CN114395528A - Preparation method of trophoblasts for culturing NK cells - Google Patents
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Abstract
The invention provides a preparation method of trophoblasts for culturing NK cells, which mainly comprises the steps of verifying the proliferation effect of the NK cells cultured by a conventional pure factor culture method and obtaining a corresponding proliferation proportion; preparing K562 cells over-expressing IL-15, 4-1 bbl; preparing trophoblast cells and the like. The preparation method of the trophoblast provided by the invention can eliminate the risk brought by K562 cancer cells as trophoblast cells, and can multiply a large number of NK cells with high purity and high survival rate.
Description
Technical Field
The invention belongs to the technical field of immune cells, and particularly relates to a preparation method of a trophoblast for culturing NK cells.
Background
Tumor immunotherapy is an emerging tumor treatment mode in tumor clinic, and is a novel autoimmune anticancer therapy. The immunotherapy is to enhance the anti-tumor immunity of the tumor microenvironment by stimulating or mobilizing the immune function of the organism, thereby achieving the purpose of controlling and killing tumor cells. At present, in the research of tumor immunotherapy, a plurality of killer cells activated by lymphokines, tumor infiltrating lymphocytes, natural killer cells, killer cells induced by cytokines and dendritic cells are applied. Among them, natural killer cells (NK cells) are unique lymphocytes, which, unlike B cells and T cells, play a role in connecting innate immunity and acquired immunity, have the ability to kill tumor cells and virus-infected cells, and play an important role in innate immunity and adoptive immunotherapy in the body. With the recent advent of immune checkpoint inhibitors and CAR-T technology, new opportunities, such as CAR-NK, have been found for NK cells.
In healthy subjects, NK cells account for only 5% -10% of Peripheral Blood Mononuclear Cells (PBMCs), since NK cells range from 5 x 10^6 to 5 x 10^7/kg body weight per dose in clinical infusions, thus obtaining sufficient numbers of NK cells to meet clinical needs, especially when planning multiple infusions, is challenging. Whereas feeder cells commonly used for NK culture are irradiated K562 cells. After irradiation treatment, K562 cells lost proliferative capacity. However, in clinical NK cell culture, there is a safety risk in irradiation-treated K562 cells, and this safety risk is that irradiation cannot guarantee that K562 cells are completely dead, many cells are in a living but non-dividing senescent state, and there is a possibility of returning to the dividing state again. Therefore, the problem of how to eliminate the risk brought by K562 cancer cells as trophoblast cells and massively proliferate high-purity and high-activity NK cells becomes the primary solution.
Disclosure of Invention
Because the traditional pure factor culture method has the problems of low purity, small quantity, low activity and the like of the obtained NK cells; by adding the trophoblasts, more efficient NK cell culture can be obtained, the purity and the survival rate of NK cells can be effectively improved, and the number of cultured cells can be greatly increased. The invention provides a preparation method of trophoblasts for culturing NK cells, which comprises the following steps:
s1, obtaining newly recovered PBMC, performing mixed culture on the PBMC and a corresponding pure factor culture formula, adding heat-inactivated fetal bovine serum, and placing the mixture in a culture flask for culture; after 14 days of culture, the cells were photographed and counted; taking cells for dyeing, and detecting the proportion and the concentration of NK cells by an up-flow cytometer;
s2, constructing an adenovirus vector for expressing siRNA/miRNA; digesting the purified plasmid with PacI; transfecting 293A cells by the digested adenovirus expression vector, and harvesting the cells to prepare a virus crude extract;
s3, infecting 293A cells with the virus crude extract to amplify viruses; after 24h of transfection, the transfection efficiency was observed under a fluorescence microscope; harvesting virus-containing supernatants 48h and 72h after transfection; centrifuging, filtering, and removing cell precipitate;
s4, infecting K562 engineering cells by using the transfected adenovirus vectors; sorting by a flow cytometer to screen K562 cells expressing IL-15 and 4-1bbl proteins;
s5, counting K562 cells, suspending the cells in a freezing solution, putting the cells in a refrigerator, directly putting the cells in liquid nitrogen for quick freezing, and then recovering the cells;
s6, through PI staining, observing that all the quick-frozen K562 cells die; taking the dead K562 cells, placing the cells in a 96-well plate, tracking and observing for 3 days, and observing the cell morphology;
s7, resuspending the K562 cells with an NK culture medium to be used as trophoblast cells for later use.
Further, the preparation method of the pure factor culture formula in step S1 is as follows: respectively adding CD16, CD3 and PBS into different culture bottles, wherein the final concentration of the antibody is 50 ng/ml; placing the culture bottle in a refrigerator at 4 ℃ for more than 24 hours; adding the PBMC obtained by separation into culture bottles coated with monoclonal antibodies in advance, adding IL-2, IFN-gamma, IL-15 and human serum, and culturing at constant temperature; observing the growth state of the cells after 3 days, and adding fresh culture solution (GT-551), IL-2 and human serum; and observing the cell state every 3 days, counting, supplementing an equal-volume fresh culture solution and IL-2 to keep the concentration of the IL-2 in the culture medium unchanged, and culturing until the 14 th day to obtain the final product.
Further, when the proportion and concentration of NK cells were measured by the cell-taking staining up-flow cytometer of step S1, the cells were suspended in 300ul of stabilizing buffer and mounted for 60S at a medium speed.
Further, the transfection efficiency was observed under a fluorescence microscope at step S3, and the transfection efficiency was 70% or more.
Further, supernatants containing virus were harvested 48h and 72h after transfection in step S3, respectively; centrifuging at 3000 rpm for 10-30 min, and filtering with 0.45um filter membrane.
Further, in step S5, the refrigerator temperature is 4 ℃, the standing time is 0.2-2 h, and the liquid nitrogen quick-freezing time is 5-7 h.
Compared with the prior art, the preparation method of the trophoblasts for culturing the NK cells has the following beneficial effects:
1. the risk caused by culturing NK cells by taking K562 cells as trophoblast cells is eliminated;
2. can obtain more efficient NK cell culture, can effectively improve the purity and the survival rate of NK cells, and greatly improve the number of cells obtained by culture;
3. a sufficient number of NK cells are available to meet clinical needs, especially when multiple infusions are planned.
Drawings
FIG. 1 shows the proportion of NK cells in PBMC before amplification in example 2
FIG. 2 is the proportion of NK cells after pure factor-formulated culture after amplification in example 2
FIG. 3 shows K562 cells after rapid-thawing in example 2
FIG. 4 is the imaging of K562 cells after rapid-thawing recovery in example 2 under the light microscope
FIG. 5 shows K562 cells after PI staining of example 2
Detailed Description
Example 1 a method for preparing feeder cells for culturing NK cells, comprising the steps of:
s1, obtaining newly recovered PBMC, performing mixed culture on the PBMC and a corresponding pure factor culture formula, adding heat-inactivated fetal bovine serum, and placing the mixture in a culture flask for culture; after 14 days of culture, the cells were photographed and counted; taking cells for dyeing, and detecting the proportion and the concentration of NK cells by an up-flow cytometer;
s2, constructing an adenovirus vector for expressing siRNA/miRNA; digesting the purified plasmid with PacI; transfecting 293A cells by the digested adenovirus expression vector, and harvesting the cells to prepare a virus crude extract;
s3, infecting 293A cells with the virus crude extract to amplify viruses; after 24h of transfection, the transfection efficiency was observed under a fluorescence microscope; harvesting virus-containing supernatants 48h and 72h after transfection; centrifuging, filtering, and removing cell precipitate;
s4, infecting K562 engineering cells by using the transfected adenovirus vectors; sorting by a flow cytometer to screen K562 cells expressing IL-15 and 4-1bbl proteins;
s5, counting K562 cells, suspending the cells in a freezing solution, putting the cells in a refrigerator, directly putting the cells in liquid nitrogen for quick freezing, and then recovering the cells;
s6, through PI staining, observing that all the quick-frozen K562 cells die; taking the dead K562 cells, placing the cells in a 96-well plate, tracking and observing for 3 days, and observing the cell morphology;
s7, resuspending the K562 cells with an NK culture medium to be used as trophoblast cells for later use.
Wherein, the preparation method of the pure factor culture formula in the step S1 comprises the following steps: sucking CD16, CD3 and PBS, respectively adding the sucked CD16, CD3 and PBS into different culture bottles to ensure that the final concentration of the antibody is 50ng/ml, placing the culture bottles in a refrigerator at 4 ℃ and standing for 24 hours; adding the PBMC obtained by separation into culture bottles coated with monoclonal antibody in advance, adding 1000U/ml IL-2 and 1000U/ml IFN-gamma, 15ug/L IL-15, 10% human serum, placing at 37 deg.C and 5% CO2Culturing in a constant-temperature incubator; observing the growth state of the cells after 3 days, and adding fresh culture solution (GT-551) with equal volume, IL-2 with concentration of 1000U/ml and 10% human serum; thereafter, the cell status was observed every 3 days and counted, and the medium was supplemented with an equal volume of fresh medium and 1000U/ml of IL-2 to maintain the IL-2 concentration constant, and cultured for the 14 th day. When the cell-taking staining up-flow cytometer of the step S1 detects the proportion and the concentration of NK cells, the cells are all suspended in 300ul of stationary buffer and are operated at a medium speed for 60S; observing the transfection efficiency under a fluorescence microscope in the step S3, wherein the transfection efficiency is more than 70%; harvesting supernatants containing viruses respectively 48h and 72h after transfection in step S3; centrifuging at 3000 rpm for 10min,filtering with 0.45um filter membrane; in step S5, the refrigerator temperature is 4 ℃, the standing time is 0.2h, and the liquid nitrogen quick-freezing time is 5 h.
Example 2 a method for preparing feeder cells for culturing NK cells, comprising the steps of:
s1, obtaining newly recovered PBMC, performing mixed culture on the PBMC and a corresponding pure factor culture formula, adding heat-inactivated fetal bovine serum, and placing the mixture in a culture flask for culture; after 14 days of culture, the cells were photographed and counted; taking cells for dyeing, and detecting the proportion and the concentration of NK cells by an up-flow cytometer;
s2, constructing an adenovirus vector for expressing siRNA/miRNA; digesting the purified plasmid with PacI; transfecting 293A cells by the digested adenovirus expression vector, and harvesting the cells to prepare a virus crude extract;
s3, infecting 293A cells with the virus crude extract to amplify viruses; after 24h of transfection, the transfection efficiency was observed under a fluorescence microscope; harvesting virus-containing supernatants 48h and 72h after transfection; centrifuging, filtering, and removing cell precipitate;
s4, infecting K562 engineering cells by using the transfected adenovirus vectors; sorting by a flow cytometer to screen K562 cells expressing IL-15 and 4-1bbl proteins;
s5, counting K562 cells, suspending the cells in a freezing solution, putting the cells in a refrigerator, directly putting the cells in liquid nitrogen for quick freezing, and then recovering the cells;
s6, through PI dyeing, observing that all K562 cells die after quick freezing; taking the dead K562 cells, placing the cells in a 96-well plate, tracking and observing for 3 days, and observing the cell morphology;
s7, resuspending the K562 cells with an NK culture medium to be used as trophoblast cells for later use.
Wherein the pure factor culture formulation in step S1 was prepared in a manner similar to that of example 1. When the cell-taking staining up-flow cytometer of the step S1 detects the proportion and the concentration of NK cells, the cells are all suspended in 300ul of stationary buffer and are operated at a medium speed for 60S; observing the transfection efficiency under a fluorescence microscope in the step S3, wherein the transfection efficiency is more than 70%; harvesting supernatants containing viruses respectively 48h and 72h after transfection in step S3; centrifuging at 3000 rpm for 20min, and filtering with 0.45um filter membrane; in step S5, the refrigerator temperature is 4 ℃, the standing time is 0.1h, and the liquid nitrogen quick-freezing time is 6 h.
Example 3a method for preparing feeder cells for culturing NK cells, comprising the steps of:
s1, obtaining newly recovered PBMC, performing mixed culture on the PBMC and a corresponding pure factor culture formula, adding heat-inactivated fetal bovine serum, and placing the mixture in a culture flask for culture; after 14 days of culture, the cells were photographed and counted; taking cells for dyeing, and detecting the proportion and the concentration of NK cells by an up-flow cytometer;
s2, constructing an adenovirus vector for expressing siRNA/miRNA; digesting the purified plasmid with PacI; transfecting 293A cells by the digested adenovirus expression vector, and harvesting the cells to prepare a virus crude extract;
s3, infecting 293A cells with the virus crude extract to amplify viruses; after 24h of transfection, the transfection efficiency was observed under a fluorescence microscope; harvesting virus-containing supernatants 48h and 72h after transfection; centrifuging, filtering, and removing cell precipitate;
s4, infecting K562 engineering cells by using the transfected adenovirus vectors; sorting by a flow cytometer to screen K562 cells expressing IL-15 and 4-1bbl proteins;
s5, counting K562 cells, suspending the cells in a freezing solution, putting the cells in a refrigerator, directly putting the cells in liquid nitrogen for quick freezing, and then recovering the cells;
s6, through PI staining, observing that all the quick-frozen K562 cells die; taking the dead K562 cells, placing the cells in a 96-well plate, tracking and observing for 3 days, and observing the cell morphology;
s7, resuspending the K562 cells with an NK culture medium to be used as trophoblast cells for later use.
Wherein the pure factor culture formulation in step S1 was prepared in a manner similar to that of example 1. When the cell-taking staining up-flow cytometer of the step S1 detects the proportion and the concentration of NK cells, the cells are all suspended in 300ul of stationary buffer and are operated at a medium speed for 60S; observing the transfection efficiency under a fluorescence microscope in the step S3, wherein the transfection efficiency is more than 70%; harvesting supernatants containing viruses respectively 48h and 72h after transfection in step S3; centrifuging at 3000 rpm for 30min, and filtering with 0.45um filter membrane; in step S5, the refrigerator temperature is 4 ℃, the standing time is 2h, and the liquid nitrogen quick-freezing time is 7 h.
Claims (7)
1. A method for preparing feeder cells for culturing NK cells, comprising the steps of:
s1, obtaining newly recovered PBMC, performing mixed culture on the PBMC and a corresponding pure factor culture formula, adding heat-inactivated fetal bovine serum, and placing the mixture in a culture flask for culture; after 14 days of culture, the cells were photographed and counted; taking cells for dyeing, and detecting the proportion and the concentration of NK cells by an up-flow cytometer;
s2, constructing an adenovirus vector for expressing siRNA/miRNA; digesting the purified plasmid with PacI; transfecting 293A cells by the digested adenovirus expression vector, and harvesting the cells to prepare a virus crude extract;
s3, infecting 293A cells with the virus crude extract to amplify viruses; after 24h of transfection, the transfection efficiency was observed under a fluorescence microscope; harvesting virus-containing supernatants 48h and 72h after transfection; centrifuging, filtering, and removing cell precipitate;
s4, infecting K562 engineering cells by using the transfected adenovirus vectors; sorting by a flow cytometer to screen K562 cells expressing IL-15 and 4-1bbl proteins;
s5, counting K562 cells, suspending the cells in a freezing solution, putting the cells in a refrigerator, directly putting the cells in liquid nitrogen for quick freezing, and then recovering the cells;
s6, through PI staining, observing that all the quick-frozen K562 cells die; taking the dead K562 cells, placing the cells in a 96-well plate, tracking and observing for 3 days, and observing the cell morphology;
s7, resuspending the K562 cells with an NK culture medium to be used as trophoblast cells for later use.
2. The method of claim 1, wherein the pure factor culture formulation of step S1 is prepared by the steps of: respectively adding CD16, CD3 and PBS into different culture bottles, placing the culture bottles in a refrigerator at 4 ℃ and placing for more than 24 hours; adding the PBMC obtained by separation into culture bottles coated with monoclonal antibodies in advance, adding IL-2, IFN-gamma, IL-15 and human serum, and culturing at constant temperature; observing the growth state of the cells after 3 days, and adding fresh culture solution (GT-551), IL-2 and human serum; and observing the cell state every 3 days, counting, supplementing an equal-volume fresh culture solution and IL-2 to keep the concentration of the IL-2 in the culture medium unchanged, and culturing until the 14 th day to obtain the final product.
3. The method of claim 2, wherein the pure factor culture formulation of step S1 is prepared in such a way that the final concentration of antibody is 50 ng/ml.
4. The method of claim 1, wherein the proportion and concentration of NK cells are measured by the cell-staining up-flow cytometer of step S1, and the cells are suspended in 300ul of stationary buffer and cultured for 60S at a medium speed.
5. The method of claim 1, wherein the transfection efficiency of step S3 is 70% or more by observing the transfection efficiency under a fluorescence microscope.
6. The method for preparing feeder cells for culturing NK cells according to claim 1, wherein the virus-containing supernatants are harvested 48h and 72h after transfection in step S3; centrifuging at 3000 rpm for 10-30 min, and filtering with 0.45um filter membrane.
7. The method according to claim 1, wherein in step S5, the refrigerator temperature is 4 ℃, the standing time is 0.2-2 h, and the liquid nitrogen quick freezing time is 5-7 h.
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