In vitro expansion method of umbilical cord blood NK cells and its kit and application

Abstract

The present invention discloses an in vitro expansion method of cord blood NK cells and a kit and application thereof. The in vitro expansion method of cord blood NK cells provided by the present invention includes the process of activating and culturing cord blood NK cells from separated cord blood mononuclear cells and proliferating cord blood NK cells. Compared with the existing methods for separating and expanding cord blood NK cells, the present invention does not require cell sorting or trophoblast cells, is simple to operate, has good versatility, and is low in cost. The reagents used do not contain animal-derived components, have good safety, and have a high NK cell yield. The purity of the cord blood NK cells obtained is more than 90%, and the total number of cells can reach 10 10‑11 The umbilical cord blood has high killing power on tumor cells. The present invention will play a role in the preparation of tumor immunotherapy drugs and tumor immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell engineering, and in particular relates to an in vitro amplification method of cord blood NK cells and a kit thereof, and applications thereof in the preparation of tumor immunotherapy drugs and tumor immunotherapy. Background Art

[0002] Natural killer cells (NK) are the third type of lymphocytes besides T and B cells. Their cell morphology is also different from that of T and B cells. NK cells are large granular lymphocytes with azure granules in the cytoplasm. They are widely present in lymphoid organs and peripheral tissues. They account for about 5%-10% of the total number of lymphocytes in normal peripheral blood, 1%-2% in the spleen, and NK cells are also present in lymph nodes and bone marrow. As an important component of human natural immunity, NK cells are important immune regulatory cells that resist infection and prevent cell malignant transformation. They play a very important role in immune surveillance and immune defense. Long-term studies have shown that in the occurrence of certain diseases, such as tumors, autoimmune diseases, aging-related diseases and HIV infection, the proportion, number and function of NK cells will decrease. In 2012, the Centers for Disease Control and Prevention in the United States reported that the activity and number of NK cells are important guarantees for human health. The onset of almost all diseases is related to the obvious lack of NK cell activity. When people suffer from a certain disease, whether it is chronic, sporadic or acute, the activity of NK cells is below the average level. Restoring NK cells to a high level of activity is a better way to treat diseases. Unlike T lymphocytes, NK cells can directly dissolve and destroy tumor cells and virus-infected cells without prior stimulation. They exert cytolytic effects by secreting perforins, serine proteases such as granzymes A and B, chondroitin sulfate proteoglycans and other molecules to degrade cell membranes and destroy the integrity of target cells. FasL (Fas ligand) and TRAIL (TNF-related opoptosis-inducing ligand) expressed on the surface of NK cells can cause apoptosis of target cells by binding to receptors on target cells. After activation, NK cells will release a large amount of cytokines such as interferon-γ and tumor necrosis factor-α. In addition, they bind to the Fc segment of antibodies through cell membrane FcγRIII (CD16) to mediate antibody-dependent cytotoxicity. With the understanding of the biological functions and activation mechanisms of NK cells, immunotherapy using NK cells as targets is of great significance for the early prevention, control and treatment of diseases.

[0003] In recent years, immunotherapy has become the fourth treatment mode after surgery, chemoradiotherapy and endocrine therapy, and has gradually received attention. As a type of immunotherapy, adoptive cell immunotherapy has made great progress in clinical research. At present, clinical studies on the application of autologous NK cell adoptive immunotherapy in blood tumors (acute myeloid leukemia, acute lymphocytic leukemia, etc.) and solid tumors (glioma, renal cancer and malignant melanoma) have been carried out, and the safety has been confirmed, but the results of clinical trials are not satisfactory. After autologous peripheral blood is sorted and re-infused, it is observed that the killing of tumor cells by NK cells in vivo is enhanced, but the survival rate and recurrence rate are not improved compared with the control group, which may be related to the easy formation of immune escape of autologous NK cells in the process of tumor development. In addition, the subjects of clinical trials of NK cell adoptive immunotherapy are often extremely advanced patients who have failed multiple chemotherapy, with heavy tumor load, poor body immunity, and serious defects in NK cell function. To improve the clinical treatment efficacy of NK cells, it is necessary to change the previous treatment strategy. In order to overcome these defects, allogeneic NK cells are a better choice. Some scholars have studied the treatment of leukemia patients by transplanting allogeneic NK cells and found that it can delay relapse and does not cause graft-versus-host disease, which has attracted people's attention to the allogeneic reactivity of NK cells. According to the results of current clinical studies, allogeneic NK cells are significantly superior to autologous NK cells in terms of complete remission rate, event-free survival, relapse and mortality, and allogeneic NK cells are not accompanied by graft-versus-host reaction. The killing ability of NK cells against tumor cells depends on the signal balance regulation between activating receptors and inhibitory receptors on the surface of NK cells. The killer cell immunoglobulin-like receptor (KIR) is composed of a family of receptors that bind to major histocompatibility class I (MHC-I), which plays an important role in regulating the activation threshold of human NK cells. Recently, more and more studies have shown that the use of KIR receptor / MHC-I ligand mismatch to screen the best donor can greatly enhance the killing ability of NK cells against recipient tumor cells and improve the effect of NK cell adoptive immunotherapy. At present, the research on KIR-based donor selection to improve the transplantation of hematopoietic stem cells / bone marrow and adoptive immunotherapy of tumors has attracted much attention. Allogeneic NK infusion has become a new strategy for NK cell adoptive immunotherapy in clinical practice (Lim O, Jung MY, Hwang YK, Shin EC: Present and Future of Allogeneic Natural Killer Cell Therapy. Frontiers in immunology 2015, 6: 286.).Umbilical cord blood is a readily available heterologous resource with no ethical issues and abundant sources. It is easier to find the best donor. In addition, the T lymphocytes in umbilical cord blood are immature and mostly immature cells. The probability of acute and chronic graft-versus-host disease after transplantation is low and the degree is mild. Therefore, umbilical cord blood has become a very promising source of heterologous NK cells (Shaim H, Yvon E: Cord blood: a promising source of allogeneic natural killer cells for immunotherapy. Cytotherapy 2015, 17(1): 1-2.). Clinical infusion of NK cells requires sufficient cell purity and cell number, but the proportion of NK cells in umbilical cord blood cells is low and the number is also small. It is necessary to separate and culture amplify in vitro to obtain a large number of high-purity NK cells to meet clinical applications. Therefore, how to obtain high-purity and high-quality NK cells has become the key to NK cell adoptive immunotherapy (Klingemann H: Challenges of cancer therapy with natural killer cells. Cytotherapy 2015, 17(3): 245-249.).

[0004] There are currently two main methods for isolating and expanding NK cells from cord blood (e.g. Figure 1 1. NK cells account for about 10%-20% of lymphocytes in cord blood, and their functions are immature, with low ability to kill tumor cells. NK cells can be sorted and enriched by flow cytometry or magnetic bead sorting, and then the number of NK cells and their ability to kill tumors can be increased by in vitro amplification. Celgene Cellular Therapeutics uses lymphocyte separation fluid to separate mononuclear cells from frozen and thawed cord blood. NK enrichment kit (StemCell Technologies, the kit contains CD3, CD4, CD14, CD19, CD20, CD36, CD66b, CD123, HLA-DR and glycophorin A antibodies) was used to separate CD56 + CD3 - NK cells, purity can reach 71%, cell number is 1.5×10 7Each portion of cord blood was then supplemented with trophoblast cells (peripheral blood mononuclear cells and K562 cells treated with mitomycin C) using a starting culture medium (including: IMDM, 10% FBS, 35 mg / mL transferrin, 5 μg / mL insulin, 20 μM ethanolamine, 1 μg / mL unsaturated fatty acids, 1 μg / mL linoleic acid, 0.2 μg / mL palmitic acid, 2.5 μg / mL bovine serum albumin, 0.1 μg / mL phytohemagglutinin, 1% penicillin-streptomycin, 200 IU / mL interleukin-2) and incubated at 37°C with 5% CO 2 After culturing NK cells for 5-7 days under the same conditions, they were transferred to maintenance medium (IMDM, 10% FBS, 2% human AB serum, 1% penicillin-streptomycin, 200 IU / mL interleukin-2) and cultured for 21 days. CD56 + CD3 - NK cell purity>80%, average total cell number is 1.2×10 9 / cord blood (Kang L, Voskinarian-Berse V, Law E, Reddin T, Bhatia M, Hariri A, Ning Y, Dong D, Maguire T, Yarmush M et al: Characterization and ex vivoExpansion of Human Placenta-Derived Natural Killer Cells for Cancer Immunotherapy. Frontiers in immunology 2013, 4: 101.). 2. NK cells originate from hematopoietic stem cells. Umbilical cord blood is rich in hematopoietic stem cells. Hematopoietic stem cells are sorted and enriched using flow cytometry or magnetic bead sorting, and a certain number of mature NK cells are obtained through in vitro differentiation. Glycostem Therapeutics uses the CliniMACS CD34 magnetic bead sorting kit to sort and enrich CD34 + The purity of hematopoietic stem cells was 67% and the number of cells was 3.8×10 6 / part of cord blood, in cell expansion medium I (including: The cells were cultured in medium with 10% human serum, high-dose cytokine combination (SCF, Flt3L, TPO, IL-7, low molecular weight heparin) and low-dose factor combination (GM-CSF, G-CSF, IL-6) for 9 days, then expanded in cell expansion medium II (TPO replaced with IL-15, other components were the same as medium I) for 14 days, and finally differentiated in NK cell differentiation medium (low-dose factor combination replaced with (IL-7, SCF, IL-15 and IL-2, other components were the same as medium I) for 35 days, obtaining a purity of >90% and an average number of 2×10 9 / Cord blood NK cells (Spanholtz J, Preijers F, Tordoir M, Trilsbeek C, Paardekooper J, de Witte T, Schaap N, DolstraH: Clinical-grade generation of active NK cells from cord blood hematopoietic progenitor cells for immunotherapy using a closed-system culture process. PloSone 2011, 6(6):e20740.).

[0005] In summary, the current method for preparing NK cells from cord blood (existing technology) has the following disadvantages:

[0006] 1. Cells need to be sorted. The sorting technology affects the activity of NK cells, and sorting instruments are also required;

[0007] 2. Trophoblast cells are required, and the introduction of K562 cells greatly affects the safety of expanded NK cells;

[0008] 3. Complex operation and poor technical versatility;

[0009] 4. The separation and amplification stages are costly;

[0010] 5. Use animal-derived additives, which are unsafe;

[0011] 6. Low NK cell production. Summary of the invention

[0012] The first object of the present invention is to provide a simple and safe method for in vitro expansion of cord blood NK cells.

[0013] The in vitro expansion method of cord blood NK cells provided by the present invention may include the following steps:

[0014] 1) Activate and culture NK cells from cord blood: Use lymphocyte culture medium to adjust the density of cord blood mononuclear cells to 0.5-5×10 6 At least 1-10 μg / mL zoledronic acid and 200-2000 IU / mL recombinant human interleukin-2 were added and incubated at 36-40°C and 5% CO 2 Culture in a saturated humidity environment for 1 to 5 days and collect the cell fluid;

[0015] 2) Proliferation and culture of NK cells from umbilical cord blood: Obtain cells from the cell fluid in step 1) and use GMP S&XFM TM -CD lymphocyte culture medium to adjust the cell density to 0.5-5×10 6 200-2000 IU / mL recombinant human interleukin-2 was added and the mixture was incubated at 36-38°C and 5% CO 2 Culture in a saturated humidity environment, add fresh culture medium every 2-3 days and adjust the cell density to 0.5-5×10 6 / mL, culture for 14 to 35 days, and harvest the cord blood NK cells.

[0016] The cord blood mononuclear cells used in step 1) are separated from fresh anticoagulated cord blood or frozen resuscitated cord blood, and the separation process is as follows:

[0017] 1.1 Take fresh anticoagulated cord blood or frozen resuscitated cord blood and dilute it with 1-2 times the volume of PBS;

[0018] 1.2 Slowly add an equal volume of diluted cord blood to the lymphocyte separation solution, keeping the interface between the two liquids clear;

[0019] 1.3 Centrifuge at room temperature at 980g for 20-30 minutes;

[0020] 1.4 After centrifugation, the second layer of mononuclear cells in the form of a white film is aspirated and washed with PBS to obtain umbilical cord blood mononuclear cells.

[0021] The lymphocyte culture medium in step 1) can be AIM Medium CTS TM (purchased from LifeTechnology, USA) or GMP S&XFM TM -CD cell culture medium, preferably GMP S&XFM TM -CD cell culture medium.

[0022] When the lymphocyte culture medium in step 1) is AIM Medium CTS TM When the activation culture is preferably: using AIM Medium CTS TMThe lymphocyte culture medium was used to adjust the cell density to 1-3×10 6 1-5 μg / mL zoledronic acid and 500-1500 IU / mL recombinant human interleukin-2 were added and incubated at 36-38°C and 5% CO 2 Cultivate in saturated humidity environment for 2 to 4 days; activation culture is better: use AIM Medium CTS TM The cell density was adjusted to 2 × 10 6 2 μg / mL zoledronic acid and 1000 IU / mL recombinant human interleukin-2 were added and incubated at 37°C and 5% CO 2 Incubate in saturated humidity environment for 3 days.

[0023] When the lymphocyte culture medium in step 1) is GMP S&XFM TM -CD, the activation culture is preferably one of the following:

[0024] A. Use GMP S&XFM TM -CD lymphocyte culture medium to adjust the cell density to 1-3×10 6 1-5 μg / mL zoledronic acid and 500-1500 IU / mL recombinant human interleukin-2 were added and incubated at 36-38°C and 5% CO 2 Cultivate in saturated humidity environment for 2 to 4 days; activation culture is better: use GMP S&XFM TM -CD lymphocyte culture medium to adjust the cell density to 2 × 10 6 2 μg / mL zoledronic acid and 1000 IU / mL recombinant human interleukin-2 were added and incubated at 37°C and 5% CO 2 Cultivate in saturated humidity environment for 3 days;

[0025] B. Use GMP S&XFM TM -CD lymphocyte culture medium to adjust the cell density to 1-3×10 6 / mL, then add 1-5 μg / mL zoledronic acid and 500-1500 IU / mL recombinant human interleukin-2, 1-100 ng / mL (preferably 1-20 ng / mL) recombinant human interleukin-15 and 1-100 ng / mL (preferably 1-20 ng / mL) recombinant human interleukin-18, and incubate at 36°C-38°C and 5% CO 2 Cultivate in saturated humidity environment for 2 to 4 days; activation culture is better: use GMP S&XFM TM -CD lymphocyte culture medium to adjust the cell density to 2 × 10 62 μg / mL zoledronic acid and 1000 IU / mL recombinant human interleukin-2, 10 ng / mL recombinant human interleukin-15, and 10 ng / mL recombinant human interleukin-18 were added and incubated at 37°C and 5% CO 2 Cultivate in saturated humidity environment for 3 days;

[0026] C. Use GMP S&XFM TM -CD lymphocyte culture medium to adjust the cell density to 1-3×10 6 / mL, then add 1-5 μg / mL zoledronic acid and 500-1500 IU / mL recombinant human interleukin-2, 1-100 ng / mL (preferably 1-20 ng / mL) recombinant human interleukin-15 and 1-100 ng / mL (preferably 1-20 ng / mL) recombinant human interleukin-18, at 38.5℃-39.5℃, 5% CO 2 Cultivate in saturated humidity environment for 0.5 to 1 day; activation culture is better: use GMP S&XFM TM -CD lymphocyte culture medium to adjust the cell density to 2 × 10 6 2 μg / mL zoledronic acid and 1000 IU / mL recombinant human interleukin-2, 10 ng / mL recombinant human interleukin-15, and 10 ng / mL recombinant human interleukin-18 were added and incubated at 39°C and 5% CO 2 Incubate in saturated humidity environment for 1 day.

[0027] The step 2) of proliferating and culturing NK cells from cord blood is preferably as follows: using a pipette to transfer the cell solution to a centrifuge tube, centrifuging at 150g for 10 minutes, discarding the supernatant, and using GMP S&XFM TM -CD lymphocyte culture medium was adjusted to a cell density of 0.5 to 2×10 6 / mL (more preferably 1×10 6 / mL), and then add 500-1500 IU / mL (more preferably 1000 IU / mL) of recombinant human interleukin-2, and incubate at 37°C and 5% CO 2 Culture in a saturated humidity environment, add fresh culture medium every 2-3 days to adjust the cell density to 0.5-2×10 6 / mL (more preferably 1×10 6 / mL), and add 500-1500IU / mL (more preferably 1000IU / mL) of recombinant human interleukin-2, and culture for 18-24 days (more preferably 21 days) to obtain cord blood NK cells.

[0028] The dedicated activation medium used in step 1) of the above-mentioned in vitro expansion method of cord blood NK cells also belongs to the content of the invention. The dedicated activation medium is one of the following combinations:

[0029] AIM supplemented with 1-10 μg / mL (preferably 1-5 μg / mL, optimally 2 μg / mL) zoledronic acid and 200-2000 IU / mL (preferably 500-1500 IU / mL, optimally 1000 IU / mL) recombinant human interleukin-2 Medium CTS TM Lymphocyte culture medium;

[0030] GMP S&XFM with 1-10 μg / mL (preferably 1-5 μg / mL, optimally 2 μg / mL) zoledronic acid and 200-2000 IU / mL (preferably 500-1500 IU / mL, optimally 1000 IU / mL) recombinant human interleukin-2 TM -CD lymphocyte culture medium;

[0031] GMP S&XFM with 1-10 μg / mL (preferably 1-5 μg / mL, optimally 2 μg / mL) zoledronic acid and 200-2000 IU / mL (preferably 500-1500 IU / mL, optimally 1000 IU / mL) recombinant human interleukin-2, 1-100 ng / mL (preferably 1-20 ng / mL, optimally 10 ng / mL) recombinant human interleukin-15 and 1-100 ng / mL (preferably 1-20 ng / mL, optimally 10 ng / mL) recombinant human interleukin-18 TM -CD lymphocyte culture medium.

[0032] The special proliferation medium used in step 2) of the in vitro expansion method of cord blood NK cells described above also belongs to the content of the present invention. The special proliferation medium is GMP S&XFM supplemented with 200-2000 IU / mL (preferably 1000 IU / mL) of recombinant human interleukin-2 TM -CD lymphocyte culture medium.

[0033] The cord blood NK cells harvested by the above-mentioned in vitro expansion method also belong to the content of the present invention. The purity of the cells exceeds 90%.

[0034] The use of umbilical cord blood NK cells obtained by the method of the present invention in the preparation of tumor immunotherapy drugs or in tumor immunotherapy also belongs to the content of the present invention.

[0035] Another object of the present invention is to provide an in vitro expansion kit for cord blood NK cells.

[0036] The in vitro expansion kit for cord blood NK cells provided by the present invention mainly includes the following reagents:

[0037] 1) Sample density separation fluid for cord blood NK cells: sample density separation fluid (patent number: ZL 201110456878.2, medical device registration number: Jinghai Xiebei 20150002), or other commercially available lymphocyte separation fluid;

[0038] 2) Umbilical cord blood NK cell activation medium: AIM Medium CTS TM (Life Technology, USA) culture medium or GMP S&XFM TM -CD culture medium (patent application number: 201310082166.8; medical device registration number: Jinghai Xiebei 20150008), as well as zoledronic acid, recombinant human interleukin-2, recombinant human interleukin-15 and recombinant human interleukin-18;

[0039] 3) Umbilical cord blood NK cell proliferation medium: GMP S&XFM TM -CD cell culture medium (patent application number: 201310082166.8; medical device registration number: Jinghai Xiebei 20150008) and recombinant human interleukin-2.

[0040] By adopting the above scheme, the present invention provides an in vitro expansion method of cord blood NK cells and a related kit that are simple and safe to operate. Compared with the existing method for isolating and expanding cord blood NK cells (prior art), the present invention has the following beneficial effects:

[0041] 1) No cell sorting step is required (no need to sort NK cells or hematopoietic stem cells), which simplifies the cell preparation process and greatly reduces the preparation cost of cord blood NK cells;

[0042] 2) No trophoblast cells are required, thus avoiding the safety risks of introducing trophoblast cells to prepare cord blood NK cells;

[0043] 3) The cell culture medium used to prepare NK cells is completely free of animal-derived ingredients, has good safety, and has a clear chemical composition, which increases the stability between batches;

[0044] 4) High NK cell yield, the purity of cord blood NK cells is over 90%, and the total number of cells can reach 10 10-11 Cord blood.

[0045] The in vitro expansion method and kit of umbilical cord blood NK cells of the present invention will play an important role in the preparation of tumor immunotherapy drugs and tumor immunotherapy, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The present invention provides a method for isolating and expanding NK cells from cord blood;

[0047] Figure 2 The present invention is a flow chart of the in vitro expansion method of umbilical cord blood NK cells;

[0048] Figure 3 The cell morphology of fresh cord blood and frozen and resuscitated cord blood NK cells before and after in vitro expansion;

[0049] Figure 4 This is the growth curve of in vitro expansion of fresh cord blood and frozen and resuscitated cord blood NK cells;

[0050] Figure 5 The results of flow cytometry test of the purity of fresh cord blood and frozen and resuscitated cord blood NK cells before and after in vitro expansion;

[0051] Figure 6 The tumor target cell killing effect of fresh cord blood NK cells expanded in vitro and frozen and revived cord blood NK cells. DETAILED DESCRIPTION

[0052] In view of the deficiencies in the prior art of isolating and amplifying NK cells from cord blood, the present invention aims to provide an in vitro method for preparing NK cells from cord blood, and to provide a special activation culture medium and a special proliferation culture medium used in the method.

[0053] The in vitro expansion method of cord blood NK cells of the present invention may include the following steps:

[0054] 1) Activate and culture NK cells from cord blood: Use lymphocyte culture medium (described in detail below) to adjust the density of cord blood mononuclear cells to 0.5-5×10 6 / mL (preferably 1 to 3 × 10 6 / mL, the most preferred is 2×10 6 / mL), inoculate the cell suspension into a cell culture flask; then add 1-10 μg / mL (preferably 1-5 μg / mL, most preferably 2 μg / mL) zoledronic acid and 200-2000 IU / mL (preferably 500-1500 IU / mL, most preferably 1000 IU / mL) recombinant human interleukin-2 (to form a special activation medium), incubate at 36°C-40°C, 5% CO 2 Cultivate in a saturated humidity environment for 1 to 5 days;

[0055] The lymphocyte culture medium in step 1) can be AIM Medium CTS TM (purchased from Life Technology, USA) or GMP S&XFM TM -CD cell culture medium (patent application number: 201310082166.8; medical device registration number: Jinghai Xiebei 20150008), preferably GMP S&XFMTM -CD cell culture medium. Depending on the type of lymphocyte culture medium selected, different special activation culture media are obtained accordingly.

[0056] 1) Choose AIM Medium CTS TM Lymphocyte culture medium, special activation culture medium composed of: AIM supplemented with 1-10 μg / mL (preferably 1-5 μg / mL, most preferably 2 μg / mL) zoledronic acid and 200-2000 IU / mL (preferably 500-1500 IU / mL, most preferably 1000 IU / mL) recombinant human interleukin-2 Medium CTS TM Lymphocyte culture medium.

[0057] Using the dedicated activation culture medium, the activation culture method in step 1) is preferably: using AIM Medium CTS TM The density of cord blood mononuclear cells was adjusted to 0.5-5×10 6 / mL (preferably 1 to 3 × 10 6 / mL, the most preferred is 2×10 6 / mL), the cell suspension was inoculated into a cell culture flask, and then AIM Medium CTS TM The lymphocyte culture medium is supplemented with 1-10 μg / mL (preferably 1-5 μg / mL, most preferably 2 μg / mL) of zoledronic acid and 200-2000 IU / mL (preferably 500-1500 IU / mL, most preferably 1000 IU / mL) of recombinant human interleukin-2 and incubated at 36°C-38°C (preferably 37°C) and 5% CO 2 The culture is carried out in a saturated humidity environment for 1 to 5 days (preferably 2 to 4 days, most preferably 3 days).

[0058] 2) Choose GMP S&XFM TM -CD lymphocyte culture medium, special activation culture medium composed of: GMP S&XFM supplemented with 1-10 μg / mL (preferably 1-5 μg / mL, most preferably 2 μg / mL) zoledronic acid and 200-2000 IU / mL (preferably 500-1500 IU / mL, most preferably 1000 IU / mL) recombinant human interleukin-2 TM -CD lymphocyte culture medium.

[0059] Using the dedicated activation culture medium, the activation culture method in step 1) is more preferably: using GMP S&XFM TM -CD lymphocyte culture medium was used to adjust the density of single cord blood cells to 0.5-5×10 6 / mL (preferably 1 to 3 × 10 6 / mL, the most preferred is 2×10 6 / mL), the cell suspension was inoculated into a cell culture flask, and then GMP S&XFM TM -CD lymphocyte culture medium is supplemented with 1-10 μg / mL (preferably 1-5 μg / mL, most preferably 2 μg / mL) of zoledronic acid and 200-2000 IU / mL (preferably 500-1500 IU / mL, most preferably 1000 IU / mL) of recombinant human interleukin-2, and incubated at 36°C-38°C (preferably 37°C) and 5% CO 2 The culture is carried out in a saturated humidity environment for 1 to 5 days (preferably 2 to 4 days, most preferably 3 days).

[0060] 3) Choose GMP S&XFM TM -CD lymphocyte culture medium, the special activation culture medium is composed of: GMP S&XFM supplemented with 1-10 μg / mL (preferably 1-5 μg / mL, most preferably 2 μg / mL) zoledronic acid and 200-2000 IU / mL (preferably 500-1500 IU / mL, most preferably 1000 IU / mL) recombinant human interleukin-2, 1-100 ng / mL (preferably 1-20 ng / mL, most preferably 10 ng / mL) recombinant human interleukin-15 and 1-100 ng / mL (preferably 1-20 ng / mL, most preferably 10 ng / mL) recombinant human interleukin-18 TM -CD lymphocyte culture medium.

[0061] Using the dedicated activation culture medium, the activation culture method in step 1) is more preferably: using GMP S&XFM TM -CD lymphocyte culture medium was used to adjust the density of cord blood mononuclear cells to 0.5-5×10 6 / mL (preferably 1 to 3 × 10 6 / mL, the most preferred is 2×10 6 / mL), the cell suspension was inoculated into a cell culture flask, and then GMP S&XFM TM -CD lymphocyte culture medium is supplemented with 1-10 μg / mL (preferably 1-5 μg / mL, most preferably 2 μg / mL) zoledronic acid and 200-2000 IU / mL (preferably 500-1500 IU / mL, most preferably 1000 IU / mL) recombinant human interleukin-2, 1-100 ng / mL (preferably 1-20 ng / mL, most preferably 10 ng / mL) recombinant human interleukin-15 and 1-100 ng / mL (preferably 1-20 ng / mL, most preferably 10 ng / mL) recombinant human interleukin-18, and incubated at 36°C-38°C (preferably 37°C) and 5% CO 2The culture is carried out in a saturated humidity environment for 1 to 5 days (preferably 2 to 4 days, most preferably 3 days).

[0062] Using this special activation medium, change the culture conditions, such as at 38°C to 40°C (preferably 38.5°C to 39.5°C, most preferably 39°C), 5% CO 2 Cultivating in a saturated humidity environment for 0.5 to 3 days (preferably 0.5 to 1 day, most preferably 1 day) will obtain the optimal conditions for activating and culturing cord blood NK cells in step 1).

[0063] 2) Proliferation and culture of NK cells from umbilical cord blood: Use a pipette to transfer the cell solution obtained in step 1) into a centrifuge tube, centrifuge at 150g for 10 min, discard the supernatant, and use GMP S&XFM TM -CD lymphocyte culture medium (patent application number: 201310082166.8; medical device registration number: Jinghai Xiebei 20150008) adjust the cell density to 0.5-5×10 6 / mL (preferably 0.5~2×10 6 / mL, preferably 1×10 6 / mL), inoculate the cell suspension into a cell culture flask, add 200-2000IU / mL (preferably 500-1500IU / mL, preferably 1000IU / mL) of recombinant human interleukin-2 (to form a dedicated proliferation medium), and incubate at 36°C-38°C (preferably 37°C) and 5% CO 2 Cultivate in a saturated humidity environment and add fresh GMP S&XFM every 2 to 3 days TM -CD lymphocyte culture medium to adjust the cell density to 0.5-5×10 6 / mL (preferably 0.5~2×10 6 / mL, preferably 1×10 6 / mL), and add 200-2000IU / mL (preferably 500-1500IU / mL, preferably 1000IU / mL) of recombinant human interleukin-2, culture for 14-35 days (preferably 18-24 days, preferably 21 days), and harvest the cord blood NK cells.

[0064] The special proliferation culture medium in step 2) is composed of: GMP S&XFM with 200-2000 IU / mL (preferably 500-1500 IU / mL, preferably 1000 IU / mL) of recombinant human interleukin-2 TM -CD lymphocyte culture medium.

[0065] Step 2) The process of proliferating and culturing NK cells from cord blood is preferably as follows: the cell solution is transferred to a centrifuge tube using a pipette, centrifuged at 150 g (centrifugal force unit) for 10 min, the supernatant is discarded, and the mixture is centrifuged using GMP S&XFM TM-CD lymphocyte culture medium was used to adjust the cell density to 1×10 6 The cell suspension was inoculated into a cell culture flask, and 1000 IU / mL recombinant human interleukin-2 was added to the cell culture flask. The flask was incubated at 37°C and 5% CO 2 Culture in a saturated humidity environment, add fresh culture medium every 2 to 3 days and adjust the cell density to 1×10 6 / mL, cultured for 21 days, and then cord blood NK cells were harvested.

[0066] In the above method, the cord blood mononuclear cells used in step 1) are separated from umbilical cord blood by the following separation method:

[0067] 1.1 Take 100 mL of fresh anticoagulated cord blood or frozen resuscitated cord blood and add 100 mL of PBS to dilute the cord blood;

[0068] 1.2 Add 20 mL of sample density separation solution (patent number: ZL201110456878.2, medical device registration number: Jinghai Xiebei 20150002; other commercially available lymphocyte separation solution can also be added) to 10 50 mL centrifuge tubes respectively, and slowly add 200 mL of diluted umbilical cord blood to the surface of the separation solution. Add 20 mL of diluted umbilical cord blood to each centrifuge tube to keep the interface between the two liquid surfaces clear;

[0069] 1.3 Centrifuge at room temperature, 400-1200g (preferably 980g, which is a unit of centrifugal force. Other commercially available lymphocyte separation solutions should be operated according to the instructions) for 20-40min (preferably 30min);

[0070] 1.4 After centrifugation, the centrifuge tube is divided into four layers with clear interfaces from top to bottom, which are: light yellow plasma layer, white film-like mononuclear cell layer, transparent separation liquid layer and red red blood cell layer. Carefully pipette the white film-like mononuclear cell layer into another centrifuge tube, wash it with PBS, and obtain umbilical cord blood mononuclear cells.

[0071] According to the above method, the present invention also provides an in vitro expansion kit for cord blood NK cells, which mainly includes the following reagents:

[0072] 1) Sample density separation fluid for cord blood mononuclear cells: sample density separation fluid (patent number: ZL201110456878.2, medical device registration number: Jinghai Xiebei 20150002), or other commercially available lymphocyte separation fluid;

[0073] 2) Umbilical cord blood NK cell activation medium: AIM Medium CTS TM (purchased from Life Technology, USA) culture medium or GMP S&XFM TM-CD medium; and other reagents described in 1) to 4) above, including zoledronic acid, recombinant human interleukin-2, recombinant human interleukin-15 and recombinant human interleukin-18;

[0074] 3) Umbilical cord blood NK cell proliferation medium: GMP S&XFM TM -CD cell culture medium (patent application number: 201310082166.8; medical device registration number: Jinghai Xiebei 20150008), and the reagent recombinant human interleukin-2 described above.

[0075] The present invention is further described below with reference to examples. The methods used in the examples are conventional methods unless otherwise specified.

[0076] Unless otherwise specified, the percentage concentrations are mass / mass (W / W, unit: g / 100 g) percentage concentrations, mass / volume (W / V, unit: g / 100 mL) percentage concentrations or volume / volume (V / V, unit: mL / 100 mL) percentage concentrations.

[0077] The methods for obtaining various biological materials described in the examples are only provided as an experimental method to achieve the specific disclosed purpose, and should not be construed as limiting the sources of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological material that can be obtained without violating laws and moral ethics can be replaced and used according to the instructions in the examples.

[0078] The embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given. The embodiments will help to understand the present invention, but the protection scope of the present invention is not limited to the following embodiments.

[0079] Example 1: In vitro expansion and detection of umbilical cord blood NK cells

[0080] 1. In vitro expansion of umbilical cord blood NK cells (activation culture protocol 1)

[0081] like Figure 1 As shown, the in vitro expansion method of cord blood NK cells of the present invention comprises the following steps:

[0082] 1) Isolation of cord blood mononuclear cells

[0083] 1.1 Take 100 mL of fresh anticoagulated cord blood (sample 1) and frozen resuscitated cord blood (sample 2, both cord blood were provided by the Department of Obstetrics and Gynecology of the General Hospital of the Armed Police Force and approved by the hospital ethics committee), add 100 mL of PBS (formula: NaCl 8.0 g, KCl 0.2 g, Na 2 HPO 4 1.44 g, KH 2 PO4 0.24 g, add distilled water to 1000 mL, adjust pH to 7.4) to dilute the cord blood;

[0084] 1.2 Add 20 mL of sample density separation liquid (patent number: ZL201110456878.2, medical device registration number: Jinghai Xiebei 20150002) to 10 50 mL centrifuge tubes respectively, and slowly add the diluted umbilical cord blood to the surface of the separation liquid. Add 20 mL of diluted umbilical cord blood to each centrifuge tube to keep the interface between the two liquid surfaces clear;

[0085] 1.3 Centrifuge at room temperature, 980g for 30 min;

[0086] 1.4 After centrifugation, the centrifuge tube is divided into four layers with clear interfaces from top to bottom, namely: light yellow plasma layer, white film-like NK cell layer, transparent separation fluid layer and red red blood cell layer. Carefully pipette the white film-like mononuclear cell layer into another centrifuge tube and wash it with PBS for 2 to 3 times to obtain mononuclear cells.

[0087] 2) Activate and culture NK cells from cord blood

[0088] Using AIM Medium CTS TM Lymphocyte culture medium (purchased from Life Technology, USA) was used to adjust the mononuclear cell density to 2×10 6 / mL (0.5~5×10 6 / mL, preferably 1~3×10 6 / mL. Less than 0.5×10 6 / mL or higher than 5×10 6 / mL, which is not conducive to the activation of NK cells), the mononuclear cell suspension was inoculated into a cell culture flask, and then AIM Medium CTS TM The lymphocyte culture medium was added with 2 μg / mL (1-10 μg / mL is acceptable, 1-5 μg / mL is preferred. Below 1 μg / mL, NK cell activation is affected; above 10 μg / mL, NK activation has little effect) zoledronic acid (Zometa, Novartis Pharmaceuticals) and 1000 IU / mL (200-2000 IU / mL is acceptable, 500-1500 IU / mL is preferred. Below 200 IU / mL, NK cell activation is affected; above 2000 IU / mL, cells are prone to death) recombinant human interleukin-2 (DeLusheng, Beijing Sihuan Biotechnology) at 37°C (36°C-38°C is acceptable, below 36°C or above 38°C, cells cannot grow normally) and 5% CO. 2Culture in a saturated humidity environment for 3 days (1 to 5 days, preferably 2 to 4 days. If it is less than 1 day, the NK cell activation is low; if it is more than 5 days, it has little effect on NK activation).

[0089] 3) Proliferation and culture of NK cells from cord blood

[0090] Use a pipette to transfer the cell solution to a centrifuge tube, centrifuge at 150g for 10 minutes, discard the supernatant, and use GMP S&XFM TM -CD lymphocyte culture medium (patent application number: 201310082166.8; medical device registration number: Jinghai Xiebei 20150008) was used to adjust the cell density to 1×10 6 / mL (0.5~5×10 6 / mL, preferably 0.5~2×10 6 / mL. Less than 0.5×10 6 / mL or higher than 5×10 6 / mL, which is not conducive to the proliferation of NK cells), inoculated into a cell culture flask, and then added 1000IU / mL (200-2000IU / mL, preferably 500-1500IU / mL. Below 200IU / mL, it affects the proliferation of NK cells; above 2000IU / mL, cells are prone to death) recombinant human interleukin-2, 37℃ (36℃-38℃, below 36℃ or above 38℃, cells cannot grow normally), 5% CO 2 Culture in a saturated humidity environment, and add fresh lymphocyte culture medium every 2 to 3 days to adjust the cell density to 0.5 to 5 × 10 6 / mL (preferably 1×10 6 / mL, preferably 0.5~2×10 6 / mL. The cell density is less than 0.5×10 6 / mL, the cells will proliferate slowly; when the cells are higher than 5×10 6 / mL, cells tend to form contact inhibition quickly, affecting cell proliferation), and add 1000IU / mL (200-2000IU / mL, preferably 500-1500IU / mL) recombinant human interleukin-2, culture for 14-35 days (preferably 18-24 days, most preferably 21 days) to obtain cord blood NK cells. Since the number of cells proliferated is large at 21 days and the killing activity is the highest, the cells enter the stable period after 21 days, and the killing activity of the cells decreases, so 21 days is preferably used as the harvesting point of cord blood NK cells.

[0091] 2. Detection of in vitro expanded NK cells from cord blood

[0092] 1. Umbilical cord blood NK cell count

[0093] 1) Gently blow the cord blood NK cell suspension to make a NK single cell suspension;

[0094] 2) Take 20 μl of NK single cell suspension, add it to 20 μl of 0.2% trypan blue staining solution (purchased from SIGMA), and gently mix it by repeated pipetting;

[0095] 3) Take 20 μl of the mixture and add it into the counting chamber of Countstar (IC1000) cell counter (purchased from Shanghai Ruiyu Biotechnology Co., Ltd.);

[0096] 4) Let it stand for 1 minute and read the value.

[0097] Results: After activation and expansion culture in vitro (obtained by the above method), the NK cells isolated from fresh anticoagulated cord blood or frozen resuscitated cord blood were observed under a microscope. The cell morphology changed from round and different sizes before expansion to uniform irregular cell morphology. The cell body and nucleus increased in size (cells expanded and cultured for 21 days, as shown in Figure 1). Figure 3 The number of cells increased from the initial 4.36×10 8 / mL (fresh cord blood), 3.25×10 8 / mL (frozen and resuscitated cord blood) proliferated to 5.08×10 after 21 days of expansion culture 10 / mL (fresh cord blood), 3.94×10 10 / mL (frozen and resuscitated cord blood), such as Figure 4 shown. Figure 4 It also shows that the rapid proliferation period of cells is from 14 to 21 days of expansion culture, and the slow apoptosis period is from then to 35 days. Therefore, cord blood NK cells can be harvested within the 14-35 day culture period according to the situation.

[0098] 2. Detection of Purity of Umbilical Cord Blood NK Cells by Flow Cytometry

[0099] 1) Take the single cell suspension and centrifuge at 150g for 10 minutes;

[0100] 2) Resuspend the cell pellet with PBS and adjust the cell concentration to 1×10 6 pcs / 100μl, placed in flow detection tube;

[0101] 3) Add antibodies CD56 and CD3 (purchased from BD), gently pipette to mix, incubate at 4°C in the dark for 30 minutes, and set up isotype controls at the same time;

[0102] 4) Centrifuge at 1500 g for 5 minutes and discard the supernatant;

[0103] 5) Add 100 μl PBS, pipette gently to mix, and then test on the instrument.

[0104] Results: The purity of NK cells expanded and cultured for 21 days increased from the initial 1.23% (fresh cord blood) and 0.79% (frozen and resuscitated cord blood) to 94.58% (fresh cord blood) and 94.37% (frozen and resuscitated cord blood). Figure 5 shown.

[0105] 3. CCK-8 method to determine the tumoricidal activity of NK cells in cord blood

[0106] 1) Take well-growing A549 cells (human lung adenocarcinoma cells, from ATCC) as target cells and digest them with 0.25% trypsin;

[0107] 2) Count the cells by trypan blue staining and adjust the cell concentration to 5×10 4 Pieces / mL;

[0108] 3) Place 100ul per well in a 96-well plate and place at 37°C with 5% CO 2 Incubate overnight.

[0109] 4) Take NK cell suspension from cord blood as effector cells and adjust the cell concentration to 2×10 6 Pieces / mL;

[0110] 5) Add the cells to a 96-well plate at effector-target ratios of 5:1, 10:1, and 20:1, with 3 replicate wells in each group;

[0111] 6) 37°C 5% CO 2 Incubate in an incubator for 4 h;

[0112] 7) Add 15 μl CCK-8 (purchased from Bio-Tech) to each well and continue incubation for 2 h;

[0113] 8) Use an ELISA reader to detect the OD value at a wavelength of 450 nm;

[0114] 9) Calculate the killing rate: Killing rate (%) = [1-(OD value of the experimental group-OD value of the effector cells alone) ÷ OD value of the target cells alone] × 100%.

[0115] Results: Fluorescence microscope observation showed that the killing ability of cord blood NK cells expanded and cultured in vitro for 21 days on A549 tumor cells was greatly improved. Figure 6 shown.

[0116] The above test results show that the cord blood NK cells expanded in vitro by the method of the present invention are high in number, high in purity, and have high killing activity against tumor cells.

[0117] Example 2: In vitro expansion of umbilical cord blood NK cells (activation culture scheme 2)

[0118] like Figure 1 As shown, the in vitro expansion of cord blood NK cells includes the following processes:

[0119] 1) Isolation of cord blood mononuclear cells from frozen and revived cord blood

[0120] 10 ml of frozen and resuscitated umbilical cord blood (sample 3) was taken in the same manner as in Example 1.

[0121] 2) Activation and culture of umbilical cord blood NK cells (control group: Example 1)

[0122] GMP S&XFM TM -CD lymphocyte culture medium (patent application number: 201310082166.8; medical device registration number: Jinghai Xiebei 20150008) was used to adjust the mononuclear cell density to 2×10 6 / mL (0.5~5×10 6 / mL, preferably 1~3×10 6 / mL), the cell suspension was inoculated into a cell culture flask, and then GMP S&XFM TM -CD lymphocyte culture medium was supplemented with 2 μg / mL (1-10 μg / mL, preferably 1-5 μg / mL) zoledronic acid (Zometa, Novartis Pharmaceuticals) and 1000 IU / mL (200-2000 IU / mL, preferably 500-1500 IU / mL) recombinant human interleukin-2 (Delusheng, Beijing Sihuan Biotechnology) at 37°C (36°C-38°C) and 5% CO 2 Culture in a saturated humidity environment for 1 to 5 days (preferably 2 to 4 days, and preferably 3 days. If it is less than 1 day, the NK cell activation is low; if it is more than 5 days, it has little effect on NK activation).

[0123] The effect of the same added reagents on cell activation is the same as described in Example 1 and will not be repeated here.

[0124] 3) Proliferation and culture of NK cells from cord blood

[0125] Same as Example 1.

[0126] Example 3: In vitro expansion and detection of NK cells from umbilical cord blood (activation culture scheme 3)

[0127] like Figure 1 As shown, the in vitro expansion of cord blood NK cells includes the following processes:

[0128] 1) Isolation of cord blood mononuclear cells from frozen and revived cord blood

[0129] 10 ml of frozen and resuscitated umbilical cord blood (sample 3) was taken in the same manner as in Example 1.

[0130] 2) Activate and culture NK cells from cord blood

[0131] GMP S&XFMTM -CD lymphocyte culture medium (patent application number: 201310082166.8; medical device registration number: Jinghai Xiebei 20150008) was used to adjust the mononuclear cell density to 2×10 6 / mL (0.5~5×10 6 / mL, preferably 1~3×10 6 / mL), the cell suspension was inoculated into a cell culture bottle, and then GMP S&XFM TM -CD lymphocyte culture medium was added with 2μg / mL (1-10μg / mL, preferably 1-5μg / mL) zoledronic acid (Zometa, Novartis Pharmaceuticals), 1000IU / mL (200-2000IU / mL, preferably 500-1500IU / mL) recombinant human interleukin-2 (Delusheng, Beijing Sihuan Biotechnology), 10ng / mL (1-100ng / ml, preferably 1-20ng / ml). When the concentration is lower than 1ng / ml, it has no effect on NK activation. When the concentration is lower than 1 ng / ml, it has no effect on NK activation, and when it is higher than 100 ng / mL, it has little effect on NK activity) recombinant human interleukin-15 (purchased from Peprotech) and 10 ng / mL (1-100 ng / ml is acceptable, preferably 1-20 ng / ml. When it is lower than 1 ng / ml, it has no effect on NK activation, and when it is higher than 100 ng / mL, it has little effect on NK activity) recombinant human interleukin-18 (purchased from Peprotech) at 37°C (36°C-38°C) and 5% CO 2 Culture in a saturated humidity environment for 3 days (1 to 5 days, preferably 2 to 4 days. If it is less than 1 day, the NK cell activation is low; if it is more than 5 days, it has little effect on NK activation).

[0132] The effect of the same added reagents on cell activation is the same as described in Example 1 and will not be repeated here.

[0133] 3) Proliferation and culture of NK cells from umbilical cord blood

[0134] Same as Example 1.

[0135] Example 4: In vitro expansion of umbilical cord blood NK cells (activation culture scheme 4)

[0136] like Figure 1 As shown, the in vitro expansion of cord blood NK cells includes the following processes:

[0137] 1) Isolation of cord blood mononuclear cells from frozen and revived cord blood

[0138] 10 ml of frozen and resuscitated umbilical cord blood (sample 3) was taken in the same manner as in Example 1.

[0139] 2) Activate and culture NK cells from cord blood

[0140] The same activation medium as in activation culture scheme 3 was used, and the culture conditions were changed to 39°C ± 0.5°C (38°C to 40°C is acceptable. Raising the culture temperature is beneficial to accelerate the activation of cord blood NK cells and improve the activation efficiency. On the other hand, it can increase the expansion multiple, purity and biological activity of cord blood NK cells. However, when the temperature is higher than 40°C, NK cells cannot adapt and die), 5% CO 2 Culture in a saturated humidity environment for 1 day (0.5 to 3 days is acceptable; if it is less than 0.5 days, the activation of NK cells is low; if it is more than 3 days, NK cells will die).

[0141] 3) Proliferation and culture of NK cells from umbilical cord blood

[0142] Same as Example 1.

[0143] Umbilical cord blood NK cell detection

[0144] The following tests were performed on cord blood NK cells expanded in vitro from 10 ml of frozen and revived cord blood (sample 3) using the method described in Example 1 (parallel operations were performed using the methods of Examples 1 to 4 with sample 3 as the starting blood source):

[0145] 1. Umbilical cord blood NK cell count

[0146] After 21 days of in vitro activation and expansion culture of cord blood mononuclear cells isolated from frozen and thawed cord blood, the cell expansion results are shown in Tables 1-1 and 1-2.

[0147] The present invention is to obtain high-purity NK cells from cord blood mononuclear cells (at this time, the proportion of NK cells in mononuclear cells is relatively low, see Table 2, the proportion of NK cells on day 0 is only 9.56%), through two steps of activation and amplification. Table 1-1 shows that the counts before and after amplification are the total number of cells (including amplified NK cells and other cells). The absolute number of NK cells is the total number of cells × the proportion of NK cells, that is: Table 1-1 data × Table 2 = Table 1-2.

[0148] The 0-day cord blood mononuclear cells are cells before activation and amplification of the present invention (including a small amount of NK cells and other cells), which are equivalent to the control; the 0-day cord blood NK cells refer to the NK cells therein. The 21-day cord blood NK cells are the total number of cells after the mononuclear cells have been amplified for 21 days, but because the NK purity is very high at this time, most of them are NK cells, so the cells amplified for 21 days are called NK cells (strictly speaking, mononuclear cells mainly composed of NK cells).

[0149] The results show that the schemes of Examples 1 to 4 all expanded the total number of cord blood cells by hundreds of times (Table 1-1), among which the absolute number of NK cells expanded by thousands of times (Table 1-2), and the expansion effect of Example 4>Example 3>Example 2>Example 1.

[0150] Table 1-1 Total number and expansion multiples of cord blood cells (10 ml cord blood volume)

[0151]

[0152] Table 1-2 Absolute number and expansion multiple of NK cells in cord blood (10 ml cord blood volume)

[0153]

[0154] 2. Detection of Purity of Umbilical Cord Blood NK Cells by Flow Cytometry

[0155] After the cord blood mononuclear cells isolated from the frozen and resuscitated cord blood were activated and expanded in vitro for 21 days, the NK cell purity results are shown in Table 2.

[0156] Table 2 Purity of NK cells from cord blood

[0157] Example 1 Example 2 Example 3 Example 4 Purity of NK cells from cord blood on day 0 9.56％ 9.56％ 9.56％ 9.56％ Purity of NK cells from cord blood on day 21 90.69％ 92.46％ 93.75％ 95.39％

[0158] The data in Table 2 show that the proportion of NK cells in cord blood mononuclear cells increased from the original 9.56%. After amplification in each example, the purity of NK cells could reach more than 90%, and Example 4>Example 3>Example 2>Example 1.

[0159] 3. CCK-8 method to determine the tumoricidal activity of NK cells in cord blood

[0160] The experiment used 0-day cord blood mononuclear cells (cells before activation and expansion) as negative control, and CIK cells (a type of immune cell that kills tumors) as positive control. The culture of CIK cells was obtained according to the literature (Adoptive immunotherapy with cytokine-induced killer cells generated with a new good manufacturing practice-grade protocol. Cytotherapy, 2012; Early Online: 1–10. DOI: 10.3109 / 14653249.2012.681038).

[0161] The results are shown in Table 3. Under a fluorescence microscope, the cord blood NK cells harvested after 21 days of in vitro expansion in each example all showed strong killing ability against A549 tumor cells. Compared with the positive control CIK cells, the NK cells expanded by the present invention had stronger activity. Under the same effect-target ratio, Example 4> Example 3> Example 2> Example 1, indicating that the cells expanded by Example 4 have the strongest killing ability.

[0162] Table 3 Cytotoxicity of NK cells from umbilical cord blood against A549 tumor cells

[0163]

[0164] It can be seen from the above examples that the in vitro expansion method of cord blood NK cells provided by the present invention is simple and safe to operate, and a comparison with the existing method for isolating and expanding cord blood NK cells (existing technology) is shown in Table 4.

[0165] In summary, the present invention has the following advantages:

[0166] 1) No cell sorting required;

[0167] 2) No need for trophoblast cells;

[0168] 3) Simple operation and good versatility;

[0169] 4) Low cost;

[0170] 5) The reagents used do not contain animal-derived ingredients and are safe;

[0171] 6) High NK cell yield: The purity of NK cells obtained from cord blood is over 90%, and the total number of cells can reach 10 10-11 Each cord blood portion (according to Example 1, the sample is 100 ml);

[0172] 7) High killing effect on tumor cells.

[0173] Table 4 Comparison between the prior art and the technology of the present invention

[0174]

[0175] Note: Each portion of umbilical cord blood has a volume of 100 ml (usually the volume of each portion of umbilical cord blood is approximately between 50 ml and 120 ml).

[0176] Example 5. In vitro expansion kit for umbilical cord blood NK cells

[0177] The in vitro expansion kit of cord blood NK cells of the present invention mainly includes the following reagents:

[0178] 1) Sample density separation fluid for cord blood NK cells: sample density separation fluid (patent number: ZL 201110456878.2, medical device registration number: Jinghai Xiebei 20150002), or other commercially available lymphocyte separation fluid;

[0179] 2) Umbilical cord blood NK cell activation medium: AIM Medium CTS TM (Purchased from Life Technology, USA) culture medium or GMP S&XFM TM -CD culture medium (patent application number: 201310082166.8; medical device registration number: Jinghai Xiebei 20150008), as well as zoledronic acid, recombinant human interleukin-2, recombinant human interleukin-15 and recombinant human interleukin-18;

[0180] 3) Umbilical cord blood NK cell proliferation medium: GMP S&XFM TM -CD cell culture medium (patent application number: 201310082166.8; medical device registration number: Jinghai Xiebei 20150008) and recombinant human interleukin-2.

[0181] The kit can be used according to the method of Examples 1-4.

[0182] Industrial Applicability

[0183] The present invention obtains cord blood NK cells from isolated cord blood mononuclear cells through activation culture and proliferation culture, does not require cell sorting, does not require trophoblast cells, is simple to operate, has good versatility, and is low in cost. The reagents used do not contain animal-derived components and have good safety. The yield of harvested NK cells is high, and the total number of cells can reach 10 10-11 The cord blood has a purity of more than 90% and a high killing power to tumor cells. The invention can be applied to the preparation of tumor immunotherapy drugs.

Claims

1. A method for in vitro expansion of cord blood NK cells, The following steps are involved: 1) Activate and culture cord blood NK cells: Adjust the density of cord blood mononuclear cells to 0.5 - 5×10 6 cells / mL with lymphocyte medium, then add 1 - 10 μg / mL zoledronic acid and 200 - 2000 IU / mL recombinant human interleukin-2, and do not sort the cord blood NK cells, or add 1 - 10 μg / mL zoledronic acid, 200 - 2000 IU / mL recombinant human interleukin-2, 1 - 100 ng / mL recombinant human interleukin-15 and 1 - 100 ng / mL recombinant human interleukin-18, and do not sort the cord blood NK cells. Culture in an environment of 36°C - 40°C, 5% CO 2 saturated humidity for 1 - 5 days, and collect the cell fluid; the lymphocyte medium is Medium CTS TM or GMP S&XFM TM -CD cell medium; 2) Proliferation and culture of NK cells from umbilical cord blood: Obtain cells from the cell fluid in step 1) and use GMP S&XFM TM -CD lymphocyte culture medium to adjust the cell density to 0.5-5×10 6 200-2000 IU / mL recombinant human interleukin-2 was added and the mixture was incubated at 36-38°C and 5% CO 2 Culture in a saturated humidity environment, add fresh culture medium every 2-3 days and adjust the cell density to 0.5-5×10 6 / mL, culture for 14 to 35 days, and harvest the cord blood NK cells.

2. The in vitro expansion method of cord blood NK cells according to claim 1, Features: The cord blood mononuclear cells used in step 1) are separated from fresh anticoagulated cord blood or frozen resuscitated cord blood, and the separation process is as follows: 1.1 Take fresh anticoagulated cord blood or frozen resuscitated cord blood and dilute it with 1-2 times the volume of PBS; 1.2 Slowly add an equal volume of diluted cord blood to the lymphocyte separation solution, keeping the interface between the two liquids clear; 1.3 Centrifuge at room temperature at 980g for 20-30 minutes; 1.4 After centrifugation, the second layer of mononuclear cells in the form of a white film is aspirated and washed with PBS to obtain umbilical cord blood mononuclear cells.

3. The in vitro expansion method of cord blood NK cells according to claim 1, Features: The lymphocyte culture medium in step 1) is Medium CTS TM , and the activation culture is as follows: Using Medium CTS TM , adjust the cell density of the lymphocyte culture medium to 1 - 3×10 6 cells / mL, then add 1 - 5 μg / mL zoledronic acid and 500 - 1500 IU / mL recombinant human interleukin - 2, and culture in an environment of 36°C - 38°C and 5% CO 2 with saturated humidity for 2 - 4 days.

4. The in vitro expansion method of cord blood NK cells according to claim 3, Features: Activation culture: Medium CTS TM The cell density was adjusted to 2 × 10 6 2 μg / mL zoledronic acid and 1000 IU / mL recombinant human interleukin-2 were added and incubated at 37°C and 5% CO 2 Incubate in saturated humidity environment for 3 days.

5. The in vitro expansion method of cord blood NK cells according to claim 1, Features: The lymphocyte culture medium in step 1) is GMP S&XFM TM -CD, activated culture is one of the following: A. Use GMP S&XFM TM -CD lymphocyte culture medium to adjust the cell density to 1-3×10 6 1-5 μg / mL zoledronic acid and 500-1500 IU / mL recombinant human interleukin-2 were added and incubated at 36-38°C and 5% CO 2 Cultivate in a saturated humidity environment for 2 to 4 days; A1. Use GMP S&XFM TM -CD lymphocyte culture medium to adjust the cell density to 2 × 10 6 2 μg / mL zoledronic acid and 1000 IU / mL recombinant human interleukin-2 were added and incubated at 37°C and 5% CO 2 Cultivate in saturated humidity environment for 3 days; B. Use GMP S&XFM TM -CD lymphocyte culture medium to adjust the cell density to 1-3×10 6 1-5 μg / mL zoledronic acid and 500-1500 IU / mL recombinant human interleukin-2, 1-100 ng / mL recombinant human interleukin-15 and 1-100 ng / mL recombinant human interleukin-18 were added and incubated at 36-38°C and 5% CO 2 Cultivate in a saturated humidity environment for 2 to 4 days; B1. Use GMP S&XFM TM -CD lymphocyte culture medium to adjust the cell density to 2 × 10 6 2 μg / mL zoledronic acid and 1000 IU / mL recombinant human interleukin-2, 10 ng / mL recombinant human interleukin-15, and 10 ng / mL recombinant human interleukin-18 were added and incubated at 37°C and 5% CO 2 Cultivate in saturated humidity environment for 3 days; C. Using GMP S&XFM TM - Adjust the cell density of the CD lymphocyte medium to 1 - 3×10 6 cells / mL, then add 1 - 5 μg / mL zoledronic acid, 500 - 1500 IU / mL recombinant human interleukin-2, 1 - 100 ng / mL recombinant human interleukin-15, and 1 - 100 ng / mL recombinant human interleukin-18, and culture in an environment of 38.5°C - 39.5°C and 5% CO 2 saturated humidity for 0.5 - 1 day; C1. Use GMP S&XFM TM -CD lymphocyte culture medium to adjust the cell density to 2 × 10 6 2 μg / mL zoledronic acid and 1000 IU / mL recombinant human interleukin-2, 10 ng / mL recombinant human interleukin-15, and 10 ng / mL recombinant human interleukin-18 were added and incubated at 39°C and 5% CO 2 Incubate in saturated humidity environment for 1 day.

6. The in vitro expansion method of cord blood NK cells according to any one of claims 1 to 5, Features: The step 2) proliferation and culture of cord blood NK cells is as follows: using a pipette to transfer the cell solution to a centrifuge tube, centrifuging at 150g for 10 minutes, discarding the supernatant, and using GMP S&XFM TM -CD lymphocyte culture medium was adjusted to a cell density of 0.5 to 2×10 6 500-1500 IU / mL recombinant human interleukin-2 was added and incubated at 37°C and 5% CO 2 Culture in a saturated humidity environment, add fresh culture medium every 2-3 days to adjust the cell density to 0.5-2×10 6 / mL, and add 500-1500IU / mL recombinant human interleukin-2, culture for 18-24 days to obtain cord blood NK cells.

7. The in vitro expansion method of cord blood NK cells according to any one of claims 1 to 5, wherein the dedicated activation culture medium used in step 1) is one of the following combinations: Add 1-10 μg / mL zoledronic acid and 200-2000 IU / mL recombinant human interleukin-2 Medium CTS TM Lymphocyte culture medium; GMP S&XFM supplemented with 1-10 μg / mL zoledronic acid and 200-2000 IU / mL recombinant human interleukin-2 TM -CD lymphocyte culture medium; GMP S&XFM supplemented with 1-10 μg / mL zoledronic acid, 200-2000 IU / mL recombinant human interleukin-2, 1-100 ng / mL recombinant human interleukin-15, and 1-100 ng / mL recombinant human interleukin-18 TM -CD lymphocyte culture medium.

8. The in vitro expansion method of cord blood NK cells according to claim 7, wherein in the dedicated activation medium, the amount of zoledronic acid added is 2 μg / mL; the amount of recombinant human interleukin-2 added is 1000 IU / mL; the amount of recombinant human interleukin-15 added is 10 ng / mL; and the amount of recombinant human interleukin-18 added is 10 ng / mL.

9. The in vitro expansion method of cord blood NK cells according to any one of claims 1 to 5, wherein the dedicated proliferation medium used in step 2) is GMP S&XFM supplemented with 200 to 2000 IU / mL recombinant human interleukin-2 TM -CD lymphocyte culture medium. 10 . The in vitro expansion method of cord blood NK cells according to claim 9 , wherein in the dedicated proliferation medium, the amount of recombinant human interleukin-2 added is 1000 IU / mL.

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