CN116426475A - NK cell in-vitro activation and amplification method - Google Patents
NK cell in-vitro activation and amplification method Download PDFInfo
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Abstract
The invention relates to an NK cell in-vitro activation and amplification method. Specifically, mononuclear cells PBMC are separated and extracted from human peripheral blood, the extracted mononuclear cells are specifically activated by adopting nanoparticle suspension coupled with biotin-labeled 2B4 antibody and biotin-labeled IL-1 beta antibody on the day of NK cell activation culture, and the NK cells with high purity, high amplification efficiency and high killing power can be obtained after 14 days of amplification culture. The combined cell expansion factor IL-15 and IL-2 are used together, so that the purity of NK cells in PBMC and the expansion efficiency of the NK cells can be obviously improved, the amplified NK cells are in a better activation state, the purity of the NK cells can reach more than 85% when the NK cells are cultured on the 8 th day, the NK cells have stronger killing potential, the NK cells have better killing activity against tumor cells K562, and the killing rate of the NK cells can reach more than 80% under the condition that the effective target ratio is 5:1.
Description
Technical Field
The invention relates to the technical field of culture media, in particular to the field of an NK cell in-vitro activation amplification method.
Background
Natural killer cells, namely NK cells (Natural Killer Cell), are lymphocytes which are identical to but different from T cells and B cells, and have certain regulation effect on T lymphocytes and B lymphocytes in human bodies. NK cells are a class of immune cells unrelated to specific immune responses and are characterized by the cell phenotype CD3 - CD56 + The mediated cell lysis is not influenced by a main tissue compatible complex (MHC), and NK cells are divided into two subgroups of NK cells with killing activity and NK cells secreting immune regulation factors according to the difference of the density of CD56 molecule expression of the NK cells, wherein the NK cells with killing activity account for more than 90% of the NK cells, mainly have cytotoxicity and have very strong killing activity; NK cells secreting immune modulating factors can produce a large number of cytokines, primarily playing an immune modulating role. As a first defense line of organisms, NK cells not only can remove parasitic bacteria, viruses and aging variant cells in cells, but also have extremely strong removal effect on cancer cells, can realize rapid activation under the condition of not generating pre-sensitization, and have strong anti-infection and anti-tumor effects. Therefore, the clinical use of NK cell adoptive immunotherapy is now becoming an important tool for tumor cell immunotherapy. Obtaining a sufficient number of NK cells of sufficient purity is thus a crucial step in developing NK cell drug development. However, NK cells obtained by culturing several current techniques have the following problems: (1) NK cells obtained by adding soluble recombinant growth factors, IL-2, IL-15 and other conventional NK cell culture methods have the defects of low purity, poor amplification efficiency and low killing activity of the NK cells. (2) NK cells obtained by using K562 as trophoblast cells in NK cell expansion culture have high purity, good expansion, but general killing activity and are not in compliance with GMP standards, including safety questioning. (3) Bottom of cell culture flask coated with certain stimulatory monoclonal antibodiesAnd the method is troublesome to operate, easy to cause pollution, and the obtained NK cells are limited in amplification factor and low in purity. (4) The magnetic beads are used for coupling various stimulatory factors, including interleukin cytokines IL-15, IL-21, IL-18 and the like, and various antibody molecules 4-1BB ligand, CD86, MICA and the like, so that the operation is troublesome, repeated addition is required in the whole cell culture process, the instability factors are more, and the obtained NK cells have different qualities.
Therefore, those skilled in the art have been devoted to research on an NK cell activation amplification method capable of rapidly and effectively activating NK cells in vitro, obtaining NK cells with high purity, high amplification efficiency, and high killing activity through long-term culture, and meeting the requirements of GMP standards and clinical use standards.
Disclosure of Invention
In view of the problems and disadvantages of the prior art, the present invention is directed to an in vitro activated expansion method of NK cells, which solves the problems set forth in the background art.
In order to solve the technical problems, the invention provides the following technical scheme:
an NK cell in vitro activation and amplification method,
separating and extracting mononuclear cell PBMC from human peripheral blood, adding nanoparticle suspension coupled with biotin-labeled 2B4 antibody and biotin-labeled IL-1 beta antibody into cell culture suspension for activation culture on the day of activation culture of NK cells in NK cell culture medium;
the number ratio of the nano particles to the NK cells is 1:2;
the concentration of the nanoparticle suspension is 1mg/mL;
the concentration of the biotin-labeled 2B4 antibody is 0.1-0.15mg/mL, and the concentration of the biotin-labeled IL-1 beta antibody is 0.1-0.15mg/mL.
More optimized scheme, the concentration of the biotin-marked 2B4 antibody is 0.1mg/mL, and the concentration of the biotin-marked IL-1 beta antibody is 0.1mg/mL.
In a more optimized scheme, the nano particles are biodegradable matrix material dextran.
In a more optimized scheme, the biodegradable matrix material dextran is streptavidin nano particles.
In an optimized scheme, the NK cell culture medium is any one of SCGM culture medium, corning KBM581 lymphocyte serum-free culture medium and Bao Ri medical NK cell serum-free culture medium.
In a more optimized scheme, IL-2 with the final concentration of 500IU/mL, IL-15 with the final concentration of 500IU/mL and 10% heat-inactivated autologous plasma are added into the NK cell culture medium.
More optimized scheme, the effective target ratio of NK cells to target cells is as follows: 1:1 to 10:1.
The more optimized scheme is characterized in that the effective target ratio of the NK cells to the target cells is as follows: 10:1.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a nanoparticle coupled with biotin-labeled 2B4 antibody and biotin-labeled IL-1 beta antibody, which is added into a cell culture suspension on the day of NK cell activation culture, is matched with a common NK cell culture medium on the market, and is used together with soluble cytokines IL-15 and IL-2, so that NK cells can be rapidly activated and greatly amplified while the high activity rate of the NK cells is ensured, NK cells with high purity and high killing activity are obtained, the purity of NK cells cultured for 8 days can reach more than 85%, and the conventional culture needs at least 14 days, thereby greatly shortening the CD3 of the NK cells - CD56 + The positive proportion rises in time, and simultaneously the killing capacity of NK cells is also obviously improved. The adopted nano-scale particles can be biodegraded without a magnetic separator, and the nano-scale particles can be completely removed from a culture system by a centrifugal method when the cells are harvested on the 14 th day, so that the method is simple to operate and is beneficial to controlling the quality of cell medicines.
The action mechanism of the composition of the invention is as follows:
2B4 (CD 244) is a member of the signaling lymphocyte activating molecule (SLAM/CD 150), expressed on all NK cells, and the use of 2B4 antibodies to bind to the NK cell surface 2B4 protein not only stimulates activating NK cells, but also mediates expression of CD48 expressing target cellsNatural cytotoxicity, and induces release of intracellular calcium, enhancing killing potential of NK cells. IL-1β is an important member of the IL-1 family of lymphocyte stimulators, and is involved in a variety of autoimmune inflammatory responses and a variety of cellular activities, including mediating NK cell activation, proliferation and differentiation, and in synergy with IL-2, can enhance NK cell activity, and IL-1β has potent pro-inflammatory activity, and activation of cells with IL-1β antibodies can induce a variety of pro-inflammatory mediators, such as cytokines and chemokines, and can induce NK cell lysis to virally infected cells. The streptavidin-Biotin (SA-Biotin) system has extremely high binding affinity and has wide application in the biological field. SA is covalently connected to the surface of the solid-phase carrier nano-particles, so that the SA can be efficiently combined with biotin-labeled antibody molecules to continuously and efficiently activate NK cells, and the activated NK cells kill target cells. Thus, one skilled in the art would be able to detect the activated NK cell marker molecule CD3 - CD56 + The purity of NK cells and the killing efficiency of NK cells on the target cell line K562 are evaluated to judge the killing potential of NK cells. From this, it can be demonstrated that the purity, amplification efficiency and killing potential of NK cells were significantly improved by adding a nanoparticle suspension coupled to the 2B4 and IL-1 β antibodies.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 is a graph showing the cell viability of example 1/2/3/4;
FIG. 2 is a graph of cell expansion for example 1/2/3/4;
FIG. 3 is a graph of NK cell killing activity of example 1/2/3/4;
FIG. 4 is a schematic representation of the proportion of CD56+ cells on day 8 of culture in example 1;
FIG. 5 is a schematic representation of the proportion of CD56+ cells on day 14 of culture in example 1;
FIG. 6 is a schematic representation of the proportion of CD56+ cells on day 8 of culture in example 2;
FIG. 7 is a schematic representation of the proportion of CD56+ cells on day 14 of culture in example 2;
FIG. 8 is a schematic representation of the proportion of CD56+ cells on day 8 of culture in example 3;
FIG. 9 is a schematic representation of the proportion of CD56+ cells on day 14 of culture in example 3;
FIG. 10 is a schematic representation of the proportion of CD56+ cells on day 8 of culture in example 4;
FIG. 11 is a schematic of the proportion of CD56+ cells on day 14 of culture in example 4.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
And (3) a step of: experimental materials:
SCGM NK cell culture medium (manufacturer CellGenix (Sadoriis), cat# 130-090-0500), conning KBM581 lymphocyte serum-free medium (manufacturer Corning (Conning) cat# 88-581-CM), baozi NK cell serum-free medium (manufacturer Takara (Baozi), cat# GT-T561), IL-2 (manufacturer: prime, cat# 101-02), IL-15 (manufacturer: prime, cat# 101-15), streptavidin nanoparticle (manufacturer: meta. Mu., cat# 130-090-485), biotin-labeled 2B4 antibody (manufacturer: biolegend (hundred organism), cat# 302004), biotin-labeled IL-1 beta antibody (manufacturer: biolegend (hundred organism), cat# 511703), CD3 antibody (Biolegend (hundred organism), cat# 300439), CD56 antibody (Biolegend (hundred organism), cat# 362508), K cell line (ATCC (manufacturer: prime# 130-090-485), biotin-labeled IL-1 beta antibody (manufacturer: 62), cat# 40-carrier (HY-40), and stock solution (ChemMexico-40).
And II: the experimental method comprises the following steps:
(1) Preparation of nanoparticle suspensions coupling 2B4 with IL-1 beta antibodies
Adding 1mL of nano-particles with the diameter of 100nm and biodegradable matrix material dextran, specifically 1mg/mL of streptavidin nano-particle suspension into a centrifuge tube, wherein each milliliter of streptavidin nano-particle suspension contains 1X 10 8 Directly adding 0.1mg of biotin-labeled 2B4 antibody and 0.1mg of biotin-labeled IL-1 beta antibody into a centrifuge tube containing nanoparticle suspension, placing the centrifuge tube on a vortex oscillator for shaking for 20 seconds, placing the centrifuge tube on a rotary mixer, rotatably mixing for 30 minutes at room temperature, and placing the mixture into a refrigerator at 2-8 ℃ for standing and incubating for 30 minutes to obtain nanoparticle suspension of coupled biotin-labeled 2B4 and IL-1 beta antibody; wherein the concentration of nanoparticle suspension coupled with biotin-labeled 2B4 and IL-1β antibody is 1mg/mL.
(2) NK cell culture
PBMCs were freshly isolated from human whole blood using density gradient centrifugation for activated expansion of NK cells and autologous plasma was collected and inactivated at 56 ℃ for 30 min for cell culture. On day 0, the isolated PBMC cell suspension was transferred to a T75 flask and cell density was adjusted to 2X 10 using NK cell medium 6 Adding IL-2 with a final concentration of 500IU/mL, IL-15 with a final concentration of 500IU/mL and heat-inactivated autologous plasma accounting for 10% of the volume of NK cell culture medium, and simultaneously adding nanoparticle suspension coupled with 2B4 and IL-1 beta antibody, wherein the whole cell culture period only needs to add nanoparticle suspension coupled with 2B4 and IL-1 beta antibody once, and the ratio of microsphere particles to cell number is 1:2, namely the culture medium volume per milliliter contains 2X 10 6 The 10 mu L nanoparticle suspension of the coupled biotin-labeled 2B4 and IL-1 beta antibody is added into cells, and the cells are placed into a 37 ℃ and 5% carbon dioxide incubator for cell activation. On day 3 after activation, NK cell culture medium added with the IL-2, IL-15 and 10% heat inactivated autologous plasma is supplemented in the same volume as that on day 0, then cell viability and density are tested every 2 days, and cell density is regulated to 1-1.5X10 by fluid replacement 6 Culture was continued until day 14 at each mL to obtain cells to be treated.
(3) NK cell viability and proliferation efficiency detection
The cells were sampled and counted on days 0,5,7,9, 11 and 14 of the culture, respectively, to obtain data of cell proliferation curve and cell viability.
(4) NK cell identification
Cell immunophenotyping assays were performed on day 8 and day 14 of cell culture. About 1X 10 is taken respectively 6 The individual cells were centrifuged at 2000rpm for 5 minutes, the supernatant removed and resuspended in PBS, and the fluorescently labeled CD3 antibody and CD56 antibody were used to incubate the PBS-resuspended cells under the following conditions: at 4℃for 15 min, then washed 2 times with 1mL of PBS, the supernatant discarded, and resuspended in 0.5mL of PBS. And detecting and analyzing the treated cells by using a flow cytometer.
(5) NK cell killing Activity assay (CCK 8 method)
Taking K562 cell strain with vigorous growth as target cell, washing with RPMI-1640 culture solution for 2 times, and adjusting cell density to 1×10 6 And each mL. 50 μl of each well was plated in a 96-well plate. The NK cells cultured until day 14 were collected and the cell density was adjusted to 1X 10 6 Per mL, 2X 10 6 Per mL, 5X 10 6 Per mL, 1X 10 7 50 mu L of each/mL is taken and added into a 96-well plate, so that the effective target ratios are respectively as follows: 1:1, 2:1, 5:1, 10:1, into four groups, respectively experimental group (K562 + NK), control group (NK + RPMI-1640), maximum release group (K562 + RPMI-1640) and blank group (RPMI-1640), wherein the control group does not contain K562 cells, and the obtained absorbance is the value of K562 cell 0 killing; the maximum release group was subjected to K562 cell lysis, and the absorbance obtained was a value of 100% killing; the blank is medium, no cells are contained, and the absorbance obtained is the background value. Each group had 3 duplicate wells. After the inoculated cells are placed in an incubator for co-culture for 12 hours, 10 mu LCCK-8 reagent is added into each hole, and the cells are uniformly shaken for continuous culture for 3 hours. The enzyme label instrument detects the light absorption value at the wavelength of 450nm, and the killing rate is calculated according to the calculation mode: 100 are 1- [ experimental (OD value) -control (OD value) -blank (OD value) ]/[ maximum release (OD value) -blank (OD value) ].
Example 1:
culturing and identifying NK cells completely according to the experimental method, adding nanoparticle suspension coupled with 2B4 and IL-1 beta antibody into SCGM culture medium on the day of activation (D0), and continuing culturing until the 14 th day (D14) after NK activation;
the experimental results are shown in fig. 1: the nanoparticle suspension coupled with the 2B4 and the IL-1 beta antibody is added, and the NK cells keep a high-activity state in the whole cell culture period, and the activity rate of the NK cells is 97% when harvested on the 14 th day.
The experimental results are shown in fig. 2: with the addition of the nanoparticle suspension coupled with the 2B4 and IL-1 beta antibody, NK cells are rapidly amplified after the 5 th day, and the NK cells are amplified up to 1600 times when harvested on the 14 th day.
The experimental results are shown in fig. 3: the nanoparticle suspension coupled with the 2B4 and IL-1 beta antibody can obviously enhance the killing activity of the harvested NK cells, and the method comprises the following steps of: under the effective target ratio of 1, the NK cell killing rate on the 14 th day can reach 82%, and the NK cell killing rate is as high as 10:1, the NK cell killing rate on the 14 th day can reach 100%.
The experimental results are shown in fig. 4 and 5: when the nanoparticle suspension of the invention, which is coupled with the 2B4 and the IL-1 beta antibody, is added, the positive rate of NK cells (CD 3-CD56+) rises fast, 86% can be reached on the 8 th day of culture, and the positive rate of NK cells (CD 3-CD56+) is 97.4% when the culture is harvested on the 14 th day.
Example 2:
unlike example 1, SCGM NK medium was replaced with corning KBM581 lymphocyte serum-free medium.
The test according to the above experimental method shows that:
the experimental results are shown in fig. 1: the nanoparticle suspension coupled with the 2B4 and the IL-1 beta antibody is added, and the NK cells keep a high-activity state in the whole cell culture period, and the activity rate of the NK cells is 92% when harvested on the 14 th day.
The experimental results are shown in fig. 2: with the addition of the nanoparticle suspension coupled with the 2B4 and IL-1 beta antibody, NK cells are rapidly amplified after the 5 th day, and the NK cells are amplified by 1250 times when harvested on the 14 th day.
The experimental results are shown in fig. 3: the nanoparticle suspension coupled with the 2B4 and IL-1 beta antibody can obviously enhance the killing activity of the harvested NK cells, and the method comprises the following steps of: 1, the NK cell killing rate on the 14 th day can reach 78%, and the NK cell killing rate is as follows: 1, the NK cell killing rate on the 14 th day can reach 100%.
The experimental results are shown in fig. 6 and 7: when the nanoparticle suspension of the invention, which is coupled with the 2B4 and the IL-1 beta antibody, is added, the positive proportion of NK cells (CD 3-CD56+) rises fast, 78.1% can be reached on 8 th day of culture, and the positive proportion of NK cells (CD 3-CD56+) is 92.2% when the culture is harvested on 14 th day.
Example 3:
unlike in examples 1 and 2, SCGM NK medium was replaced with baodial NK cell serum-free medium.
The test according to the above experimental method shows that:
the experimental results are shown in fig. 1: the nanoparticle suspension coupled with the 2B4 and the IL-1 beta antibody is added, and the NK cells keep a high-activity state in the whole cell culture period, and the activity rate of the NK cells is 94% when harvested on the 14 th day.
The experimental results are shown in fig. 2: with the addition of the nanoparticle suspension coupled with the 2B4 and IL-1 beta antibody, NK cells are rapidly amplified after the 5 th day, and the NK cells are amplified up to 1400 times when harvested on the 14 th day.
The experimental results are shown in fig. 3: the nanoparticle suspension coupled with the 2B4 and IL-1 beta antibody can obviously enhance the killing activity of the harvested NK cells, and the method comprises the following steps of: 1, the NK cell killing rate on the 14 th day can reach 81% under the effective target ratio of 10:1, the NK cell killing rate on the 14 th day can reach 100%.
The experimental results are shown in fig. 8 and 9: the nanoparticle suspension coupled with the 2B4 and the IL-1 beta antibody has high rising speed of NK cell positive proportion (CD 3-CD56+) which can reach 82.8% after 8 days of culture, and the NK cell positive proportion (CD 3-CD56+) is 91.6% when the culture is harvested on 14 days.
Example 4:
unlike example 1, a nanoparticle suspension of biotin-labeled 2B4 and IL-1 β antibody was used at a level of 0.15mg per ml of coupled 2B4 and IL-1 β antibody;
the test according to the above experimental method shows that:
the experimental results are shown in fig. 1: the nanoparticle suspension coupled with the 2B4 and the IL-1 beta antibody is added, and the NK cells keep a high-activity state in the whole cell culture period, and the activity rate of the NK cells is 90% when harvested on the 14 th day.
The experimental results are shown in fig. 2: with the addition of the nanoparticle suspension coupled with the 2B4 and IL-1 beta antibody, NK cells are rapidly amplified after the 5 th day, and the NK cells are amplified by 1100 times when harvested on the 14 th day.
The experimental results are shown in fig. 3: the nanoparticle suspension coupled with the 2B4 and IL-1 beta antibody can obviously enhance the killing activity of the harvested NK cells, and the method comprises the following steps of: 1, the NK cell killing rate on the 14 th day can reach 71%, and the NK cell killing rate is as high as 10:1, the NK cell killing rate on the 14 th day can reach 100%. Example 4 cultured NK cells were less killing against K562 cells than example 1.
The experimental results are shown in fig. 10 and 11: the nanoparticle suspension coupled with the 2B4 and the IL-1 beta antibody has the advantages that the positive rate of NK cells (CD 3-CD56+) rises fast, 74.4% can be achieved on 8 th day of culture, and 89.6% of NK cells are obtained on 14 th day of harvest. Example 4 cultured NK cells were not as pure as example 1.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. An NK cell in vitro activation and amplification method is characterized in that,
separating and extracting mononuclear cell PBMC from human peripheral blood, adding nanoparticle suspension coupled with biotin-labeled 2B4 antibody and biotin-labeled IL-1 beta antibody into cell culture suspension for activation culture on the day of activation culture of NK cells in NK cell culture medium;
the number ratio of the nano particles to the NK cells is 1:2;
the concentration of the nanoparticle suspension is 1mg/mL;
the concentration of the biotin-labeled 2B4 antibody is 0.1-0.15mg/mL, and the concentration of the biotin-labeled IL-1 beta antibody is 0.1-0.15mg/mL.
2. The method for in vitro activation and expansion of NK cells of claim 1 wherein,
the concentration of the biotin-labeled 2B4 antibody is 0.1mg/mL, and the concentration of the biotin-labeled IL-1 beta antibody is 0.1mg/mL.
3. The method for in vitro activated expansion of NK cells according to claim 1, wherein said nanoparticle is a biodegradable matrix material dextran.
4. The method for in vitro activated expansion of NK cells according to claim 3, wherein said biodegradable matrix material dextran is streptavidin nanoparticle.
5. The method for in vitro activation and amplification of NK cells according to claim 1, wherein the NK cell culture medium is any one of SCGM culture medium, corning KBM581 lymphocyte serum-free medium and Bao Ri Yi NK cell serum-free medium.
6. The method for in vitro activation and expansion of NK cells according to claim 1, wherein IL-2 with a final concentration of 500IU/mL, IL-15 with a final concentration of 500IU/mL, 10% heat-inactivated autologous plasma are added to the NK cell culture medium.
7. The method for in vitro activation and expansion of NK cells according to claim 1, wherein the NK cells to target cells have an effective target ratio of: 1:1 to 10:1.
8. The method for in vitro activation and expansion of NK cells according to claim 7, wherein the NK cells to target cells have an effective target ratio of: 10:1.
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