CN107400660B - A method of activation and amplification NKT cell - Google Patents
A method of activation and amplification NKT cell Download PDFInfo
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Abstract
The invention discloses a kind of methods of activation and amplification NKT cell, comprising the following steps: step 1: PBMC cell is isolated from peripheral blood；Step 2: by the PBMC cell isolated after culture medium is resuspended, being transferred in the culture vessel being coated with through anti-CD3antibody and recombinant human fibronectin polypeptide, and cultivated after IFN-γ is added into culture medium；Step 3: continuing to cultivate after continuously adding vitamin C, human serum albumins, IL-2 and OK432 into culture medium, obtain NKT cell.The present invention can significantly improve the purity and and yield of NKT cell；And NKT cultural method provided by the invention is easy to operate, spent cost is also relatively low.CD3 antibody and fibronectin are used in the initial phase of culture NKT cell, this can significantly improve the induced efficiency of NKT.IFN-γ and OK432 are added inside the culture medium of NKT cell, can significantly increase the killing ability of NKT cell, and OK432 can significantly promote the proliferative capacity of NKT cell.
Description
Technical field
The present invention relates to technical field of cell biology, and in particular to a method of activation and amplification NKT cell.
Background technique
Classical Immunology is thought there are three types of the lymphocytes of body: 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 cell is one
Group's existing T cell receptor of cell surface (T cell receptor, TCR), and have the special T cell subgroup of NK cell receptor.
NKT cell is distributed mainly in liver, marrow, thymus gland, spleen and peripheral blood, also there is NKT cell in Cord blood.
The biological function of NKT cell mainly includes immunological regulation and cytotoxic effect, after NKT cell is stimulated,
It can secrete a large amount of interleukin 4 (interleukin-4, IL-4), gamma interferon (Interferon- γ, IFN-
γ), granulocyte-macrophage colony stimutaing factor (granulocyte-macrophage colony stimulating
Factor, GM-CSF), interleukin-13 (interleukin-13, IL-13) and other cell factors and chemotactic factor (CF), hair
Immunoregulation effect is waved, NKT cell is connection one of inherent immunity and the bridge of acquired immunity.Have after NKT cell activation
Cytotoxicity can dissolve target cell, and main effects molecule is perforin, FasL and IFN-γ.NKT cell, which is used as, to be had by force
A kind of cell mass of lethality, the antitumor action with wide spectrum, has begun and is widely used in clinical treatment, and toxicity
It is lower.The NKT cell obtained through in vitro culture has good anti-tumor activity and health-care effect.
The conventional method of culture NKT cell is in peripheral blood mononuclear cells (peripheral blood at present
Mononuclear cell, PBMC) culture medium in activating antibodies and cell factor is added, obtained greatly by culture in 2-3 weeks
The NKT cell of amount.But the NKT cell yield that conventional culture methods obtain is relatively low, has a large amount of T cell inside immunocyte.
The purity that the presence of a large amount of T cells not only reduces NKT cell also affects the proliferation of NKT cell, so needing to improve at present
The technique of existing culture NKT cell.
Summary of the invention
The object of the present invention is to provide a kind of methods of activation and amplification NKT cell, to solve the routine cell culture side NKT
The not high problem of low output and purity existing for method.
In order to achieve the above objectives, the present invention provides a kind of methods of activation and amplification NKT cell, comprising the following steps:
Step 1: PBMC cell is isolated from peripheral blood；
Step 2: by the PBMC cell isolated after culture medium is resuspended, being transferred to fine through anti-CD3antibody and recombined human
In the culture vessel that even albumen was coated with, and cultivated after IFN-γ is added 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, obtains NKT cell.
Above-mentioned activation and the method for expanding NKT cell, wherein in step 2, the anti-CD3antibody and recombined human
Fibronectin is coated with culture vessel after being prepared by culture medium.
Above-mentioned activation and the method for expanding NKT cell, 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 activation and the method for expanding NKT cell, wherein in step 2, the coated time is 8-24h, temperature
Degree condition is 0-10 DEG C.
Above-mentioned activation and the method for expanding NKT cell, wherein in step 2, the final concentration of 1- of the IFN-γ
1000U/ml。
Above-mentioned activation and the method for expanding NKT cell, wherein in step 3, the ascorbic final concentration of 1-
10μg/ml；The final concentration of 0.5-10mg/ml of the human serum albumins；The final concentration of 1-1000U/ml of the IL-2；Institute
State the final concentration of 1-10 μ g/ml of OK432.
Above-mentioned activation and the method for expanding NKT cell, wherein in step 3, according to the growing state of cell, every
1-3 days supplemented mediums, vitamin C, human serum albumins and IL-2.
Above-mentioned activation and the method for expanding NKT cell, wherein after adding every time, cell is transferred to not anti-human
It is cultivated in the culture vessel that CD3 antibody and recombinant human fibronectin polypeptide were coated with.
Above-mentioned activation and the method for expanding NKT cell, wherein the time of culture described in step 2 is 12-48h；Step
The time of culture described in 3 is 12-30 days.
Above-mentioned activation and the method for expanding NKT cell, wherein the culture medium is 15 serum free medium of X-VIVO.
Compared with the existing technology, the invention has the following advantages:
(1) contain natural NKT cell inside PBMC cell, ratio is probably in 2-3%.The present invention is conducive to by manufacture
The condition of culture of NKT cell Proliferation allows NKT cell to be proliferated as much as possible.The result shows that the present invention can significantly improve NKT
The purity of cell and and yield；And NKT cultural method provided by the invention is easy to operate, spent cost is also relatively low.
(2) present invention uses CD3 antibody and fibronectin in the initial phase of culture NKT cell, this can be largely
Raising NKT cell induced efficiency, conducive to the amplification of NKT cell.
(3) IFN-γ and OK432 is added in the present invention inside the culture medium of NKT cell, can significantly increase NKT cell
Killing ability, and OK432 can significantly promote the proliferative capacity of NKT cell.
Detailed description of the invention
Fig. 1 is the killing-efficiency schematic diagram of the NKT cells against tumor cells of different effect target ratios.
Specific embodiment
Below in conjunction with attached drawing, by specific embodiment, the invention will be further described, these embodiments are merely to illustrate
The present invention is not limiting the scope of the invention.
The present invention provides a kind of methods of activation and amplification NKT cell, comprising the following steps:
Step 1: PBMC cell is isolated from peripheral blood；
Step 2: by the PBMC cell isolated after culture medium is resuspended, being 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 cell after addition IFN-γ into culture medium, is cultivated
12-48h；
Step 3: continuing to cultivate 12- after continuously adding vitamin C, human serum albumins, IL-2 and OK432 into culture medium
30 days, obtain NKT cell.
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
Stating the coated time is 8-24h, and temperature condition is 0-10 DEG C.The final concentration of 1-1000U/ml of the IFN-γ.The culture
Base is 15 serum free medium of X-VIVO.
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 to and is not cultivated in culture vessel that anti-CD3antibody and recombinant human fibronectin polypeptide were coated with.The culture medium is X-
15 serum free medium of VIVO.
The method for illustrating above-mentioned activation below by way of a specific embodiment and expanding NKT cell:
One, PBMC cell is separated
Firstly, 10 milliliters of peripheral bloods of separation.Human lymphocyte separating liquid Ficoll-Hypaque, (density is in advance
1.077g/ml) equilibrate to room temperature.10ml Ficoll is slowly added into the sterile centrifugation tube of 50ml volume, tilts centrifuge tube
45 degree, isometric peripheral blood is added slowly to above Ficoll at 1cm on from Ficoll liquid level.Centrifuge tube is put into level
Centrifuge is centrifuged 30 minutes with the speed of 400g, also shuts off the deceleration valve of centrifuge.
Intermediate PBMC cellular layer is drawn inside from centrifuge tube, is added in new 50ml centrifuge tube.It is fixed with physiological saline
Hold to 40ml, uniformly, 400g is centrifuged 10 minutes for piping and druming.Supernatant is abandoned after centrifugation, is added 40ml physiological saline piping and druming cell and is extremely mixed,
Again with 400g centrifugation 10 minutes, abandons supernatant and obtain peripheral blood mononuclear cells.Cell is resuspended with 6-7ml serum free medium,
Cell count simultaneously.
Two, culture bottle is coated with anti-CD49d McAb and fibronectin
Anti-human CD3 monoclonal antibody and recombinant human fibronectin polypeptide are diluted with serum free medium, contains 20 μ g/ml's 1.5 milliliters
CD3 monoclonal antibody and the serum free medium of 5 μ g/ml recombinant human fibronectin polypeptides are added in the culture bottle of a T25.It is paved with liquid
Bottom of bottle lies in culture bottle in 4 DEG C of refrigerators and is coated with overnight.
Three, the incubation of NKT cell
PBMC cell is added in the culture bottle being coated, while the volume of culture solution is supplied to 10 milliliters；Then
IFN-γ is added, its final concentration is made to reach 1000U/ml.
After 24 hours, vitamin C, human serum albumins, OK432 and IL-2 is added toward cell the inside；Guarantee 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% volume (contains 10 μ
The IL-2 of the vitamin C of g/ml, the human serum albumins of 0.5mg/ml and 1000U/ml), cell is transferred to the culture bottle of T75
In.Average every fluid infusion in two days is primary, with increasing for nutrient solution volume, then cell is transferred to the culture bottle not being coated
Middle culture.One co-cultures 21 days, collects NKT cell.
Four, NKT cell count and phenotypic analysis
Cell count is carried out to the PBMC cell being just separated to, and takes a small amount of cell for flow cytometry analysis.Carefully
The anti-CD45 antibody of born of the same parents, anti-cd 3 antibodies and the dyeing of anti-CD56 antibody.
Cell count equally is carried out to the NKT cell of culture 21 days, and takes a small amount of cell for flow cytometry analysis.
In order to examine the ability of practical amplification NKT cell of the invention, the peripheral blood of multiple Healthy Peoples is acquired in experiment
For testing, everyone takes out 10 milliliters of fresh peripheral bloods for testing.Table 1 is the single core of peripheral blood for cultivating 5 blood donors
The obtained data of cell.The average purity of the obtained NKT cell of 5 groups of experiments is being averaged for 67.3 ± 8.2%, NKT cell
Amplification times are that 2923 ± 902,10 milliliters of peripheral bloods can averagely amplify (1.9 ± 0.68) × 109A NKT cell.
The interpretation of result of 1. peripheral blood mononuclear cells of table after cultivation
Five, the killing experiments of NKT cells against tumor cells
Hepatoma H22 cells culture takes in the DMEM culture medium containing 10% fetal calf serum in logarithmic growth phase
HepG2 cell count and for testing.
The culture medium used in entire killing experiments is the DMEM culture medium of the fetal calf serum containing 1% inactivation.It uses
Kit is on-radiation citotoxicity detection kit (Promega, G1780).
HepG2 cell is made 1 × 10 with fresh culture medium5The cell suspension of a/ml is added to 96 orifice plates of round bottom
In, every 100 μ l of hole.A series of concentration that NKT cells are adjusted with same culture medium, 80 μ l NKT cells are thin according to effect
Born of the same parents: the ratio of target cell 1:1,5:1,10:1,20:1,40:1 are added in HepG2 cell；NKT cell controls are set simultaneously
With HepG2 cell controls group, the final volume correction of medium controls and HepG2 cell maximum release group, every hole is 200 μ
L is all provided with three multiple holes, and 250g is centrifuged 4 minutes, 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.After reaction from every
A 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 value in each hole is measured with microplate reader.
Calculate the formula of NKT cell killing activity: killing activity %=(experimental group OD value-NKT cell controls group OD value-
HepG2 cell controls group OD value)/(HepG2 cell maximum release group OD value-HepG2 cell controls group OD value) × 100%.
As a result as shown in Figure 1, NKT cell obtained by the present invention has very strong killing ability to HepG2 cell.When effect target
When than being 10:1, killing ratio is (59.9 ± 10) %；When imitate target ratio be 40:1 when, killing ratio be (96.2 ±
2) %.
In conclusion the present invention can significantly improve the purity and and yield of NKT cell；And NKT training provided by the invention
The method of supporting is easy to operate, and spent cost is also relatively low.Connected in the initial phase of culture NKT cell using CD3 antibody and fibre
Albumen, this can significantly improve the induced efficiency of NKT.Inside the culture medium of NKT cell be added IFN-γ and
OK432 can significantly increase the killing ability of NKT cell, and OK432 can significantly promote the proliferative capacity of NKT cell.
It is discussed in detail although the contents of the present invention have passed through 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 above content, for of the invention
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 (3)
1. a kind of method of activation and amplification NKT cell, which comprises the following steps:
Step 1: PBMC cell is isolated from peripheral blood；
Step 2: by the PBMC cell isolated after culture medium is resuspended, being transferred to and connect egg through anti-CD3antibody and recombined human fibre
In the white culture vessel being coated with, and cultivated after IFN-γ is added into culture medium；The culture vessel uses 1.5 milliliters
CD3 monoclonal antibody containing 20 μ g/ml and the serum free medium of 5 μ g/ml recombinant human fibronectin polypeptides are coated with；The IFN-γ
Final concentration reaches 1000U/ml；
After step 3:24 hours, vitamin C, human serum albumins, IL-2 and OK432 are continuously added into culture medium, are guaranteed
Ascorbic final concentration of 10 μ g/ml, human serum albumins final concentration of 0.5mg/ml, OK432 final concentration of 1 μ g/ml
With the final concentration of 1000U/ml of IL-2；After cell continues culture 2 days, the serum free medium of 30-50% volume is added, it should
Serum free medium contains the vitamin C, the human serum albumins of 0.5mg/ml and the IL-2 of 1000U/ml of 10 μ g/ml；It is average
Every fluid infusion in two days is primary, with increasing for nutrient solution volume, then cell is transferred in the culture vessel not being coated and is trained
It supports；One co-cultures 21 days, obtains NKT cell.
2. the method for activation as described in claim 1 and amplification NKT cell, which is characterized in that in step 2, the coating
Condition are as follows: 4 DEG C coating overnight.
3. the method for activation as described in claim 1 and amplification NKT cell, which is characterized in that the culture medium is X-VIVO
15 serum free mediums.
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| CN102597223A (en) * | 2009-09-11 | 2012-07-18 | 宝生物工程株式会社 | Process for production of natural killer cells |
| EP2666466A1 (en) * | 2011-01-21 | 2013-11-27 | Biotherapy Institute Of Japan | Process for production of nk-cell-enriched blood preparation |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102597223A (en) * | 2009-09-11 | 2012-07-18 | 宝生物工程株式会社 | Process for production of natural killer cells |
| EP2666466A1 (en) * | 2011-01-21 | 2013-11-27 | Biotherapy Institute Of Japan | Process for production of nk-cell-enriched blood preparation |
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| Christoph Härtel等.Effects of vitamin C on intracytoplasmic cytokine productionin human whole blood monocytes and lymphocytes.《Cytokine》.2004,第27卷第101-106页，参见摘要、图1和2. * |
| Fibronectin promotes the proliferation of cytotoxic T lymphocytes generated from cancer patients;S Mizobata等;《British Journal of Cancer》;19961231;第74卷;第1598-1604页，参见全文 * |
| 不同细胞因子培养体系对CIK、NK及NKT细胞等诱导产率的影响;黎阳等;《中山大学学报（医学科学版）》;20030731;第24卷(第4期);第363-367页，具体参见摘要、第367页左栏第1段 * |
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