CN112029722A - NK cell culture medium and application thereof - Google Patents

NK cell culture medium and application thereof Download PDF

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CN112029722A
CN112029722A CN202010941836.7A CN202010941836A CN112029722A CN 112029722 A CN112029722 A CN 112029722A CN 202010941836 A CN202010941836 A CN 202010941836A CN 112029722 A CN112029722 A CN 112029722A
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culture medium
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汤朝阳
秦乐
吴迪
魏志辉
王翠花
杨凯纯
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Guangdong Zhaotai In Vivo Biomedical Technology Co ltd
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    • C12N2501/50—Cell markers; Cell surface determinants
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/59—Lectins

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Abstract

The invention provides an NK cell culture medium and application thereof, wherein the culture medium is a basic culture medium containing MICA and CD 80. The invention adopts the basal culture medium containing MICA and CD80 to culture the NK cells, not only improves the amplification capacity of the NK cells, but also keeps good tumor cell killing toxicity of the NK cells, and is applied to tumor biotherapy.

Description

NK cell culture medium and application thereof
Technical Field
The invention belongs to the technical field of cell culture, and relates to an NK cell culture medium and application thereof.
Background
NK cells (natural killer cells), also known as large granular lymphocytes, perform innate and adaptive immune functions in conjunction with gamma T cells and NKT cells. On one hand, when the organism is infected or wounded, the NK cells can rapidly, widely and specifically recognize antigens in a non-peptide-MHC (major histocompatibility complex) -mode to prevent the identity of a person, remove pathogenic microorganisms and variant cells in time and play a role in innate immunity; on the other hand, NK cells are involved in adaptive immune responses, affecting the effector functions of α β T cells and B cells.
In the process of generating and developing tumors, NK cells can recognize that tumor cells are activated through an activating receptor and can also be activated by accessory cells such as monocyte, macrophage and dendritic cell, the accessory cells respond to the change of internal and external environments through a pattern recognition receptor and transmit signals to the NK cells through secreting soluble factors or directly contacting.
At present, the main obstacle restricting the clinical application of NK cells is that sufficient NK cells are difficult to obtain, and the realization of large-scale amplification of NK cells in vitro is a key problem to be solved in NK cell treatment. The number of NK cells in peripheral blood is small, while the number and activity of NK cells in peripheral blood of tumor patients are obviously reduced, and the nature of NK cells of different people is greatly different. The Chimeric Antigen Receptor (CAR) is used for modifying NK cells to treat tumors, and the requirement on the number of the NK cells is high.
The prior art has the problems of short service life, insufficient activity and the like of the amplified NK cells. Therefore, the search for a more efficient NK cell large-scale amplification method has great significance for clinical application of NK cells.
Disclosure of Invention
Aiming at the defects and actual requirements of the prior art, the invention provides the NK cell culture medium and the application thereof, wherein the culture medium has a plurality of ligands for activating NK cells, so that the number of the NK cells can be obviously increased, the service life of the NK cells is prolonged, and the activity of the NK cells is improved.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides an NK cell culture medium, which is a basal medium comprising MICA and CD 80.
In the invention, MICA is the ligand of the most main activating receptor NKG2D on the surface of NK cells, has obvious activating effect on the NK cells, and CD80 is expressed on the surfaces of activated B cells, dendritic cells and mononuclear macrophages and transmits auxiliary signals to the NK cells; the invention adopts the basal culture medium containing MICA and CD80 to culture the NK cells, not only improves the amplification quantity of the NK cells, but also keeps good tumor cell killing toxicity of the NK cells, and is applied to tumor biotherapy.
Preferably, the MICA comprises the amino acid sequence shown as SEQ ID NO. 1;
SEQ ID NO:1：
MGLGPVFLLLAGIFPFAPPGAAAEPHSLRYNLTVLSWDGSVQSGFLTEVHLDGQPFLRCDRQKCRAKPQGQWAEDVLGNKTWDRETRDLTGNGKDLRMTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGELFLSQNLETKEWTMPQSSRAQTLAMNVRNFLKEDAMKTKTHYHAMHADCLQELRRYLKSGVVLRRTVPPMVNVTRSEASEGNITVTCRASGFYPWNITLSWRQDGVSLSHDTQQWGDVLPDGNGTYQTWVATRICQGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQSHWQTFHVSAVAAAAIFVIIIFYVRCCKKKTSAAEGPELVSLQVLDQHPVGTSDHRDATQLGFQPLMSDLGSTGSTEGA。
preferably, the CD80 comprises an amino acid sequence shown as SEQ ID NO. 2;
SEQ ID NO:2：
MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV。
preferably, the MICA concentration is 1-100 ng/mL, for example, 1ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL or 100 ng/mL.
Preferably, the concentration of the CD80 is 1-100 ng/mL, for example, 1ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL or 100 ng/mL.
Preferably, the medium further comprises IL-2.
Preferably, the concentration of IL-2 is 20-40U/mL, for example, 20U/mL, 21U/mL, 22U/mL, 23U/mL, 24U/mL, 25U/mL, 26U/mL, 27U/mL, 28U/mL, 29U/mL, 30U/mL, 31U/mL, 32U/mL, 33U/mL, 34U/mL, 35U/mL, 36U/mL, 37U/mL, 38U/mL, 39U/mL or 40U/mL.
Preferably, the medium further comprises serum.
Preferably, the serum is present in a volume percentage of 1% to 5%, for example 1%, 2%, 3%, 4% or 5%.
Preferably, the basal medium comprises any one of Eagle's broth, RPMI-1640 medium, or Ham's F-10, or a combination of at least two thereof.
In a second aspect, the present invention provides a method for culturing NK cells, the method comprising the step of culturing NK cells using the medium according to the first aspect.
Preferably, the seeding density of the NK cells is (1-5) multiplied by 104/mL, for example, may be 1X 104/mL、2×104/mL、3×104/mL、4×104Per mL or 5X 104Perml, preferably (1 to 3). times.104Perml, more preferably 2X 104/mL。
Preferably, the temperature of the culture is 35-40 ℃, for example, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃ or 40 ℃, preferably 37 ℃.
Preferably, the humidity of the culture is 90% to 98%, and may be, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, preferably 92% to 97%, and more preferably 95%.
As a preferred technical scheme, the invention provides an NK cell culture method, which comprises the following steps:
resuspending NK cells in a basal medium containing 1-100 ng/mL MICA, 1-100 ng/mL CD80, 20-40U/mL IL-2 and 1-5% serum so that the density of NK cells is (1-5) x 104/mL；
And (3) placing the cell suspension into a cell culture dish, culturing at the humidity of 90-98% and the temperature of 35-40 ℃, and performing NK cell amplification.
In a third aspect, the present invention provides an NK cell prepared by the method of the second aspect.
In a fourth aspect, the present invention provides the use of the NK cell of the third aspect for the preparation of a medicament for the treatment and/or prevention of tumors.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the invention, the NK cells are cultured by adopting a basal medium containing MICA and CD80, the MICA and CD80 jointly activate the NK cells, the amplification number of the NK cells is increased, and the number of the cells is increased to 3200 times after 2 weeks of culture;
(2) the NK cells cultured by the invention have good tumor cell killing toxicity and wide application prospect in the field of biological treatment of tumors.
Drawings
FIG. 1 shows the expansion efficiency of NK cells;
FIG. 2 shows the killing efficiency of NK cells.
Detailed Description
To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Example 1
This example resuspends NK cells in RPMI-1640 medium containing 50ng/mL MICA, 50ng/mL CD80, 30U/mL IL-2 and 5% serum to a density of 3X 104/mL；
The cell suspension was placed in a cell culture dish, cultured at 37 ℃ with 95% humidity, and NK cell expansion was performed.
Example 2
Resuspending NK cells in RPMI-1640 medium containing 80ng/mL MICA, 75ng/mL CD80, 25U/mL IL-2 and 4% serum to a density of 2X 104/mL；
The cell suspension was placed in a cell culture dish, cultured at a temperature of 36 ℃ with a humidity of 92%, and NK cell expansion was performed.
Example 3
Resuspending NK cells in RPMI-1640 medium containing 20ng/mL MICA, 25ng/mL CD80, 35U/mL IL-2 and 3% serum to a density of 4X 104/mL；
The cell suspension was placed in a cell culture dish, cultured at a humidity of 97% and a temperature of 38 ℃ to expand NK cells.
Example 4
Resuspending NK cells in Eagle Medium containing 1ng/mL MICA, 1ng/mL CD80, 20U/mL IL-2 and 2% serum to a density of 5X 104/mL；
The cell suspension was placed in a cell culture dish, cultured at a temperature of 35 ℃ and humidity of 90%, and NK cell expansion was performed.
Example 5
Resuspend NK cells with Ham's F-10 containing 100ng/mL MICA, 100ng/mL CD80, 40U/mL IL-2 and 1% serum to a density of 1X 104/mL；
The cell suspension was placed in a cell culture dish, cultured at a humidity of 98% and a temperature of 40 ℃ to perform NK cell expansion.
Comparative example 1
Compared with example 1, the culture medium does not contain MICA, specifically as follows:
resuspending NK cells in RPMI-1640 medium containing 50ng/mL CD80, 30U/mL IL-2 and 5% serum to a density of 3X 104/mL；
The cell suspension was placed in a cell culture dish, cultured at 37 ℃ with 95% humidity, and NK cell expansion was performed.
Comparative example 2
Compared with the example 1, the culture medium does not contain CD80, and the specific steps are as follows:
this example resuspends NK cells to a density of 3X 10 NK cells using RPMI-1640 medium containing 50ng/mL MICA, 30U/mL IL-2 and 5% serum4/mL；
The cell suspension was placed in a cell culture dish, cultured at 37 ℃ with 95% humidity, and NK cell expansion was performed.
Comparative example 3
Compared to example 1, the medium did not contain MICA and CD80, as follows:
this example resuspends NK cells to a density of 3X 10 NK cells using RPMI-1640 medium containing 30U/mL IL-2 and 5% serum4/mL；
The cell suspension was placed in a cell culture dish, cultured at 37 ℃ with 95% humidity, and NK cell expansion was performed.
Amplification efficiency of NK cells
The numbers of NK cells of examples and comparative examples were counted on days 0 and 14, respectively, and the amplification factor of NK in 14 days was calculated.
The results are shown in fig. 1, the expansion efficiency of NK cells of the example is significantly higher than that of the comparative example, indicating that MICA and CD80 cooperate to promote the expansion of NK cells; the culture medium of example 1 is optimally formulated and the culture conditions are optimally set, and the number of cells increases by about 3200 times after 14 days, compared with the highest expansion efficiency of NK cells of other examples and comparative examples.
Killing efficiency of NK
The NK prepared in examples and comparative examples were compared with 1X 104Mixing individual myeloid leukemia cell line KG1 at E: T ratio of 1:1, adding into 96-well plate with 3 multiple wells in each group, centrifuging at 250g for 5min, standing at 37 deg.C and 5% CO2Co-culturing for 18h in an incubator;
after 18h, 100. mu.L/well Luciferase substrate (1X) was added to a 96-well plate, the cells were resuspended and mixed well, RLU (relative light unit) was immediately measured by a multifunctional microplate reader for 1 second, and the killing effect of NK prepared in different examples and comparative examples on KG1 was compared in vitro by the Luciferase (Luciferase) quantitative killing efficiency evaluation method, and the killing ratio calculation formula was as follows:
100% × (control well reading-experimental well reading)/control well reading (blank reading without cells negligible)
The results are shown in fig. 2, the killing efficiency of the NK cells of the example is also significantly higher than that of the comparative example, which shows that the combination of MICA and CD80 not only promotes the expansion of NK cells, but also enhances the killing effect of NK cells; the culture medium of example 1 is optimal in formulation and culture conditions, and the killing efficiency of NK cells is highest compared to other examples and comparative examples.
In conclusion, the NK cells are cultured by adopting the basal medium containing MICA and CD80, and the number of the cells is increased by 3200 times after 2 weeks of culture; the cultured NK cells have good tumor cell killing toxicity and have wide application prospect in the field of biological treatment of tumors.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Sequence listing
<110> Guangdong Shoutai biomedical science and technology Co., Ltd
<120> NK cell culture medium and application thereof
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Val His Leu Asp Gly Gln Pro Phe Leu Arg Cys Asp Arg Gln Lys Cys
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Leu Ala Met Asn Val Arg Asn Phe Leu Lys Glu Asp Ala Met Lys Thr
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Lys Thr His Tyr His Ala Met His Ala Asp Cys Leu Gln Glu Leu Arg
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Arg Tyr Leu Lys Ser Gly Val Val Leu Arg Arg Thr Val Pro Pro Met
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Val Asn Val Thr Arg Ser Glu Ala Ser Glu Gly Asn Ile Thr Val Thr
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Cys Arg Ala Ser Gly Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg
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Claims (10)

1. An NK cell culture medium, characterized in that the culture medium is a basal medium containing MICA and CD 80.
2. The culture medium of claim 1, wherein the MICA comprises the amino acid sequence set forth in SEQ ID No. 1;
preferably, the CD80 comprises the amino acid sequence shown as SEQ ID NO. 2.
3. The culture medium according to claim 1 or 2, wherein the MICA has a concentration of 1 to 100 ng/mL;
preferably, the concentration of the CD80 is 1-100 ng/mL.
4. The culture medium according to any one of claims 1 to 3, wherein the culture medium further comprises IL-2;
preferably, the concentration of the IL-2 is 20-40U/mL.
5. The culture medium according to any one of claims 1 to 4, wherein the culture medium further comprises serum;
preferably, the volume percentage of the serum is 1-5%.
6. The culture medium according to any one of claims 1 to 5, wherein the basal medium comprises any one of Eagle's broth, RPMI-1640 medium, or Ham's F-10, or a combination of at least two thereof.
7. A method for culturing NK cells, comprising the step of culturing NK cells using the medium according to any one of claims 1 to 6;
preferably, the seeding density of the NK cells is (1-5) multiplied by 104Perml, preferably (1 to 3). times.104/mL；
Preferably, the temperature of the culture is 35-40 ℃;
preferably, the humidity of the culture is 90% to 98%.
8. The method of claim 7, wherein the method comprises:
resuspending NK cells in a basal medium containing 1-100 ng/mL MICA, 1-100 ng/mL CD80, 20-40U/mL IL-2 and 1-5% serum so that the density of NK cells is (1-5) x 104/mL；
And (3) placing the cell suspension into a cell culture dish, culturing at the humidity of 90-98% and the temperature of 35-40 ℃, and performing NK cell amplification.
9. An NK cell produced by the method of claim 7 or 8.
10. Use of the NK cell of claim 9 for the preparation of a medicament for the treatment and/or prevention of tumors.
CN202010941836.7A 2020-09-09 2020-09-09 NK cell culture medium and application thereof Pending CN112029722A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112626028A (en) * 2020-12-21 2021-04-09 广东昭泰体内生物医药科技有限公司 Engineered cell for activating NK-like cell and preparation method and application thereof

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