CN107400660A - A kind of new method of efficient activation and amplification NKT cells - Google Patents
A kind of new method of efficient activation and amplification NKT cells Download PDFInfo
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Abstract
The invention discloses a kind of efficient activation and the new method of amplification NKT cells, comprise the following steps：Step 1：PBMC cells are isolated from peripheral blood；Step 2：By the PBMC cells isolated after culture medium is resuspended, it is transferred in the culture vessel being coated with through anti-CD3antibody and recombinant human fibronectin polypeptide, and is cultivated after adding IFN γ into culture medium；Step 3：Continue to cultivate after continuously adding vitamin C, human serum albumins, IL 2 and OK432 into culture medium, obtain NKT cells.The present invention can significantly improve the purity and and yield of NKT cells；And NKT cultural methods provided by the invention are simple to operate, spent cost is also than relatively low.CD3 antibody and fibronectin are used in the initial period of culture NKT cells, this can significantly improve NKT induced efficiency.IFN γ and OK432 are added inside the culture medium of NKT cells, can significantly increase the killing ability of NKT cells, and OK432 can significantly promote the multiplication capacity of NKT cells.
Description
Technical field
The present invention relates to technical field of cell biology, and in particular to the new side of a kind of efficient activation and amplification NKT cells
Method.
Background technology
Classical Immunology thinks that the lymphocyte of body has three kinds：T cell, B cell and NK (Natural
Killer) cell, and NKT (Natural Killer T) cell is the 4th kind of lymphocyte of later discovery.NKT cells are one
Group's existing φt cell receptor of cell surface (T cell receptor, TCR), there is the special T cell subgroup of NK cell receptors again.
NKT cells are distributed mainly in liver, marrow, thymus gland, spleen and peripheral blood, also there is NKT cells in Cord blood.
The biological function of NKT cells mainly includes immunological regulation and cytotoxic effect, after NKT cells are upset,
Substantial amounts of interleukin 4 (interleukin-4, IL-4), gamma interferon (Interferon- γ, IFN- can be secreted
γ), granulocyte-macrophage colony stimutaing factor (granulocyte-macrophage colony stimulating
Factor, GM-CSF), IL-13 (interleukin-13, IL-13) and other cell factors and chemotactic factor (CF), hair
Immunoregulation effect is waved, NKT cells are one of bridges for contacting inherent immunity and acquired immunity.Have after NKT cell activations
Cytotoxicity, target cell is can dissolve, main effects molecule is perforin, FasL and IFN-γ.NKT cells, which are used as, to be had by force
A kind of cell mass of lethality, there is the antitumor action of wide spectrum, have begun to be widely used in clinical treatment, and toxicity
It is relatively low.The NKT cells obtained through in vitro culture have good antitumor activity and health-care effect.
The conventional method of culture NKT cells is in PMNC (peripheral blood at present
Mononuclear cell, PBMC) culture medium in add activating antibodies and cell factor, obtained greatly by the culture of 2-3 weeks
The NKT cells of amount.But the NKT cell yields that conventional culture methods obtain are relatively low, there is substantial amounts of T cell inside immunocyte.
The purity that the presence of a large amount of T cells not only reduces NKT cells also have impact on the propagation of NKT cells, so need to improve at present
The technique of existing culture NKT cells.
The content of the invention
It is an object of the invention to provide a kind of efficient activation and the new method of amplification NKT cells, to solve conventional NKT cells
Yielded poorly existing for cultural method with purity it is not high the problem of.
It is including following the invention provides a kind of efficient activation and the new method of amplification NKT cells to reach above-mentioned purpose
Step：
Step 1：PBMC cells are isolated from peripheral blood；
Step 2：By the PBMC cells isolated after culture medium is resuspended, it is transferred to fine through anti-CD3antibody and recombined human
In the culture vessel that even albumen was coated with, and cultivated after adding IFN-γ into culture medium；
Step 3：Vitamin C, human serum albumins, interleukin 2 are continuously added into culture medium
Continue to cultivate after (interleukin-2, IL-2) and OK432, obtain NKT cells.
The new method of above-mentioned efficient activation and amplification NKT cells, wherein, in step 2, the anti-CD3antibody and
Recombinant human fibronectin polypeptide is coated with after being prepared by culture medium to culture vessel.
Above-mentioned efficient activation and the new method of amplification NKT cells, wherein, the concentration of the anti-CD3antibody is 1-40 μ
g/ml；The concentration of the recombinant human fibronectin polypeptide is 1-10 μ g/ml.
Above-mentioned efficient activation and the new method of amplification NKT cells, wherein, in step 2, the coated time is 8-
24h, temperature conditionss are 0-10 DEG C.
Above-mentioned efficient activation and the new method of amplification NKT cells, wherein, in step 2, the final concentration of the IFN-γ
For 1-1000U/ml.
Above-mentioned efficient activation and the new method of amplification NKT cells, wherein, in step 3, the ascorbic end is dense
Spend for 1-10 μ g/ml；The final concentration of 0.5-10mg/ml of the human serum albumins；The final concentration of 1- of the IL-2
1000U/ml；The final concentration of 1-10 μ g/ml of the OK432.
Above-mentioned efficient activation and the new method of amplification NKT cells, wherein, in step 3, according to the growth feelings of cell
Condition, every 1-3 days supplemented mediums, vitamin C, human serum albumins and IL-2.
Above-mentioned efficient activation and the new method of amplification NKT cells, wherein, after adding every time, cell, which is transferred to, not to be had
Cultivated in the culture vessel being coated with by anti-CD3antibody and recombinant human fibronectin polypeptide.
Above-mentioned efficient activation and the new method of amplification NKT cells, wherein, the time cultivated described in step 2 is 12-
48h；The time cultivated described in step 3 is 12-30 days.
Above-mentioned efficient activation and the new method of amplification NKT cells, wherein, the culture medium is trained for X-VIVO15 serum-frees
Support base.
Relative to prior art, the invention has the advantages that：
(1) natural NKT cells are contained inside PBMC cells, ratio is probably in 2-3%.The present invention is advantageous to by manufacture
The condition of culture of NKT cells propagation, allows NKT cells to breed as much as possible.As a result show, the present invention can significantly improve NKT
The purity of cell and and yield；And NKT cultural methods provided by the invention are simple to operate, spent cost is also than relatively low.
(2) present invention uses CD3 antibody and fibronectin in the initial period of culture NKT cells, and this can be largely
Raising NKT cells induced efficiency, beneficial to the amplification of NKT cells.
(3) present invention adds IFN-γ and OK432 inside the culture medium of NKT cells, can significantly increase NKT cells
Killing ability, and OK432 can significantly promote the multiplication capacity of NKT cells.
Brief description of the drawings
Fig. 1 be it is different effect targets than NKT cells against tumor cells killing-efficiency schematic diagram.
Embodiment
Below in conjunction with accompanying drawing, by specific embodiment, the invention will be further described, and these embodiments are merely to illustrate
The present invention, it is not limiting the scope of the invention.
The invention provides a kind of efficient activation and the new method of amplification NKT cells, comprise the following steps：
Step 1：PBMC cells are isolated from peripheral blood；
Step 2：By the PBMC cells isolated after culture medium is resuspended, it is transferred to fine through anti-CD3antibody and recombined human
Connect in the culture vessel that albumen was coated with, and increase the toxicity of NKT cells after addition IFN-γ into culture medium, cultivated
12-48h；
Step 3：Continue to cultivate 12- after continuously adding vitamin C, human serum albumins, IL-2 and OK432 into culture medium
30 days, obtain NKT cells.
In step 2, culture vessel is carried out after the anti-CD3antibody and recombinant human fibronectin polypeptide are prepared by culture medium
Coating.The concentration of the anti-CD3antibody is 1-40 μ g/ml；The concentration of the recombinant human fibronectin polypeptide is 1-10 μ g/ml.Institute
It is 8-24h to state the coated time, and temperature conditionss are 0-10 DEG C.The final concentration of 1-1000U/ml of the IFN-γ.The culture
Base is the serum free mediums of X-VIVO 15.
In step 3, the ascorbic final concentration of 1-10 μ g/ml；The human serum albumins it is final concentration of
0.5-10mg/ml；The final concentration of 1-1000U/ml of the IL-2；The final concentration of 1-10 μ g/ml of the OK432.According to thin
The growing state of born of the same parents, every 1-3 days supplemented mediums, vitamin C, human serum albumins and IL-2.After adding every time, by cell
It is transferred in the culture vessel not being coated with by anti-CD3antibody and recombinant human fibronectin polypeptide and cultivates.The culture medium is X-
The serum free mediums of VIVO 15.
Above-mentioned efficient activation and the new method of amplification NKT cells are illustrated below by way of a specific embodiment：
First, PBMC cells are separated
First, 10 milliliters of peripheral bloods are separated.Human lymphocyte separating liquid Ficoll-Hypaque, (density is in advance
1.077g/ml) equilibrate to room temperature.10ml Ficoll are slowly added into the sterile centrifugation tube of 50ml volumes, tilt centrifuge tube
45 degree, isometric peripheral blood is added slowly to above Ficoll at 1cm on from Ficoll liquid levels.Centrifuge tube is put into level
Centrifuge, with 400g centrifugation 30 minutes, also shut off the deceleration valve of centrifuge.
PBMC cellular layers among being drawn inside from centrifuge tube, are added in new 50ml centrifuge tubes.Determined with physiological saline
Hold to 40ml, piping and druming is uniform, and 400g is centrifuged 10 minutes.Supernatant is abandoned after centrifugation, 40ml physiological saline piping and druming cell is added and extremely mixes,
Centrifuged 10 minutes with 400g again, abandon supernatant and obtain PMNC.Cell is resuspended with 6-7ml serum free mediums,
Cell count simultaneously.
2nd, it is coated with blake bottle with anti-CD49d McAb and fibronectin
Anti-human CD3 monoclonal antibodies and recombinant human fibronectin polypeptide are diluted with serum free medium, contains 20 μ g/ml's 1.5 milliliters
CD3 monoclonal antibodies and the serum free medium of 5 μ g/ml recombinant human fibronectin polypeptides are added in T25 blake bottle.It is paved with liquid
Bottom of bottle, blake bottle is lain in 4 DEG C of refrigerators and is coated with overnight.
3rd, the incubation of NKT cells
PBMC cells are added in the blake bottle being coated, while the volume of nutrient solution is supplied to 10 milliliters；Then
IFN-γ is added, its final concentration is reached 1000U/ml.
After 24 hours, vitamin C, human serum albumins, OK432 and IL-2 are added toward cell the inside；Ensure vitamin C
Final concentration of 10 μ g/ml, human serum albumins final concentration of 0.5mg/ml, OK432 final concentration of 1 μ g/ml and IL-2
Final concentration of 1000U/ml.
For cell after incubator continues culture 2 days, the fresh serum-free media for adding 30-50% volumes (contains 10 μ
The IL-2 of g/ml vitamin C, 0.5mg/ml human serum albumins and 1000U/ml), cell is transferred to T75 blake bottle
In.Average every fluid infusion in two days once, with increasing for nutrient solution volume, then is transferred to cell on the blake bottle not being coated
Middle culture.One co-cultures 21 days, collects NKT cells.
4th, NKT cell counts and phenotypic analysis
Cell count is carried out to the PBMC cells being just separated to, and takes a small amount of cell to be used for flow cytometry analysis.Carefully
Born of the same parents are with anti-CD45 antibody, anti-cd 3 antibodies and anti-CD56 antibody stainings.
Cell count equally is carried out to the NKT cells of culture 21 days, and takes a small amount of cell to be used for flow cytometry analysis.
In order to examine the ability of the actual amplification NKT cells of the present invention, the peripheral blood of multiple Healthy Peoples is acquired in experiment
For testing, everyone takes out 10 milliliters of fresh peripheral bloods and is used to test.Table 1 is the single core of peripheral blood for cultivating 5 blood donors
Data obtained by cell.The average purity of NKT cells obtained by 5 groups of experiments is being averaged for 67.3 ± 8.2%, NKT cells
Amplification times are that 2923 ± 902,10 milliliters of peripheral blood average energies amplify (1.9 ± 0.68) × 109Individual NKT cells.
Interpretation of result of the PMNC of table 1. after culture
5th, the killing experiments of NKT cells against tumor cells
Hepatoma H22 cells culture is taken in exponential phase in the DMEM culture mediums containing 10% hyclone
HepG2 cell counts and for testing.
The culture medium used in whole killing experiments is the DMEM culture mediums of the hyclone containing 1% inactivation.Use
Kit is on-radiation citotoxicity detection kit (Promega, G1780).
HepG2 cells are made 1 × 10 with fresh culture medium5Individual/ml cell suspension, it is added to 96 orifice plates of round bottom
In, per the μ l of hole 100.A series of concentration of NKT cells is adjusted with same culture medium, 80 μ l NKT cells are thin according to effect
Born of the same parents：Target cell is 1:1、5:1、10:1、20:1、40:1 ratio is added in HepG2 cells；NKT cell controls are set simultaneously
With HepG2 cell controls groups, medium controls and HepG2 cell maximum release groups, the final volume correction per hole is 200 μ
L, three multiple holes are all provided with, 250g is centrifuged 4 minutes, and cell is placed on 12 hours of culture in 37 degree of incubator.
45 minutes before reaction terminates, 20 μ l lysates are added to target cell maximum release group.React after terminating from every
Individual hole siphons away 50 μ l cell conditioned mediums to another 96 new orifice plate, adds 50 μ l LDH enzyme reaction solutions, mixes 30 seconds, at room temperature
Avoid light place 30 minutes, 50 μ l terminate liquids are added, the OD values in each hole are measured with ELIASA.
Calculate the formula of NKT cell killing activities：Killing activity %=(experimental group OD value-NKT cell controls group OD values-
HepG2 cell controls group OD values)/(HepG2 cell maximum release group OD value-HepG2 cell controls group OD values) × 100%.
As a result as shown in figure 1, the NKT cells obtained by the present invention have very strong killing ability to HepG2 cells.When effect target
Than being 10：When 1, killing ratio is (59.9 ± 10) %；When effect target ratio is 40：When 1, killing ratio be (96.2 ±
2) %.
In summary, the present invention can significantly improve the purity and and yield of NKT cells；And NKT trainings provided by the invention
The method of supporting is simple to operate, and spent cost is also than relatively low.Connected in the initial period of culture NKT cells using CD3 antibody and fibre
Albumen, this can significantly improve NKT induced efficiency.Inside the culture medium of NKT cells add IFN-γ and
OK432, it can significantly increase the killing ability of NKT cells, and OK432 can significantly promote the multiplication capacity of NKT cells.
Although present disclosure is discussed in detail by above preferred embodiment, but it should be appreciated that above-mentioned
Description is not considered as limitation of the present invention.After those skilled in the art have read the above, for the present invention's
A variety of modifications and substitutions all will be apparent.Therefore, protection scope of the present invention should be limited to the appended claims.
Claims (10)
1. the new method of a kind of efficient activation and amplification NKT cells, it is characterised in that comprise the following steps：
Step 1：PBMC cells are isolated from peripheral blood；
Step 2：By the PBMC cells isolated after culture medium is resuspended, it is transferred to through the fine even egg of anti-CD3antibody and recombined human
In the white culture vessel being coated with, and cultivated after adding IFN-γ into culture medium；
Step 3：Continue to cultivate after continuously adding vitamin C, human serum albumins, IL-2 and OK432 into culture medium, obtain
NKT cells.
2. the new method of efficient activation as claimed in claim 1 and amplification NKT cells, it is characterised in that in step 2, institute
State anti-CD3antibody and recombinant human fibronectin polypeptide prepared by culture medium after culture vessel is coated with.
3. the new method of efficient activation as claimed in claim 2 and amplification NKT cells, it is characterised in that the anti-human CD3 resists
The concentration of body is 1-40 μ g/ml；The concentration of the recombinant human fibronectin polypeptide is 1-10 μ g/ml.
4. the new method of efficient activation as claimed in claim 1 and amplification NKT cells, it is characterised in that in step 2, institute
It is 8-24h to state the coated time, and temperature conditionss are 0-10 DEG C.
5. the new method of efficient activation as claimed in claim 1 and amplification NKT cells, it is characterised in that in step 2, institute
State the final concentration of 1-1000U/ml of IFN-γ.
6. the new method of efficient activation as claimed in claim 1 and amplification NKT cells, it is characterised in that in step 3, institute
State ascorbic final concentration of 1-10 μ g/ml；The final concentration of 0.5-10mg/ml of the human serum albumins；The IL-2's
Final concentration of 1-1000U/ml；The final concentration of 1-10 μ g/ml of the OK432.
7. the new method of efficient activation as claimed in claim 1 and amplification NKT cells, it is characterised in that in step 3, root
According to the growing state of cell, every 1-3 days supplemented mediums, vitamin C, human serum albumins and IL-2.
8. the new method of efficient activation as claimed in claim 7 and amplification NKT cells, it is characterised in that, will after adding every time
Cell is transferred in the culture vessel not being coated with by anti-CD3antibody and recombinant human fibronectin polypeptide and cultivated.
9. the new method of efficient activation as claimed in claim 1 and amplification NKT cells, it is characterised in that trained described in step 2
The foster time is 12-48h；The time cultivated described in step 3 is 12-30 days.
10. the new method of efficient activation as claimed in claim 1 and amplification NKT cells, it is characterised in that the culture medium is
The serum free mediums of X-VIVO 15.
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| CN114957441A (en) * | 2022-05-20 | 2022-08-30 | 南京涵台余生物科技有限公司 | Transgenic NK (natural killer) cell and application thereof in antitumor drugs |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109666639A (en) * | 2018-12-24 | 2019-04-23 | 浙江大学 | A kind of NK cell and preparation method thereof of killing activity enhancing |
| CN114957441A (en) * | 2022-05-20 | 2022-08-30 | 南京涵台余生物科技有限公司 | Transgenic NK (natural killer) cell and application thereof in antitumor drugs |
| CN114957441B (en) * | 2022-05-20 | 2023-05-09 | 江苏康合生物科技有限公司 | Transgenic NK cells and application thereof in antitumor drugs |
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