CN113388579A - A culture medium containing cytokine for differentiating hematopoietic stem cell into natural killer cell and its differentiation method - Google Patents
A culture medium containing cytokine for differentiating hematopoietic stem cell into natural killer cell and its differentiation method Download PDFInfo
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Abstract
The invention discloses a culture medium containing cell factors and capable of differentiating hematopoietic stem cells into natural killer cells, which belongs to the technical field of biology and comprises the following components: IMDM medium, DMEM-F12 medium and stemline II hematopoietic stem cell medium, glucose, L-glutamine, sodium pyruvate, sodium selenite, non-essential amino acids and cytokines. The invention uses a pure cell factor method to prepare the culture medium, thereby avoiding the use of serum and reducing pollution sources so as to avoid pollution; the invention avoids using other cell lines as the feeding induction conditions, so that the differentiated cells are safer and the unknown risk is reduced.
Description
Technical Field
The invention belongs to the field of biotechnology, and particularly relates to a culture medium containing cytokines and capable of differentiating hematopoietic stem cells into natural killer cells and a differentiation method thereof.
Background
Natural killer cells (NK) are important immune cells in the body, involved not only in anti-tumor, anti-viral infection and immune regulation, but also in some cases in hypersensitivity reactions and autoimmune diseases.
Natural Killer cells (NK), which are named because their killing of tumor cells or virus-infected cells do not require pre-sensitization, are the first line of defense in human immune surveillance, have strong anti-tumor effects and good safety, NK cells appear in central and peripheral immune organs as well as multiple non-immune organs with multiple Cell phenotypes, playing an important surveillance role in the early stages of the body's immunity: in the liver, the liver stellate cells can be killed through direct cytotoxicity and the production of gamma-interferon equivalent reactive molecules, so that the liver fibrosis resistance is obvious, the liver is damaged to a certain extent, physiological or pathological changes of NK cells have important influence on the pregnancy process, and an immune system plays an important role in controlling and eradicating tumor cells.
Natural Killer (NK) cells are the effector cells of the body's innate immune system and can recognize and kill tumor cells without antigen presentation.
NK cells have cytotoxic effects on various tumors, and the combined application of the NK cells and other clinical tumor treatment methods, particularly the application of the NK cells in the clinical transformation of anti-tumor in recent years, is increasing, so that how to obtain the NK cells meeting clinical requirements through in vitro culture becomes a hot problem for researchers to study in recent years.
Disclosure of Invention
The invention aims to provide a culture medium containing cytokines and capable of differentiating hematopoietic stem cells into natural killer cells and a differentiation method thereof, so as to solve the defects in the prior art.
The technical scheme adopted by the invention is as follows:
a culture medium containing cytokine for differentiating hematopoietic stem cells into natural killer cells, comprising the following components:
IMDM medium, DMEM-F12 medium and stemline II hematopoietic stem cell medium.
Further, the feed also comprises the following components:
glucose, L-glutamine, sodium pyruvate, sodium selenite, non-essential amino acids, and cytokines.
Further, the volume ratio of the IMDM culture medium, the DMEM-F12 culture medium and the stemline II hematopoietic stem cell culture medium is 1:2: 1.
Further, the concentration content of the glucose is 1000-2500 mg/l; the concentration content of the L-glutamine is 0.5-5 mM; the concentration content of the sodium pyruvate is 10-50 ng/l; the concentration content of the sodium selenite is 10-50 ng/l; the concentration content of the non-essential amino acid is 10-100 ug/l.
Preferably, the concentration content of the glucose is 1500 mg/l; the concentration content of the L-glutamine is 2 mM; the concentration content of the sodium pyruvate is 20 ng/l; the concentration content of the sodium selenite is 20 ng/l; the concentration content of the non-essential amino acid is 20 ug/l.
Further, the cytokines include: IL-7, IL-15, SCF, Flt3L, SR-1, UM 171.
Further, the concentration content of the IL-7 is 10-100 ng/ml; the concentration content of the IL-15 is 5-50 ng/ml; the concentration content of the SCF is 10-100 ng/ml; the concentration content of Flt3L is 5-50 ng/ml; the concentration content of the SR-1 is 0.5-1 nmlo/ml; the concentration of UM171 is 10-50 nmlo/ml.
A method for differentiating hematopoietic stem cells into natural killer cells, comprising a first stage culture and a second stage culture, comprising the steps of, using the medium according to any one of claims 1 to 7, specifically:
in the first stage, cells were cultured in the above medium for 7 days using a low adsorption plate coated overnight with IL-3 at a concentration of 20ug/ml, during which half-changes were made to the cells at 3 day intervals;
in the second stage, cells are transferred to the culture medium, and the cells are cultured on an ultra-low adsorption culture plate for 7 days, and half liquid change is carried out on the cells every 3 days.
Further, the hematopoietic stem cells include hematopoietic progenitor cells.
The invention has the following beneficial effects:
the invention uses a pure cell factor method to prepare the culture medium, thereby avoiding the use of serum and reducing pollution sources so as to avoid pollution; the invention avoids using other cell lines as the feeding induction conditions, so that the differentiated cells are safer and the unknown risk is reduced.
Drawings
FIG. 1 is a graph showing the results of flow-assay of the positive rates of CD3-CD56+ in example 1 and its comparative group;
FIG. 2 is a graph showing the results of flow-assay of the positive rates of CD3-CD56+ in example 2 and its comparative group;
FIG. 3 is a graph showing the results of the positive rates of CD3-CD56+ in the negative group of flow assay;
FIG. 4 is a bar graph of the positive rates of example 1 and its comparative group, and example 2 and its comparative group.
Detailed Description
The following will clearly and completely describe the technical solutions of the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The formula of the culture medium adopted in this example takes a 100m system as an example: 25ml of IMDM culture medium, 50ml of DMEM-F12 culture medium and 25ml of stemline II hematopoietic stem cell culture medium, wherein the glucose concentration is 1500mg/L, the L-glutamine concentration is 2mM, the sodium pyruvate concentration is 20ng/L, the sodium selenite concentration is 20ng/L, the unnecessary amino acid concentration is 20ug/L, the IL-7 concentration is 50ng/ml, the IL-15 concentration is 25ng/ml, the SCF concentration is 50ng/ml, the Flt3L concentration is 25ng/ml, the SR-1 concentration is 35uM, and the UM171 concentration is 35 uM.
Example 1 six well low adsorption plates were coated one day ahead with DPBS containing IL-3 at a concentration of 20ug/ml and refrigerated overnight at 4 ℃. The coated culture plate is washed by DPBS for 3 times, and the inoculation number of the hematopoietic stem cells inoculated into the differentiation culture medium is 12 ten thousand/ml; the day of inoculation was recorded as day 0, hematopoietic stem cells were cultured under these conditions for 7 days, during which half-fluid changes were made to the cells at 3-day intervals; on day 8, cells were transferred to new ultra low adsorption plates with medium and culture conditions unchanged to day 14 of culture.
For comparison, this example also provided a control group, which differed from this example only in that the cytokine concentration was halved, the IL-15 concentration was 12.5ng/ml, the SCF concentration was 12.5ng/ml, the Flt3L concentration was 25ng/ml, the SR-1 concentration was 17.5uM, and the UM171 concentration was 17.5 uM.
Example 2
This example is a variation of example 1, only in that hematopoietic progenitor cells are selected for differentiation.
The formula of the culture solution adopted in this example takes a 100m system as an example: 25ml of IMDM culture medium, 50ml of DMEM-F12 culture medium and 25ml of stemline II hematopoietic stem cell culture medium, wherein the glucose concentration is 1500mg/L, the L-glutamine concentration is 2mM, the sodium pyruvate concentration is 20ng/L, the sodium selenite concentration is 20ng/L, the unnecessary amino acid concentration is 20ug/L, the IL-7 concentration is 50ng/ml, the IL-15 concentration is 25ng/ml, the SCF concentration is 50ng/ml, the Flt3L concentration is 25ng/ml, the SR-1 concentration is 35uM, and the UM171 concentration is 35 uM.
Example 1 six well low adsorption plates were coated one day ahead with DPBS containing IL-3 at a concentration of 20ug/ml and refrigerated overnight at 4 ℃. The coated culture plate is washed by DPBS for 3 times, and the inoculation number of the hematopoietic stem cells inoculated into the differentiation culture medium is 12 ten thousand/ml; the day of inoculation was recorded as day 0, hematopoietic stem cells were cultured under these conditions for 7 days, during which half-fluid changes were made to the cells at 3-day intervals; on day 8, cells were transferred to new ultra low adsorption plates with medium and culture conditions unchanged to day 14 of culture.
For comparison, this example also provided a control group, which differed from example 2 only in that the cytokine concentration was halved, the IL-15 concentration was 12.5ng/ml, the SCF concentration was 12.5ng/ml, the Flt3L concentration was 25ng/ml, the SR-1 concentration was 17.5uM, and the UM171 concentration was 17.5 uM.
Results of examples 1-2
Detection of CD3-CD56+ positivity: the cells finally obtained in the examples and the comparative groups thereof are collected, centrifuged at 1000r/min for 5min in a 50mL centrifuge tube, the supernatant is discarded and then resuspended in 1mL PBS containing 2% (v/v) serum, the antibodies CD3-FITC and CD56-APC are added, and the mixture is incubated at 4 ℃ for 30min and washed once with PBS containing 2% serum. The cells were then resuspended at a density of 5X 106/ml and the differentiation efficiency was examined and analyzed using an Agilent NovoCyte flow cytometer. In the detection process, the common hematopoietic stem cells are used as a negative group, namely, the cells which are not subjected to NK induced differentiation or the cells which are subjected to a differentiation experiment but are not stained by a flow antibody.
Results the test results of example 1 and the comparative group are shown in fig. 1 and fig. 4, and the flow cytometry analysis shows that the positive rate of CD3-CD56+ of example 1 is increased to 96.55% (the comparative group is 81.14%); the test results of example 2 and the comparative group are shown in fig. 2 and fig. 4, the positive rate of CD3-CD56+ is increased to 87.25% (the comparative group is 74.41%), and the differentiation efficiency is obviously increased. The negative control group is shown in FIG. 3.
The cells adopted in the embodiment are NK cells obtained by differentiation of hematopoietic stem cells by Protist Biotech, Inc., and the positive rate of CD3-CD56+ is over 90 percent.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention, and the present invention is not limited to the illustrated embodiments, and all the modifications and equivalents of the embodiments may be made without departing from the spirit of the present invention.
Claims (9)
1. A medium containing cytokines for differentiating hematopoietic stem cells into natural killer cells, comprising the following components:
IMDM medium, DMEM-F12 medium and stemline II hematopoietic stem cell medium.
2. The medium according to claim 1, further comprising the following components:
glucose, L-glutamine, sodium pyruvate, sodium selenite, non-essential amino acids, and cytokines.
3. The cytokine-containing medium for differentiating hematopoietic stem cells into natural killer cells according to claim 2, wherein the IMDM medium, the DMEM-F12 medium and the stemline ii hematopoietic stem cell medium correspond to a volume ratio of 1:2: 1.
4. The medium containing cytokines for differentiating hematopoietic stem cells into natural killer cells as claimed in claim 3, wherein the concentration of glucose is 1000-2500 mg/l; the concentration content of the L-glutamine is 0.5-5 mM; the concentration content of the sodium pyruvate is 10-50 ng/l; the concentration content of the sodium selenite is 10-50 ng/l; the concentration content of the non-essential amino acid is 10-100 ug/l.
5. The cytokine-containing medium for differentiating hematopoietic stem cells into natural killer cells according to claim 4, wherein the concentration of glucose is 1500 mg/l; the concentration content of the L-glutamine is 2 mM; the concentration content of the sodium pyruvate is 20 ng/l; the concentration content of the sodium selenite is 20 ng/l; the concentration content of the non-essential amino acid is 20 ug/l.
6. The medium according to claim 4, wherein the cytokine comprises: IL-7, IL-15, SCF, Flt3L, SR-1, UM 171.
7. The medium according to claim 6, wherein the concentration of IL-7 is 10-100 ng/ml; the concentration content of the IL-15 is 5-50 ng/ml; the concentration content of the SCF is 10-100 ng/ml; the concentration content of Flt3L is 5-50 ng/ml; the concentration content of the SR-1 is 0.5-1 nmlo/ml; the concentration of UM171 is 10-50 nmlo/ml.
8. A method for differentiating hematopoietic stem cells into natural killer cells, comprising the first stage culture and the second stage culture, comprising the steps of, using the medium according to any one of claims 1 to 7, specifically:
in the first stage, cells were cultured in the above medium for 7 days using a low adsorption plate coated overnight with IL-3 at a concentration of 20ug/ml, during which half-changes were made to the cells at 3 day intervals;
in the second stage, cells are transferred to the culture medium, and the cells are cultured on an ultra-low adsorption culture plate for 7 days, and half liquid change is carried out on the cells every 3 days.
9. The method of differentiating hematopoietic stem cells into natural killer cells according to claim 8, wherein said hematopoietic stem cells comprise hematopoietic progenitor cells.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109777773A (en) * | 2019-02-26 | 2019-05-21 | 北京呈诺医学科技有限公司 | It is a kind of to break up the method and its culture medium for generating NK cell from candidate stem cell |
| CN111235105A (en) * | 2020-03-06 | 2020-06-05 | 安徽中盛溯源生物科技有限公司 | Method for differentiating human pluripotent stem cells into natural killer cells and application |
| CN112662627A (en) * | 2021-01-19 | 2021-04-16 | 上海爱萨尔生物科技有限公司 | Culture solution for differentiating pluripotent stem cells into natural killer cells and differentiation method |
| KR102292843B1 (en) * | 2020-12-29 | 2021-08-25 | 주식회사 온코인사이트 | Induced pluripotent stem cell(iPSC) derived natural killer cell and its use |
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| CN109777773A (en) * | 2019-02-26 | 2019-05-21 | 北京呈诺医学科技有限公司 | It is a kind of to break up the method and its culture medium for generating NK cell from candidate stem cell |
| CN111235105A (en) * | 2020-03-06 | 2020-06-05 | 安徽中盛溯源生物科技有限公司 | Method for differentiating human pluripotent stem cells into natural killer cells and application |
| KR102292843B1 (en) * | 2020-12-29 | 2021-08-25 | 주식회사 온코인사이트 | Induced pluripotent stem cell(iPSC) derived natural killer cell and its use |
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Application publication date: 20210914 |