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CN117286098A - Preparation scheme of high-purity NK cells - Google Patents

Preparation scheme of high-purity NK cells Download PDF

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CN117286098A
CN117286098A CN202210159187.4A CN202210159187A CN117286098A CN 117286098 A CN117286098 A CN 117286098A CN 202210159187 A CN202210159187 A CN 202210159187A CN 117286098 A CN117286098 A CN 117286098A
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cancer
cell
concentration
cells
carcinoma
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CN117286098B (en
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王立燕
景浩然
张晓艳
杨月峰
郭雷鸣
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Beijing Jingda Biotechnology Co ltd
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Abstract

The invention belongs to the field of biology and biotechnology, and particularly relates to a preparation scheme of high-purity NK cells. To solve the problem that high purity NK cells are difficult to prepare, the present invention provides a method for preparing high purity NK cells, which comprises culturing mononuclear cells in an activation medium containing IL-2, IL-15, IL-12, CD137, and plasma; the culture was performed in a cell culture vessel pretreated with CD137, CD28, CD 3.

Description

Preparation scheme of high-purity NK cells
Technical Field
The invention belongs to the field of biology and biotechnology, and particularly relates to a preparation scheme of high-purity NK cells.
Background
Human Natural Killer (NK) cells account for 15% of all circulating lymphocytes. NK cells were found in the 70 s of the 20 th century, mainly associated with killing infected microorganisms and malignantly transformed allogeneic and autologous cells. NK cells exhibit anti-tumor cytotoxicity without the need for prior sensitization and cytokine production and chemokines that regulate various immune responses. Human peripheral blood NK cells can be classified into CD56 bright And CD56 dim Two main categories. CD56 bright NK cells are generally considered as a low cytotoxicity subtype, acting primarily through cytokine production, while CD56 dim NK cell subtypes are potentially cytotoxic and can act by releasing perforin and granzyme, interferon gamma, and the like. NK cell-based tumor immunotherapy has become an important direction for tumor treatment because of its ability to recognize and lyse tumor cells.
After the 21 st century, NK cells developed rapidly in the basic and clinical studies of the tumor therapeutic field, and chimeric antigen receptors (Chermic antigen receptor, CAR) are expected to become a further important breakthrough in tumor cell immunotherapy following CAR-T. NK cells are free from MHC restriction, and foreign body reinfusion is free from the risk of immune rejection, so that NK cells and CAR-NK cells are expected to develop into foreign body spot products.
Although NK cells do not have immune rejection, if NK cells are directly amplified using peripheral blood mononuclear cells, the purity is difficult to reach 99% or more. And the T lymphocytes, B lymphocytes, monocytes, etc. remaining in the final amplified product risk to initiate immune rejection. Thus, establishing a method for preparing high purity NK cells is critical for the development of NK and CAR-NK cell pharmaceuticals. The MACS (magnetic-activated cell sorting) separation is based on that cell surface antigen can be combined with specific monoclonal antibody connected with magnetic beads, cells connected with the magnetic beads through antibodies in an external magnetic field are adsorbed and retained in a separation column, and cells without the surface antigen can not be combined with the specific monoclonal antibody connected with the magnetic beads and have no magnetism, and the cells can not stay in the separation column, so that the cells can be separated. Compared with flow separation, the magnetic bead separation has the advantages of simpler operation, mild treatment, less flow, short time and high efficiency. However, pure NK cells after magnetic bead sorting have limited expansion capacity, often more than 100 times. The invention is distinguished from pure NK cell culture, establishes a high-purity NK cell preparation scheme based on magnetic bead negative sorting, and greatly improves the amplification capability of NK cells on the premise of ensuring the purity of the final product to be more than 99 percent.
Disclosure of Invention
In order to solve the problem that high-purity NK cells are difficult to prepare, especially NK cells with purity of more than 98%, the invention provides a preparation method of the high-purity NK cells, and provides cells prepared by the method and application thereof, thereby providing an advanced scheme for clinical application.
In one aspect, the invention provides a combination of magnetic beads comprising one or more of CD3, TCR, CD14, CD19, CD1 beta and their use in the preparation of NK cells.
In another aspect, the invention provides a method of negative selection of NK cells comprising the use of the aforementioned magnetic bead combination.
Preferably, the concentration of the magnetic beads is 5-50. Mu.L/10 7 A cell; specifically comprises 5, 10, 20, 30, 40, 50 mu L/10 7 And (3) cells.
Preferably, the concentration of the magnetic beads is 20. Mu.L/10 7 And (3) cells.
The negative selection method of the invention can be more specifically called 'magnetic bead separation', 'magnetic bead negative selection'.
Preferably, the method further comprises a step of filtering with a cell screen before the magnetic bead sorting, and a step of washing with physiological saline after the magnetic bead sorting.
Preferably, the mesh pore size is 70 μm.
As demonstrated in example 3 of the present invention, NK cell content is raised from about 94% (about 94% including 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to 99% or more by the negative selection, i.e. NK cells with high purity can be prepared by the negative selection method provided by the present invention.
In another aspect, the present invention provides a method for preparing high purity NK cells, the method comprising the steps of selecting negatively, and:
1) Pretreatment of cell culture vessels with CD137, CD28 and CD 3;
2) Culturing mononuclear cells (which may also be referred to as "seeding" in the present invention) in the cell culture vessel obtained in 1) using an activation medium;
3) Supplementation with the first medium (preferably, within 7 days of inoculation, more preferably, 2 times);
4) The second medium is supplemented (preferably, on days 7-15 of inoculation, more preferably, 4 times).
Preferably, the cells are harvested on days 15-17.
The method of the invention is limited to a cell culture container when cells are inoculated, and a proper container can be selected at any time according to the volume of the culture medium after inoculation, for example, in the specific embodiment of the invention, the cells are subpackaged and transferred by limiting the volume of the culture container on the 9 th and 13 th days.
More preferably, the final volume of the medium is doubled after each medium supplementation as described above. For example, in embodiments of the invention the time and volume changes of adding the first amplification medium, adding the second amplification medium are as follows:
1) On day 3, the first amplification medium was added with a final volume of 2 volumes;
2) On day 5, the first amplification medium was added with a final volume of 4 volumes;
3) On day 7, the second amplification medium was added with a final volume of 8 volumes;
4) On day 9, the second amplification medium was added with a final volume of 16 volumes;
5) On day 11, the second amplification medium was added with a final volume of 32 volumes;
6) On day 13, the second amplification medium was added to a final volume of 64 volumes.
Preferably, the high purity NK cells have a purity above 95%, 96%, 97%, 98%, 99%, more specifically a purity of 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more.
Preferably, the initial cell concentration of the culture (i.e., the cell concentration after inoculation) is 1.0X10 6 individual/ml-5.0X10 6 And each ml. Specifically 1.0X10 6 Per ml, 1.5X10) 6 Per ml, 2.0X10 6 Per ml, 2.5X10) 6 Per ml, 3.0X10) 6 Per ml, 3.5X10) 6 Per ml, 4.0X10 6 Per ml, 4.5X10) 6 Per ml, 5.0X10 6 And each ml.
Preferably, the initial cell concentration of the culture is 2.0X10 6 And each ml.
Preferably, the mononuclear cells are derived from blood (preferably peripheral blood), cord blood, bone marrow.
Preferably, the mononuclear cells are peripheral blood derived mononuclear cells (PBMCs).
Preferably, the mononuclear cells can be prepared by any method; preferably, the particles are prepared by a Ficoll density gradient centrifugation method.
Preferably, the negative selection may be performed at any time during the above culture: including performing the negative selection method one or more times on any of days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15. Cell negative selection was performed on day 0, day 7, and day 15 as in the specific examples of the present invention.
The pretreatment method of the invention is as follows: adding physiological saline solution containing CD137, CD28 and CD3 into a cell culture container, dispersing the liquid thoroughly, and standing for use.
Preferably, the concentration of CD137 in the physiological saline is 0-10 mug/ml; more preferably 8. Mu.g/ml;
preferably, the concentration of CD28 in the physiological saline is 0-10 mug/ml; more preferably 8. Mu.g/ml;
preferably, the concentration of CD3 in the physiological saline is 0-10 mug/ml; more preferably 8. Mu.g/ml.
The CD137, CD28 and CD3 can be human monoclonal antibodies independently.
The activation culture medium contains IL-2, IL-15, IL-12, CD137 and plasma;
preferably, the IL-2 concentration is 1000-10000IU/mL; more preferably 2000IU/mL.
Preferably, the IL-15 concentration is 500-2000IU/mL; more preferably 1000IU/mL.
Preferably, the IL-12 concentration is 50-500IU/mL; more preferably, 100IU/mL.
Preferably, the CD137 concentration is 0-10 μm/ml; more preferably 5. Mu.g/ml.
Preferably, the plasma percentage is 3-10% (3, 4, 5, 6, 7, 8, 9, 10); preferably 5% (volume percent).
The first amplification culture medium contains IL-2, IL-15, IL-12 and plasma;
preferably, the IL-2 concentration is 1000-10000IU/mL; more preferably 2000IU/mL.
Preferably, the IL-15 concentration is 500-2000IU/mL; more preferably 1000IU/mL.
Preferably, the IL-12 concentration is 50-500IU/mL; more preferably, 100IU/mL.
Preferably, the plasma volume ratio is 3-10% (3, 4, 5, 6, 7, 8, 9, 10); preferably 5% (volume percent).
The second amplification culture medium contains IL-2, IL-15 and IL-21;
preferably, the IL-2 concentration is 1000-10000IU/mL; more preferably 2000IU/mL.
Preferably, the IL-15 concentration is 500-2000IU/mL; more preferably 1000IU/mL.
Preferably, the IL-21 concentration is 20-100IU/mL; more preferably, 50IU/mL.
Meanwhile, the invention provides application of the magnetic bead combination, the activation culture medium, the first amplification culture medium and the second amplification culture medium in preparation of NK cells; more particularly, in the preparation of high purity (at least 99%) NK cells.
Meanwhile, the invention also provides application of the pretreated cell culture vessel in preparing high-purity (at least 99%) NK cells. The pretreated culture vessel may be stored at room temperature for 12 hours or at 4℃for 7 days.
In another aspect, the invention provides NK cells treated by the above negative selection method, the method for preparing high purity NK cells, and applications thereof.
Preferably, the use includes, but is not limited to, the use of any NK cell, in particular, in the manufacture of a medicament for cell therapy, in the manufacture of a medicament for antiviral infection, in the manufacture of a medicament for the treatment of cancer or autoimmune disease; the application in NK cells and antibody drugs, nucleic acid drugs, small molecule drugs, oncolytic virus drugs and cell drugs combined treatment; the application of the NK cells in combination with therapeutic means such as radiotherapy, chemotherapeutics, stem cell transplantation operation, interventional therapy, ablation therapy and the like.
In another aspect, the invention provides a method of using a composition comprising the above-described cells, including cells in combination with a drug.
Preferably, the compositions include, but are not limited to, pharmaceutical compositions, cells, and pharmaceutical compositions, which may further include a pharmaceutically acceptable carrier, diluent, or excipient.
The mode of drug combination includes the mode of combining different cell doses with the drug at different time points.
In another aspect, the invention provides a method of treatment for cancer or autoimmune disease using the above NK cells, compositions. By "treating" is meant delivering an effective amount of NK cells, composition for the purpose of preventing the development of any symptom or disease condition or for the purpose of preventing or delaying progression, alleviating, ameliorating or eliminating such symptoms or disease condition that have developed.
General concepts
The NK cells of the present invention include modified NK cells such as CAR-NK cells.
"plasma" as used herein includes inactivated plasma, or human albumin, or cord blood inactivated plasma; preferably, the plasma is autologous plasma, as used in the specific embodiments of the present invention is plasma obtained during blood separation when isolating mononuclear cells. Alternatively, any plasma prepared by methods conventional in the art may be used.
The basal medium of the activation medium, the first amplification medium, the second amplification medium according to the present invention, each independently, may be any general cell culture medium.
The "universal cell culture medium" may be a self-formulated medium, or a commercial product. The universal cell culture medium may be selected from a variety of conventional cell culture media such as DMEM, RPMI1640, MEM, DEME/F12, F10, CD293, medium231, medium106. However, fetal bovine serum or human serum must be added to culture cells such as RPMI-1640 or DMEM. However, the addition of serum will present problems such as pathogen contamination from the incoming serum source, possible differences between different batches of serum, immune rejection risk, etc. Therefore, in practical use, it is more preferable to use a basal medium such as X-VIV015, texMACS or IMSF 100.
Preferably, the basal medium of the activation medium, the first amplification medium, and the second amplification medium is X-Vivo15 (manufacturer: lonza).
By "pharmaceutically acceptable" as used herein is meant that the NK cells, compositions, when properly administered to animals (or humans), do not produce adverse, allergic or other untoward reactions. Specific examples of some substances which may be pharmaceutically acceptable carriers or components thereof are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifying agents, such as wetting agents, e.g., sodium lauryl sulfate; a colorant; a flavoring agent; tabletting and stabilizing agent; an antioxidant; a preservative; non-thermal raw water; isotonic saline solution; and phosphate buffer, etc.
The NK cells and the composition can be prepared into various dosage forms according to the needs, and a doctor can determine the beneficial dosage for patients according to the types, ages, weights, general disease conditions, administration modes and other factors of the patients. The composition may be in any dosage form and applied in any manner. The preparation comprises the preparation of tablets, capsules, granules, oral liquid, suspension, injection, microspheres or liposome and the like according to the conventional method. The modes of use include oral or parenteral administration, wherein the parenteral mode is intravenous, intramuscular, intraperitoneal or subcutaneous injection, and the like.
Preferably, the antibody drug of the present invention, exemplary, includes but is not limited to: trastuzumab, cetuximab, daclizumab, tanigzumab, ab Fu Shan, aldamuzumab, alfutuzumab, albizumab, pegzhuzumab, alstuzumab, bavisuzumab, bei Tuo momab, belimumab, bevacizumab, mobivalizumab, belantuximab-vildazole, mo Kantuo bead mab, lakamtuzumab, carlo-mizosin, cartuzumab, posituzumab, cetuximab, coronamumab, daclizumab, pohuperuzumab darunazumab, deluximab, exemesenchymab, edelomab, enozumab, enkeximab, epazumab, er Ma Suoshan, eguzumab, falexizumab, phenytoin mab, gancicximab, gemtuzumab, ji Ruixi mab, glaucoumab-vildazole, temozolomab, icovomab, lamodex mab, etomium, italopram, lamitumomab, la Bei Zhushan, cissamumab, lintuzumab, mo Luofu pearl mab, including antigen binding fragments thereof.
In another aspect, the present invention provides a composition comprising NK cells treated by the above preparation method, and a method for the combined use of NK cells and an antibody drug.
Other drugs that may be used in combination with the NK cells, compositions of the present invention include, but are not limited to, chemotherapeutic drugs, exemplary for example: doxorubicin, vincristine, vinorelbine, taxol, cisplatin, actinomycin, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, epirubicin, etoposide, fluoroarabinoic acid; fluorouracil, gemcitabine (Gemcitabine); herceptin, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, cyclohexanitrourea, melphalan, levophenylalanine nitrogen mustard, mercaptopurine, methotrexate, mitomycin, mitoxantrone, dihydroxyanthrone, oxaliplatin, procarbazine, methyl (yl) benzyl hydrazine, rituximab (Rituxan), steroids, streptozocin, taxotere (Taxotere), thioguanine, thiotepa, triaminophos, tetroxide, topotecan, qushu Vaseline, 5-fluorouracil, hiruda (Xeloda), vinblastine, vindesine, gleevec, hydroxycamptothecin, lapatinib (Tykerb, lapatinib), torisel (temsirolimus), sunitinib (sutiniib, sutent), iressa (Iressa, gefitinib), tazizania (Tarceva ), herceptin (Herceptin, trastuzumab), avastin (Bevacizumab), arsenic trioxide, trans-retinoic acid, velcade (Velcade, bortezomib), temozolomide (Temodar), erbitux (Erbitux, cetuximab), dasatinib (dasatinib Sprycel), sorafenib (neoxavar), towel Bai Nishan anti (velctix, panitumumab), ti Ji (TS-1), ixempra (ixabepilone), rituximab (RTX, trade name); rituximab), temozolomide (trade name: zevalin, zevalin), tositumomab (trade name: BEXXAR), ofatumumab (trade name: arzerra), tosimomab (trade name: arzerra), oxlizumab (trade name: ocrelizumab), atozuzumab (trade name: gazyva), daclizumab (Daratumumab), ibritumomab (i.e., E Sha Tuo ximab, isatuximab), zolbetuximab (IMAB 362, claudixmab).
The term "cancer" as used herein encompasses any type of cancer, including solid cancers and hematopoietic cancers. Representative cancers include Acute Lymphoblastic Leukemia (ALL), acute Myelogenous Leukemia (AML), adrenocortical carcinoma, anal carcinoma, appendicular carcinoma, astrocytoma, atypical teratoid/rhabdoid tumor, cholangiocarcinoma, extrahepatic bladder carcinoma, bone carcinoma, brain stem glioma, brain carcinoma, breast carcinoma, bronchial tumor, burkitt Lymphoma (Burkitt Lymphoma), carcinoid tumor, cardiac tumor (Cardioc/Heart Tumour), central nervous system Cancer (including atypical teratoid/rhabdoid tumor, embryonic tumor, germ Cell tumor, lymphoma), cervical carcinoma, chordoma, chronic Lymphocytic Leukemia (CLL), chronic Myelogenous Leukemia (CML), chronic myeloproliferative disorder, colon carcinoma, colorectal carcinoma, craniopharyngeal pipe tumor skin T Cell Lymphoma, cholangiocarcinoma, embryonic tumor, endometrial carcinoma, ependymoma, esophageal carcinoma, nasal glioma, ewing's sarcoma, extragonadal germ Cell tumor, extrahepatic cholangiocarcinoma, ocular carcinoma (including intraocular melanoma and retinoblastoma), bone fibroblastic tumor, gallbladder carcinoma, gastric carcinoma (Gastric/Cancer), germ Cell tumor, gestational trophoblastic disease, glioma, head and neck Cancer, heart Cancer, hepatocellular (liver) carcinoma, histiocytosis, langerhans Cell, hodgkin Lymphoma, hypopharyngeal carcinoma, intraocular melanoma, islet Cell tumor, pancreatic neuroendocrine tumor, kaposi's sarcoma, renal carcinoma, langerhans Cell histiocytosis, langerhans disease, laryngeal carcinoma, leukemia (including Acute Lymphoblastic Leukemia (ALL), acute Myelogenous Leukemia (AML), chronic Lymphocytic Leukemia (CLL), chronic Myelogenous Leukemia (CML)), lip and Oral cancers, liver Cancer (primary), lobular Carcinoma In Situ (LCIS), lung Cancer, lymphoma, melanoma, mesothelioma, occult primary metastatic squamous neck Cancer, oral Cancer, childhood multiple endocrine neoplasia Syndrome, multiple myeloma/plasmacytoid neoplasm, mycosis fungoides, myelodysplastic Syndrome, myelodysplastic/myeloproliferative neoplasm, multiple myeloma, myeloproliferative disorders, nasal and paranasal sinus Cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung Cancer Oral Cancer (Oral Cancer/OralCavity Cancer), osteosarcoma, ovarian Cancer, pancreatic neuroendocrine tumor (islet cell tumor), papillomatosis, paraganglioma, paranasal and nasal cavity Cancer, parathyroid Cancer, penile Cancer, pharyngeal Cancer, pituitary tumor, plasma cell neoplasm/multiple myeloma, pleural lung blastoma, gestational and breast Cancer, primary Central Nervous System (CNS) lymphoma, prostate Cancer, rectal Cancer, renal cell (renal) Cancer, renal pelvis and ureteral transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoma, sezary Syndrome (Sezary syncrome), skin Cancer, small cell lung Cancer, small intestine Cancer, soft tissue sarcoma, squamous cell carcinoma, latent primary metastatic squamous neck Cancer, stomach Cancer (stomachs/Cancer), T cell lymphomas, testicular Cancer, laryngeal Cancer, thymoma and transitional cell carcinoma of the thymus, thyroid Cancer, renal pelvis and ureter, cancer of the urethra, uterine Cancer, endometrial, uterine sarcoma, vaginal Cancer, vulvar Cancer Wei Erm schamomum.
Exemplary "autoimmune diseases" as described herein include, but are not limited to, addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, crohn's disease, diabetes (type 1), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, graves ' disease, gillin-barre syndrome, hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxoedema, pernicious anemia, ulcerative colitis, and the like.
Drawings
FIG. 1 is a graph of NK cell flow results before and after magnetic bead sorting on day 0, FIG. 1A before sorting, and FIG. 1B after sorting.
FIG. 2 is a graph showing NK cell flow type results after 15 days of culture after magnetic bead sorting on day 0.
FIG. 3 is a graph showing the statistical result of the killing effect of NK cells on tumors after 15 days of amplification culture after the 0 th day of magnetic bead sorting.
FIG. 4 is a graph of 2.0X10 6 Peripheral blood mononuclear cell culture inoculated at a density of individual/ml NK cell flow charts before and after magnetic bead sorting for 7 days, fig. 4A before sorting, fig. 4B after sorting, and fig. 4C up to day 15.
FIG. 5 is a graph showing the statistical result of the killing effect of NK cells on tumors obtained after the culture to the 15 th day after the magnetic bead sorting at the 7 th day.
FIG. 6 is a graph of 2.0X10 6 Mononuclear cells inoculated at a density of individual/ml, NK cell flow charts before and after magnetic bead sorting on day 15, FIG. 6A before sorting, and FIG. 6B after sorting.
FIG. 7 is a graph of 2.0X10 6 Statistics of killing effect of NK cells on tumor before and after magnetic bead sorting at 15 days.
Detailed Description
The present invention will be described in detail below with reference to the drawings and examples to facilitate understanding and practice of the invention and to further realize advantages thereof by those skilled in the art. Unless otherwise defined in the specification of the present invention, all technical terms herein are used according to conventional definitions commonly used and understood by those of ordinary skill in the art. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.
The following description is of the preferred embodiments of the invention and is not intended to limit the invention in any way, but rather to enable any person skilled in the art to make and use the invention as disclosed above with equivalent embodiments. Any simple modification or equivalent variation of the following embodiments according to the technical substance of the present invention falls within the scope of the present invention.
The general method comprises the following steps: tumor inhibition assay
Taking K562 cell strain in logarithmic growth phase as target cell, and taking the target cell strain as 5.0X10 5 Each ml was plated in 96-well plates at 50. Mu.L per well. At the same time, 50. Mu.L of 2.5X10 concentration was added to each well 6 Per ml, 5.0X10 6 Per ml, 1.0X10) 7 NK cells to be tested were individually per ml so that the effective target ratio (the ratio of NK cells to K562 prepared in example 1) was 5:1, 10:1, 20:1, n=3, respectively.
1) The specific inoculation mode is as follows:
experimental group (a value):K562(100ul，5.0×10 5 per ml) +prepared NK (100 μl, 2.5X10) 6 Per ml, 5.0X10 6 Per ml, 1.0X10) 7 Personal/ml)
LDH maximum release group:K562(100ul,5.0×10 5 LDH-releasing reagent in total volume of (50. Mu.l) +10% of serum-free medium of X-VIVO (ml) +
LDH natural release group:K562(100ul,5.0×10 5 serum-free medium of X-VIVO (100. Mu.l)
Background blank control group:X-VIVO serum-free medium (200 μl)
NK cells (potency), K562 cells (target) with potency target ratio = 5:1, 10:1, 20:1 were added to 96 well round bottom plates; and simultaneously setting a background blank control hole, an LDH natural release hole and an LDH maximum release hole. Each group is provided with 3 compound holes.
2) Centrifuging at 500rpm/5min to closely combine effective target cells, and placing at 37deg.C and 5% CO 2 Culturing in an incubator for 4 hours;
3) 1 hour before the predetermined detection time point, the cell culture plate was removed from the cell culture incubator, and an LDH releasing agent was added to [ LDH maximum releasing hole ] in an amount of 10% of the original volume. Blowing uniformly, and then placing the mixture into an incubator for continuous culture;
4) After reaching the preset time, centrifuging the culture plate for 400g/5min, respectively taking 120 mu L of supernatant of each hole, adding the supernatant into a new culture plate, and immediately detecting a sample;
5) Sample measurement:
(1) 60 mu L of LDH detection solution is added to each hole;
(2) mixing, and incubating at room temperature for 30min in dark place (shaking table after wrapping with tinfoil);
(3) the absorbance at 490nm (if the microplate reader does not have this wavelength, 450nm is chosen) was determined using >
Dual wavelength measurements were made at any wavelength of 600 nm;
(4) calculate NK cell activity, NK killing activity =
[ (Experimental well OD-natural release well OD)/(maximum release well OD-natural release well OD) ]. 100%
EXAMPLE 1 NK cell culture with magnetic bead sorting (negative selection) on day 0
1.1 pretreatment of cell culture flask
6.5mL of physiological saline solution containing 8. Mu.g/mL of CD137 and 8. Mu.g/mL of CD28 and 8. Mu.g/mL of CD3 was added to 75cm 2 The bottom area of the cell culture flask (Nunc) was filled with the liquid and the flask was allowed to spread well at the bottom and left to stand at 4℃overnight.
1.2 isolation of Peripheral Blood Mononuclear Cells (PBMC)
For example, 40ml of peripheral blood may be used, and the amount of blood may be adjusted accordingly. The sterile 40ml of patient peripheral blood after plate bacteria detection was subjected to differential centrifugation (Hunan) at room temperature for 30 minutes in a horizontal low speed heart machine, and plasma and blood cells were separated by increasing 9 and decreasing 7 (i.e., a decreasing time from 1800rpm to 0rpm was 10 minutes).
Transferring the upper plasma into a centrifuge tube, inactivating at 56 ℃ for 30min, centrifuging at 2000rpm for 10min, and taking the supernatant for later use at 4 ℃.
The blood cell pellet was mixed with an equal volume of physiological saline and Peripheral Blood Mononuclear Cells (PBMCs) were isolated by Ficoll density gradient centrifugation. Specifically, the above mixture was carefully added to a 50ml centrifuge tube containing a Ficoll layer, and centrifuged at room temperature at differential speed for 20 minutes (9 drops of 0, i.e., 2000rmp to 0rmp,30 min). Sucking the PBMC layer, sucking the cell layer at the junction of the two liquid surfaces as far as possible, adding normal saline, blowing and mixing uniformly, and centrifuging at 1500rpm for 10 minutes at room temperature. Cells were washed again with physiological saline.
After discarding the supernatant, the cells were resuspended in 10ml Buffer and the volume was fixed to 20ml. A small cell count was aspirated. A small amount of cell suspension was simultaneously taken for flow detection and the NK (CD 3-CD56+) ratio was found to be 7.12%.
1.3 magnetic bead sorting
Filtering the separated PBMC cell suspension with a 70 μm cell screen to remove cell clusters, sampling and counting, calculating the dosage of Macs-buffer and magnetic beads according to the counting result, and performing negative sorting by using one or more of CD3, TCR, CD14, CD19 and CD1 beta; specifically, the invention uses CD3 magnetic beads (Miltenyi Biotec, cat: 130-097-043) and CD14 magnetic beads (Miltenyi Biotec, cat: 130-124-420) to carry out negative sorting according to the proportion of 1:1 of the magnetic bead volume, wherein the magnetic bead dosage is 20 mu L/10 7 The cells were washed once with physiological saline after sorting.
After discarding the supernatant, the cells were resuspended in 5ml X-Vivo basal medium and the volume was fixed to 10ml. A small cell count was aspirated. A small amount of cell suspension was simultaneously taken for flow detection, with a proportion of NK (CD 3-CD56+) of 17.45%.
1.4 inoculation
According to 2.0X10 6 Cell concentration per mL PBMC cells obtained in step 1.2 were inoculated into the coating flask obtained in step 1.1 containing 2000IU/mL IL-2, 1000IU/mL IL-15, 100IU/mL IL-12, 5ug/mL CD137 and 5% of the 10mL medium (X-VIVO 15) of patient-inactivated plasma obtained in step 1.3. In an incubator (37 ℃, CO) 2 Concentration 5%, humidity: 45% -55%) of the culture.
1.5 first fluid replacement of NK cells (amplification Medium1, i.e. first amplification Medium)
On day 3 of culture, the cell pellet density was detected to be > 50%. At this time, the flask was supplemented with an amplification medium (basal medium X-Vivo 15) containing 5% of patient-inactivated plasma, IL-2 2000IU/mL, IL-15 1000IU/mL, and IL-12 100IU/mL, to ensure a final volume of 20mL. Note that the cells were not blown.
1.6 second NK cellSecondary fluid infusion (amplification medium 1)
On day 5, the cell mass density was measured to be > 50%, and the culture flask was continuously supplemented with an amplification medium (basal medium X-Vivo 15) containing 5% of patient-inactivated plasma, IL-2 2000IU/mL, IL-15 1000IU/mL, and IL-12 100IU/mL, to ensure that the final volume of the medium was 40mL. Note that the cells were not blown.
1.7 third fluid infusion and roller bottle for NK cells (amplification Medium2, i.e. second amplification Medium)
On day 7, the cell concentration was measured to be 2.66×10 6 And each ml. The cells at the bottom of the flask were gently tapped (about 40 mL) and mixed with 40mL of X-VIVO serum-free cell culture medium containing IL-2 2000IU/mL, IL-15 1000IU/mL, IL-21 50IU/mL and 5% inactivated plasma, and the mixture was packed into T225cm 2 In the cell culture flask, a final volume of 80ml was ensured. Cell counting is performed simultaneously to detect the production condition of cells.
1.8 fourth fluid replacement (amplification Medium 2)
On day 9, the cell concentration was measured to be 2.98X10 6 And each ml. The flask was taken from the cell incubator (37 ℃, CO) 2 5%) was removed from the sample, and the amplification medium2 (X-VIVO serum-free cell culture medium containing 2000IU/mL of IL-2, 1000IU/mL of IL-15, and 50IU/mL of IL-21) was supplemented in equal volumes to ensure a final volume of 160mL. The cell culture flask was placed in an incubator for continuous culture. Cell counts were performed simultaneously to determine the production status of the cells.
1.9 fifth fluid infusion (amplification Medium 2)
On day 11, the cell concentration was measured to be 3.04×10 6 And each ml. The flask was taken from the cell incubator (37 ℃, CO) 2 5%) was removed from the sample, and the amplification medium2 (X-VIVO serum-free cell culture medium containing 2000IU/mL of IL-2, 1000IU/mL of IL-15, and 50IU/mL of IL-21) was supplemented in equal volumes to ensure a final volume of 320mL. The cell culture flask was placed in an incubator for continuous culture. Cell counts were performed simultaneously to determine the production status of the cells.
1.10 sixth fluid infusion(amplification Medium 2)
On day 13, the cell concentration was measured to be 3.12X10) 6 And each ml. The flask was taken from the cell incubator (37 ℃, CO) 2 5% concentration) was removed, and the amplification medium2 (X-VIVO serum-free cell culture medium containing IL-2 2000IU/mL, IL-15 1000IU/mL, and IL-21 50 IU/mL) was supplemented in equal volume, and the mixture was evenly mixed and split-packed into 2 pieces of T225cm on average 2 In the cell culture flask, a final volume of 640ml was ensured. Two cell culture flasks were placed in an incubator for continued culture. Cell counts were performed simultaneously to determine the production status of the cells.
1.11 bacteria detection
FIG. 1 shows NK cell flow results before and after magnetic bead sorting on day 0. Cells were harvested from day 15 of cell culture and examined at a cell concentration of 2.76X10 6 (cell flow results are shown in FIG. 2).
The cell suspension is subjected to bacteria detection and endotoxin detection, and the results show that the cell suspension is sterile and the endotoxin is less than 0.25EU/ml.
During the above culture, cell density was monitored during the culture, and cell expansion of the mononuclear cells during the culture was as shown in table 1:
TABLE 1 NK cell expansion Meter (D0 sorting)
Tumor inhibition tests were performed on the K562 cell line according to the general method, NK cells had killing effect on tumors at different target ratios, and the results are shown in Table 2 and FIG. 3.
TABLE 2 NK cell killing experiments on K562 cell line tumor, absorbance values results (OD 490nm -OD 620nm )
EXAMPLE 2 NK cell culture protocol for cell negative selection on day 7
In this example, by the same method as in example 1, no bead sorting was performed on day 0, negative sorting of CD3 beads (Miltenyi Biotec, cat: 130-097-043) was performed on day 7, amplification culture was continued after sorting, and amplified NK cells were detected on day 15.
Briefly, flasks were pre-treated and peripheral blood mononuclear cells isolated following the procedure of 1.1 and 1.2 in example 1. At 2.0X10 6 Seed concentration of mononuclear cells of peripheral blood origin per ml cells were inoculated into culture flasks pretreated according to step 1.1 of example 1. Then, after performing amplification twice by the same procedure as 1.5 to 1.6 in example 1, CD3 beads (Miltenyi Biotec, cat: 130-097-043) and CD14 beads (Miltenyi Biotec, cat: 130-124-420) were subjected to negative sorting at a ratio of 1:1 by volume of the beads according to 1.4 in example 1, and the amounts of the beads were 20. Mu.L/10 7 Cells were further amplified 4 times according to the same procedure as 1.7 to 1.10, and examined according to the procedure of 1.11 in example 1.
The purity of NK cells was measured before and after the magnetic bead sorting on day 7 (fig. 4A, fig. 4B), and fig. 4C is a flow-through result of NK cells cultured until day 15 after the magnetic bead sorting.
Tumor inhibition tests were performed on the K562 cell line according to the general method, NK cells had killing effect on tumors at different target ratios, and the results are shown in Table 3 and FIG. 5.
TABLE 3 NK cell killing experiments on K562 cell line tumor, absorbance values results (OD 490nm -OD 620nm )
EXAMPLE 3 NK cell culture protocol for cell negative selection on day 15
In this example, mononuclear cells were subjected to 2.0X10-th protocol in the same manner as in examples 1 and 2, without bead sorting on day 0 6 After 15 days of amplification culture after inoculation of the individual/mL, the cells were cultured at 15 thNegative sorting of CD3 magnetic beads (Miltenyi Biotec, cat: 130-097-043) was performed on day, and flow detection was performed after sorting.
Briefly, flasks were pre-treated and peripheral blood mononuclear cells isolated following the procedure of 1.1 and 1.2 in example 1. At 2.0X10 6 Mononuclear cell seeding concentration of peripheral blood source per ml cells were seeded into flasks pretreated according to step 1.1 of example 1.
Then, after six amplifications were performed according to the same procedure as 1.5 to 1.10 in example 1, CD3 beads (Miltenyi Biotec, cat: 130-097-043) and CD14 beads (Miltenyi Biotec, cat: 130-124-420) were negatively sorted according to a ratio of 1:1 by volume of the beads according to 1.4 in example 1, and the amounts of the beads were 20. Mu.L/10 7 Cells were examined by following the procedure of 1.11 in example 1.
Finally, NK cells before and after sorting were detected by flow cytometry, as shown in FIG. 6A before sorting and as shown in FIG. 6B after sorting.
Tumor inhibition tests are carried out on the K562 cell line according to a general method, and NK cells have killing effect on tumors at different effect target ratios before and after sorting, and the results are shown in Table 4 and FIG. 7.
TABLE 4 NK cell killing experiments on K562 cell line tumor, absorbance values results (OD 490nm -OD 620nm )

Claims (10)

1. A magnetic bead or its use in the preparation of NK cells, wherein the magnetic bead comprises one or more of CD3, TCR, CD14, CD19, cd1β;
preferably, the NK cells have a purity of 95%, 96%, 97%, 98%, or 99% or more.
2. A method of screening NK cells, the method comprising screening cells using the magnetic bead combination of claim 1;
preferably, the concentration ratio of each magnetic bead is 1:1
Preferably, the concentration of the magnetic beads is 5-50. Mu.L/10 7 A cell;
preferably, the concentration of the magnetic beads is 20. Mu.L/10 7 A cell;
preferably, the NK cell proportion in the cells obtained by the method is above 95%, 96%, 97%, 98%, or 99%.
3. A method of preparing NK cells, the method comprising the method of claim 2 and the steps of:
1) Pretreating a cell culture vessel with the magnetic beads of claim 1; preferably, the concentration of the magnetic beads is 5-50. Mu.L/10 7 A cell; preferably, the concentration of the magnetic beads is 20. Mu.L/10 7 A cell;
2) Culturing mononuclear cells in the cell culture vessel obtained in 1) using an activation medium; the activation culture medium contains IL-2, IL-15, IL-12, CD137 and plasma;
preferably, the concentration ratio of IL-2, IL-15, IL-12 is 20:10:1;
3) Supplementing a first culture medium, wherein the first amplification culture medium contains IL-2, IL-15, IL-12 and plasma; preferably, it is performed within 7 days of inoculation;
preferably, the concentration ratio of IL-2, IL-15, IL-12 is 20:10:1;
4) Supplementing a second culture medium, wherein the second amplification culture medium contains IL-2, IL-15 and IL-21; preferably, the inoculation is carried out on days 7-15 of inoculation;
preferably, the concentration ratio of IL-2, IL-15, IL-21 is 40:20:1;
preferably, the plasma is inactivated plasma or human serum albumin;
preferably, the plasma is autologous plasma;
preferably, the mononuclear cells are derived from blood, cord blood, and bone marrow; preferably, the blood is peripheral blood;
preferably, the mononuclear cells are prepared by a Ficoll density gradient centrifugation method;
preferably, the NK cells have a purity of 95%, 96%, 97%, 98%, or 99% or more.
4. The method of claim 3, wherein the method of claim 2 is performed on days 0, 7, and 15.
5. The method of claim 3, wherein the activation medium has an IL-2 concentration of 1000-10000IU/mL, an IL-15 concentration of 500-2000IU/mL, an IL-12 concentration of 50-500IU/mL, a CD137 concentration of 0-10 μg/mL, and a plasma percentage of 1-10%;
preferably, the IL-2 concentration is 1000-5000IU/mL;2000IU/mL;
preferably, the IL-15 concentration is 1000IU/mL;
preferably, the IL-12 concentration is 100IU/mL;
preferably, the CD137 concentration is 5 μg/ml;
preferably, the plasma percentage is 5%.
6. The method of claim 3, wherein the concentration of IL-2 in the first amplification medium is 1000-10000IU/mL, the concentration of IL-15 is 500-2000IU/mL, the concentration of IL-12 is 50-500IU/mL, and the plasma volume ratio is 3-10%;
preferably, the IL-2 concentration is 1000-5000IU/mL;2000IU/mL;
preferably, the IL-15 concentration is 1000IU/mL;
preferably, the IL-12 concentration is 100IU/mL;
optionally, the plasma volume is 5%.
7. The method of claim 3, wherein the second amplification medium has an IL-2 concentration of 1000-10000IU/mL, an IL-15 concentration of 500-2000IU/mL, and an IL-21 concentration of 20-100IU/mL;
preferably, the IL-2 concentration is 1000-5000IU/mL;2000IU/mL;
preferably, the IL-15 concentration is 1000IU/mL;
preferably, the IL-21 concentration is 50IU/mL.
8. An NK cell, wherein said NK cell is selected by the method of claim 2 and prepared by the method of claim 3;
preferably, the NK cells have a purity of 95%, 96%, 97%, 98%, or 99% or more.
9. A composition comprising the NK cell of claim 8;
preferably, the composition is a pharmaceutical composition;
preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.
10. Use of the NK cell of claim 8, the composition of claim 9, for the manufacture of a medicament for cell therapy, for the manufacture of a medicament for antiviral infection, for the manufacture of a medicament for the treatment of cancer or autoimmune disease;
the application also comprises the application in combination therapy with antibody drugs, nucleic acid drugs, small molecule drugs, oncolytic virus drugs and cell drugs; the application in the combination of NK cells and therapeutic means such as radiotherapy, chemotherapeutics, stem cell transplantation operation, interventional therapy, ablation therapy and the like;
preferably, the method comprises the steps of, the cancer comprises acute lymphoblastic leukemia, acute myelogenous leukemia, adrenocortical carcinoma, anal carcinoma, appendicular carcinoma, astrocytoma, atypical teratoid/rhabdoid tumor, cholangiocarcinoma, extrahepatic bladder carcinoma, bone carcinoma, brain stem glioma, brain carcinoma, breast carcinoma, bronchogenic tumor, burkitt lymphoma, carcinoid tumor, cardiac tumor, central nervous system cancer, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, colon cancer, colorectal cancer, craniopharyngeal tumor, cutaneous T-cell lymphoma, cholangiocarcinoma, embryo tumor, endometrial carcinoma, ependymoma, esophagus carcinoma, nasal glioma, ewing's sarcoma, extrahepatic cholangiocarcinoma, eye carcinoma, bone fibromatoid tumor, gallbladder carcinoma, gastric cancer germ cell tumors, gestational trophoblastic disease, glioma, head and neck cancer, heart cancer, hepatocellular carcinoma, histiocytosis, langerhans cells, hodgkin's lymphoma, hypopharynx cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kaposi's sarcoma, renal cancer, langerhans cell histiocytoma, laryngeal cancer, lip and oral cancer, liver cancer, lobular carcinoma in situ, lung cancer, lymphoma, melanoma, mesothelioma, occult primary metastatic squamous neck cancer, oral cancer, childhood multiple endocrine neoplasia syndrome, multiple myeloma/plasmacytoid neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferative neoplasm, multiple myeloma, myeloproliferative disorders, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, mesothelioma, latent primary metastatic squamous neck cancer, non-hodgkin's lymphoma, non-small cell lung cancer, oral cancer, osteosarcoma, ovarian cancer, pancreatic neuroendocrine tumor, papillomatosis, paraganglioma, paranasal and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary tumor, plasmacytoid neoplasm/multiple myeloma, pleural lung blastoma, gestational and breast cancer, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cancer, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoma, cerclash syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, occult primary metastatic squamous neck cancer, gastric cancer, T cell lymphoma, testicular cancer, laryngeal cancer, thymoma and thymus cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, carcinoma of the uterus, endometrium, sarcoma, vaginal carcinoma, vulval carcinoma Wei Erm s tumor;
preferably, the autoimmune disease comprises addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, crohn's disease, type 1 diabetes, dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, graves ' disease, gillin-barre syndrome, hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxoedema, pernicious anemia, ulcerative colitis, and the like.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1706938A (en) * 2004-06-09 2005-12-14 哈尔滨医科大学肿瘤病防治研究所 A-NK cell separating culture method
US20120121544A1 (en) * 2008-10-24 2012-05-17 Medicell Corporation Method for Efficiently Proliferating and Differentiating Natural Killer Cells from Umbilical Cord Blood
CN107475196A (en) * 2017-10-09 2017-12-15 天津长和生物技术有限公司 The amplification cultivation method of NK culture matrix and NK
CN109666640A (en) * 2019-01-14 2019-04-23 武汉睿健医药科技有限公司 The method of the external pure culture of natural killer cells
KR20200143578A (en) * 2019-06-13 2020-12-24 한국원자력의학원 Method for Isolating NK cells Directly from whole blood and Method for Mass Proliferation of the NK cells

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1706938A (en) * 2004-06-09 2005-12-14 哈尔滨医科大学肿瘤病防治研究所 A-NK cell separating culture method
US20120121544A1 (en) * 2008-10-24 2012-05-17 Medicell Corporation Method for Efficiently Proliferating and Differentiating Natural Killer Cells from Umbilical Cord Blood
CN107475196A (en) * 2017-10-09 2017-12-15 天津长和生物技术有限公司 The amplification cultivation method of NK culture matrix and NK
CN109666640A (en) * 2019-01-14 2019-04-23 武汉睿健医药科技有限公司 The method of the external pure culture of natural killer cells
KR20200143578A (en) * 2019-06-13 2020-12-24 한국원자력의학원 Method for Isolating NK cells Directly from whole blood and Method for Mass Proliferation of the NK cells

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
陶家龙 等: "CD137单抗联合IL-15体外扩增NK细胞及其对肺癌细胞的杀伤活性", 中国肿瘤生物治疗杂志, no. 06, 31 December 2012 (2012-12-31) *

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