CN116694569A - Intracellular lysate and cytokine composition and method for efficiently amplifying NK cells in vitro - Google Patents
Intracellular lysate and cytokine composition and method for efficiently amplifying NK cells in vitro Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0646—Natural killers cells [NK], NKT cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/82—Undefined extracts from animals from invertebrates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2315—Interleukin-15 (IL-15)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2321—Interleukin-21 (IL-21)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a cell endolysate and cytokine composition and a method for efficiently amplifying NK cells in vitro, belonging to the technical field of immunology; a composition of intracellular lysate and cytokine is provided, the composition components comprising: intracellular lysates, cytokines; the intracellular lysate is K562 intracellular lysate; the cytokines are: IL15 and IL21; the preparation method of the composition of the intracellular lysate and the cytokine comprises the following steps: dissolving cell factors IL15 and IL21 by using 1XPBS to prepare mother liquor, and mixing the prepared mother liquor with the cell lysate prepared by the method to prepare a composition; a kit for preparing NK cells; use of said composition in the preparation of NK cells; the invention adopts the combination of the cell endolysates and the cytokines to efficiently amplify NK cells in vitro, has active cell growth, high growth speed, homogeneous and transparent cells and stronger killing effect.
Description
Technical Field
The invention belongs to the technical field of immunology, and particularly relates to a composition of intracellular solubles and cytokines and a method for efficiently amplifying NK cells in vitro.
Background
Studies have shown that Natural Killer (NK) cells induce potent anti-tumor, anti-infective immune responses without pre-stimulation, and without MHC-restricted killing of tumor cells and virus-infected cells by the use of perforin and granzyme, among other mechanisms. Allogeneic NK cell transplantation hardly causes graft versus host disease and cytokine storm, and is expected to be an 'off-the-shelf' product. Clinical results show that NK therapy treats recurrent or refractory non-hodgkin lymphoma patients, mostly with clinical remission, and without major toxic effects.
One of the bottlenecks limiting its clinical application is that NK cells are difficult to expand in large amounts in vitro. The K562 cells or the engineering K562 cells can activate and amplify NK cells, the main technology at present is to irradiate the K562 cells or the engineering K562 cells, the K562 cells or the engineering K562 cells are used as feeder cells to be co-cultured with peripheral blood mononuclear cells, and the cultured end product has the potential risk of introducing exogenous cells and has lower safety.
Cytokines play an important role in regulating NK cell activity. Among them, IL-2, IL-12, IL-15, IL-18, IL-21 are important regulating factors for NK cell proliferation and promoting its differentiation and maturation and exerting cytotoxicity, and can stimulate proliferation and activity of NK cells in vitro culture. But the purity of NK cells obtained by the factor method culture is low.
The NK cell amplification kit product prepared by using K562 as feeder cells has high requirement on storage conditions and needs liquid nitrogen storage, and the kit product prepared by the invention can be stored in a refrigerator below minus 20 ℃ without liquid nitrogen storage.
There is no report of K562 cell endolysates and their effects found in the prior art.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a composition of intracellular solubles and cytokines and a method for efficiently amplifying NK cells in vitro.
The invention discovers that a great amount of high-purity NK cells can be obtained by using the cell content and cytokine composition for in vitro amplification of NK cells for the first time, and the risk of K562 cell residue easily caused when K562 is taken as feeder cells is effectively avoided.
The cell content and cytokine composition provided by the invention is used for amplifying NK cells, is easy to prepare into a commercialized kit, does not need liquid nitrogen storage, and is favorable for market popularization and application.
The technical scheme of the invention is as follows:
a composition of an intracellular lysate and a cytokine, the composition components comprising: intracellular lysates, cytokines; the intracellular lysate is K562 intracellular lysate; the cytokines are: IL15 and IL21.
According to the invention, the cytokines IL15 and IL21 preferably have a composition mass ratio of (0.8-1.2): 1.
According to the invention, the protein concentration of the intracellular lysate in the composition is preferably 1-5 mg/ml, and the concentration of the cytokine is preferably 30-100 ug/ml.
The preparation method of the intracellular lysate comprises the following steps:
(1) Lysing K562 cells, and centrifuging to obtain a supernatant;
(2) And (3) carrying out ultrafiltration of 3 KD-10 KD on the supernatant obtained in the step (1), re-suspending substances obtained by ultrafiltration by using a phosphoric acid buffer solution, and carrying out ultrafiltration of 3 KD-10 KD for more than 1 time, wherein the substances obtained by ultrafiltration are intracellular solutions.
According to the invention, in the step (1), K562 cells are mixed with the lysate for lysis, and then centrifuged for 10000 r/min-12000 r/min and 10-15 min to obtain supernatant.
Further preferably, the K562 cells are mixed with the lysate and lysed under low temperature conditions.
More preferably, K562 cells are mixed with the lysate and lysed under ice bath conditions for 3-5 min.
Further preferably, the composition of the lysate: 50mM Tris,150mM NaCl, mass fraction 1% Triton X-100, mass fraction 1% SDS.
The lysate is a conventional commercial product.
According to a preferred embodiment of the invention, in step (2), the ultrafiltration is performed using a 10KD ultrafiltration tube.
Further preferably, the ultrafiltration is performed using a 10KD ultrafiltration tube, centrifuged, 4000g, and 1-2 min.
According to a preferred embodiment of the invention, in step (2), the repetition is carried out 1 to 2 times.
According to the preferred embodiment of the present invention, in the step (2), the obtained intracellular lysate is dissolved by using a 1XPBS solution to prepare intracellular lysate solutions with different concentrations.
The preparation method of the composition of the intracellular lysate and the cytokine comprises the following steps:
(1) the cytokines IL15 and IL21 were solubilized using 1XPBS to prepare a mother liquor;
(2) mixing the mother solution prepared in the step (1) with the intracellular solution to obtain the composition.
According to a preferred embodiment of the present invention, in step (2), the intracellular lysate is prepared by the above-described method.
According to the preferred embodiment of the present invention, in step (2), the concentration of the cytokine in the resulting composition is 30-100 ug/ml and the protein concentration of the intracellular lysate is 1-5 mg/ml.
A kit for preparing NK cells comprising the above cell contents.
According to a preferred embodiment of the invention, the kit comprises the above-mentioned composition.
Use of the above composition for the preparation of NK cells.
A method of preparing NK cells comprising the steps of:
dissolving cell factor IL2 in serum-free culture medium to make IL2 concentration 100-1000 IU/mInoculating peripheral blood mononuclear cells into a serum-free culture medium containing IL2, and making the cell density in the culture medium be (1-3) x10 6 Adding the composition or the cell lysate and the cell factor IL15 and IL21 to make the final concentration of the cell factor in the culture medium be 30-100 ng/mL, the final concentration of the protein in the cell lysate be 3-5 ug/mL, adding 5-10% of cell culture additive or plasma by volume fraction, supplementing serum-free culture medium containing IL2 and 2.5-10% of cell culture additive or plasma by volume fraction every 2-3 days, and keeping the cell density (0.8-1) multiplied by 10 after fluid infusion 6 Culture was continued until NK cells were harvested at a volume of per ml.
According to a preferred embodiment of the invention, the added plasma is autologous plasma.
According to the invention, the serum-free medium is preferably GT-T551H 3 medium.
According to the present invention, NK cells are preferably harvested by culturing until 14 to 21 days.
According to the invention, the cytokines IL15 and IL21 preferably have a composition mass ratio of (0.8-1.2): 1.
The cell culture additives used in the above method are conventional commercial products.
Advantageous effects
The invention discovers for the first time that the combination of K562 cell lysate, cytokines IL15 and IL21 is used for in vitro amplification of NK cells, a large amount of high-purity NK cells can be obtained, the risk of K562 cell residue is easily caused when K562 is taken as feeder cells is effectively avoided, and the method provided by the invention has good safety.
The cell content and cytokine composition provided by the invention is used for amplifying NK cells, is easy to prepare into a commercialized kit, does not need liquid nitrogen storage, and is favorable for market popularization and application.
Drawings
FIG. 1 is a flow chart of NK cell fraction (CD 3) for example 5, example 6, comparative example 1, comparative example 2, comparative example 3, comparative example 4 - CD56 + ) Results of (3) are shown.
FIG. 2 is a graph showing the results of observation of NK cells of the inverted microscope of 40X in examples 5, 6 and 1, 2, 3 and 4.
FIG. 3 is a bar graph of NK amplification factors for examples 5, 6 and comparative examples 1, 2, 3 and 4.
Fig. 4 is a graph showing the in vitro killing effect of K562 in example 5, example 6 and comparative example 1, comparative example 2, comparative example 3, comparative example 4.
Detailed Description
The following describes the invention further in connection with examples, but the invention is not limited thereto.
The experimental procedures referred to in the examples, unless otherwise specified, are conventional in the art.
Sources of the principal materials
K562 cells were purchased from the Withanbozier Life technologies Co.
Cytokines IL12, IL15, IL21 were purchased from su-state offshore protein technologies inc.
1XPBS was purchased from burning.
Lysates were purchased from Biyun Tian biotechnology.
GT-T551H 3 medium was purchased from TAKARA.
Cell culture additives were purchased from daceae as a stock of biotechnology.
Lactate dehydrogenase cytotoxicity assay kits are purchased from Biyun Tian Biotechnology.
Peripheral Blood Mononuclear Cells (PBMC) may be prepared or may be obtained using conventional commercial products.
Example 1
The preparation method of the K562 cell lysate comprises the following steps:
(1) Will be 1X10 6 Placing K562 cells into a 1.5ml sterile centrifuge tube, centrifuging, 600g for 10min, discarding supernatant, washing twice with ice-bath 1×PBS, adding 200ul of sterilized lysate, mixing well, and placing on ice for 5min;
(2) After ice bath is finished, the centrifuge tube is placed in a centrifuge at 4 ℃, and is centrifuged, 12000r/min and 15min are carried out, and the supernatant is transferred into a sterile centrifuge tube;
(3) Placing the obtained supernatant into a 10KD ultrafilter tube, centrifuging, adding 1ml of 1XPBS into the ultrafiltrate obtained by the upper centrifuge tube, mixing well, centrifuging, 4000g of 2min, repeating for 2 times, then taking the ultrafiltrate obtained by the upper centrifuge tube as an intracellular solution, adding 0.2ml of 1XPBS solution, mixing well, and obtaining the intracellular solution for standby.
Example 2
Protein content detection
(1) The intracellular lysate solution prepared in example 1 was assayed for reading at 562nm wavelength according to the BCA protein concentration assay kit protocol configuration standards.
(2) Drawing a standard curve, and calculating the protein content; the protein content of the intracellular lysate solution prepared in example 1 of the present invention was 10mg/ml.
Example 3
A method for preparing a composition of an intracellular lysate and a cytokine comprising the steps of:
(1) The cytokines IL15 and IL21 were prepared in a mass ratio of 1:1, and a cytokine solution having a mother liquor concentration of 100ug/ml was prepared by dissolving the cytokines in 1 XPBS.
(2) Mixing a certain volume of the cytokine solution obtained in step (1), a certain volume of the intracellular solution obtained in example 1 and a certain volume of 1XPBS to form a composition, and keeping the final concentration of the cytokine in the composition at 30ug/ml and the final concentration of the protein in the intracellular solution at 5mg/ml at-20deg.C for later use.
Example 4
Peripheral Blood Mononuclear Cell (PBMC) isolation comprising the steps of:
(1) 30ml of peripheral blood from healthy volunteers was placed in a separation tube containing 15ml of Ficoll separation, centrifuged, 800g,20min.
(2) After centrifugation, peripheral blood mononuclear cells are sucked into a 50ml sterile centrifuge tube, the volume is fixed to 45ml by using NaCl with the mass fraction of 0.9%, centrifugation is carried out for 600g and 10min, and the supernatant is discarded, so that Peripheral Blood Mononuclear Cells (PBMC) are obtained.
Example 5
The intracellular lysate is co-cultured with the cytokine IL15+ IL21 composition with PBMCs to expand NK cells comprising the steps of:
(1) Dissolving a cytokine IL2 in a serum-free culture medium, so that the concentration of the IL2 in the culture medium is 200IU/ml, wherein the serum-free culture medium is a GT-T551H 3 culture medium;
(2) The peripheral blood mononuclear cells isolated in example 4 were suspended in a serum-free medium containing IL2 at a cell density of 2X10 6 Each mL was added with the composition prepared in example 3 so that the final concentration of cytokine in the medium was 30ng/mL and the final concentration of protein in the intracellular lysate was 5ug/mL, and 10% by volume of cell culture additive was added to the medium at 37℃and 5% CO 2 Culturing;
(3) Serum-free medium containing IL2 and 5% by volume of cell culture additive were supplemented every 2 days, maintaining cell density at 1X10 after rehydration 6 Culturing is continued until 14 days, NK cells are harvested, the concentration of living cells is detected by using the dyeing of the phenol blue, and the purity of the NK cells is detected by using the flow type.
Example 6
The difference from example 5 is that the composition used in step (2) has a final cytokine concentration of 100ng/ml, all other things being equal.
Comparative example 1
The difference from example 5 is that the composition used in step (2) replaces cytokine IL21 with cytokine IL15, i.e. does not contain cytokine IL21, all other things being equal.
Comparative example 2
The difference from example 5 is that the composition used in step (2) had a final protein concentration of 2.5ug/ml in the intracellular solution, all other things being equal.
Comparative example 3
The difference from example 5 is that the composition used in step (2) is supplemented with cytokine IL12, the mass ratio of cytokine IL12 to IL15 and IL21 being 1:1:1, all other things being equal.
Comparative example 4
The difference from example 5 is that the intracellular lysate was replaced with irradiated feeder layer K562 cells, the irradiated feeder layer K562 cells were cultured with the cytokine IL15+ IL21, the ratio of the number of feeder layer K562 cells to the number of PBMC was 1:1, and the other was the same.
The experimental results show that the NK cells obtained in the example 5 and the example 6 have advantages in terms of purity, growth state and amplification factor compared with those of the comparative examples 1-4, the detection results are shown in the figures 1, 2 and 3, the purity of the NK cells prepared in the example 5 is 90.48%, the amplification factor is 432 times, the purity of the NK cells prepared in the example 6 is 91.27%, and the amplification factor is 442 times; the NK cell purity 86.77% prepared in comparative example 4 was amplified 340-fold; examples 5 and 6 showed active cell growth, fast growth, homogeneous and transparent cells, and more cell clusters than the other comparative examples.
The present inventors have also performed a comparison that the cell lysate contains only the cytokine IL15+ IL21 without the intracellular lysate when NK cells are prepared, and the effect is poor.
Effect example
NK cells kill human chronic myelogenous leukemia K562 cells of hematological tumor in vitro, specifically as follows:
NK cell killing power was calculated by performing operations according to the instruction of the lactate dehydrogenase cytotoxicity detection kit under conditions of 10:1 of the effector cells and the target cells K562, which were obtained by culturing in example 5, example 6, comparative example 1, comparative example 2, comparative example 3 and comparative example 4.
The experimental results show that the in vitro killing K562 reaches 66% in the embodiment 5 and 68.5% in the embodiment 6 under the condition of the effective target ratio of 10:1, and is superior to the comparative examples 1-4, and 49.5% in the comparative example 4, as shown in figure 4.
The invention adopts the combination of the cell endolysate and the cytokine to efficiently amplify NK cells in vitro, has active cell growth, high growth speed, homogeneous and transparent cells and stronger killing effect.
Claims (10)
1. A composition of intracellular lysate and cytokine, wherein the composition comprises: intracellular lysates, cytokines; the intracellular lysate is K562 intracellular lysate; the cytokines are: IL15 and IL21.
2. The composition of claim 1, wherein the cytokines IL15 and IL21 comprise a mass ratio of (0.8-1.2) 1;
preferably, the protein concentration of the intracellular lysate in the composition is 1-5 mg/ml, and the concentration of the cytokine is 30-100 ug/ml.
3. The method for preparing an intracellular lysate according to claim 1, comprising the steps of:
(1) Lysing K562 cells, and centrifuging to obtain a supernatant;
(2) And (3) carrying out ultrafiltration of 3 KD-10 KD on the supernatant obtained in the step (1), re-suspending substances obtained by ultrafiltration by using a phosphoric acid buffer solution, and carrying out ultrafiltration of 3 KD-10 KD for more than 1 time, wherein the substances obtained by ultrafiltration are intracellular solutions.
4. The method according to claim 3, wherein in the step (1), K562 cells are mixed with the lysate to be lysed, and then centrifuged at 10000r/min to 12000r/min for 10 to 15min to obtain a supernatant;
preferably, K562 cells are mixed with the lysate and lysed under low temperature conditions;
preferably, K562 cells are mixed with the lysate and lysed under ice bath conditions for 3-5 min;
preferably, the composition of the lysate is: 50mM Tris,150mM NaCl, 1% Triton X-100 by mass and 1% SDS by mass;
in the step (2), the ultrafiltration is performed by using a 10KD ultrafiltration tube;
preferably, the ultrafiltration is carried out by using a 10KD ultrafiltration tube, centrifuging, 4000g and 1-2 min;
preferably, in step (2), repeating 1-2 times;
preferably, in the step (2), the obtained intracellular lysate is dissolved by using a 1XPBS solution to prepare intracellular lysate solutions with different concentrations.
5. A method of preparing the endo-lysate and cytokine composition of claim 1, comprising the steps of:
(1) the cytokines IL15 and IL21 were solubilized using 1XPBS to prepare a mother liquor;
(2) mixing the mother solution prepared in the step (1) with the intracellular solution to obtain the composition.
6. The method of claim 5, wherein in step (2), the intracellular lysate is prepared by the method of claim 3;
preferably, in step (2), the concentration of cytokines in the resulting composition is 30-100 ug/ml and the protein concentration of intracellular lysates is 1-5 mg/ml.
7. A kit for preparing NK cells, comprising the cell content of claim 1;
preferably, the kit comprises a composition as claimed in claim 1.
8. Use of the composition of claim 1 for the preparation of NK cells.
9. A method of preparing NK cells comprising the steps of:
dissolving cell factor IL2 in serum-free culture medium to make IL2 concentration be 100-1000 IU/ml, inoculating peripheral blood mononuclear cell in serum-free culture medium containing IL2, after inoculating, making cell density in culture medium be (1-3). Times.10 6 Adding the composition of claim 1 or the cell lysate and the cell factor IL15 and IL21 of claim 1 to make the final concentration of the cell factor in the culture medium be 30-100 ng/mL, the final concentration of the protein in the cell lysate be 3-5 ug/mL, adding 5-10% of cell culture additive or plasma by volume fraction, supplementing serum-free culture medium containing IL2 and 2.5-10% of cell culture additive or plasma by volume fraction every 2-3 days, and keeping the cell density (0.8-1) x10 after the fluid supplementation 6 Culture was continued until NK cells were harvested at a volume of per ml.
10. The method of claim 9, wherein the added plasma is autologous plasma;
preferably, the serum-free medium is GT-T551H 3 medium;
preferably, NK cells are harvested after culturing until 14 to 21 days;
preferably, the cytokines IL15 and IL21 are combined in a mass ratio of (0.8-1.2): 1.
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