 (final concentration: 50ng/mL) and IL-21 (final concentration: 50ng/mL) in lymphocyte serum-free medium (purchased from Corning, Inc., 88-581-CM), which had the same composition as the optimal B1 in example 6;
B2. IL-21(50ng/mL) was replaced with IL-15(15ng/mL) based on A2, i.e., a medium containing IL-2 (final concentration: 1000IU/mL), IL-12 (final concentration: 10ng/mL), IL-15 (final concentration: 15ng/mL) and IL-18 (final concentration: 50ng/mL) in lymphocyte serum-free medium (purchased from Corning, 88-581-CM);
C2. IL-12(10ng/mL) was replaced with IL-15(15ng/mL) based on A2, i.e., a medium containing IL-2 (final concentration: 1000IU/mL), IL-15 (final concentration: 15ng/mL), IL-18 (final concentration: 50ng/mL) and IL-21 (final concentration: 50ng/mL) in lymphocyte serum-free medium (purchased from Corning, 88-581-CM).
Passing through the above-mentioned A2 and B2. C2 cells treated with three activation media at 37 deg.C with 5% CO 2 And cultured under 100% humidity conditions for 5 days. The amplification medium in example 3 was changed to continue amplification for 14-16 days. Counting the cultured cells, each at least 5 × 10 5 The individual cells are ready for subsequent flow cytometry analysis and detection, and the quantity and the cell proportion of NK cells generated by stimulation are qualitatively and quantitatively detected. Among them, the results of measuring the NK cell ratio are shown in fig. 5(a), fig. 5(b), and table 5 below.
TABLE 5 NK cell proportion analysis results after stimulus combination optimization
(the unit is the percentage of the cells in all cells)
Figure BDA0003679560760000121
FIG. 5(a) is a flow chart showing the results of NK cell ratio analysis by in vitro stimulation culture with culture medium containing optimized different combinations of stimulators A2, B2, C2, in particular the results of CD3-CD56+ and CD16+ CD56+ ratios of plasma from volunteer V201 separated, magnetically sorted and cultured by adding different cytokines to day 14. FIG. 5(B) is a flow chart showing the results of NK cell ratio analysis by in vitro stimulation culture with culture medium containing optimized different combinations of stimulators A2, B2 and C2, specifically the results of CD3-CD56+ and CD16+ CD56+ ratios of plasma from volunteer V202 separated, magnetically sorted and cultured by adding different cytokines up to day 14.
TABLE 6 results of cell number analysis after in vitro stimulation
(the unit is the multiplication factor of the cells, i.e., the ratio of the number of cells after culture to the number of cells before culture.)
V201 V202
A2 treatment 779.33 1508.79
B2 treatment 86.40 209.35
C2 treatment 1302.08 3026.23
According to the embodiment, the C2 group is the optimal combination, the purity of the cultured cells in the treatment is as high as more than 98% on the 14 th day, and compared with other groups, the cultured cells have higher proliferation fold and higher number of NK cells, and more than 200 hundred million cells can be obtained according to the estimation that 2000 ten thousand cells are initially cultured in every 50mL of human peripheral blood, so that the requirement of clinical multiple treatment can be met. Meanwhile, the additive components are less, the operation is simple and convenient, and the clinical application is easier.

Claims (15)

1. A method for culturing enriched human NK cells, comprising the steps of:
1) separating PBMCs from human peripheral blood;
2) magnetically sorting said PBMCs to obtain a suspension of CD3 negative cells depleted of T lymphocytes;
3) culturing the cell suspension in a cell culture flask pre-coated with a CD16 antibody by using a lymphocyte serum-free culture medium containing 0-15% of human autologous plasma to obtain a target NK cell suspension,
the concentration of the CD16 antibody used in pre-coating was 5-20 μ g/mL,
the culture medium contains IL-2 with the concentration of 100-2000IU/mL, IL-15 with the concentration of 10-100ng/mL, IL-18 with the concentration of 10-100ng/mL and IL-21 with the concentration of 10-100 ng/mL.
2. The method according to claim 1, wherein the culture is carried out by supplementing an activated medium in an amount equal to that of the culture system on day 0 from day 0 to day 5 of the culture, and the culture is carried out by supplementing an amplification medium every 2 to 3 days from day 5 to the end of the culture, each time with the amplification medium, so that the cell density is maintained at 0.5X 10 6 /mL-1×10 6 /mL，
The activation culture medium is a lymphocyte serum-free culture medium containing 5-10% of human autologous plasma, wherein the lymphocyte serum-free culture medium contains IL-2 with the concentration of 100-2000IU/mL, IL-15 with the concentration of 10-100ng/mL, IL-18 with the concentration of 10-100ng/mL and IL-21 with the concentration of 10-100ng/mL,
the amplification culture medium is a lymphocyte serum-free culture medium containing human autologous plasma only containing IL-2 with the concentration of 100-2000IU/mL as a cytokine, wherein the amplification culture medium contains 5-10% of the human autologous plasma from day 0 to day 7, and contains 0-2% of the human autologous plasma from day 7.
3. The method of claim 2, wherein the activation medium is supplemented from day 3 to day 5 of culture.
4. The method according to claim 1 or 2, wherein the cultivation time is 10 to 20 days, preferably 12 to 16 days.
5. The method according to claim 1 or 2, wherein the activation medium comprises IL-2 at a concentration of 500-1500IU/mL, IL-15 at a concentration of 10-30ng/mL, IL-18 at a concentration of 30-70ng/mL, and IL-21 at a concentration of 30-70ng/mL, the IL-2 in the amplification medium is at a concentration of 500-1500IU/mL, and the CD16 antibody used in the pre-coating is at a concentration of 5-10 μ g/mL.
6. The method of claim 5, wherein the activation medium comprises IL-2 at a concentration of 1000IU/mL, IL-15 at a concentration of 15ng/mL, IL-18 at a concentration of 50ng/mL, and IL-21 at a concentration of 50ng/mL, the amplification medium comprises IL-2 at a concentration of 1000IU/mL, and the CD16 antibody used in the pre-coating comprises 10 μ g/mL.
7. The method of claim 1 or 2, wherein the activation medium is free of IL-12.
8. The method of claim 1 or 2, wherein the seeding density of the cell suspension is 0.5 x 10 6 /mL-2×10 6 /mL。
9. The method according to claim 1 or 2, wherein the processing of step 1) is: separating plasma from freshly collected peripheral blood, diluting the plasma by four times by using normal-temperature 0.9% sodium chloride injection, slowly adding the diluted blood sample to the upper layer of the human peripheral blood lymphocyte separation liquid in a ratio of 5:3, centrifuging the blood sample at the temperature of 20 ℃ for 20 minutes at 400g, sucking out the middle mononuclear cell layer to a new sterile centrifuge tube, diluting the blood sample by using the equal volume of 0.9% sodium chloride injection, centrifuging the blood sample at the temperature of 25 ℃ for 5 minutes at 800g, discarding the supernatant, repeatedly diluting and centrifuging the blood sample once, and adding the sodium chloride injection to resuspend the blood sample to obtain separated PBMC.
10. The method according to claim 1 or 2, wherein the cell culture flask pre-coated with the CD16 antibody is effected by: 0.9% sodium chloride injection containing CD16 antibody at a concentration of 5-10. mu.g/mL was added to the cell culture flasks and incubated at 37 ℃ for 2-6 hours or at 4 ℃ overnight.
11. The method of claim 1 or 2, wherein the sorting in step 2) is accomplished by a sorting column.
12. The method of claim 11, wherein said sorting is accomplished by placing a sorting column in a strong magnetic field.
13. The method according to claim 1 or 2, wherein the proportion of NK cells in the resulting NK cell suspension of interest to all cells is above 80%, preferably above 90%, more preferably above 92%, above 95%, above 97%.
14. The method according to claim 1 or 2, wherein the resulting NK cell suspension of interest has a proliferation multiple of NK cells of 1000 to 3500, preferably 2000 to 3000.
15. A system for culturing an enriched human NK cell suspension comprising:
IL-2 at a concentration of 500-1500 IU/mL;
IL-15 at a concentration of 10-30 ng/mL;
IL-18 at a concentration of 30-70 ng/mL; and
IL-21 at a concentration of 30-70 ng/mL.
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