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CN-122012392-A - NK cell culture kit and NK cell culture method using same

CN122012392ACN 122012392 ACN122012392 ACN 122012392ACN-122012392-A

Abstract

The invention provides an NK cell culture kit and an NK cell culture method using the same. The kit comprises a pretreatment liquid, a first culture medium, a second culture medium and a third culture medium, wherein the pretreatment liquid comprises at least one of an anti-human CD3 monoclonal antibody, an anti-human CD16 monoclonal antibody and an anti-human CD56 monoclonal antibody, a first cytokine in the first culture medium comprises at least one of IL-1 alpha, IL-2, IL-3, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, FLT3 Ligand, SCF, an anti-human CD3 monoclonal antibody, an anti-human CD16 monoclonal antibody and an anti-human CD56 monoclonal antibody, a second cytokine in the second culture medium comprises at least one of IL-1 alpha, IL-2, IL-3, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, FLT3 ligandd, SCF and an anti-human CD56 monoclonal antibody, and a third cytokine in the third culture medium comprises at least one of IL-1 alpha, IL-2, IL-7 and IL-21. The NK cell culture kit provided by the invention can be used for efficiently inducing, activating and rapidly amplifying NK cells and obtaining NK cells which are mature in function and have high killing activity on tumor cells.

Inventors

  • ZENG HAOYU
  • JIANG BIYU
  • Mao Juanxuan
  • LI XIAOQING

Assignees

  • 广东普罗凯融生物医药科技有限公司

Dates

Publication Date
20260512
Application Date
20260204

Claims (10)

  1. 1. The NK cell culture kit is characterized by comprising a pretreatment liquid, a first culture medium, a second culture medium and a third culture medium; The pretreatment liquid comprises at least one of an anti-human CD3 monoclonal antibody, an anti-human CD16 monoclonal antibody and an anti-human CD56 monoclonal antibody; the first medium comprises a first cytokine and a basal medium, the first cytokine comprising at least one of IL-1 alpha, IL-2, IL-3, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, FLT3 Ligand, SCF, anti-human CD3 monoclonal antibody, anti-human CD16 monoclonal antibody, anti-human CD56 monoclonal antibody; The second medium comprises a second cytokine and a basal medium, the second cytokine comprising at least one of IL-1 alpha, IL-2, IL-3, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, FLT3 Ligand, SCF, anti-human CD56 monoclonal antibodies; The third medium includes a third cytokine including at least one of IL-1 alpha, IL-2, IL-7, IL-15, IL-21, and a basal medium.
  2. 2. The NK cell culture kit according to claim 1, wherein the first cytokine comprises IL-2, IL-12, IL-15, IL-18 and IL-21, the concentration of IL-2 in the first culture medium is 100-2000U/mL, and the concentrations of IL-12, IL-15, IL-18 and IL-21 in the first culture medium are 5-50 ng/mL; The second cytokines comprise IL-2, IL-12, IL-15, IL-18, IL-21 and anti-human CD56 monoclonal antibodies, the concentration of the IL-2 in the second culture medium is 100-2000U/mL, and the concentrations of the IL-12, the IL-15, the IL-18 and the IL-21 in the second culture medium are 5-50 ng/mL; The third cytokines comprise IL-2, IL-15 and IL-21, the concentration of the IL-2 in the third culture medium is 100-2000U/mL, and the concentrations of the IL-15 and the IL-21 in the third culture medium are 5-50 ng/mL.
  3. 3. The NK cell culture kit of claim 2 wherein the concentration of IL-2 in the first medium is 2000U/mL, the concentration of IL-12 in the first medium is 20 ng/mL, the concentration of IL-15 in the first medium is 15 ng/mL, the concentration of IL-18 in the first medium is 30 ng/mL, the concentration of IL-21 in the first medium is 15 ng/mL, And/or the number of the groups of groups, The concentration of the IL-2 in the second medium is 2000U/mL, the concentration of the IL-12 in the second medium is 20 ng/mL, the concentration of the IL-15 in the second medium is 15 ng/mL, the concentration of the IL-18 in the second medium is 30 ng/mL, the concentration of the IL-21 in the second medium is 15 ng/mL, And/or the number of the groups of groups, The concentration of the IL-2 in the third medium is 2000U/mL, the concentration of the IL-15 in the third medium is 15 ng/mL, and the concentration of the IL-21 in the third medium is 50 ng/mL.
  4. 4. The NK cell culture kit according to claim 1, wherein the pretreatment solution comprises an anti-human CD16 monoclonal antibody and an anti-human CD56 monoclonal antibody, the concentration of the anti-human CD16 monoclonal antibody in the pretreatment solution is 8-12 μg/mL, and the concentration of the anti-human CD56 monoclonal antibody in the pretreatment solution is 14-16 μg/mL.
  5. 5. The NK cell culture kit according to claim 1, wherein the basal medium comprises at least one of BC-515NK serum-free medium, AMMS NK serum-free medium, ALyS NK-EX serum-free medium, ham's F-12 medium, IMDM medium, alpha MEM medium, RPMI 1640 medium, NK cell serum-free medium; The NK cell serum-free culture medium comprises 1 mM L-glutamine, 2mM alanyl-glutamine, 2mM alanyl-alanine, 2mM glycine, 20 mg/L folic acid, 20 mg/L nicotinamide, 0.3 mug/L sodium selenite, 0.3 mug/L manganese chloride, 0.05 mg/L5 '-trisodium phosphate hydrate, 0.5 mg/L taurine, 2 mg/L ethanolamine, 8 mg/L beta-mercaptoethanol, 5 g/L human serum albumin, 2.5 mg/L recombinant human transferrin, 10 mg/L recombinant human insulin, 7 mg/L linoleic acid, 8 mg/L lauric acid, 10 mg/L beta-sitosterol, 100 mg/L tween 20,100 mg/L polyoxyethylene lauryl ether and a basal medium, wherein the basal medium is formed by mixing a Ham's F-12 medium, an IMDM medium and a MEM alpha medium according to a volume ratio of 1:2.
  6. 6. The NK cell culture kit according to claim 1, wherein the first medium further comprises 5-15 vol% of a first nutrient, and/or the second medium further comprises 5-15 vol% of a second nutrient, and/or the third medium further comprises 0-4 vol% of a third nutrient; the first nutrient, the second nutrient and the third nutrient are independently selected from at least one of umbilical cord blood plasma, human AB serum, fetal bovine serum and serum substitutes.
  7. 7. The NK cell culture kit of claim 1 wherein the first medium further comprises trophoblast cells and/or the second medium further comprises trophoblast cells and/or the third medium further comprises trophoblast cells.
  8. 8. A method of NK cell culture comprising the steps of: s1, re-suspending mononuclear cells by using a first culture medium to obtain a cell suspension; s2, adding the NK pretreatment liquid into a culture bottle, standing at room temperature for 30-120 min or treating at 4 ℃ for 12-72 h, adding the cell suspension into the culture bottle for culture, then adding a second culture medium into a culture system for continuous culture, and then adding a third culture medium into the culture system for continuous culture to obtain NK cells; The pretreatment liquid comprises at least one of an anti-human CD3 monoclonal antibody, an anti-human CD16 monoclonal antibody and an anti-human CD56 monoclonal antibody; the first medium comprises a first cytokine and a basal medium, the first cytokine comprising at least one of IL-1 alpha, IL-2, IL-3, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, FLT3 Ligand, SCF, anti-human CD3 monoclonal antibody, anti-human CD16 monoclonal antibody, anti-human CD56 monoclonal antibody; The second medium comprises a second cytokine and a basal medium, the second cytokine comprising at least one of IL-1 alpha, IL-2, IL-3, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, FLT3 Ligand, SCF, anti-human CD56 monoclonal antibodies; The third medium includes a third cytokine including at least one of IL-1 alpha, IL-2, IL-7, IL-15, IL-21, and a basal medium.
  9. 9. The NK cell culture method according to claim 8, wherein the pretreatment solution comprises 8-12. Mu.g/mL of an anti-human CD16 monoclonal antibody and 14-16. Mu.g/mL of an anti-human CD56 monoclonal antibody; The first culture medium comprises 100-2000U/mL IL-2, 5-50 ng/mL IL-12, 5-50 ng/mL IL-15, 5-50 ng/mL IL-18, 5-50 ng/mL IL-21, 5-15 vol% of a first nutrient, trophoblasts and basic culture; the second culture medium comprises 100-2000U/mL IL-2, 5-50 ng/mL IL-12, 5-50 ng/mL IL-15, 5-50 ng/mL IL-18, 5-50 ng/mL IL-21, 5-15 vol% of a first nutrient, trophoblasts and a basic culture medium; The third culture medium comprises 100-2000U/mL IL-2, 5-50 ng/mL IL-15, 5-50 ng/mL IL-21, 0-5 vol% of a third nutrient, trophoblasts and a basic culture medium.
  10. 10. The NK cell culture method according to claim 8, wherein in the S1, the cell density of the cell suspension is (1.about.3). Times.10 6 cells/mL, And/or the number of the groups of groups, And in the step S2, adding a second culture medium into the culture system for continuous culture, wherein the operation of adjusting the cell density in the culture system to (0.6-2) multiplied by 10 6 /mL by using the second culture medium is included.

Description

NK cell culture kit and NK cell culture method using same Technical Field The invention relates to the technical field of cell culture, in particular to an NK cell culture kit and an NK cell culture method using the same. Background Natural killer cells (Natural KILLER CELL, NK cells) act as core effector cells of the innate immune system, playing an irreplaceable role in tumor immune surveillance and clearance. NK cells can recognize and kill malignant transformed cells and virus-infected cells without depending on Major Histocompatibility Complex (MHC), so that the NK cells become candidate cells with great prospect in the field of adoptive cell immunotherapy. In recent years, immunotherapy based on in vitro expansion of NK cells shows good safety and primary curative effect in clinical researches of blood tumors and partial solid tumors, and promotes rapid development of NK cell preparation technology. At present, NK cells are cultured and expanded in vitro mainly by taking mononuclear cells derived from peripheral blood or umbilical cord blood and the like as initial cells, and are stimulated and expanded by adding various cytokines. Interleukin-2 (IL-2) has long been considered as a basic cytokine for the maintenance and expansion of NK cells. However, conventional, high-dose IL-2-based single culture systems have obvious limitations in that, firstly, IL-2 strongly stimulates NK cell proliferation and simultaneously non-specifically expands a large number of coexisting T cells (especially regulatory T cells) in PBMC, resulting in low purity of final cell products (CD 56 +CD3- ratio), and potential T cell contamination may bring about the risk of graft versus host disease (GvHD), secondly, continuous high-intensity IL-2 stimulation tends to cause NK cell function exhaustion or aging, manifesting as decreased killing activity and reduced secretion capacity of effector, and furthermore, a culture strategy lacking stepwise regulation is difficult to simulate natural development process of NK cells from activation, expansion to functional maturation in vivo, resulting in limited expansion factors of NK cells. Therefore, the mononuclear cells from peripheral blood or umbilical cord blood are used as the initial cells, and the problems of limited expansion times of NK cells and low killing activity of the obtained NK cells exist in the process of carrying out induction culture on the mononuclear cells by utilizing a single culture system of high-concentration IL-2, so that the clinical transformation and application of NK cell immunotherapy are greatly limited. Disclosure of Invention In order to solve the problems that in the existing single culture system containing high-concentration IL-2, the mononuclear cells are subjected to induction culture to obtain NK cells, the amplification times of the NK cells are limited, and the killing activity of the obtained NK cells is low, the invention provides an NK cell culture kit and an NK cell culture method using the same. According to a first aspect of the present invention, there is provided an NK cell culture kit comprising a pretreatment liquid, a first medium, a second medium and a third medium; The pretreatment liquid comprises at least one of an anti-human CD3 monoclonal antibody, an anti-human CD16 monoclonal antibody and an anti-human CD56 monoclonal antibody; The first medium comprises a first cytokine comprising at least one of IL-1 alpha, IL-2, IL-3, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, FLT3 Ligand, stem cell growth factor (SCF), anti-human CD3 monoclonal antibody, anti-human CD16 monoclonal antibody, anti-human CD56 monoclonal antibody, and a basal medium; The second medium comprises a second cytokine and a basal medium, the second cytokine comprising at least one of IL-1 alpha, IL-2, IL-3, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, FLT3 Ligand, stem cell growth factor (SCF), anti-human CD56 monoclonal antibody; the third medium includes a third cytokine and a basal medium, the third cytokine including at least one of IL-1 alpha, IL-2, IL-7, IL-15, IL-21. The pretreatment solution containing the components, the first culture medium, the second culture medium and the third culture medium are combined to be used as an NK cell culture kit, the first culture medium, the second culture medium and the third culture medium are respectively used for different growth periods of cells by adopting the specific pretreatment solution and three different functional and cytokine combinations, the first culture medium is mainly used for an activation period of NK cells, the second culture medium is mainly used for an activation period of NK cells, the third culture medium is mainly used for an expansion period of NK cells, the NK cell culture kit is used for carrying out culture on Peripheral Blood Mononuclear Cells (PBMC) or umbilical Cord Blood Mononuclear Cells (CBMC), firstly, the first culture medium, the second culture medium and the third culture medium can ada